Maurizio Lestani

Aberrant Wnt/β-Catenin Pathway Activation in Idiopathic Pulmonary Fibrosis

To investigate the molecular events that may underpin dysfunctional repair processes that characterize idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), we analyzed the expression patterns of β-catenin on 20 IPF/UIP lung samples, together with two downstream target genes of Wnt signaling, cyclin-D1, and matrilysin. In 18 of 20 cases of IPF/UIP investigated on serial sections, nuclear β-catenin immunoreactivity and abnormal levels of cyclin-D1 and matrilysin were demonstrated in proliferative bronchiolar lesions (basal-cell hyperplasia, squamous metaplasia, bronchiolization, honeycombing). The nature of these lesions was precisely defined using specific markers (ΔN-p63, surfactant-protein-A, cytokeratin-5). Interestingly, nuclear β-catenin accumulation was also demonstrated in fibroblast foci in most (16 of 20) IPF/UIP samples, often associated with bronchiolar lesions. Similar features were not observed in normal lung and other fibrosing pulmonary diseases (diffuse alveolar damage, organizing pneumonia, nonspecific interstitial pneumonia, desquamative interstitial pneumonia). Sequence analysis performed on DNA extracted from three samples of IPF/UIP did not reveal abnormalities affecting the β-catenin gene. On the basis of these findings new models for IPF/UIP pathogenesis can be hypothesized, centered on the aberrant activation of Wnt/β-catenin signaling, with eventual triggering of divergent epithelial regeneration at bronchiolo-alveolar junctions and epithelial-mesenchymal-transitions, leading to severe and irreversible remodeling of the pulmonary tissue.

Intravascular lymphoma: clinical presentation, natural history, management and prognostic factors in a series of 38 cases, with special emphasis on the 'cutaneous variant'.

Despite its recognition as a distinct, extremely rare entity, no large studies of intravascular lymphoma (IVL) have been reported. The clinico‐pathological characteristics of 38 human immunodeficiency virus‐negative patients with IVL diagnosed in Western countries were reviewed to better delineate clinical presentation, clinical variants, natural history and optimal therapy. The IVL is an aggressive and usually disseminated disease (Ann Arbor stage IV in 68% of cases) that predominantly affects elderly patients (median age 70 years, range: 34–90; male:female ratio 0·9), resulting in poor Eastern Cooperative Oncology Group Performance Status (ECOG‐PS >1 in 61%), B symptoms (55%), anaemia (63%) and high serum lactate dehydrogenase level (86%). The brain and skin are the most common sites of disease. In contrast to previous reports, hepatosplenic involvement (26%) and bone marrow infiltration (32%) were found to be common features in IVL, while nodal disease was confirmed as rare (11% of cases). Patients with disease limited to the skin (‘cutaneous variant’; 26% of cases) were invariably females with a normal platelet count, and exhibited a significantly better outcome than the remaining patients, which deserves further investigation. Overall survival was usually poor; however, the early use of intensive therapies could improve outcome in young patients with unfavourable features. ECOG‐PS >1, ‘cutaneous variant’, stage I and chemotherapy use were independently associated with improved survival.

bcl-2 EXPRESSION IN PLEURAL AND EXTRAPLEURAL SOLITARY FIBROUS TUMOURS

This study evaluated the immunoreactivity for bcl‐2, a molecule involved in the control of programmed cell death, in cases of pleural (14) and extrapleural (2) solitary fibrous tumour (SFT), malignant mesotheliomas of different histological types, and a variety of extrapleural CD34‐positive and CD34‐negative spindle‐cell tumours. In all SFTs, strong and diffuse immunostaining was demonstrated with anti‐bcl‐2 antibody, sharply contrasting with the complete lack of staining observed in all mesotheliomas. The specificity of immunodetection of bcl‐2 in SFT was confirmed by immunoblot analysis, showing a band consistent with the bcl‐2 protein. At extrapleural locations, strong bcl‐2 immunoreactivity was observed in Schwannoma (2/3 cases), synovial sarcoma (4/4 cases), and all cases of CD34‐positive gastrointestinal stromal tumour (GIST; 10/10 cases). Most sarcomas were bcl‐2‐negative. Lack of bcl‐2 expression was demonstrated in tumours which can pose problems in the differential diagnosis of SFT and can exhibit haemangiopericytoma‐like features, including haemangiopericytoma (3 cases), dermatofibrosarcoma protuberans (16 cases), and deep‐seated fibrous histiocytoma (3 cases). The constitutive expression of bcl‐2 in SFT widens the spectrum of available markers for these tumours, providing a useful adjunct to their differential diagnosis in difficult cases at pleural and extrapleural sites, and contributing to the understanding of their histogenesis and molecular pathogenesis.

Abnormal Re-epithelialization and Lung Remodeling in Idiopathic Pulmonary Fibrosis: The Role of ΔN-p63

Products of the p63 gene, a recently described member of the p53 family, are constitutively expressed in the basal cells of human bronchi and bronchioli. The truncated isoforms of the p63 gene (ΔN-p63 proteins) counteract the apoptotic and cell cycle inhibitory functions of p53 after DNA damage, and this property is likely to be central in the cell renewal strategy of stratified epithelial tissues. To investigate the dysfunctional repair processes that characterize idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), we immunohistochemically analyzed the expression of the transactivating and dominant-negative isoforms of the p63 gene on 16 tissue samples obtained from patients suffering from this disorder. In most IPF cases herein investigated, epithelial cells expressing ΔN-p63 were observed at sites of abnormal proliferation at the bronchiolo-alveolar junctions, characterized by epithelial hyperplasia, squamous metaplasia, bronchiolization, and abnormal p53 nuclear accumulation. Similar features were not observed in normal lung and in samples taken from other pulmonary diseases used as controls, including acute interstitial pneumonia, idiopathic bronchiolitis obliterans organizing pneumonia, nonspecific interstitial pneumonia, and desquamative interstitial pneumonia. On the basis of these findings, we can hypothesize a new model for UIP pathogenesis, involving a deregulated development of mesenchymal-epithelial interactions and abnormal proliferation of epithelial cells at the bronchiolo-alveolar junction after cell injury. In our view, the progressive loss of alveolar tissue and lung remodeling after injury in IPF/UIP is concomitantly produced by pneumocyte loss and alveolar collapse on one hand and by progressive bronchiolar proliferation and architectural distortion on the other.

Mediastinal large-cell lymphoma with sclerosis: Genotypic analysis establishes its B nature

The Southern blot hybridization technique has been applied to study the configuration of immunoglobulin and T-cell receptor genes in 6 cases of the so called mediastinal large cell lymphoma with sclerosis. This lymphoma has been recently recognized as a separate entity among non-Hodgkin lymphomas mainly affecting young adult patients. The B-cell origin of this neoplasm was suggested by means of immunohistochemical analysis. However, the immunophenotypical B-cell related markers used do not always exhibit lineage fidelity. The Southern blot analysis demonstrated the presence of unique heavy and k-light chain immunoglobulin gene rearrangements, establishing genotypically their B-cell origin.

Constitutive expression of ΔN-p63α isoform in human thymus and thymic epithelial tumours

Abstractp63, a member of the p53 family, is involved in the survival and differentiation of reserve/stem cells in different epithelia. To unveil the possible role of p63 in thymic physiology and pathology, we investigated the expression of p63 isoforms in normal thymus, thymomas and other mediastinal tumours. All samples were analysed using immunohistochemistry with three different antibodies: 4A4 antibody recognising all p63 isoforms, p40 antibody reacting only with truncated dominant-negative isoforms (ΔN-p63) and H-129 antibody recognising all alpha-isoforms. Reverse-transcription polymerase chain reaction (RT-PCR), and real-time PCR analyses were performed on RNA extracted from frozen samples of four thymomas and two primary-mediastinal large-B-cell lymphoma (PMLBCL). In normal thymus, ΔN-p63α was expressed in all cortical and medullary epithelial cells, with decreasing intensity in Hassalls corpuscles. This phenotype was conserved in neoplastic transformation since all 54 investigated thymomas (World Health Organization types A, AB, B1, B2, B3, C) expressed ΔN-p63α (virtually 100% cells). The predominance of ΔN-p63α isoform mRNA was confirmed by real-time PCR. Among other mediastinal tumours, ΔN-p63α was only expressed in those displaying either a stratified epithelial component (teratomas) or epidermoid differentiation (lung carcinoma). Among lymphomas, T-cell-precursor lymphomas did not express p63, whereas most PMLBCL expressed TA-p63α (7/8).

ZAP-70 expression, as detected by immunohistochemistry on bone marrow biopsies from early-phase CLL patients, is a strong adverse prognostic factor

Zeta-associated protein-70 (ZAP-70), mostly assessed by flow-cytometry (FC), recently emerged as reliable prognostic factor in chronic lymphocytic leukaemia (CLL) at presentation. We evaluated ZAP-70 expression in 156 CLL patients by immunohistochemistry (IHC) on formalin-fixed bone marrow (BM) biopsies at diagnosis. At presentation, 117 patients (75%) were with Binet stage A, 27 (17%) stage B and 12 (8%) stage C. Median follow-up was 61 months (range 6–242). ZAP-70 was expressed in neoplastic lymphocytes of 69 patients (44%). Concordance between ZAP-70 by IHC and ZAP-70 by FC, immunoglobulin heavy chain variable genes (IGHV) mutational status and CD38 expression was found in 41/46 (89%), 41/49 (80%) and in 60/88 (68%) tested cases, respectively. ZAP-70 expression significantly correlated with advanced Binet stage (B–C), diffuse BM infiltration, increased lactate dehydrogenase (LDH) and β2-microglobulin serum levels and lymphocyte doubling time <12 months. ZAP-70 positivity was significantly related to poorer time to progression (median 16 months vs 158 of ZAP-70-negative cases) (P<0.0001) and overall survival (median 106 months vs not reached) (P=0.0002); this correlation was confirmed at multivariate analysis. ZAP-70 expression correlated with poorer outcome also when evaluated only in the 117 stage A patients. In conclusion, immunohistological detection of ZAP-70 on formalin-fixed BM biopsies at diagnosis appears a useful methodological approach to identify patients with poor prognosis in CLL.

The impact of P53 and P21(waf1) expression on the survival of patients with the germinal center phenotype of diffuse large B-cell lymphoma

Purpose: To prospectively investigate the prognostic significance of p21 and p53 expression in diffuse large B-cell lymphoma in the context of the U.S. Intergroup trial comparing conventional cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy to rituximab-CHOP (R-CHOP) induction, with or without maintenance rituximab. Experimental Design: Immunohistochemical staining of 197 paraffin-embedded biopsy specimens was scored by an independent panel of experts. Results: The cyclin-dependent kinase inhibitor, p21, was expressed in 55% of cases examined. In a multivariable analysis adjusting for International Prognostic Index score and BCL2 status, p21 expression was a significant, independent, favorable predictive factor for failure-free survival (relative risk, 0.3; P = 0.001) and overall survival (relative risk, 0.3; P = 0.003) for patients treated with R-CHOP. Expression of p21 was not predictive of outcome for CHOP-treated patients. Only p21-positive cases benefited from the addition of rituximab to CHOP. Among p21-positive patients, treatment with R-CHOP was associated with a higher failure-free survival rate at 5 years compared with CHOP (61% versus 24%; P = 0.01). In contrast, no significant differences were detected in failure-free survival according to treatment arm for p21-negative patients. Expression of p53, alone or in combination with p21, did not predict for outcome in univariable or multivariable analyses. Conclusions: In this study, p21 protein expression emerged as an important independent predictor of a favorable clinical outcome when rituximab was added to CHOP therapy. These data suggest that rituximab-related effects on lymphoma survival pathways may be functionally linked to p21 activity. Clin Cancer Res; 16(8); 2435–42. ©2010 AACR.Immunohistochemically detected over-expression of P53-related protein (P53+++) and absence of P21(waf1) expression (P21-) correspond to loss of function of the P53-gene in diffuse large B-cell lymphoma (DLBCL) patients. Using immunohistochemistry we examined 80 patients with DLBCL and found that 23% had the P53+++/P21- phenotype while 51% had a germinal center (GC) pattern. Both the P53+++/P21- phenotype and the non-GC pattern were associated with inferior outcome. Notably, the prognostic power of the P53+++/P21- phenotype was restricted to patients with a GC pattern, without effect on outcome of patients with a non-GC phenotype. Our results show that immunohistochemistry can parallel gene expression profiling in addressing clinical variability of DLBCL patients.

The risk of false-positive histology according to the reason for colposcopy referral in cervical cancer screening: a blind revision of all histologic lesions found in the NTCC trial.

All cervical intraepithelial neoplasia (CIN) diagnoses identified during the New Technologies for Cervical Cancer trial (ISRCTN81678807) were blindly reviewed by 2 pathologists. Original diagnoses based on colposcopy-guided biopsies were compared with those made by the reviewers who had access to all clinical histologic samples (including postsurgical). Cases downgraded from CIN 2+ by the reviewers were considered indicative of unnecessary treatments. The analyses are presented according to the molecular (high-risk human papillomavirus [HPV]) and/or cytologic diagnosis used to refer the women for colposcopy. We reviewed 812 CIN 1 and 364 CIN 2 + diagnoses. The specificity of colposcopy-guided biopsy was 98% and the sensitivity, 84%. The probability of unnecessary treatment was 27% for women with atypical squamous cells of undetermined significance cytologic findings and 8% for women with low-grade squamous intraepithelial lesion or worse, 10% for HPV+ and positive cytologic findings, and 16% for HPV+ alone. The positive predictive value of the first-level screening test was inversely associated with probability of a histologic false-positive result (P = .015). In screening, a low positive predictive value of the colposcopy-referring test may result in unnecessary treatments.

Neoplastic epithelial cells in a subset of human thymomas express the B cell-associated CD20 antigen.

A series of 36 human thymomas have been immunohistochemically analyzed using a panel of antibodies recognizing B-cell markers including CD20. Most thymomas exhibiting the cortical pattern, according to the criteria of Marino and Muller-Hermelink, were characterized by areas of medullary differentiation containing variable numbers of CD20+ B lymphocytes, thus mimicking the medulla of normal thymus. On the other hand, B cells were absent or rare in thymomas recognized as mixed using the same morphological criteria. Surprisingly, we observed in most mixed thymomas variable numbers of CD20+ spindle cells, characterized by long slender processes. Using double-marker analysis we could demonstrate the epithelial nature of these cells (expression of keratin and lack of lymphoid and B-cell-related markers). The immunoreactivity of thymoma epithelial cells with L26, an antibody widely used in the characterization of B-cell lymphomas, can represent a drawback of practical relevance in the differential diagnosis of mediastinal tumors.