Emanuela Bonoldi

Prognostic and predictive value of tumour angiogenesis in ovarian carcinomas

Experimental studies suggest that angiogenesis plays an important role in the pathogenesis of ascites and progression of ovarian cancer. To evaluate the association of intratumoral microvessel density (IMD) with the conventional clinicopathologic features and to determine the capability of these factors in predicting responsiveness to platinum‐based chemotherapy and overall survival (OS) we studied 112 ovarian carcinomas. IMD was determined using the anti‐CD31 antibody and immunocytochemistry. In the entire series, we correlated IMD with the other features. In the subgroup of patients with FIGO stage III‐IV (60 cases), we correlated the factors studied, determined prior of treatment, with response to therapy and prognosis.

Angiogenesis Sustains Tumor Dormancy in Patients with Breast Cancer Treated with Adjuvant Chemotherapy

Experimental studies performed in Folkman laboratories suggest that angiogenesis is involved in the biology of tumor dormancy. We determined the vascular index in a series of 190 women operated of node-positive invasive breast cancer treated with adjuvant chemotherapy (CMF schedule) and we studied the relationship between vascularity of primary tumors with the behaviour in time of metastasis. The study of the hazard function of recurrence (in any site) was performed resorting to a generalized linear modelling approach with a binominal error according to Efron. A total of 80 cases developed recurrences during the period of observation. We found that the hazard function of metastasis in time presented two peaks of incidence at 20 and 60 months, respectively. We also plotted the curves of the hazard function by considering three values of microvessel counts corresponding to the quartiles of their distribution. The risk of first recurrence was associated with vascular index, and the patients of the third quartile of distribution of microvessels had the highest risk. In the final full model for the risk of recurrence at 5 years vascular index provided the highest prognostic contribution followed by the number of involved axillary lymph nodes. The observation that the patients with highly angiogenic tumors are at high risk of recurrence coupled with the identification of the second peak of incidence after 5 years which was also mainly sustained by angiogenic tumors suggest that a fraction of breast cancers promote metastasis after a period of tumor dormancy. The clinical and therapeutic implications of our results are discussed.

The combined evaluation ofp53 accumulation and of Ki-67 (MIBI) labelling index provides independent information on overall survival of ovarian carcinoma patients

BACKGROUND The prognostic implications of p53 accumulation, bcl-2 immunoreactivity and tumour proliferative fraction in ovarian carcinomas are still debated. PATIENTS AND METHODS One hundred twelve ovarian carcinomas were immunostained for p53 protein, for bcl-2 and for the cell cycle-associated Ki-67 antigen. The immunostaining results were correlated with conventional clinico-pathological variables, response to induction chemotherapy, and patient survival. RESULTS p53 accumulation and bcl-2 immunoreactivity in more than 10% of neoplastic cells were detected in 61 (54.5%) and 42 (37.5%) cases, respectively. A positive correlation between p53 accumulation and high (more than 30% neoplastic cells) MIB1 labelling index (r = 0.235; P = 0.015) was ascertained, whereas no significant association was found between bcl-2 immunoreactivity and p53 accumulation or MIB1 labeling index. Both p53 accumulation and MIB1 immunoreactivity correlated significantly with a reduced overall survival, but the association was lost in multivariate analysis. However, patients with tumours simultaneously showing p53 accumulation and MIB1 labelling index higher than 30% had significantly reduced overall survivals, in both univariate and multivariate analyses. CONCLUSION The simultaneous evaluation of p53 accumulation and MIB1 labelling index has independent prognostic implications in common epithelial malignancies of the ovary, irrespective of the disease stage.

Cutaneous CD30+ lymphoproliferative disorders: expression of bcl-2 and proteins of the tumor necrosis factor receptor superfamily

The spectrum of CD30+ cutaneous lymphoproliferative disorders is characterized by the histology of a high-grade lymphoma but frequent clinical regression of skin lesions in lymphomatoid papulosis (LyP) and occasional regression in CD30+ large cell lymphomas (LCLs). A recent study shows that apoptosis may be a significant mechanism of regression of LyP (Arch Dermatol 133:828-833, 1997). Therefore, we studied expression of proteins that induce apoptosis, including CD27, CD40, CD95, and nerve growth factor receptor (NGF-R), as well as anti-apoptotic protein bcl-2 in skin lesions from 25 patients within the spectrum of CD30+ cutaneous lymphoma. Our results show consistent expression of CD95 (APO-1/Fas), but rare or absent expression of CD27, CD40, and NGF-R on tumor cells from both regressing LyP lesions and nonregressing CD30+ lymphomas. Bcl-2 was expressed at low levels in LyP and at high levels in pleomorphic CD30+ lymphomas. These results indicate that, in addition to CD30, CD95 expression is preferentially expressed at high levels in all cutaneous CD30+ lymphomas and suggest that CD95 may play a role in the regression of CD30+ skin lesions. Expression of bcl-2 appears to protect tumor cells from apoptosis in CD30+ lymphoproliferative disorders.

Reproducibility of the WHO histological criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms

This study, performed on behalf of the Italian Registry of Thrombocythaemias (Registro Italiano Trombocitemie), aimed to test the inter-observer reproducibility of the histological parameters proposed by the WHO classification for the diagnosis of the Philadelphia chromosome-negative myeloproliferative neoplasms. A series of 103 bone marrow biopsy samples of Philadelphia chromosome-negative myeloproliferative neoplasms consecutively collected in 2004 were classified according to the WHO criteria as follows: essential thrombocythaemia (n=34), primary myelofibrosis (n=44) and polycythaemia vera (n=25). Two independent groups of pathologists reviewed the bone marrow biopsies. The first group was asked to reach a collegial ‘consensus’ diagnosis. The second group reviewed individually all the cases to recognize the main morphological parameters indicated by the WHO classification and report their results in a database. They were subsequently instructed to individually build a ‘personal’ diagnosis of myeloproliferative neoplasms subtype just assembling the parameters collected in the database. Our results indicate that high levels of agreement (≥70%) have been reached for about all of the morphological features. Moreover, among the 18 evaluated histological features, 11 resulted statistically more useful for the differential diagnosis among the different Philadelphia chromosome-negative myeloproliferative neoplasms. Finally, we found a high percentage of agreement (76%) between the ‘personal’ and ‘consensus’ diagnosis (Cohen’s kappa statistic >0.40). In conclusion, our results support the use of the histological criteria proposed by the WHO classification for the Philadelphia chromosome-negative myeloproliferative neoplasms to ensure a more precise and early diagnosis for these patients.

Increased expression of vascular endothelial growth factor receptor 1 correlates with VEGF and microvessel density in Philadelphia chromosome-negative myeloproliferative neoplasms

Aims The authors investigated vascular endothelial growth factor receptor 1 (VEGFR-1) protein expression in a series of Philadelphia chromosome-negative myeloproliferative neoplasms (Ph- MPNs) and its correlations with microvessel density (MVD) and vascular endothelial growth factor (VEGF). Methods 83 bone marrow biopsies of Ph- MPNs patients, including 27 essential thrombocythaemia (ET), 21 polycythaemia vera (PV) and 35 primary myelofibrosis (PMF), and 10 normal controls (NCs) were investigated by immunohistochemistry. Results Patients with PV and PMF showed an increased MVD (PV: 20.1±10.6; PMF: 25.8±6.5) compared with those with ET or NCs (ET: 10.4±4.6; NCs: 7±3.4). VEGFR-1 expression was increased in Ph- MPNs, particularly in PV and PMF (NCs: 0.07±0.03; ET: 0.15±0.09; PV: 0.31±0.2; PMF: 0.31±0.04). VEGF expression parallelled VEGFR-1 and resulted increased in Ph- MPNs (NCs: 0.08±0.04; ET: 0.13±0.06; PV: 0.29±0.2; PMF: 0.31±0.15) and higher in post-polycythaemic myelofibrosis and in the fibrotic stage of PMF than in the non-fibrotic phases of both diseases. VEGFR-1 protein expression correlated with MVD and VEGF expression in Ph- MPNs. VEGFR-1 and VEGF were expressed by the same bone marrow populations: megakaryocytes, macrophages and immature myeloid precursors showed a moderate to strong immunostaining intensity in both Ph- MPNs and NCs. The erythroid precursors were not immunoreactive. Conclusions VEGFR-1 and VEGF were increased and co-localised in megakaryocytes, macrophages and myeloid precursors of Ph- MPNs. This finding supports the hypothesis of a VEGF/VEGFR-1 autocrine loop in the neoplastic cells of Ph- MPNs.

MicroRNA as potential biomarker in HCV-associated diffuse large B-cell lymphoma

Aims To identify molecular characteristics to hepatitis C virus (HCV)-associated diffuse large B-cell lymphoma (DLBCL) through a comprehensive miRNAs expression profiling. Methods In this study, miRNA profiles were obtained from 37 patients with DLBCLs and 60 patients with reactive lymph nodes, equally distributed according to HCV presence. Germinal centres, from reactive lymph nodes were used as controls. Clinical features at presentation were available for all patients. Results A set of 52 miRNAs define a signature for HCV-associated DLBCL. Importantly, decreased expression of miR-138-5p and increased expression of miR-147a, miR-147b and miR-511-5p in HCV DLBCL was found to be a poor prognostic factor for HCV-positive DLBCL patients. Conclusions These data reveal molecular differences in diffuse DLBCL patients according to HCV presence, potentially useful as novel prognostic or therapeutic biomarkers.

Detection of Chlamydophila pneumoniae DNA in peripheral blood mononuclear cells of blood donors in the north-east of Italy

Abstract. Recent studies have implicated Chlamydia pneumoniae (now Chlamydophila pneumoniae) in the pathogenesis of atherosclerosis and demonstrated its presence within human peripheral blood mononuclear cells (PBMCs). In this study the presence of C. pneumoniae DNA was assessed, using nested PCR, in PBMCs from 169 active blood donors as a function of age, of specific antibodies and C-reactive protein. The results obtained demonstrated a high degree of global positivity (46.15%), which was higher in females (52%) than in males (43.7%). Seroepidemiological studies showed a high percentage of positivity both in subjects positive by PCR (65.91%) and negative by PCR (71.74%). The clinical implication of such finding are under study.

Detection of identical T-cell clonotype expansions in both the donor and recipient after allogeneic bone marrow transplantation

Using phenotypic, functional and molecular techniques, this study was performed to compare the complexity of the T‐cell receptor repertoire of a bone marrow transplanted patient with that of his HLA‐matched related donor, both of whom developed a chronic lymphocytosis sustained by CD3+CD8+CD57+CD16−CD56− granular lymphocytes 3 years after transplantation. Although Southern blot analysis revealed the presence of extra bands in both subjects, thus indicating the presence of at least one clonal T‐cell population, the study of the different T‐cell receptor Vβ (TCRBV) usage did not demonstrate discrete overexpression of any TCRBV segments. On the contrary, heteroduplex analysis of TCRBV transcripts suggested the presence of oligoclonal T‐cell expansions in the two subjects. Cloning and sequencing studies demonstrated that T‐cell clones expressing identical TCRBV chains were expanded both in the donor and in the recipient. Furthermore, clones with similar, but not identical, junctional regions were also found in the two subjects. These data indicate that, at the time of the graft, a few cells with a monoclonal/oligoclonal pattern that were present in the donor were transferred to the recipient, where they may have found the same environmental in vivo conditions and/or the antigenic pressure favouring their abnormal expansion.

Bone marrow localisation of metastatic melanoma and synchronous leukaemic evolution of low-risk myelodysplastic syndrome

Myelodysplastic syndromes (MDS) are haematopoietic stem-cell disorders characterised by peripheral cytopenia, dysplastic haematopoiesis, and a substantial risk of progression into acute myeloid leukaemia (AML). We report a case of low-risk MDS, according to the international prognostic scoring systems (IPSS), which evolved into AML simultaneously to metastatic bone marrow localisation of malignant melanoma. A 70-year-old man was diagnosed with myelodysplastic syndrome in 2006. He presented with complaints of weakness for a month. He referred arterial hypertension and myocardial infarction 3 years before, treated with coronary revascularisation. Blood analysis revealed mild macrocytic anaemia. Bone marrow biopsy and aspirate evaluation at diagnosis showed a slightly hypercellular marrow, with significant dysplasia in the erythroid lineage, less than 5% of CD34-positive haematopoietic precursors and absence of marrow fibrosis. Cytogenetic analysis showed normal karyotype. According to the updated WHO classification, a diagnosis of refractory anaemia was made, and he was stratified as low risk by means of IPSS scoring systems.1 He was included in a clinical trial with thalidomide for 9 months, which was interrupted for inefficacy, worsening of anaemia and appearance of moderate neutropenia. At this point, in January 2007, he was referred to our centre presenting with bicytopenia in the peripheral blood. Bone marrow re-evaluation was consistent with refractory cytopenia with multilineage …