Abnormalities of the P53, MDM2, BCL2 and BAX genes in acute leukemias.

Abnormalities of the P53 network have been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). The purpose of this study was to define P53 gene mutations, to detect MDM2 gene amplification and to estimate mRNA expression of P53, MDM2, BCL2 and BAX genes in patients with ALL and AML. Twenty-five patients with ALL and 65 patients with AML, both recently diagnosed, were included into this study. Exons 5-8 of the P53 gene with flanking intronic sequence were amplified by the polymerase chain reaction (PCR) method and subjected to mutation screening by single-strand conformation polymorphism analysis (SSCP). Mutation of the P53 gene was found in one patient of the 25 with ALL and in five patients of the 65 with AML. Sequence analysis was subsequently performed. One mutation in intronic sequence in ALL and four missense mutations and one silent nucleotide substitution in AML were identified. Amplification of MDM2 gene was detected by multiplex-PCR analysis in only one sample from patient with ALL, but was not observed in any case of AML. To gain further insight into the role of P53 network in the evolution of acute leukemias, the P53, MDM2, BCL2 and BAX mRNAexpressions in portion samples from patients with ALL and AML were analyzed using multiplex RT-PCR. Although a low frequency of molecular disturbances of the P53 and the MDM2 genes was detected in this study, there was a high percentage of cases with increased mRNA level of P53 and MDM2. A high frequency of BCL2 mRNA overexpression and a relatively low frequency of BAX mRNA overexpression detected in both analyzed leukemias in this study, indicate that altered transcription of these genes may be involved in leukemogenesis.

Activity of cladribine combined with etoposide in heavily pretreated patients with indolent lymphoid malignancies.

We determined the effectiveness and toxicity of combined chemotherapy consisting of etoposide 100 mg/m(2)/day i.v. and cladribine (2-CdA) 0.12 mg/kg/day i.v. each for 5 days (EC regimen) in the treatment of refractory or relapsed low-grade non-Hodgkin's lymphoma and chronic lymphocytic leukemia. The cycles were repeated every 28 days, reaching a maximum of six courses. Twenty patients entered the study. All patients had received three or more cycles of chemotherapy before the EC regimen (median 8, range 3-19). Thirteen patients received 2-CdA before the EC regimen. Seven out of 20 patients (35%) responded, including one complete response. Median overall survival time of responding patients was 22 months (range 3-30). Myelosuppression and infections were the major toxicity of the EC regimen.

Second malignancies and Richter's syndrome in patients with chronic lymphocytic leukaemia treated with cladribine.

The increased frequency of second malignancies in chronic lymphocytic leukaemia (CLL) is well known. Moreover, antineoplastic therapy additionally increases the risk of secondary cancers. In this study, we analysed whether treatment with cladribine (2-chlorodeoxyadenosine, 2-CdA) during the course of CLL had an impact on the subsequent occurrence of either secondary solid tumours or Richter's syndrome. There were 1487 eligible patients, 251 treated with 2-CdA alone, 913 treated with alkylating agents (AA)-based regimens alone and 323 treated with both 2-CdA and AA. Median time from the start of CLL treatment to the diagnosis of secondary malignancy was 1.9 years (0.5-5.1 years) for the 2-CdA group, 1.8 years (0.3-7.9 years) for the AA group and 3.9 years (0.3-8.4 years) for the 2-CdA+AA group. A total of 68 malignancies were reported in 65 patients. Ten events were non-melanotic skin cancers and were excluded from the analysis, leaving 58 events in 58 patients. In the group of patients treated with 2-CdA alone, there were 15 (6.0%) cases, in the group of patients treated with AA alone there were 26 (2.8%) cases, and in the group treated with 2-CdA+AA there were 17 (5.3%) cases of secondary malignancies. The differences between the frequency of secondary malignancies in the 2-CdA and 2-CdA+AA versus AA alone groups were not significant (P=0.05 and P=0.06, respectively). Only lung cancers occurred significantly more frequently in the 2-CdA (2.8%) and 2-CdA+AA (2.2%) treated groups compared with the AA patients (0.3%) (P<0.001 and P<0.01, respectively). In conclusion, 2-CdA in CLL patients does not seem to increase the risk of secondary malignancies except for lung cancers. However, further studies are necessary to establish the real risk of lung cancer in CLL patients treated with 2-CdA.

Expression of gene encoding P65 oncofetal protein in acute and chronic leukemias.

In this study we have established conditions for p65 gene expression analysis by reverse transcriptase polymerase chain reaction (RT-PCR). On the basis of this technique we analyzed p65 gene expression in various types of leukemia: acute myeloblastic leukemia (AML) (n=26); acute lymphoblastic leukemia (ALL) (n=26) and chronic lymphocytic leukemia (CLL) (n=40). The highest frequency of p65 gene expression was found in the patients with CLL (66%). No relationship between the expression of p65 gene and clinical stage of leukemia was observed. The lower percentage of positivity (presence of gene transcript) was seen in patients with ALL (42%) and AML (46%).

Cladribine decreases the level of angiogenic factors in patients with chronic lymphocytic leukemia.

We investigated the serum concentration of basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGFbeta1), using an enzyme linked immunosorbent assay (ELISA) in a group of 18 chronic lymphocytic leukemia (CLL) patients, before and after a successful treatment with cladribine (2-chlorodeoxyadenosine, 2-CdA) and 16 healthy volunteers. The serum level of bFGF was found to be significantly lower in the control group (median 0.15 pg/ml, range 0.0-15.7 pg/ml), when compared to the untreated CLL patients (median 41.4 pg/ml, range 2.1-292.6 pg/ml) (p=0.0002). After a successful 2-CdA treatment we observed a significantly lower level of this cytokine (median 10.55 pg/ml, range 0.4-140.4 pg/ml) (p=0.0019) in the same patients. However, the level of bFGF in this group was still higher than in the control group (p=0.003). The levels of TGFbeta1 were higher in the group of untreated CLL patients (median 31.36 ng/ml, range 14.36-75.71 ng/ml) than in the control group (median 28.35 ng/ml, range 10.85-70.10 ng/ml) (p=0.029). After the 2-CdA treatment serum concentration of this cytokine decreased significantly (median 20.34 ng/ml, range 3.02-43.85 ng/ml) (p=0.031) with similar levels present to that of the healthy control group (p=0.3). In conclusion, we have shown that the serum concentration of bFGF and TGFbeta1 in CLL patients were significantly reduced after 2-CdA chemotherapy that resulted in remission. The level of these factors might correlate with the activity of the disease.

Molecular diversity of gammadelta T cells in peripheral blood from patients with B-cell chronic lymphocytic leukaemia.

To characterize circulating gammadelta T cell subpopulations in B chronic lymphocytic leukaemia patients (n=30), TCR Vgamma and Vdelta gene-segment use was analyzed by RT-PCR using a panel of subfamily-specific oligonucleotide primers. All results were compared with those obtained with specimens from healthy donors (n=10). The cells expressing Vdelta1+ TCR displayed the highest relative increase in B-CLL patients (particularly observed in 60% of cases), but Vdelta3+ T lymphocytes also expanded in leukaemic peripheral blood (10% of studied cases). Both mentioned gammadelta T cell subsets were significantly more frequent in the most severe stages of disease--Rai III+IV. The analysis of Vgamma region usage in TCR formation revealed that gammadelta T cells from B-CLL patients predominantly expressed a Vgamma9 segment (26 of 30 cases), usually linked to Cgamma1 region. It should be noticed that the dominant TCR genes expression in a 50% of healthy donors was Vdelta2+/Vgamma9+, however, Vgamma4 and Vgamma8 transcripts were also observed (2 and 3 of 10 cases, respectively). The above results indirectly indicate that gammadelta T lymphocyte expansion was driven by the oligo- or polyclonal proliferation and can reflect specific response against the autologous tumor cells.

Richter's syndrome following cladribine therapy for chronic lymphocytic leukemia first manifested as pathologic fracture of the femur.

Richter's syndrome (RS) refers to the development of aggressive non-Hodgkin's lymphoma (NHL) during the course of chronic lymphocytic leukaemia (CCL). It occurs in approximately 3% of patients with CLL. The isolated form of this complication in bone is extremely rare and, so far, has not been described in a patient treated with cladribine (2-CdA). We report a case of CLL treated successfully with 2-CdA, where isolated diffuse large B-cell lymphoma (LBCL) developed 2 years after the diagnosis of CLL Rai II and one year after the completion of 2-CdA treatment. RS was first manifested as a pathologic fracture of the left femur. The LBCL was clonally distinct from the original CLL cells. The patient was successfully treated with CHOP and radiotherapy and obtained complete response of the LBCL.

Lymphocyctes Tgammadelta in clinically normal skin and peripheral blood of patients with systemic lupus erythematosus and their correlation with disease activity.

Human Tgammadelta lymphocytes constitute from 1 to 15% of all peripheral blood lymphocytes. Recent work has demonstrated that this population plays a major role in the pathogenesis of infectious and immune diseases. Increased numbers of gammadelta T cells have been found in affected skin from systemic sclerosis and chronic cutaneous lupus erythematosus patients. In our study, we have determined the numbers of Tgammadelta lymphocytes and their subpopulations in peripheral blood from 29 patients with systemic lupus erythematosus (SLE) and in 19 healthy volunteers using flow cytometry and specific monoclonal antibodies. The same cells in uninvolved skin from SLE patients and human controls using immunohistochemical analysis were estimated. T-Cell receptor (TCR) delta chain gene rearrangement was identified with primers for Vdelta1, Vdelta2 and Vdelta3 by the polymerase chain reaction. Statistical analysis showed a significantly decreased number of gammadelta T cells in SLE patients (26.4+/-16.9/microl) compared with the control group (55.3+/-20.6/microl (p < 0.001). The number of Vdelta2 TCR+ and Vgamma9 TCR+ subpopulations was also lower in SLE patients than in healthy persons. No statistical correlation between disease activity and the number of gammadelta T cells was demonstrated. The percentage of Tgammadelta lymphocytes in clinically normal skin from SLE patients was twice (22.0+/-9.4%) that found in the skin from healthy persons (11.1+/-5.5%) (p < 0.002). Higher percentages of the Vdelta2 TCR+ and Vgamma9 TCR+ subpopulation of lymphocytes were found in the skin from SLE patients. We have also found positive correlation between the percentage of Tgammadelta lymphocytes in skin and the activity of SLE (r=0.594, p < 0.001), and between subpopulation Vdelta3 TCR+ and disease activity (r=0.659, p< 0.001). In conclusion, the results of our studies demonstrate that, in patients with SLE, accumulation of Tgammadelta lymphocytes can be seen in clinically normal skin, and the percentage of these cells correlates with the activity of the disease.

Cladribine combined with cyclophosphamide and mitoxantrone as front-line therapy in chronic lymphocytic leukemia.

The objective of the study was to determine the effectiveness and the toxicity of a combined chemotherapy consisting of cladribine (2-CdA), mitoxantrone and cyclophosphamide (CMC regimen) in the treatment of previously untreated B cell chronic lymphocytic leukemia (B-CLL). From August 1998 to December 2000 2-CdA was administered at a dosage of 0.12 mg/kg for 3 (CMC3) or 5 (CMC5) consecutive days, mitoxantrone at 10 mg/m2 on day 1 and cyclophosphamide at 650 mg/m2 on day 1 to 62 patients with advanced or progressive B-CLL. The cycles were repeated at 4 week intervals or longer if severe myelosuppression occurred. Twenty patients received CMC5 and 42 patients CMC3. Within the analyzed group an overall response (OR) rate (CR+PR) of 64.5% (95% CI: 52.7-76.3%) was reported, including 29.0% CR. There was no difference in the CR rate between the patients treated with CMC5 (30%) and CMC3 (28.6%) (P = 0.9), nor in the OR rate (55.0% and 69.0%, respectively, P = 0.3). Residual disease was identified in seven out of 18 (38.9%) patients who were in CR, including two treated with CMC5 and five treated with CMC3 protocols. CMC-induced grade III or IV thrombocytopenia occurred in 12 (19.4%) of patients, including four (20%) CMC5-treated and eight (19%) CMC3-treated patients (P= 0.8). Neutropenia grade III or IV was observed in seven (35%) and 11 (26.2%) patients, respectively (P = 0.8). Severe infections, including pneumonia and sepsis, occurred more frequently after CMC5 (11 patients, 55.0%) than CMC3 (10 patients, 28.6%) (P = 0.03) Fourteen patients died, including six treated with CMC5 and eight treated with CMC3 (30% and 19%, respectively). Infections were the cause of death in nine patients, including four in the CMC5 group and five in the CMC3 group. In conclusion, our results indicate that the CMC programme is an active combined regimen in previously untreated B-CLL patients; its efficiency seems to be similar to that observed earlier in B-CLL patients treated with 2-CdA as a single agent. However, toxicity, especially after CMC5 administration, is significant. Therefore, we recommend the CMC3 but not the CMC5 programme for further evaluation.

Enzyme-linked immunosorbent assay in screening of leukemia-associated nuclear proteins.

Our previous data revealed some diversities in electrophoretic characteristics of nuclear fraction proteins isolated from peripheral blood mononuclear cells of B-cell chronic lymphocytic leukemia (B-CLL) patients and healthy donors. Two electrophoretically-specific nuclear non-histones in the molecular mass zone of 38/39 and 44/46 kDa of leukemic mononuclear cells were used as immunogens to produce rabbit antisera. The Western blot analysis indicated that both nuclear components are expressed only in mononuclear cells isolated from peripheral blood of B-CLL patients, but not in those isolated from the blood of healthy donors. For further investigations of nuclear fraction from normal and B-CLL mononuclear cells, an enzyme-linked immunosorbent assay (ELISA) was used. The results obtained by ELISA with the antisera raised against both electrophoretically-specific B-CLL nuclear polypeptides revealed a different extend of cross-reactivity of nuclear fraction preparations isolated from normal cells and those isolated from leukemic ones. We noticed that nuclear fraction preparations which originated from leukemic mononuclear cells are much more reactive than normal ones with both antisera (at a broad range of antisera dilutions).

Nuclear non-histone protein p36 associated with colorectal tumours.

Rabbit serum raised against electrophoretically specific nuclear polypeptides with molecular weights of 35-40 kD from colon adenocarcinoma has been used to detect p36 antigen in 83.3% (40 of 48) of cases of large intestine tumours by means of Western blot technique. Immunological analysis revealed that this antiserum cross-reacted with antigen of the same molecular weight in 83.3% (10 of 12) and 85.7% (6 of 7) nuclear protein preparations from stomach and lung tumours, respectively, but not in any control tissue samples. No cross-reactivity within the region of 36 kD was observed among nuclear proteins isolated from mononuclear cells of B-cell chronic lymphocytic leukaemia patients as well as healthy donors.

Two-dimensional gel electrophoresis of nuclear proteins of different stages of B-chronic lymphocytic leukaemia.

Two-dimensional polyacrylamide gel electrophoresis was used to compare the composition of nuclear proteins from normal and B-chronic lymphocytic leukaemia (B-CLL) mononuclear cells. Some differences in the electrophoretic behaviour of these proteins from normal and transformed cells, especially with molecular weights/pI of 14-16 kD/5.9-7.4; 28-32 kD/4.9-5.5; 38-39 kD/5.4-6.1; 44-46 kD/5.1-5.6; 47-52 kD/5.0-5.6; 64-69 kD/5.1-5.7, and 95-105 kD/5.2-5.5, were observed. The comparative analysis of nuclear proteins, obtained from mononuclear cells of patients with B-CLL at different stages of development, indicated that the expression of some protein components might be correlated with the progression of this disease.

Cladribine in combination with mitoxantrone and cyclophosphamide(CMC) in the treatment of heavily pre-treated patients with advanced indolent lymphoid malignancies.

The aim of our study was to determine the effectiveness and toxicity of combined chemotherapy consisting of cladribine (2-chloro-deoxyadenosine, 2-CdA), mitoxantrone and cyclophosphamide (CMC regimen) in the treatment of refractory or relapsed indolent lymphoproliferative disorders. The treatment course consisted of 2-CdA given at a dose of 0.12 mg/kg/24 h in a 2-h intravenous infusion for 5 (CMC5) or 3 (CMC3) consecutive days, mitoxantrone 10 mg/m2 on day 1 and cyclophosphamide 650 mg/m2/iv on day 1. Thirty-three patients (19 with B-CLL and 14 with LG-NHL) entered the study and all of them were eligible. Twenty patients (60.6%) were recurrent after prior therapy and 13 (39.4%) had refractory disease. All patients received 5 or more cycles of chemotherapy before CMC treatment. Twenty-one patients were treated with CMC5 regimen and 12 with CMC3 regimen. The overall response rate, including CR and PR, was 48.6% (95% CI 32-66). There were no differences in the frequency of responses between the CMC3 and CMC5 treated groups (p>0.05). One patient with B-CLL and three patients with lymphocytic lymphoma achieved CR (12.1%). Among 12 patients (36.4%) who achieved PR there were 6 CLL patients, and 6 lymphoma patients. The major toxicity was myelosuppression. Severe neutropenia was seen in 11/33 (33.3%) patients, more frequently in patients who received CMC5 than in the patients who received CMC3, both in the CLL (50.0% and 28.5%, respectively) and in the LG-NHL group (22.2% and 0%, respectively). The rate of thrombocytopenia was similar in both groups. Infections and fever of unknown origin complicated the treatment with CMC5 more often than with CMC3: five episodes were seen in 3 patients treated with CMC3 when compared to 15 episodes in 12 patients treated with CMC5. In conclusion, the CMC programme is an active combined regimen in heavily pre-treated CLL and LG-NHL patients. However, its toxicity is significant and we suggest a shortening of 2-CdA infusion from 5 to 3 d in further studies. Whether a combination of 2-CdA with cyclophosphamide and mitoxantrone would result in improved outcome as compared to 2-CdA alone, is being investigated in a prospective, randomised trial.

Altered expression of nuclear non-histone protein (p44/46) in different stages of B-chronic lymphocytic leukemia.

Our previous data have shown some differences in electrophoretic characteristics of proteins from cellular fractions (nuclear, mitochondrial, microsomal and cytosolic) isolated from peripheral blood mononuclear cells of B-cell chronic lymphocytic leukemia (B-CLL), acute lymphoblastic leukemia (ALL) patients and healthy donors. The main differences were found in electrophoretic patterns of nuclear proteins from normal and leukemia cells, especially in the nuclear mass regions of 36-52, 58-85, and 120-180 kDa. Electrophoretically-specific nuclear non-histone protein in the molecular mass zone 44/46 kDa of cells obtained from the peripheral blood of a B-CLL patient was used to produce rabbit polyclonal antiserum. SDS-polyacrylamide gel electrophoresis as well as immunological techniques (Western blot and immunocytochemistry) indicate that the nuclear protein with a molecular mass of 44/46 kDa is specifically expressed in mononuclear cells from B-CLL patients. The expression of this particular nuclear protein seems to correlate with the progression of the leukemia.

Low-grade non-Hodgkin's lymphoma in a patient with systemic lupus erythematosus.

Coexistence of systemic lupus erythematosus (SLE) with low-grade non-Hodgkin's lymphoma (LGNHL) has been described occasionally in the literature with the potential pathogenetic role of monoclonal B CD5+/CD19+ cells. We report a case of LGNHL which developed 18 months after diagnosis of SLE. The monoclonal population of lymphocytes in the peripheral blood and bone marrow was CD5/CD19 negative but CD19/CD22 positive. The SLE responded well to treatment with prednisone and the course of the LGNHL was stable and cytotoxic treatment was not required.