Gianluigi Arrigoni

The C-C chemokine receptors CCR4 and CCR8 identify airway T cells of allergen-challenged atopic asthmatics

In vitro polarized human Th2 cells preferentially express the chemokine receptors CCR3, CCR4, and CCR8 and migrate to their ligands: eotaxin, monocyte-derived chemokine (MDC), thymus- and activation-regulated chemokine (TARC), and I-309. We have studied the expression of chemokines and chemokine receptors in the airway mucosa of atopic asthmatics. Immunofluorescent analysis of endobronchial biopsies from six asthmatics, taken 24 hours after allergen challenge, demonstrates that virtually all T cells express IL-4 and CCR4. CCR8 is coexpressed with CCR4 on 28% of the T cells, while CCR3 is expressed on eosinophils but not on T cells. Expression of the CCR4-specific ligands MDC and TARC is strongly upregulated on airway epithelial cells upon allergen challenge, suggesting an involvement of this receptor/ligand axis in the regulation of lymphocyte recruitment into the asthmatic bronchi. In contrast to asthma, T cells infiltrating the airways of patients with chronic obstructive pulmonary disease and pulmonary sarcoidosis produce IFN-gamma and express high levels of CXCR3, while lacking CCR4 and CCR8 expression. These data support the role of CCR4, of its ligands MDC and TARC, and of CCR8 in the pathogenesis of allergen-induced late asthmatic responses and suggest that these molecules could be considered as targets for therapeutic intervention.

Lack of CD 29 (β1 integrin) and CD 54 (ICAM-1) adhesion molecules in intravascular lymphomatosis*

Intravascular Lymphomatosis (IL) is a rare and usually aggressive form of non-Hodgkins lymphoma characterized by the growth of neoplastic cells within vascular lumina that usually presents with skin or central nervous system (CNS) involvement. The mechanism(s) for the selective intravascular growth of this neoplasm remain(s) unexplained. We now report clinical and immunohistologic data on surgical material from 6 cases of IL; in 4 of 6 cases, autopsies were performed. Our IL cases shared the following features: (1) B-cell lineage; (2) lack of skin involvement at presentation; (3) aggressive behavior; and (4) lack of extravascular lymphomatous masses; in addition, 1 case had an associated gastric low-grade MALT lymphoma. We studied by immunohistochemistry formalin-fixed, paraffin-embedded sections with monoclonal antibodies to molecules known to be involved in lymphocyte and endothelial adhesion phenomena, that is, CD29 (beta1 integrin subunit), CD43 (leukosialin), CD44 (H-CAM), CD54 (ICAM-1), embryonal N-CAM (e-NCAM), and EMA (episialin). In all cases, the surfaces of IL aggregates reacted for CD44 but were consistently negative for CD29; also absent was CD54. Conversely, the integrity of the endothelial cells was underscored by their even reactivity for CD29, CD44, and CD54. Given that CD29 is currently regarded as critical for lymphocyte trafficking in general and for transvascular migration in particular, and CD54 is also involved in transvascular lymphocyte migration, we conclude that their consistent absence in IL may contribute to its intravascular and disseminated distribution pattern. The rather frequent association of IL with various conventional lymphomas is known; yet, one of our cases appears to be the first report of IL associated with a low-grade MALT lymphoma.

Prune cAMP phosphodiesterase binds nm23-H1 and promotes cancer metastasis.

We identify a new enzymatic activity underlying metastasis in breast cancer and describe its susceptibility to therapeutic inhibition. We show that human prune (h-prune), a phosphoesterase DHH family appertaining protein, has a hitherto unrecognized cyclic nucleotide phosphodiesterase activity effectively suppressed by dipyridamole, a phosphodiesterase inhibitor. h-prune physically interacts with nm23-H1, a metastasis suppressor gene. The h-prune PDE activity, suppressed by dipyridamole and enhanced by the interaction with nm23-H1, stimulates cellular motility and metastasis processes. Out of 59 metastatic breast cancer cases analyzed, 22 (37%) were found to overexpress h-prune, evidence that this novel enzymatic activity is involved in promoting cancer metastasis.

Proliferating cell nuclear antigen expression in central nervous system neoplasms

Proliferating cell nuclear antigen (PCNA) is a cell-cycle-regulated protein, which can be demonstrated in routinely fixed specimens. Studies on various tissues, cell cultures and neoplasms have shown that PCNA labelling index (LI) correlates with flow cytometry, tritiated thymidine LI, bromodeoxyuridine (BrdU) incorporation and Ki67 LI. PCNA LI may have prognostic value in various neoplasms. The present study concerns PCNA immunostaining in a series of neuroglial tumours. We demonstrate that there is a relation between PCNA LI and histological grade, and between PCNA LI and reported thymidine LI, BrdU LI and Ki67 LI. Pleomorphic xanthoastrocytomas and lowgrade astrocytomas had the lowest LI, whereas metastases of small cell lung cancer and medulloblastomas had the highest LI. Glioblastomas sometimes showed a certain degree of intratumoral heterogeneity of distribution of immunostained cells. Intratumoral heterogeneity underscores the critical importance of representative sampling of central nervous system neoplasms for kinetic studies. As expected, PCNA LI are somewhat higher than tritiated thymidine LI, BrdU LI and Ki67 LI because PCNA is a marker of G1, S, G2 and M-phases of the cell cycle and not of S-phase only. In addition, because of its long half-life, PCNA may be detected immunohistochemically in cells that have recently left the cell cycle. The immunohistochemical evaluation of PCNA LI is easy to perform on routinely processed material, allowing retrospective studies. PCNA LI may be a useful tool in grading gliomas. However, its prognostic value must be validated by comparing PCNA LI with the follow-up of the neoplasms, and possibly with the responsiveness to anti-proliferative therapy.

Independent value of fascin immunoreactivity for predicting lymph node metastases in typical and atypical pulmonary carcinoids

Immunoreactivity for fascin, an actin-bundling protein related to cell motility, has been reported in breast, ovary, pancreas, skin, and non-small cell carcinomas, and associated with more advanced disease stage and poorer prognosis. Data on pulmonary neuroendocrine (NE) tumors, however, are lacking. We evaluated the expression of fascin by immunohistochemistry--using two different monoclonal antibodies--in surgical specimens of pulmonary NE tumors of all the diverse histological types from 128 consecutive patients recruited between 1987 and 2001, and investigated its relationship with the presence of lymph node metastases. Overall, fascin immunoreactivity was detected in 5% of 38 typical carcinoids (TC), 35% of 23 atypical carcinoids (AC), 83% of 40 large-cell neuroendocrine carcinomas (LCNEC), and 100% of 27 small-cell lung carcinomas (SCLC) (P<0.001), Normal NE cells or hyperplastic NE tumorlets were consistently unreactive. No statistically significant differences in fascin immunoreactivity were found between the two antibodies. In TC and AC but not high-grade NE tumors, fascin immunoreactivity closely correlated with the occurrence of lymph node metastases, the pN class and the number of involved lymph nodes (P<0.001). It was also significantly associated with an increased proliferative activity (Ki-67 labeling index >5%) (P=0.020), and with either down-regulation or altered subcellular compartmentalization of E-cadherin (P<0.001) and CD99 (P=0.030), two cell adhesion complexes in pulmonary NE tumors. At multivariate analysis, only fascin emerged as an independent predictor of lymph node metastases in this tumor group (HR 30.28; 95% confidence intervals: 1.59-574.49; P=0.023). This study indicates that fascin immunoreactivity may identify subsets of pulmonary carcinoid patients with different metastatic potential to regional lymph nodes. Targeting the fascin pathway could be a novel therapeutic strategy of pulmonary carcinoids.

Aberrant methylation in the promoter region of the reduced folate carrier gene is a potential mechanism of resistance to methotrexate in primary central nervous system lymphomas

We investigated the prevalence and prognostic role of CpG island methylation of the reduced folate carrier (RFC) gene promoter region in primary central nervous system lymphoma (PCNSL) in immunocompetent patients. Genomic DNA from 40 PCNSL was used for methylation‐specific polymerase chain reaction and bisulphite genomic sequencing of the RFC promoter region. Human immunodeficiency virus‐negative systemic diffuse large B‐cell lymphomas (DLBCL) were used as controls (n = 50). The impact on outcome of RFC promoter methylation was assessed in 37 PCNSL patients treated with high‐dose methotrexate (HD‐MTX)‐based chemotherapy ± radiotherapy. RFC promoter methylation occurred in 12 of 40 (30%) PCNSL and in four of 50 (8%) DLBCL (P = 0·01). Of 37 PCNSL treated with HD‐MTX‐based chemotherapy, methylation occurred in nine cases (24%, M‐PCNSL), while 28 cases (76%, U‐PCNSL) were negative. Three M‐PCNSL (33%) and 15 U‐PCNSL (54%) achieved complete remission (CR) after primary chemotherapy. Logistic regression confirmed the independent association between CR rate and International Extranodal Lymphoma Study Group score (P = 0·03), RFC promoter methylation (P = 0·07) and use of cytarabine (P = 0·08). The 3‐year failure‐free survival (FFS) and overall survival for M‐PCNSL and U‐PCNSL was 0% vs. 31 ± 9% (P = 0·34) and 0% vs. 31 ± 9% (P = 0·35) respectively. This is the first study to assess the methylation status of the RFC promoter in human tumour samples. RFC methylation is more common in PCNSL compared with systemic DLBCL, and is associated with a lower CR rate to HD‐MTX‐based chemotherapy. If confirmed in prospective trials on PCNSL treated with HD‐MTX alone, these data may suggest the necessity for alternative strategies in M‐PCNSL considering the increased risk of MTX resistance by tumour cells.

Natural history of imatinib-naive GISTs: a retrospective analysis of 929 cases with long-term follow-up and development of a survival nomogram based on mitotic index and size as continuous variables.

Gastrointestinal stromal tumor (GIST) natural history per se has not been extensively investigated yet, with most data being drawn from large studies with a relevant referral bias. Hence, the estimation of prognosis still remains a critical issue. We retrospectively evaluated 929 GISTs resected between 1980 and 2000 in 35 Italian institutions. A total of 526 patients were found to be suitable for refining risk assessment through the development of a survival nomogram. Median follow-up was 126 months. On testing for potential prognostic parameters, age, tumor site, size, and mitotic index proved to be predictors of OS on both univariable and multivariable Cox model analyses, whereas necrosis and cytonuclear atypia were significant on univariable analysis only. The discriminative ability of the model, including the parameters selected after a backward procedure (C=0.72), improved compared with the National Institutes of Health 2002 (C=0.64) and the National Comprehensive Cancer Network 2007 (C=0.63). On the basis of these data we developed a prognostic nomogram for survival that considers site, size, and mitotic index as continuous variables, providing estimates stratified for patients aged ⩽65 and >65 years. This nomogram is a tool based on survival. It overcomes problems that result from artificial categorization of continuous variables. We believe that in the future this should also be attempted by nomograms based on the risk of relapse.

Amplification and overexpression of PRUNE in human sarcomas and breast carcinomas - a possible mechanism for altering the nm23-H1 activity

PRUNE, the human homologue of the Drosophila gene, is located in 1q21.3, a region highly amplified in human sarcomas, malignant tumours of mesenchymal origin. Prune protein interacts with the metastasis suppressor nm23-H1, but shows impaired affinity towards the nm23-H1 S120G mutant associated with advanced neuroblastoma. Based on these observations, we previously suggested that prune may act as a negative regulator of nm23-H1 activity. We found amplification of PRUNE in aggressive sarcoma subtypes, such as leiomyosarcomas and malignant fibrous histiocytomas (MFH) as well as in the less malignant liposarcomas. PRUNE amplification was generally accompanied by high mRNA and moderate to high protein levels. The sarcoma samples expressed nm23-H1 mostly at low or moderate levels, whereas mRNA and protein levels were moderate to high in breast carcinomas. For the more aggressive sarcoma subtypes, 9/13 patients with PRUNE amplification developed metastases. A similar situation was observed in all breast carcinomas with amplification of PRUNE. Infection of NIH3T3 cells with a PRUNE recombinant retrovirus increased cell proliferation. Possibly, amplification and overexpression of PRUNE has the same effect in the tumours. We suggest that amplification and overexpression of PRUNE could be a mechanism for inhibition of nm23-H1 activity that affect the development or progression of these tumours.

p53 protein expression in central nervous system neoplasms.

AIMS: To demonstrate, immunohistochemically, p53 protein expression in a selection of central nervous system tumours; to investigate the relation between p53 expression and that of the proliferation related antigen, PCNA. METHODS: Surgical specimens from 86 central nervous system tumours were routinely fixed, paraffin wax embedded, and immunostained with a monoclonal (PAb 1801) and a policlonal antibody (CM1) p53 protein and a monoclonal antibody against PCNA (PC10). Normal brain samples obtained at necropsy and 10 surgically obtained samples of gliotic brain parenchyma were also immunostained. RESULTS: p53 protein expression was observed in 35 of 86 brain tumours, suggesting frequent p53 gene mutation. p53 protein alterations were associated with all grades of malignancy in tumours displaying solely astrocytic differentiation, with the exception of pilocytic astrocytomas. In those showing oligodendroglial or ependymal differentiation they appeared to be restricted almost to only high grade lesions. No p53 immunoreactivity was observed in normal or gliotic brain tissue; p53 altered expression was not related to the percentage of PCNA labelled cells. CONCLUSIONS: The use of sophisticated gene amplification techniques or highly sensitive immunohistochemical methods might be useful in distinguishing between reactive and neoplastic astrocytic lesions, and in the identification of malignant progression in other non-astrocytic glial tumours. Tumours with very similar histogenetic differentiation features might actually be a genetically heterogeneous group with possible different clinical courses.

Identification of CXCL13 as a new marker for follicular dendritic cell sarcoma

The homeostatic chemokine CXCL13 is preferentially produced in B‐follicles and is crucial in the lymphoid organ development by attracting B‐lymphocytes that express its selective receptor CXCR5. Follicular dendritic cells (FDCs) have been identified as the main cellular source of this chemokine in lymphoid organs. Recently, genome‐wide approaches have suggested follicular CD4 T‐helper cells (THF) as additional CXCL13 producers in the germinal centre and the neoplastic counterpart of THF (CD4+ tumour T‐cells in angioimmunoblastic T‐cell lymphoma) retains the capability of producing this chemokine. In contrast, no data are available on CXCL13 expression on FDC sarcoma (FDC‐S) cells. By using multiple approaches, we investigated the expression of CXCL13 at mRNA and protein level in reactive and neoplastic FDCs. In reactive lymph nodes and tonsils, CXCL13 protein is mainly expressed by a subset of FDCs in B‐cell follicles. CXCL13 is maintained during FDC transformation, since both dysplastic FDCs from 13 cases of Castlemans disease and neoplastic FDCs from ten cases of FDC‐S strongly and diffusely express this chemokine. This observation was confirmed at mRNA level by using RT‐PCR and in situ hybridization. Of note, no CXCL13 reactivity was observed in a cohort of epithelial and mesenchymal neoplasms potentially mimicking FDC‐S. FDC‐S are commonly associated with a dense intratumoural inflammatory infiltrate and immunohistochemistry showed that these lymphocytes express the CXCL13 receptor CXCR5 and are mainly of mantle zone B‐cell derivation (IgD+ and TCL1+). In conclusion, this study demonstrates that CXCL13 is produced by dysplastic and neoplastic FDCs and can be instrumental in recruiting intratumoural CXCR5+ lymphocytes. In addition to the potential biological relevance of this expression, the use of reagents directed against CXCL13 can be useful to properly identify the origin of spindle cell and epithelioid neoplasms. Copyright