Evaluation of apoptosis in classical Hodgkin's lymphoma comparing different methods.

PURPOSE: Apoptosis is a type of programmed cell death (PCD) with specific morphologic changes in the dying cell. Since classical Hodgkin's lymphoma (cHL) is characterised by abnormalities in the apoptotic pathways, apoptosis may play a central role in its pathogenesis. Our purpose was to estimate the apoptotic process in cases of cHL using 3 different, widely accepted methods, comparing their results as well as with those found in the literature. METHODS: Detection of apoptosis was performed in 76 cases of cHL, using morphological criteria, TUNEL assay (TUNEL apoptotic index; T-AI) and immunohistochemical detection of active caspase 3 (casp3-AI) on paraffin embedded sections. RESULTS: When both apoptotic (MA) and mummified (mummi-I) cells were evaluated by morphological apoptotic index (morph-AI), the median value was 10.3%, while for MA and mummi-I the results were 3.4% and 6%, respectively. T-AI and casp3-AI values were 10.9% and 1.9%, respectively. Morph-AI was significantly higher in the mixed cellularity (MC) subtype (p7equals;0.047rpar;, while MA was significantly higher in the male subgroup (p7equals;0.03). MA was strongly correlated with casp37horbar;AI (p=0.01). CONCLUSION: Detection of apoptosis has become an important parameter in understanding tumor pathology and in designing antitumor treatment. A combination of methods is proposed in order to estimate accurately this form of cell death.

Genetic polymorphisms and risk of MALT lymphoma in Greek population.

PURPOSE: MALT lymphoma is thought to have a genetic component. Genetic studies in the greek population are rare and genetic determinants remain to be established. The current study aimed to seek correlations between genetic polymorphisms and risk of MALT lymphoma in the Greek population. PATIENTS AND METHODS: 83 MALT lymphoma patients and 60 age-matched healthy outpatients were recruited. SNPs in TNFa, LTA and CTLA-4 genes and IL1RN-VNTR and GSTT1 and GSTTM1 null polymorphisms were genotyped using published PCR/PCR-RFLP methods, while two novel PCR-RFLP methods were developed for IL-22 rs7314777 and TCF19 rs7750641 SNPs. Part of the results was validated by DNA-sequencing. Statistical analysis was performed using SPSS and the SNPstats bioinformatic tool. RESULTS: The mean age of the patients and controls were 55.9 and 56.2 years respectively. The majority of patients (63) suffered gastric marzinal zone lymphoma (GMZL) and 71.1% were stage I at diagnosis. A statistically significant association was noted for the CTLA-4 49A/ G G variant (OR:2.56,p: 0.006) and the TCF19 rs7750641 SNP T variant (OR: 3.86, p:0.023). CONCLUSIONS: Our study confirmed a role for CTLA-4 49A/G and TCF19 rs7750641 SNPs in the Greek population. Additional studies could help confirm these associations and possibly link them to prognosis or response to treatment parameters.

PUVA plus interferon α2b in the treatment of advanced or refractory to PUVA early stage mycosis fungoides: a case series.

BACKGROUND: The combination of PUVA with variable doses of systemically administered interferon α2b (IFN-α2b) reduces the number of PUVA treatments and the dose of IFN-α2b required to produce remission in all mycosis fungoides (MF) stages. OBJECTIVES: To evaluate the efficacy of the combination of PUVA and IFN-α2b in patients with late stage or refractory to treatment early stage MF. METHODS: The combination of PUVA three times weekly and IFN-α2b 2-5 MU three times weekly was retrospectively reviewed in 22 patients. Kaplan-Meyer method and log-rank test was used for statistical analysis. RESULTS: Twenty-two patients were analysed, seven with refractory to PUVA early stage MF, seven with tumour stage, five with erythrodermic MF and three with Sézary syndrome (SS). The overall response rate (complete or partial response) was 68%, including 10 complete responses (CR) (45%) and five partial responses (PR) (23%). Significantly, more patients of the early stage group achieved CR compared with the advanced stage group (86% vs. 27%, P=0.03). Within the advanced stage group, CR rates were 14% vs. 37% in stage IIB and III/SS patients respectively, but the difference was not statistically significant. Patients with early stage disease had a 2-year PFS of 100% vs. 27% for the advanced stage group (P<0.001). Sustained remissions (>2 years) were achieved in five out of six complete responders in the early stage group of patients. CONCLUSION: This combination of IFN-α2b and PUVA is an effective and safe treatment for refractory to treatment early stage MF patients as well as treatment-naïve advanced stage patients. Its efficacy is more pronounced in the former patient group.

FLT3 overexpression in acute promyelocytic leukemia patients without detectable FLT3-ITD or codon 835-836 mutations: a pilot study.

BACKGROUND: Activating mutations of the FLT3 receptor tyrosine kinase are common in acute promyelocytic leukemia (APL) but have uncertain prognostic significance. Information regarding FLT3 expression levels in APL without FLT3 mutations is lacking. MATERIALS AND METHODS: Using RT-PCR, mutation analysis of the FLT3 gene, regarding internal tandem duplications (ITDs) and codon 835-836 point mutations, was performed and real-time PCR was carried out to determine the level of FLT3 expression in 11 APL patients at diagnosis and 5 in haematological remission with molecularly detectable disease. RESULTS: High levels of FLT3 transcript, at least a 10-fold increase compared to the normal controls, were found at diagnosis in all 3 mutated cases and in 2 patients without detectable FLT3 mutations. CONCLUSION: FLT3 overexpression can be documented in patients without FLT3 mutations. These patients might benefit from treatment using specific FLT3 tyrosine kinase inhibitors. Larger studies are needed to evaluate the clinical and biological significance of FLT3 overexpression in the absence of FLT3 mutations.

Angiogenesis in Hodgkin's lymphoma: a morphometric approach in 286 patients with prognostic implications.

The significance of angiogenesis in Hodgkin's lymphoma (HL) is not well defined. The aim of this study was to evaluate various morphometric characteristics of microvessels in lymph node sections of 286 patients with HL at diagnosis and investigate their relationship with clinicopathologic parameters and prognosis. Microvessel density (MVD), total vascular area (TVA) and several size- and shape-related microvascular parameters were quantitated--after anti-CD34 immunohistochemical staining--in the region of most intense vascularization, using image analysis. An increase in microvessel caliber parameters (area, perimeter, major and minor axis length) and a decrease in MVD were noted with increasing stage. An inverse relationship was recorded between MVD and the number of involved sites (NIS) and LDH. In univariate analysis, overall disease-specific survival was adversely affected by MVD and TVA, whereas inferior failure-free survival (FFS) was associated with the presence of more flattened vessel sections. Multivariate analysis disclosed that the extent of angiogenesis (MVD/TVA), age and the NIS independently affected overall survival. Accordingly, FFS was independently linked to the shape of microvessels and albumin levels or the NIS. In conclusion, our data support the view that angiogenesis in HL provides independent prognostic information, requiring the concomitant evaluation of quantitative and qualitative aspects of microvascular network.

beta(2)-microglobulin in Hodgkin's lymphoma: prognostic significance in patients treated with ABVD or equivalent regimens.

PURPOSE: Serum beta-2 microglobulin (sbeta(2)m) is an established prognostic factor for several lymphoproliferative disorders. Because its significance in Hodgkin's lymphoma (HL) is controversial, we determined sbeta(2)m levels in pretreatment serum samples of patients with HL in order to elucidate its prognostic value in this condition. PATIENTS AND METHODS: Pretreatment sbeta(2)m levels were determined in 379 HL patients who were treated with ABVD or equivalent regimens with or without radiotherapy (RT), using a radioimmunoassay (upper normal limit 2.4 mg/l). Sbeta(2)m levels were correlated with several clinical and laboratory parameters. RESULTS: Elevated sbeta(2)m levels were detected in 138/379 (36%) patients and correlated with all clinical and laboratory baseline features except gender, lung involvement and mediastinal bulk. They also correlated with serum soluble CD30 and interleukin-10 levels. The 8-year failure-free survival (FFS) was 78 -/+ 4% for patients with normal versus 65 -/+ 7% for patients with elevated sbeta(2)m levels (p=0.003). The corresponding rates among early-stage patients were 83 -/+ 53% versus 71 -/+ 9% (p=0.003), while for advanced stages they were 70 -/+ 6% versus 64 -/+ 8% (p=0.54). In multivariate analysis of the whole patient population elevation of sbeta(2)m levels was not predictive of FFS, but it was strongly predictive among early-stage patients. The 8-year overall survival (OS) rates were 91 -/+ 3% for patients with normal versus 59 -/+ 11% (p <0,0001) for patients with elevated sbeta(2)m levels, while unrelated mortality at 8 years was 1 -/+ 1% versus 27 -/+ 12% (p<0.0001). CONCLUSION: Our data suggest that sbeta(2)m levels may be a potent prognostic factor for FFS in patients with early stage HL treated with ABVD and equivalent regimens. Their effect on OS is confounded by the higher unrelated mortality in patients with elevated baseline sbeta(2)m levels, probably due to the strong association between sbeta(2)m and older age.

B-chronic lymphocytic leukemia, small lymphocytic lymphoma, and lymphoplasmacytic lymphoma, including Waldenström's macroglobulinemia: a clinical, morphologic, and biologic spectrum of similar disorders.

Among small lymphocyte cell disorders, B-chronic lymphocytic leukemia (B-CLL), small lymphocytic lymphoma (SLL), and lymphoplasmacytic lymphoma/Waldenström's macroglobulinemia (LPL/MW) are included. B-CLL patients always have blood and bone marrow (BM) involvement by a CD5+ B lymphocyte. They frequently present with lymphadenopathy and/or hepatosplenomegaly, although in a considerable number of patients, no abnormal physical findings are found. They are prone to develop hypogammaglobulinemia, autoimmune hemolysis, or autoimmune thrombocytopenia. The typical immunophenotype of the malignant cell is CD5+, surface immunoglobulin (slg)+ (weak), CD23+, CD79b-, and FMC7-. Trisomy 12 and 13q deletions are frequent chromosomal abnormalities. The bcl-2 protein is usually overexpressed. SLL patients present with lymphadenopathy, usually generalized. Lymphocytosis is by definition absent and BM involvement, usually nodular, is found in 25% to 50% of patients. The lymph node lymphocytes are CD5+ and have a similar immunophenotype with CLL, but frequently express the LFA-1 adhesion molecule. Patients are at low risk to develop hypogammaglobulinemia, autoimmune hemolysis, or autoimmune thrombocytopenia. LPL/MW patients may present either with an accidental discovery of IgM gammopathy, symptoms related to paraproteinemia, or lymphadenopathy and/or splenomegaly. The BM is frequently involved and a leukemic picture may be found. A monoclonal gammopathy of IgM class is by definition present in MW and is frequently accompanied by hypogammaglobulinemia. Immunophenotypic studies usually reveal a CD5-, slg+ (moderate), cytoplasmic immunoglobulin (clg)+, FMC7+, and CD38+ cell. A significant proportion of cases carry the translocation t(9;14)(p13;q32) involving the PAX-5 gene. All of these disorders may potentially undergo transformation to large-cell lymphoma or Richter's syndrome. Prognostic factors have been extensively studied in B-CLL, but more studies are needed for SLL and LPL/MW. These entities should be differentiated from other B-chronic small lymphocyte cell disorders, particularly when the latter are leukemic.

Application of a prediction rule to select which patients presenting with lymphadenopathy should undergo a lymph node biopsy.

We conducted the present study to develop a clinical prediction rule for discriminating which patients with peripheral lymphadenopathy require a lymph node biopsy. The clinical features of 315 patients with peripheral lymphadenopathy were analyzed to develop the prediction rule: 83 had diseases requiring a lymph node biopsy (Lymph Node Biopsy Group [BG]), while 232 had diseases that could be diagnosed without a lymph node biopsy (Non-Lymph Node Biopsy Group [NBG]). Among 23 examined clinical covariates, we identified 6 that independently predicted the need for lymph node biopsy and were graded as follows: 1) Age: x1 = 0, if < or = 40 years and 1, if > 40 years. 2) Tenderness in palpation: x2 = 0, if absent and 1, if present. 3) Size of the greatest lymph node: x3 = 0, if < 1.0 cm2, 1 if 1.0-3.99 cm2, 2 if 4.0-8.99 cm2, and 3 if > or = 9.0 cm2. 4) Generalized pruritus: x4 = 1, if present and 0, if not. 5) Supraclavicular lymphadenopathy: x5 = 1, if present and 0, if not. 6) Texture: x6 = 1, if nodes are hard and 0, if not. The prediction rule was then validated in a subsequent group of 160 patients (32 in the BG; 128 in the NBG). A score Z = 5x1 - 5x2 + 4x3 + 4x4 + 3x5 + 2x6 - 6 corresponded to every patient, according to the results of logistic regression analysis. If patients with Z > or = 1 were considered to need lymph node biopsy, the sensitivity of the prediction rule was 95.2% (95% confidence intervals [CI]: 88.1%-98.1%) and the specificity was 81.0% (95% CI: 75.4%-85.6%). Within the Validation Group of patients the prediction rule was at least equally effective. Sensitivity was 96.9% (95% CI: 83.9%-99.5%) and specificity was 91.4% (95% CI: 85.1%-95.2%). The described rule can be useful in the clinical evaluation of patients with peripheral lymphadenopathy. Further validation by other groups is required, and its cost-effectiveness has to be investigated.

Correction of disease related anaemia of B-chronic lymphoproliferative disorders by recombinant human erythropoietin: maintenance is necessary to sustain response.

Thirty three B-chronic lymphoproliferative disorder (B-CLD) patients [22 with B-chronic lymphocytic leukemia (B-CLL), 5 with small lymphocytic lymphoma (SLL) and 6 with lymphoplasmacytic lymphoma (LPL)] with anaemia (Ht <32%) of no other cause but their disease, received recombinant human erythropoietin (r-HuEPO). The treatment protocol provided r-HuEPO in a dose of 150 U/kg s.c. thrice weekly for 3 mo. After 1.5 mo of r-HuEPO administration, if response was not satisfactory, r-HuEPO dose escalation was utilised by giving incremental doses of 50 U/kg more than the previous dose up to a maximum dose of 300 U/kg tiw. After maximal response, half of the responding patients discontinued therapy, while the other half received maintenance therapy at a dose of 150 U/kg s.c./w. Oral iron was given throughout the study. Pretreatment EPO levels were determined in all patients. A complete response (CR) was defined when Ht was >38% and a partial response (PR) when there was an increase of the Ht >6% from the initial value was achieved. Sixteen of the 22 B-CLL patients had Rai stage III disease and 6 stage IV, with a median duration of anaemia 27 months (6-38); twelve of them were receiving chlorambucil while the rest were on no treatment. Of the SLL and LPL group, 4 patients had Ann Arbor stage III disease and 7 stage IV with a median duration of anaemia 24 months (5-36); 8 patients were on chlorambucil. Complete response was achieved in 50% of the B-CLL group and 54% of the SLL and LPL group, with an overall response rate of 77% and 81% respectively. All patients on maintenance therapy had a continuous response, while all patients, in whom rHuEPO was discontinued, relapsed. No correlation was found between patients: with low or high pretreatment serum EPO levels; those receiving concomitant therapy or not; those with B-symptoms or not; those with a non-diffuse or diffuse bone marrow infiltration pattern; and with splenomegaly or not. Life quality was significantly improved and no major side effects were encountered. We conclude from our study that r-HuEPO is very effective in correcting disease-related anaemia in B-CLD, resulting in down-staging of Rai stage III patients and that maintenance therapy is necessary. Whether the correction of anaemia improves patients' overall survival, still remains to be seen.

EBVD combination chemotherapy plus low dose involved field radiation is a highly effective treatment modality for early stage Hodgkin's disease.

To evaluate the efficacy of EBVD combination chemotherapy followed by low dose (LD) involved field (IF) radiation therapy (RT) in patients with clinical stage (CS) I-IIA Hodgkin's disease (HD), we analyzed 148 patients treated in our Unit from March 1988 to November 1995. EBVD consisted of Epirubicine 40 mg/m2, Bleomycin 10 mg/m2, Vinblastine 6 mg/m2 and Dacarbazine 300 mg. All drugs were administered i.v. at days 1 and 15, every 4 weeks, for a total of 4-6 cycles. LDIF RT (24-32 Gy) was scheduled for patients with complete response (CR) or >90% reduction of tumor load, after EBVD. Patients with stable or progressive disease (SD, PD) after EBVDx3 or poor compliance to the regimen received mantle or inverted Y RT at standard dose. The median follow-up of patients currently alive was 71.5 months. 129 patients achieved a CR after EBVD and 10 a >90% reduction of tumor load, for a post-CT response rate of 94%. Eight patients had SD after EBVDx3 and one had a partial response with poor compliance. All 9 patients received mantle or inverted Y RT and 8/9 achieved a CR. Nine patients relapsed at a median of 7 months from the end of treatment. At 10 years, FFS was 90% and overall survival 95%. Six patients have died so far; 5 of HD and one of stroke. One patient developed a diffuse large cell lymphoma 48 months after the diagnosis of HD. We conclude that EBVD followed by LDIF RT is a highly effective regimen for patients with CS I-IIA HD. Longer follow up is required to assess the risk of secondary malignancies, especially solid tumors.

Campath-1H (anti-CD52) monoclonal antibody therapy in lymphoproliferative disorders.

Campath-1H is a humanized monoclonal antibody targeted against the CDw52 membrane antigen of lymphocytes, which causes complement and antibody-dependent cell-mediated cytotoxicity. Campath-1H has been used in B-chronic lymphocytic leukemia (B-CLL), T-prolymphocytic leukemia (T-PLL), and low-grade non-Hodgkin's lymphoma (LGNHL). Campath-1H is administered intravenously thrice weekly for up to 12 wk, at an initial dose of 3 mg, escalated to 10 and 30 mg. The responses (complete [CR] and partial [PR]) obtained in untreated B-CLL patients are of the order of 90%. In previously treated B-CLL patients, responses are of the order of approximately 40%, with 2-4% CRs. Responses are more prominent in the blood and bone marrow compared to the lymph nodes. The median duration of response is 9-12 mo. Because of the antibody's higher activity on circulating lymphocytes, it has been used for in vivo purging of residual disease in B-CLL, followed by autologous stem-cell transplantation. In heavily pretreated advanced stage LGNHL, response is achieved only in 14% of cases with B-phenotype; a 50% response rate is noted in mycosis fungoides. In T-PLL, the CR rate is approximately 60%. Promising results have been reported in a small number of patients with refractory autoimmune thrombocytopenia of lymphoproliferative disorders. The main complications of Campath-1H treatment are caused by tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 release, usually during the first intravenous infusion, and include fever, rigor, nausea, vomiting, and hypotension responsive to steroids. These side effects are usually less severe with subsequent infusions and can be prevented by paracetamol and antihistamines. Immunosupression resulting from normal B- and T-lymphocyte depletion is frequent, resulting in an increased risk for opportunistic infections. More clinical trials in a larger number of patients are necessary to determine the exact role and indications of Campath-1H in lymphoproliferative disorders.

Primary effusion lymphoma in two HIV-negative patients successfully treated with pleurodesis as first-line therapy.

Primary effusion lymphoma (PEL) is a rare non-Hodgkin's lymphoma (NHL) mostly occurring in HIV-positive patients. It is characterized by the development of effusion in one or more body cavities, with no tumor masses and a positive human herpes virus-8 (HHV8) status. It has a poor survival profile and no optimal treatment is yet defined. We report two HIV-negative, HHV8-positive patients with PEL of the pleural cavity who achieved a durable remission after pleurodesis with bleomycin and no systemic therapy. We also perform a review of the relevant literature regarding the clinical data, treatment, and survival of PEL in HIV-negative patients.