Chronic myeloid leukemia: a prospective comparison of interphase fluorescence in situ hybridization and chromosome banding analysis for the definition of complete cytogenetic response: a study of the GIMEMA CML WP.

In chronic myeloid leukemia, different methods are available to monitor the response to therapy: chromosome banding analysis (CBA), interphase fluorescence in situ hybridization (I-FISH), and real-time quantitative polymerase chain reaction (RT-Q-PCR). The GIMEMA CML WP (Gruppo Italiano Malattie Ematologiche Adulto Chronic Myeloid Leukemia Working Party) has performed a prospective study to compare CBA and I-FISH for the definition of complete cytogenetic response (CCgR). Samples (n = 664) were evaluated simultaneously by CBA and I-FISH. Of 537 cases in CCgR, the number of positive nuclei by I-FISH was less than 1% in 444 cases (82.7%). Of 451 cases with less than 1% positive nuclei by I-FISH, 444 (98.4%) were classified as CCgR by CBA. The major molecular response rate was significantly greater in cases with I-FISH less than 1% than in those with I-FISH 1% to 5% (66.8% vs 51.6%, P < .001) and in cases with CCgR and I-FISH less than 1% than in cases with CCgR and I-FISH 1% to 5% (66.1% vs 49.4%, P = .004). I-FISH is more sensitive than CBA and can be used to monitor CCgR. With appropriate probes, the cutoff value of I-FISH may be established at 1%. These trials are registered at http://www.clinicaltrials.gov as NCT00514488 and NCT00510926.

Prognostic significance of microvessel density and vascular endothelial growth factor expression in sinonasal carcinomas.

The prognostic significance of microvessel density and proliferative activity of the neoplastic cells, evaluated respectively by CD31 and Ki-67 positivity, and immunohistochemical expression of vascular endothelial growth factor (VEGF) was retrospectively investigated in 105 cases of sinonasal carcinoma (80 surgical specimens and 25 biopsies). The most represented histologic types were intestinal-type adenocarcinoma found in 36 patients (34.3%), squamous cell carcinoma (SCC) in 34 (32.4%), mucinous adenocarcinoma (mainly made up of signet-ring cell patterns) in 15 (14.3%), and adenoid cystic carcinoma in 7 (6.7%). Microvessel density values (in vessels per square millimeter), VEGF, and Ki-67 were not dependent on histologic type but were rather correlated to the histologic grading in SCC. Clinical data were available for 92 (87.6%) of 105 patients, with minimum follow-up of 48 months. Most of the patients (81.5%) were at an advanced stage (T3-T4) at diagnosis. The values of all markers were correlated to tumor stage (P = .03). Multivariate analysis showed that both microvessel density and proliferative activity of the neoplastic cells were independent prognostic parameters (mortality hazard ratio, 1.33 and 1.60, respectively). Although VEGF expression was not correlated to prognosis on the whole series (P = .06), it was a powerful prognostic marker when the analysis was restricted to the group of SCCs (hazard ratio, 3.02; 90% confidence interval, 1.58-5.80). These results show that tumor neoangiogenesis, expressed by microvessel density, together with proliferative activity, is a pathologic marker with a strong prognostic impact in sinonasal carcinomas. Therefore, it may be a useful tool in this field so as to carry out therapeutic protocol planning, which may be further enhanced by the adoption of the more recent antiangiogenic molecules.

Oligonucleotide Array-based Comparative Genomic Hybridization Approach in Hematologic Malignancies With Normal/Failed Conventional Cytogenetics and Fluorescent In Situ Hybridization.

Oligonucleotide array-based comparative genomic hybridization (oaCGH) was used to investigate 60 cases of hematologic malignancies, mainly acute myeloid leukemias and myelodysplastic syndromes, in order to evaluate its sensitivity and specificity and to search for genomic alterations undetected by previous investigation with conventional cytogenetics (CC) and fluorescent in situ hybridization (FISH). On the basis of CC and FISH results, we subdivided the series into group A (36 cases with a normal karyotype after CC and/or FISH testing) and group B (24 cases with anomalies detected by CC and/or FISH). oaCGH did not show alterations in 21 cases of the group A (58.3%); in the remaining 15 cases (41.7%), it detected 19 new abnormalities (14 amplifications and 5 deletions). In the group B, oaCGH confirmed 32/55 aneuploidies detected by FISH (58.1%). The sensitivity increased at 27/33 confirmed aneuploidies (81.8%) by placing as a cutoff a mosaic of 50%. Moreover, in the cases of this group oaCGH revealed 36 new alterations (15 amplifications and 21 deletions). From these results it is possible to assess a strong overlap between results obtained by FISH and oaCGH. However, oaCGH is a reliable alternative where CC and FISH are not feasible and is able to identify new alterations unexplored by FISH.

High expression of cathepsin D in non-Hodgkin's lymphomas negatively impacts on clinical outcome.

The lysosomal protease Cathepsin D (CD) has been implicated in the homeostasis of lymphatic tissues. We investigated whether the level of CD expression influences the progression and the clinical outcome in Non-Hodgkin's Lymphomas (NHLs). The expression of CD was assessed by immunohistochemistry and immunofluorescence in biopsies of Diffuse Large B Cell Lymphomas (DLBCL, 35 cases), Follicular Lymphomas (FL, 9 cases of grade I-II plus 14 cases of grade IIIB), Chronic Lymphocytic Leukaemias (CLL, 17 cases) and Peripheral T-cell Lymphomas (PTCL, 5 cases). CD staining showed a cytoplasmic punctate pattern compatible with its lysosomal localization. Based on the level of CD expression and the proportion of positive cells, lymphomas were classified as 'low expressing' (< 20% of tumor cells) or 'highly expressing' (>or= 20% of tumor cells). Lymphomas highly expressing CD were associated with a worse stage (III-IV) at diagnosis (31/34 cases; p=0.002) and with a poor clinical outcome (i.e., partial remission and death; 28/34 cases; p=0.03). In the subgroup of aggressive/high grade of malignancy lymphomas (i.e., DLBCL, FL IIIB and PTCL), the Kaplan-Meier curve revealed a very low cumulative overall survival probability (approximately 20% at 5 year) for patients bearing a NHL with > 40% CD-positive cells compared to that of patients bearing a NHL with < 20% CD-positive cells ( approximately 70% at 5 year). This correlation was statistically significant (log-rank test, p=0.01). In Cox multivariate analysis CD failed to be a prognosticator independent of pathologic stage, though the hazard ratio confirmed the association of low expression with a better survival probability. These data indicate that the presence of a high percentage of CD-positive tumor cells negatively reflects on the progression of NHLs.

Deletions of the derivative chromosome 9 do not influence the response and the outcome of chronic myeloid leukemia in early chronic phase treated with imatinib mesylate: GIMEMA CML Working Party analysis.

PURPOSE: Deletions of the derivative chromosome 9 [der(9)] have been associated with a poor prognosis in chronic myeloid leukemia (CML) across different treatment modalities. In the imatinib era, the prognostic impact of der(9) deletions has been evaluated mainly in patients with late chronic-phase (CP) CML, giving partially conflicting results. Few data are available in the early CP setting. For this reason, in 2006, the European LeukemiaNet recommendations still considered der(9) deletions as a candidate adverse prognostic factor and required a careful monitoring of the patient. PATIENTS AND METHODS: To investigate the prognostic value of der(9) deletions in early CP CML, we performed an analysis of three prospective imatinib trials of the Italian Group for Hematological Malignancies of the Adult (GIMEMA) CML Working Party. RESULTS: A fluorescent in situ hybridization (FISH) analysis of bone marrow cells was performed at diagnosis; der(9) deletions were detected in 60 (12%) of 521 evaluable patients. At 60 months, the cumulative incidence of complete cytogenetic response and major molecular response-and the probability of event-free survival, failure-free survival, progression-free survival, and overall survival-in patients with and without deletions were not statistically different. CONCLUSION: Our data strongly support the notion that, when investigated by FISH, der(9) deletions are not a poor prognostic factor in patients with early CP CML treated with imatinib.

Co-expression of plexin-B1 and Met in human breast and ovary tumours enhances the risk of progression.

BACKGROUND: Plex-B1, the receptor of Sema4D, has been implicated in tumour growth, angiogenesis and metastasis. The binding of Sema4D to Plex-B1 can trigger the activation of Met tyrosine kinase, thereby promoting cell dissociation and invasive growth. We tested the hypothesis that the expression of Plex-B1, either alone or in association with Met, can be of predictive value for tumour progression. METHODS: The expression and distribution of Plex-B1 and Met were investigated by immunohistochemistry and immunofluorescence in 50 human neoplasias originating in the breast and ovary, and correlated with clinical-pathological data at diagnosis. RESULTS: Plex-B1 and Met were individually expressed in 14% and in 24% of the tumours, respectively. Plex-B1 and Met were co-expressed in 24/50 cases (48%), and in the majority of these (83%) Met was tyrosine phosphorylated. The expression of Plex-B1 or Met alone showed no significant correlation with tumour aggressiveness, whereas advanced stage tumours (III-IV) frequently showed Plex-B1-Met double-positive (9/13). Tumours co-expressing Plex-B1 and Met were characterised by worse grading and higher incidence of lymph node metastases. Out of 22 tumours with lymph node metastases, as many as 19 were Plex-B1 and Met double-positive (p=0.0008), and 17 expressed phosphorylated Met (p=0.002). CONCLUSION: Plex-B1 assumes a predictive value for unfavourable outcome when co-expressed with Met.

cFLIP expression correlates with tumour progression and patient outcome in non-Hodgkin lymphomas of low grade of malignancy.

The present study investigated whether the expression of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE) inhibitory protein (cFLIP) conveys prognostic information in non-Hodgkin lymphomas (NHLs). cFLIP expression was quantified by immunohistochemistry and immunofluorescence in biopsy specimens from 86 NHL patients for whom clinical information was available. NHL malignancy was graded as high/intermediate or low according to the World Health Organization Classification of Lymphoid Neoplasms. cFLIP was positive in 23 of 45 high-/intermediate-grade NHLs and in 25 of 41 low-grade NHLs. Negative expression of cFLIP was associated with the presence of apoptotic cells in the tumour mass, regardless of the histotype and of the malignancy grade. In NHLs positive for cFLIP, 11 of 23 (48%) high-/intermediate-grade cases and 18 of 25 (72%) low-grade cases showed a bad outcome. In NHLs negative for cFLIP, only four of 22 (18%) high-/intermediate-grade patients and 12 of 16 (75%) low-grade patients achieved complete remission. All these correlations were statistically significant. The correlation of cFLIP expression with clinical outcome was independent of therapy, whether or not it included anti-CD20 antibody (Rituximab). The present findings strongly indicate that cFLIP is a reliable predictor of tumour progression and clinical prognosis in NHLs of low grade of malignancy.

Evidence of p53 immunohistochemical overexpression in ethmoidal mucosa of woodworkers.

A high risk of neoplastic transformation of nasal and paranasal sinuses mucosa is related to the occupational exposure to wood dust, however no conclusive data have been reported up to now about morphological precursors of these tumors, mechanisms of carcinogenesis and role of p53 gene. Immunohistochemical overexpression of protein p53 (DO7 clone) by epithelial cells of ethmoidal mucosa was investigated on 60 woodworkers occupationally exposed for a minimum of 10 years, on 50 functional and/or esthetic nasal surgery patients (control group) and on 15 cases of intestinal-type adenocarcinoma, 10 of these involving subjects who had a longtime exposure to wood dust. In almost all the woodworkers (92%) the normal ciliated epithelium showed tracts of squamous metaplasia. The mean percentage of p53-positive cells in woodworkers and in controls was 28.6 and 7.97%, respectively, in metaplastic epithelium (P<0.001), 11.7 and 2.08% in ciliated epithelium (P<0.001), 12.46 and 1.03% (P<0.001) in the sero-mucous glands of the nasal stroma. Both in tracts of metaplastic epithelium and in those of ciliated epithelium, positive cells were distributed in basal and suprabasal layers. A high number of p53-positive cells was also observed in the normal ciliated epithelium close to the neoplastic cells, of intestinal-type adenocarcinomas affecting subjects with longtime exposure to wood dust. Moreover, a higher number of p53 positive neoplastic cells was showed by the cases occurring in occupationally exposed patients than by the others. The following conclusions can be drawn: (1) in the ethmoidal mucosa, a region at high risk of carcinogenesis in subjects exposed to wood dust, epithelial cells overexpress p53 protein, and this may be linked to the presence of p53 gene mutations; malignant transformation, as at other head and neck sites, may thus occur through this pathway; (2) the presence of p53 overexpression in sero-mucous glands is in keeping with the histogenesis of some tumors from these structures; (3) since tumors of nose and paranasal sinuses, mainly adenocarcinoma, are recognized as occupational neoplasias, immunohistochemical evaluation of p53, perhaps combined with molecular methods, could be the first step to detect subjects at high risk of carcinogenesis among woodworkers.