HPyV6, HPyV7 and TSPyV DNA sequences detection in skin disease patients and healthy subjects.

BACKGROUND: The discovery, from 2007, of eight new human polyomaviruses (HPyVs) has revived interest in the Polyomaviridae family and their association with human diseases and cancer. In particular, HPyV6 and HPyV7 were discovered in skin swabs of healthy donors and TSPyV was discovered in a heart transplant recipient affected by virus-associated Trichodysplasia Spinulosa (TS), a rare skin disease, exclusively found in immunocompromised patients. OBJECTIVE: The presence of HPyV6, HPyV7 and TSPyV DNA in skin biopsies from patients affected by different skin diseases (cancers and inflammatory disorders) has been evaluated to confirm their skin tropism and the possible pathological association. METHODS: DNA extracted was amplified with HPyV6, HPyV7 and TSPyV specific PCR real time on Taqman platform with standard profile. RESULTS: HPyV7 and TSPyV sequences were not found in any skin specimen analysed. HPyV6, on the other hand, was detected in 30% of samples from healthy subjects vs. 14.3% of skin cancer patients and 2.9% of inflammatory disorders. HPyV6 sequences have been detected in primary cutaneous T-cell lymphoma (CTCL) patients (in 18.6% out of Mycosis Fungoides (MF) patients and in 16.7% out of CTCL not MF/SS(Sèzary syndrome) but have not been detected in primary cutaneous B-cell lymphoma (CBCL) patients. CONCLUSION: Our preliminary data suggest that these three novel human polyomaviruses seem not to play a significant role neither in the pathogenesis of cutaneous malignancies nor in that of inflammatory disorders but, according to literature, can inhabit the skin. On the basis of our data regarding the HPyV6 DNA presence with decreasing percentages in healthy subjects, skin cancer and inflammatory disorders patients, it could be an intriguing matter to study if the activated innate immune response in inflammatory disorders can suppress the virus. Further investigations are needed to better understand their relationship with the human host and its innate immune system.

Th1, Th2, Th17 and regulatory T cell pattern in psoriatic patients: modulation of cytokines and gene targets induced by etanercept treatment and correlation with clinical response.

BACKGROUND: Psoriasis is sustained by pro-inflammatory CD4+ T helper cells mainly belonging to the Th1, Th17 and Th22 lineage. OBJECTIVE: To identify whether treatment with the anti-tumour-necrosis-factor antagonist etanercept is able to induce significant modulations in transcription factor and cytokine mRNA gene expressions related to the different T cell immune response polarization (Th1, Th2, Th17 and regulatory T cells, Treg and to correlate them with clinical response. METHODS: The study population included 19 psoriasis patients treated with etanercept and 19 healthy subjects. Blood samples were collected at baseline and every 4 weeks during treatment. Taqman quantitative real-time polymerase chain reaction was applied to analyse the expression of: Stat-4, T-bet, IL-12p35 and IFN-γ (Th1-related); GATA-3, IL-4 (Th2-related); Stat-3, RORγt, IL-23p19 (Th17-related); Foxp3, IL-2 (Treg-related). Flow cytometry was applied to analyse CD4+CD25+(bright)Foxp3+ cells in peripheral blood. RESULTS: Upregulation of Th1 and Th17 and downregulation of Treg subsets was found at baseline. The response to etanercept could be associated with a significant reversal of the Th1/Th17 activation, and a concomitant upregulation of Th2 and Treg subsets. CONCLUSION: Our data may contribute to a better understanding of the mechanisms underlying the achievement of clinical response in psoriasis and could be helpful for the identification of early predictive markers of response.

Circulating CD4+CD25 bright FOXP3+ T cells are up-regulated by biological therapies and correlate with the clinical response in psoriasis patients.

BACKGROUND: Regulatory T-cell (T(reg)) modulation is one of the potential mechanisms of anti-tumour-necrosis-factor biological agents. However, literature data on psoriasis patients are lacking. OBJECTIVE: To analyse the circulating CD4+CD25(bright)FOXP3+ subset in 30 patients with psoriasis vulgaris/arthropathic psoriasis treated with biologicals and to investigate its relationship with the clinical response. METHODS: The CD25(bright)FOXP3+ expression within the CD4+ subset was determined by multi-parameter flow cytometry at baseline and during treatment. FOXP3 mRNA expression was analysed by real-time reverse transcription PCR. RESULTS: A response was obtained in 16/17 patients (91.1%) with increased CD25(bright)FOXP3+ values and in only 3/11 patients (27.3%) who showed a CD25(bright)FOXP3+ decrease during biological treatment (p = 0.0001). Responders showed significantly higher values than did non-responders as from the first 2 months of treatment (p = 0.0032). A significantly higher posttreatment expression of mRNA FOXP3 was observed in responders compared to non-responders. CONCLUSION: Biological drugs induce a circulating T(reg) up-regulation in a significant percentage of patients; such an increase is an early predictive marker of response.

Reciprocal modulation of circulating CD4+CD25+bright T cells induced by extracorporeal photochemotherapy in cutaneous T-cell lymphoma and chronic graft-versus-host-disease patients.

The mechanisms of action of extracorporeal photochemotherapy (ECP) in cutaneous T-cell lymphoma (CTCL) are poorly understood. Recently, ECP has been shown to induce an increase in regulatory T cell (Treg) expression and functional activities in Graft-versus-host-disease (GvHD), whereas no data are available in CTCL patients. The aim of this study is to evaluate whether ECP is able to modulate the expression levels of the circulating CD4+CD25+bright subset in CTCL patients and whether these modifications are related to the disease course. The patient population included 43 CTCL and 15 chronic GvHD patients treated by ECP at our institutions since 1992. The expression of the circulating CD4+CD25+bright subset was analysed at baseline and sequentially during treatment by flow-cytometry. Fifty healthy donors were used as controls. The baseline circulating CD4+CD25+bright percentage values in CTCL (median: 4.3 percent) were similar to those of healthy donors, whereas GvHD showed significantly lower values (median: 1.5 percent; p<0.001). During treatment, CTCL patients were characterised by an early decrease (from 4.3 percent to 2.4 percent median after 6 months). The CD4+CD25+bright decrease was associated to the disease course, as it occurred in 91.3 percent of responding but in only 25 percent of PD patients (p=0.0001). On the other hand, a significant increase of CD4+CD25+bright cells was observed in GvHD. ECP induces a reciprocal modulation of the circulating CD4+CD25+bright cells in CTCL and GvHD, with a downregulation in CTCL potentially associated with the response mechanisms.

Human herpesvirus 7 detection by quantitative real time polymerase chain reaction in primary cutaneous T-cell lymphomas and healthy subjects: lack of a pathogenic role.

BACKGROUND: Primary cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of lymphomas where the tumour population emerges within a multiple subclone pattern. Mycosis fungoides (MF) and Sézary syndrome (SS) are characterized by the expansion of clonal CD4+/CD45RO+ memory T cells. Lymphomatoid papulosis (LyP) is a chronic, lymphoproliferative disorder included in the CD30+ primary CTCL spectrum. Several studies have suggested a role of viral infection for super-antigenic activation of T lymphocytes; however, evidence of their association with CTCLs is still lacking. Human herpesvirus (HHV) 7 is a CD4+ T-lymphotropic herpesvirus; its restricted cellular tropism and the ability to induce cytokine production in infected cells could make it an important pathogenic cofactor in lymphoproliferative disorders. OBJECTIVES: To investigate the presence of HHV7 DNA on CTCL and healthy skin donors (HD). METHODS: We used quantitative real time polymerase chain reaction to evaluate the potential pathogenic role of HHV7. RESULTS: Twenty-seven of 84 (32.1%) HD were positive for HHV7 DNA. Twenty-one of 148 (14.2%) patients with CTCLs were positive for HHV7 DNA: nine of 39 (23.1%) SS, six of 14 (42.9%) CD30+ CTCLs and six of 24 (25.0%) LyP, and HHV7 DNA was negative in all 71 patients with MF. CONCLUSIONS: These results seem to exclude a pathogenic role of HHV7 in CTCLs, suggesting the possibility of skin as a latency site.

Heterogeneity of circulating CD4+ memory T-cell subsets in erythrodermic patients: CD27 analysis can help to distinguish cutaneous T-cell lymphomas from inflammatory erythroderma.

BACKGROUND: Chronic dermatoses, as well as Sézary syndrome (SS), the erythrodermic and leukaemic cutaneous T-cell lymphoma, display a T-cell memory pattern. Recent findings suggest that different memory T-cell subsets can be recognized based on CD27 and CD45RO/RA expression. No data are reported as to CD27 expression in SS. OBJECTIVES: To evaluate different memory T-cell subsets, i.e. central memory (TCM), effector memory (TEM) and terminally differentiated cells in SS and inflammatory erythroderma (IE). MATERIALS AND METHODS: Forty SS and 137 IE patients were included. CD27 and CD45RO/CD45RA expression was analysed by flow cytometry on peripheral blood lymphocytes and immunohistochemistry. RESULTS: A significantly higher expression of the CD4+CD27+CD45RA- TCM subset was observed in SS whilst IE patients were characterized by increased CD4+CD27-CD45RA- TEM levels. The Vbeta-restricted population was homogeneously CD4+CD26-CD27+ in the SS subjects. CONCLUSIONS: SS and IE are characterized by a different memory T-cell subset expression; CD27 expression could be used as an additional diagnostic tool in the differential diagnosis.

T-cell receptor gamma gene rearrangement by multiplex polymerase chain reaction/heteroduplex analysis in patients with cutaneous T-cell lymphoma (mycosis fungoides/Sézary syndrome) and benign inflammatory disease: correlation with clinical, histological and immunophenotypical findings.

BACKGROUND: A dominant T-cell clone can be detected by polymerase chain reaction (PCR) in 40-90% of cutaneous samples from patients with cutaneous T-cell lymphoma (CTCL). MATERIALS AND METHODS: From 1996 to 2003 we analysed 547 cutaneous biopsies performed to exclude CTCL (mycosis fungoides, MF/Sézary syndrome, SS). The final diagnosis was benign inflammatory disease (BID) in 353 samples (64.5%) and CTCL in 194 (35.5%). T-cell receptor (TCR)-gamma gene rearrangement was studied by using a multiplex PCR/heteroduplex (HD) analysis. The PCR results were correlated with the clinical picture, the histological pattern and the presence of T-cell lineage antigen loss, using univariate and multivariate logistic regression analyses. OBJECTIVE: To determine the sensitivity and specificity of the multiplex PCR/HD analysis and to identify which are the clinical, histopathological or immunophenotypical features significantly associated with a positive T-cell clonality. RESULTS: A clonality was demonstrated in 83.5% of CTCL and in 2.3% of BID (P < 0.001). A significantly higher percentage of clonal cases was associated with the cutaneous T-score (71.4% in T1, 76.1% in T2 and 100% in nodular and erythrodermic MF samples) and with the presence of a T-cell lineage antigen loss (93.9% vs. 77.4%). Moreover, clonality was closely related to an increase in the histopathological score (51.3% in the samples with a score < 5, compared with 92% in the lesions with > or = 5). No significant difference in the percentage of clonal cases was found between T1/T2 and T3/T4 lesions with a histopathological score > or = 5. The multivariate logistic regression showed that the density and extent of the cell infiltrate, the degree of epidermotropism and the presence of cytological atypia share an independent predictive value for clonality in T1/T2 samples, even if the highest odds ratios (3.6) were associated with the density of the cell infiltrate. The disease course of T1/T2 patients was analysed according to the PCR findings. All the PCR-negative patients showed a long-standing stable disease course; on the other hand, a disease progression occurred in 12/87 (13.8%) positive patients. CONCLUSIONS: The multiplex PCR/HD analysis is associated with a high diagnostic accuracy (92.7%) in CTCL patients. The finding of a clonal T-cell rearrangement is more closely associated with the histological pattern (in particular with the density and extent of the cell infiltrate) rather than with the MF cutaneous T-score or immunophenotype.

Collagenase digestion and mechanical disaggregation as a method to extract and immunophenotype tumour lymphocytes in cutaneous T-cell lymphomas.

Various enzymatic or mechanical methods have been proposed in the past to dissociate cells from different solid tissues. An automated mechanical disaggregation device (Medimachine) has recently been proposed. Unfortunately, most of these techniques are associated with a high cellular damage and a low cell recovery and are difficult to apply to skin biopsies. In this paper, we propose a combined enzymatic and mechanical method based on Medimachine, useful for the isolation of skin infiltrating T-lymphocytes from small cutaneous biopsies. As this method is easy and allows for a more correct qualitative and quantitative cytofluorimetric analysis of the lymphocyte subsets, it may be useful in the immunophenotyping of cutaneous T-cell lymphomas.

In vitro effects of cholesteryl butyrate solid lipid nanospheres as a butyric acid pro-drug on melanoma cells: evaluation of antiproliferative activity and apoptosis induction.

Literature data show that butyric acid derivatives bear a dose-dependent differentiative anti-proliferative activity on cancer cell lines and that apoptosis induction may play a major role. Although it was recently shown that solid lipid nanospheres (SLNs) are a suitable tool for several in vivo drug administration routes, there is little available information on melanoma cell lines. This study was aimed at evaluating the anti-proliferative and apoptotic in vitro effects of cholesteryl butyrate (chol-but) SLNs on melanoma cells. Increasing concentrations of chol-but SLNs were used to test two melanoma cell lines. Both cell lines were treated with Na-butyrate (Na-but) and chol-but SLNs for viability. Those tested with chol-but SLNs were more effective than Na-butirate (3 to 72 h). The apoptotic effects of chol-but SLNs were evaluated between 3 and 72 h by annexin-V (ANX-V)/propidium iodide (PI) staining and the antiproliferative effect by PI staining. Apoptosis anti-proliferative-regulatory proteins as bcl-2, Fas/APO1 (CD95) and PCNA (PC10) were also investigated. Flow cytometric analyses evidenced a G(0/1)-S transition block and a 'sub-G(0/1)' apoptotic peak from 0.5 to 1.0 mM butyric acid. In ANX-V/PI flow cytometric staining, a dose- and time-dependent increase in the apoptotic cell percentage (ANX-V+) coupled with a down-regulation of PC10 and bcl-2 and a parallel up-regulation of Fas/APO1 (CD95) were found in both lines started after 3 to 24 h of chol-but SLNs treatment. Results show that chol-but SLNs exerts a dose/time-dependent effect in melanoma cell apoptosis induction between 3 and 24 h and a dose but not time-dependent effect after 24 h of treatment.

Trans-septal catheterization for radiofrequency catheter ablation of cardiac arrhythmias. Results and safety of a simplified method.

AIM: This study reports on the results and safety of a simplified method of trans-septal catheterization for radiofrequency catheter ablation of cardiac arrhythmias. METHODS AND RESULTS: Over 5 years, 411 patients underwent trans-septal catheterization for radiofrequency catheter ablation: 388 patients had a left-sided accessory pathway, 19 a left-sided focal atrial tachycardia, two atrial fibrillation and two post-infarction ventricular tachycardia. All but one patient with ventricular tachycardia underwent elective trans-septal catheterization. In the absence of a patent foramen ovale, puncture of the atrial septum was performed by using an 8F Mullins sheath and a Brockenbrough needle, according to the simplified method described in this paper. Trans-septal catheterization was accomplished in 383/388 patients (98.7%); in 41 patients a second trans-septal catheterization and radiofrequency catheter ablation was performed for initial failure or recurrence. Radiofrequency catheter ablation was successful in 96% of accessory pathway patients, 90% of atrial tachycardia patients, in both patients with atrial fibrillation and in both patients with ventricular tachycardia. No complication related to trans-septal catheterization was observed. CONCLUSION: In experienced hands and according to the method described in this paper, the elective use of transseptal catheterization for radiofrequency catheter ablation in a large cohort of patients with cardiac arrhythmias is feasible, safe and allows successful ablation in the vast majority of the patients.

Cell proliferation, bcl-2, c-myc, p53 and apoptosis as indicators of different aggressiveness in small lymphocytic lymphoma (SLL).

Cell proliferation activity, by MIB1 mAb, expression of bcl-2, c-myc and p53 gene proteins and apoptotic index (AI) were assessed in 54 cases of SLL and compared to the morphological subtypes of this disorder, defined by Lennert on the basis of amount and distribution of small and larger activated lymphocytes as diffuse, tumor-forming and pseudofollicular subtypes (DS, TFS, PFS). MIB1 scores showed significant differences between DS, PFS and TFS (5.5%, 16.61% and 24.14%, respectively; p < 0.0001). Worth noting, the MIB1 score did not differ significantly when comparing DS with the diffuse areas of PFS, or TFS with the pseudofollicles of PFS. The mean bcl-2 gene protein score was displayed to a high extent in all subtypes, but less extensively by larger activated lymphocytes that, conversely, expressed c-myc. MIB1 score correlated negatively with bcl-2 and positively with c-myc protein scores. These findings suggest that lymphocytes protected from apoptosis by bcl-2 would be exponed to cell activation and growth acceleration provided by c-myc. This condition would account for a different aggressiveness of morphologically activated subtypes, such as TFS and PFS with larger pseudofollicles. The survival analysis, performed in 23 cases, showed a trend of association of cell proliferation and c-myc expression with a more aggressive progression of the disease. Overexpression of p53 and apoptosis were found only in a minority of cases, unrelated to the subtypes. In conclusion, cell growth fraction, bcl-2 and c-myc assessment may be of help in predicting the aggressiveness of different subtypes of SLL. This approach should be most conveniently applied to PFS, which represents a continuum between DS and TFS, in order to distinguish, in this heterogeneous subtype, more indolent from more aggressive disorders.

DNA ploidy and p53 expression correlate with survival and cell proliferative activity in male breast carcinoma.

DNA flow cytometry and the monoclonal antibody DO7 were applied in formalin-fixed, paraffin-embedded specimens from 34 primary male breast carcinomas to verify whether DNA ploidy and p53 expression were associated with survival and proliferative activity. They were compared with tumor clinicopathologic features, sex steroid hormone receptors and cell proliferative activity, assessed by the counts of the argyrophilic nucleolar organizer regions (AgNORs), the monoclonal antibody PC10 against the proliferating cell nuclear antigen and the monoclonal antibody MIB-1. A significant correlation was found between survival and tumor ploidy (median survival, 77 months for diploid but only 38 months for aneuploid cases; P = .03) and p53 expression (median survival, 95 months for cases with p53 scores < or = 14.06% versus 33 for cases with P53 scores > 14.06%; P = .0004; median survival, 99 months for p53 negative vs 39 for positive cases; P = .007). Tumor histological grade (P = .006), AgNOR counts (P = .0001), PC10 scores (P = .002), and MIB-1 scores (P = .001) were also associated with prognosis. In the multivariate analysis, only p53 scores (P = .001) or p53 immunopositivity (P = .003) and AgNOR counts (P = .022) retained an independent prognostic significance. Aneuploid tumors had higher AgNOR counts (P = .002), PC10 (P = .007), MIB-1 (P = .006), and p53 scores (P = .01) than diploid cases. A linear relationship was observed between p53 scores and AgNOR counts (r = .41; P = .014), PC10 (r = .46; P = .005), and MIB-1 scores (r = .44; P = .011). These results indicate that DNA ploidy and p53 expression are associated with survival and cell proliferative activity in male breast carcinoma. Quantitative parameters, such as DNA ploidy, p53 scores, AgNOR counts, PC10, and MIB-1 scores substantially improve the prognostic significance of the traditional parameters in male breast carcinoma.

p53 expression and proliferative activity predict survival in non-invasive thymomas.

We performed p53 immunohistochemistry, DNA flow cytometry and analysis of the argyrophilic nucleolar organizer regions (AgNORs) in formalin-fixed, paraffin-embedded sections from 46 non-invasive thymomas and correlated the results with the traditional clinicopathologic features of the tumor. p53 immunopositivity was detected in 21 of 46 cases; it was not associated with any clinicopathologic features nor DNA content but significantly correlated with AgNOR counts. On univariate analysis, 10-year survival rates were 100% for p53-negative cases but only 71% for p53-positive cases and 93% for patients with low AgNOR counts but only 77% for patients with high AgNOR counts. Age, sex, histologic type, myasthenia gravis and DNA content did not correlate with survival. Our results indicate that p53 staining and evaluation of proliferative activity allow assessment of prognosis in non-invasive thymomas, when all of the other parameters are insufficient. Furthermore, the high rate of p53 expression in non-invasive thymomas suggests that abnormal p53 immunoreactivity may occur early in the neoplastic process.

Biological heterogeneity of diffuse mixed small and large cell non-Hodgkin's lymphomas assessed by DNA flow cytometry and Ki67.

The cell proliferative activity of the clinico-pathologically heterogeneous non-Hodgkin's lymphomas (NHL) included in the intermediate grade F category of the Working Formulation (WF) was investigated. S-phase fraction with flow cytometry on cell suspensions, and Ki67 on frozen tissue sections were performed in 42 F NHL. An avidin-biotin immunocomplex method was used and 1000 cells from 10 representative fields were counted. DNA content, S-phase and Ki67 were also detected in 194 NHL covering the whole spectrum of the WF. DNA content anomalies were found in 52 of 194 NHL. Their incidence, like that of S-phase fraction and Ki67 positive cells, progressively increased from low- to high-grade. A linear correlation was found between Ki67 and S-phase (r = .59). Using the median value of proliferating cells obtained with both procedures as a cut off, two very different groups of lymphomas could be distinguished within a series of 42 F-intermediate NHL: with low and high proliferative cell activity (p < .0001) that were termed F(low) and F(high), respectively. A intermediate group was placed between them. It differed significantly from the others if Ki67 was used but only from the F(high) group if the S-phase fraction analysis was applied. No significant differences were seen when comparing F(low) with the single categories of low-grade NHL and F(high) with H high-grade NHL; no significant differences were found between F(high) and G, and between G and H categories. The existence of distinct groups of NHL in the F category, as defined by biological parameters assessing the cell proliferative activity, indicates that this category includes biologically heterogeneous lymphoma subtypes with different grades of aggressiveness. The results also indicate that the G intermediate category displays proliferation indices similar to those of H high grade category.

Long-term survival of thymoma patients by histologic pattern and proliferative activity.

We performed DNA flow cytometry and analysis of the argyrophilic nucleolar organizer regions (AgNORs) in formalin-fixed, paraffin-embedded sections from 60 surgically resected thymomas. The results were correlated with histologic pattern, stage, associated clinical features, and survival to assess which parameters could best predict prognosis. On univariate analysis, the 10-year survival rates were 86% for predominantly lymphocytic type but only 42% for predominantly epithelial, mixed lymphoepithelial, or spindle cell thymomas (p = 0.006); survival rates were 85% for noninvasive but only 34% for invasive thymomas (p = 0.0002); 73% for diploid but only 38% for aneuploid cases (p = 0.005); 88% for thymomas with 5.75 AgNORs per cell or fewer but only 34% for thymomas with more than 5.75 AgNORs per cell (p < 0.0001). On multivariate survival analysis, tumor stage (p < 0.001) and AgNOR counts (p = 0.009) retained independent prognostic significance. The 16 patients with predominantly lymphocytic type and 5.75 AgNORs per cell or fewer were all alive at the end of the observation period. In conclusion, the histologic type of the American classification and the proliferative activity evaluated by AgNOR analysis are the best predictors of long-term survival for patients with thymoma. Both predictors can be easily evaluated in the same histologic section, are highly reproducible, and permit identification of a group of patients with a favorable outcome regardless of other clinicopathological features.

Identification of granulocyte-macrophage colony stimulating factor receptor mRNA by non-isotopic in situ hybridization in bone marrow biopsies.

BACKGROUND: In the last few years many studies have been published on GM-CSF receptors, focusing on molecular structure, function and distribution. Nevertheless, protocols for detecting GM-CSF receptors on formalin-fixed paraffin-embedded histological sections, to our knowledge, have not been described. METHODS: A method based on non-isotopic in situ hybridization (ISH) using a 21-base antisense DNA oligoprobe whose 3'-end was labeled with digoxigenin 11-dUTP was devised. The probe was applied on 20 routinely processed bone marrow trephine biopsies which were considered as positive controls. RESULTS: The hybridization signal was seen in myeloid cells, erythroid progenitors and rare megakaryocytes. CONCLUSIONS: Non-isotopic ISH represents an alternative to current methodologies for the assessment of GM-CSF receptor expression; since it is suitable for routinely processed samples, it can be regarded as a helpful tool for diagnostic determination of GM-CSF receptors in tumors from patients receiving GM-CSF and for retrospective studies on archival material.

MIB-1, Ki67, and PCNA scores and DNA flow cytometry in intermediate grade malignant lymphomas.

AIMS: To verify the correlation between MIB-1, Ki67, and proliferating cell nuclear antigen (PCNA-PC10) scores and S-phase fraction in intermediate grade non-Hodgkin's lymphomas (Working Formulation F); and their reliability in differently processed tissues. METHODS: Forty one non-Hodgkin's lymphomas were classified as (F) intermediate grade malignant lymphomas according to the Working Formulation; mitotic counts and percentage of large cells were assessed for each case. Sections from formalin fixed, paraffin wax embedded tissues were stained with anti MIB-1 monoclonal antibody, after microwave oven processing, and anti-PCNA (PC10) monoclonal antibody using an avidin-biotin immunoperoxidase (ABC) method. One thousand cells from 10 representative fields were scored. Frozen sections from surgical specimens were stained with Ki67 monoclonal antibody using the ABC method; the fraction of Ki67 positive cells was calculated scoring 1000 cells. Flow cytometry analysis (FCM) was performed on cell suspensions from fresh tissues. Correlations between data were estimated using linear regression. RESULTS: A linear correlation was found between MIB-1 and Ki67 scores (r = 0.92; p < 0.00001); between MIB-1 and PCNA scores (r = 0.79; p < 0.00001); and between MIB-1 score and S-phase fraction (r = 0.51; p = 0.0006). A linear correlation was also found between Ki67 and PCNA scores (r = 0.85; p < 0.00001); between Ki67 score and S-phase fraction (r = 0.6; p = 0.0002); and between PCNA score and S-phase fraction (r = 0.74; p < 0.00001). A correlation was found between mitotic counts and MIB-1 (r = 0.56; p = 0.0001), PCNA (r = 0.51; p = 0.0007), or Ki67 scores (r = 0.47; p = 0.002); between the percentage of large cells and MIB-1 (r = 0.49; p = 0.0009), PCNA (r = 0.6; p = 0.00003), and Ki67 scores (r = 0.53; p = 0.0003) and S-phase fraction (r = 0.55; p = 0.0002). CONCLUSION: MIB-1, Ki67, and PCNA (PC10) scores and S-phase fraction are highly correlated and equally well represent the proliferative activity of intermediate grade non-Hodgkin's lymphomas in differently processed material. MIB-1 and PCNA stains can be applied even on small biopsy specimens. MIB-1 produces homogenous staining without background; it also strongly stains mitotic figures. It can be performed on routinely processed tissues, permitting the simultaneous evaluation of the morphology and tumour cell kinetics. The wide standard deviations of the proliferative indices found for intermediate grade NHL suggest that this category probably includes various degrees of malignancy.

An immunoenzyme technique for the identification of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors using digoxigenated-GM-CSF.

A method for detecting granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors has been devised using human macrophages and a GM-CSF/IL-3-dependent human megakaryoblastic leukemia cell line (M-07e). Recognition of the factor-binding site was accomplished by linking recombinant human (rh) unglycosylated GM-CSF previously labeled with digoxigenated compounds. Digoxigenates were able to link amino and sulphydryl groups of the soluble factor and an immunoperoxidase technique using monoclonal anti-digoxigenin antibody was employed to demonstrate the interaction. To support morphological data cross-linking analysis was performed with M-07e cells using digoxigenated-rh-GM-CSF. Macrophages and M-07e cells incubated with digoxigenated-rh-GM-CSF showed intense positivity by the immunoperoxidase technique. In cross-linking, M07e cells showed a 96 kDa band corresponding to receptor plus bound factor. This technique permits a high degree of specificity in the detection of GM-CSF receptors with good morphological preservation of cellular detail.

Heterogeneous immunoglobulin gene rearrangement in a B-chronic lymphocytic leukemia progressing into non-Hodgkin lymphoma (Richter syndrome).

BACKGROUND: The relationship between chronic lymphocytic leukemia (CLL) and supervening non-Hodgkin lymphoma is debated, as is whether a particular genomic pattern is related to the emergence of the terminal lymphoma. To investigate these features, the molecular organization of the immunoglobulin (Ig) gene region in a case during both the B-CLL and Richter transformation phase was studied. METHODS: B-CLL and non-Hodgkin lymphoma cells were processed for Southern blot analysis of Ig heavy- and light-chain gene configuration. RESULTS: Molecular studies of B-CLL cells revealed the presence of a single Ig heavy-chain rearrangement with both kappa and lambda light-chain rearranged genes, which was consistent with the occurrence of multiple mutational events during the development of the B-CLL clone. Molecular analysis of the lymphoma DNA showed new Ig heavy- and kappa light-chain rearrangements in addition to the original ones related to the CLL phase, indicating that the lymphoma tissue consisted of two genotypically distinct populations of cells. CONCLUSIONS: On the basis of the overall molecular configuration, this heterogeneous pattern of Ig gene rearrangement was interpreted as an inherent genetic instability of the CLL clone, in which multiple mutational events allowed a selective pressure toward more aggressive subclones, resulting in the emergence of the terminal lymphoma.

[Indications and results of transcatheter ablation and surgery in paroxysmal reentry nodal tachycardia]. / Indicazioni e risultati dell'ablazione transcatetere e della chirurgia nelle tachicardie parossistiche da rientro "nodale".

In patients with nodal tachycardia refractory to medical therapy, transcatheter or surgical ablation is necessary. From January 1989 to December 1990, in 26/42 patients with nodal tachycardia, referred to our institution for electrophysiologic evaluation, transcatheter ablation by radiofrequency (20 patients) or surgical ablation by perinodal cryo (6 patients) was performed. In all these cases, a total refractoriness to several antiarrhythmic drugs alone or in association had been observed. The radiofrequency current, generated by the Osypka HAT 100 device, was administered through a tripolar USCI 7 F catheter. The ideal site for energy delivery was defined on the basis of a mapping, performed in the A-V junction area. In order to find out the most premature retrograde atrial activation, the following areas were explored: right bundle, atrial His bundle, peri-nodal region, proximal His bundle and coronary sinus ostium. Local atrial activation time was evaluated during nodal tachycardia by delivering a premature ventricular extrastimulus to discover the atrial deflection from the ventricular one. In the selected area, 5 applications (range 1-12) of 20-25 W power radiofrequency energy for 5-30 s were delivered on average. A complete prevention of nodal tachycardia was achieved in 18/20 patients (90%). Only in 2 patients a total A-V block was induced. The pre- and post-procedure values are as follows: AH: pre 71 +/- 19, post 113.6 +/- 50; HV: pre 45.5 +/- 8, post 47 +/- 6; aWP: pre 353 +/- 57, post 391 +/- 87; rWP: pre 322 +/- 58, post 411 +/- 58. In 10/18 cases the AH interval was normal after radiofrequency application.(ABSTRACT TRUNCATED AT 250 WORDS)