Clinical implications of glutathione S-transferase genotyping in patients with diffuse large B-cell lymphoma.

PURPOSE: Polymorphic deletions in glutathione S-transferase (GST) genes are recognized as a risk factor for lymphoma, other hematological and non-hematological malignancies. The purpose of the present study was to investigate whether deletions of GSTT1 and GSTM1 as well as GSTP1 Ile- 105Val single nucleotide polymorphism influence clinical presentation, response to therapy and outcome in patients with diffuse large B-cell lymphoma (DLBCL). METHODS: The study included a total of 82 DLBCL patients treated with rituximab-CHOP (R-CHOP) therapy (6-8 cycles). GST genes were analyzed with PCR-based methodology. RESULTS: The obtained frequencies of GSTT1 and GSTM1 null genotypes were 24 and 63%, respectively. The variant GSTP1 Val allele was present in 76% of the patients. No association between GST genotypes and clinical presentation was found. However, a higher frequency of GSTM1 null genotype was observed in patients who developed DLBCL before the age of 60 [odds ratio (OR) 3.12, 95% confidence interval (CI) 1.11-9.17; p=0.03]. Patients carrying at least one GSTP1 Val allele achieved remission in a shorter time period than patients with GSTP1 Ile/Ile genotype (p=0.05). GST genotypes didn't influence the incidence of relapse and survival. There were no toxic effects, life-threatening infections or significant delay in immunochemotherapy in the analyzed group of patients. CONCLUSION: The present study showed the association of GSTM1 null genotype and DLBCL development before the age of 60 (prognostic cutoff). GST genotypes didn't influence survival, but patients with at least one low-producing GSTP1 Val allele achieved clinical remission in a shorter time period.

Kidney failure as an unusual initial presentation of biclonal gammopathy (IgD multiple myeloma associated with light chain disease)--a case report.

INTRODUCTION: Immunoglobulin D (IgD) myeloma is a rare disease, about 2% of all myelomas, even rarer when accompanied with another multiple myeloma in biclonal gammopathy. We presented a case of biclonal gammopathy-as-sociated manifestation of IgD myeloma and light chain disease in a patient who initially had renal failure. CASE REPORT: 37-year-old male approximately one month before hospitalization began to feel malaise and fatigue along with decreased urination. Laboratory analysis revealed azotemia. A dialysis catheter was placed and hemodialysis started. The patient was then admitted to our hospital for further tests and during admission, objective examination revealed pronounced paleness with hepatosplenomegaly and hypertension (170/95 mmHg). Laboratory analysis showed erythrocyte sedimentation rate 122 mm/h, expressed anemic syndrome (Hb 71 g/L) and renal failure dialysis rank: creatinine 1,408 micromol/L, urea 31.7 mmol/L. There was two M components in serum protein electrophoresis: IgD lambda and free light chain lambda. Proteinuria was nephrotic rank (5.4 g/24 h), whose electrophoresis revealed 2 M components--massive in alpha 2 fraction of 71%; 7% in the discrete beta fraction, beta 2M / serum 110 mg / L, in urine 1.8 mg/L--extremely high; IgL kappa I lambda index 1:13 (reference value ratio 2:1). The findings pointed to double myeloma disease: IgD myeloma and Bence Jones lambda myeloma. Bone biopsy confirmed IgD myeloma lambda 100% infiltration medulla predominantly plasmablasts. The treatment continued with hemodialysis 3 times per week with chemotherapy protocol bortezomib, doxorubicin, dexamethasone. After 4 cycles of chemotherapy, there was a decrease of IgD, lamda-light chains, reduction in proteinuria (1.03 g/24 h), so hemodialysis was reduced to once per week. Six months after treatment initiation the patient underwent autologous bone marrow transplantation. In a 2-year follow-up period double myeloma disease showed complete remission. CONCLUSION: The presented rare form of double myeloma disease with initial renal insufficiency underscores the importance of careful observation and teamwork that can alter the course of this serious disease.

Multiple myeloma invasion of the central nervous system.

INTRODUCTION: Multiple myeloma (MM) is characterized by the presence of neoplastic proliferating plasma cells. The tumor is generally restricted to the bone marrow. The most common complications include renal insufficiency, hypercalcemia, anemia and reccurent infections. The spectrum of MM neurological complications is diverse, however, involvement of MM in the cerebrospinal fluid (CSF) and leptomeningeal infiltration are rare considered. In about 1% of the cases, the disease affects the central nervous system (CNS) and presents itself in the form of localized intraparenchymal lesions, solitary cerebral plasmocytoma or CNS myelomatosis (LMM). CASE REPORT: We presented the clinical course of a 55-year-old man with MM and LMM proven by malignant plasma cells in the CSF, hospitalized with the pain in the thoracic spine. His medical history was uneventful. There had been no evidence of mental or neurological impairment prior to the seizures. Physical examination showed no abnormalities. After a complete staging, the diagnosis of MM type biclonal gammopathia IgG lambda and free lambda light chains in the stage III was confirmed. The treatment started with systemic chemotherapy (with vincristine, doxorubicin plus high-dose dexamethasone--VAD protocol), radiotherapy and bisphosphonate. The patient developed weakness, nausea, febrility, dispnea, bilateral bronchopneumonia, acute renal insufficiency, confusions, headaches and soon thereafter sensomotor aphasias and right hemiparesis. The patient was treated with the adequate therapy including one hemodyalisis. His neurological status was deteriorated, so Multislice Computed Tomography (MSCT) of the head was performed and the findings were normal. Analysis of CSF showed pleocytosis, 26 elements/mL and increased concentrations of proteins. Cytological analysis revealed an increased number of plasma cells (29%). Electrophoretic analysis of proteins disclosed the existance of monoclonal components in the serum, urine and CSF. Immunofixation electrophoretic and quantitative nephelometric tests confirmed Biclonal multiple myeloma of IgG lambda and light chain lambda isotypes. Analysis of neurothropic viruses with ELISA methods was negative. Once the presence of LMM was confirmed, the patient received intrathecal chemotherapy with methotrexate, cytosine arabinoside, dexamethasone three times a week, and systemic high doses of dexamethasone iv like a single agent without craniospinale irradiations. Despite the treatment, the patient died one month after the diagnosis. Autopsy was not performed. CONCLUSION: Presented patient, as well as most other patients with MM progressing to CN Sinfiltration was in the stage III. In addition to the detailed clinical examination, and all investigations required for MM diagnosis and staging of the disease, we introduced the additional CSF examination and calculation of kappa lambda ratio, that helped us make an early diagnosis and prognosis of MM with LMM. Although LMM had a low prevalence, it could be more frequent than expected especially in patients with high risk. CSF examination with positive plasma cells and abnormal morphology remains the hallmark for diag nosing CNS infiltration.

Allogeneic stem cell transplant for chronic myeloid leukemia as a still promising option in the era of the new target therapy.

BACKGROUND/AIM: Introducing tyrosine kinase inhibitors (TKIs) has essentially changed curative approach, to be precise, clearly improved treatment efficacy for chronic myeloid leukemia (CML). Thus, the place and usage of allogeneic stem cell transplant (SCT) in CML treatment--as a former "nearly monopolistic" therapeutic manner--is nowadays controversial. The objective of this retrospective study was to evaluate the results obtained in the treatment of CML patients, with a particular attempt to define parameters critical for clinical benefit and superior overall outcome following allogeneic SCT. METHODS: A total of 32 CML patients (27 in chronic phase and 5 with advanced disease), with female/male ratio 11/21, aged from 9 to 54 (32 in average) years, underwent allogeneic SCTs (1993 to 2009). The initial treatment for 25 patients was interferon alpha (IFN-alpha) with or without ARA-C, and additional 7 patients with no response to imatinib mesylat (IM). The time from diagnosis to SCT was approximately 12 (range 3-37) months. The patient were categorized according to the risk for the disease, transplant-related mortality (TRM) scoring system, and stem cell (SC) source. The basic conditioning regimen was a combination of busulphan and cyclophosphamide (BuCy-2). Graft-versus-host disease (GvHD) was typically prevented with cyclosporine-A (CsA) and methotrexate (MTX). RESULTS: Engraftment was observed in 26 (84.4%) patients, with polymorphonuclear (PMNs) and platelet (Plt) recovery on the 15th (range 10-22) and 19th (range 11-29) posttranspalnt days, respectively. Acute GvHD (aGvHD) had 13/26 (50%), and chronic GvHD (cGvHD) 10/21 (47.1%) patients. The incidence of overall TRM was 46.8% (15/32), while early death was noticed in 4 (12.5%) patients. A cause of death in 9 (28.1%) patients was cGvHD, in 2 (6.25%) patients infection, and in 3 (9.35%) cases disease-relapse was occurred. Fourteen (43.7%) of the patients are still alive, 9 from the low-risk group for TRM, with long-term survival from 1 to 16 years. Patients who received SCs from peripheral blood (PB) vs bone marrow (BM) had significantly faster engraftment (p < 0.05), lower oropharingeal mucositis rate (25% vs 70%; p < 0.05), but more frequent cGvHD (83.3% vs 30.3%; p < 0.05). A significantly improved (log-rank = 2.39; p < 0.01) overall survival (OS) was obtained in BM-setting. CONCLUSION: The results obtained in this study are in accordance with data from analogous clinical trials. Exactly, in the era of the new target therapy (TKI application), allogeneic SCT can be still a convenient therapeutic approach for well-selected CML-patients, especially for those with initial high-risk disease and lower probability of TRM.

[Autologous stem cell transplantation in the treatment of multiple myeloma--single center experience].

BACKGROUND/AIM: In comparison to standard therapy autologous stem cell transplant (ASCT) with high doses mel-phalane has improved treatment of multiple myeloma (MM) patients. The aim of this study was to evaluate the results of treatment of MM patients in our center with ASCTconditioning with melphalane or combining busulphane, cyclophosphamide and melphalane. METHODS: We performed 62 ASCT procedures in 47 patients from 1998 till 2008. Single ASCT were performed in 32 patients (68%), after 3-6 cycles of (26% patients. RESULTS: Median engraftment was on 12th day. In a 50-month follow-up period 64% patients were alive. The overall response rate (ORR), wich was reached in 38 (80%) patients, was better in the group of patients treated in the early phase of MM. Totally 25 (53%) patients were without progression in a 25-month follow-up period. Twenty patients met criteria for CR + VGPR (very good partial remission), that was 5 patients more than in the period before ASCT. Fourteen (30%) patients died and median time till death was 17 months. CONCLUSION: The ASCT perfomed in early phase of MM after V A D induction had a significant influence onthe treatment of MM patients. Reaching CR + VGPR before and after the ASCT is predictive factor for overall survival (OS) or prolongation of period till recidive appears, progression, therapy withdrowal or death.

Impact of stem cell source on allogeneic stem cell transplantation outcome in hematological malignancies.

BACKGROUND/AIM: Peripheral blood (PB) is used more frequently as a source of stem cells (SCs) for allogeneic transplantation. However, the influence of cell source on the clinical outcome of SC transplantation is not yet well established. The aim of this study was to compare the results of PBSC transplantation (PBSCT) with bone marrow transplantation (BMT) on the basis of engraftment, frequency and severity of immediate (mucositis, acute Graft versus Host Disease--aGvHD) and delayed (chronic GvHD--cGvHD) complications, as well as transplant-related mortality (TRM), transfusion needs, relapses and overall survival (OS). METHODS: We analyzed 158 patients, women/men ratio 64/94 median age 29 (range 9-57), who underwent allogeneic SC transplantation between 1989 and 2009. All included patients had diseases as follows: acute myeloid leukemia (AML)--39, acute lymphoblastic leukemia (ALL) 47, chronic myeloid leukemia (CML)--32, myelodysplastic syndrome (MDS)--10, Hodgkin's lymphoma (HL)- 2, multiple myeloma (MM) 3, granulocytic sarcoma (GrSa) 3, severe aplastic anemia (sAA)--22. The patients underwent transplantations were divided into two groups: BMT group (74 patients) and PBSCT group (84 patients). Each recipient had HLA identical sibling donor. SCs from bone marrow were collected by multiple aspirations of iliac bone and from PB by one "Large Volume Leukapheresis" (after recombinant human granulocyte colony stimulating factor, rHuG-CSF) application (5-12 microg/kgbm, 5 days). Conditioning regimens were applied according to primary disease, GvHD prophylaxis consisted of combination of a cyclosporine A and methotrexate. Results. Engraftment, according to the count of polymorphonuclear and platelets, were significantly (p < 0.001) faster in the PBSCT vs BMT group. The needs for transfusion support were significantly (P < 0.01) higher in the BMT group. Those patients had more frequently oropharingeal mucositis grade 3/4 (33.3% vs 10.0%, p < 0.05). There were no significant differences in the incidence of aGvHD and cGvHD between the two groups. The patients who underwent PBSCT had more frequently extensive cGvHD in comparison with the BMT group (29.1% vs 11.29%, p < 0.05). SC source (SCS) had no significant influence on the TRM (21.62% vs 23.8%, p = 0.64) and the incidence of relapses (21.6% vs 29.7%, p = 0.32). Finally, the patients treated by BMT hd a significantly better OS (logrank 2.33, p < 0.05). Conclusion. SCs harvesting from PB resulted in improved cell yield, faster engraftment, as well as in a decrease of immediate transplantation related complications with a reduced treatment cost. Allogeneic PBSCT were associated with more frequent extensive cGvHD, while the influence of SCS in TRM and relapses was not observed. Finally, the long-term OS was better in the patients treated by BMT. To verify impact of SC source on transplantation (PBSCT vs BMT) overall efficacy, more larger randomized clinical studies are needed.

[Autologous stem cell transplantation in the treatment of Hodgkin's disease].

BACKGROUND/AIM: High-dose chemotherapy with autologous stem cell transplantation (ASCT) has shown to produce long-term disease-free survival in patients with chemotherapy-sensitive Hodgkin disease. The aim of the study was to evaluate efficacy of ASCT in the treatment of Hodgkin's disease. METHODS: Between May 1997 and September 2008, 34 patients with Hodgkin's disease in median age of 25 (range 16-60) years, underwent ASCT. Autologous SCT were performed as consolidation therapy in one poor-risk patients with complete response (CR) and in 10 patients in partial remission (PR) after induction chemotherapy (32.5%), for chemosensitive relapse (CSR 1 and CSR 2) in 47% patients and in 20.5% patients with chemoresistant disease (CRD). All except one patient were in stage III/IV, extranodal site of disease had 24 patients and bulky disease had 10 patients. All the patients received a uniform preparatory regimen (BEAM). RESULTS: An overall response was achieved in 30 of 32 evaluated patients, with 62.5% in CR and 31.25% in PR. After applying radiotherapy, two patients with PR after ASCT reached CR. Median follow-up was 15.5 months (range 3-133 months). The probability of overall survival (OS) and progression-free survival (PFS) at a 3-year period for all patients was 51.9 % and 48.9%, respectively. For 22 patients in CR after ASCT, a 3-year DFS was 66.5%. Estimates of 2.5-year survival were 14.3%, 61.9% and 100% for CRD, CSR and for patients with CR/PR, respectively (p < 0.01). However, when patients undergoing consolidation were analyzed separately from those in CSR, no significant difference in OS and PFS was observed according to the disease status at ASCT. In univariate analysis for OS, PFS i DFS, extranodal site of disease and disease bulk had no predictive value. Twelve patients died. The main cause of death was Hodgkin's disease. Transplant-related mortality was 3.1%. One patient with CRD developed secondary acute myeloid leukemia and died 28 months after the transplantation. CONCLUSION: Autologous SCT is efficient as consolidation therapy in high-risk patients and in chemosensitive relapse, but it has no benefit in patients with chemoresistant disease.

Primary gastric mucosa associated lymphoid tissue lymphoma: clinical data predicted treatment outcome.

AIM: To determine clinical characteristics and treatment outcome of gastric lymphoma after chemotherapy and immuno-chemotherapy. METHODS: Thirty four patients with primary gastric mucosa associated lymphoid tissue (MALT) lymphoma (Ann Arbor stages I to IV) were enrolled. All had upper gastric endoscopy, abdominal ultrasonography, CT and H pylori status assessment (histology and serology). After anti-H pylori treatment and initial chemotherapy, patients were re-examined every 4 mo. RESULTS: Histological regression of the lymphoma was complete in 22/34 (64.7%) and partial in 9 (26.5%) patients. Median follow up time for these 31 responders was 60 mo (range 48-120). No regression was noted in 3 patients. Among the 25 (73.5%) H pylori positive patients, the eradication rate was 100%. CONCLUSION: Using univariate analysis, predictive factors for overall survival were international prognostic index (IPI) score, hemoglobin level, erythrocyte sedimentation rate (ESR), and platelet numbers (P < 0.005). In addition to this, Cox proportion hazard model differentiate IPI score, ESR, and platelets as predictors of survival.

[Skin necrosis of penis after chemotherapy regimen in patients with resistant nonseminomatous testicular tumor].

The majority of patients with resistant nonseminomatous testicular tumor can be cured with standard chemotherapy based on cisplatin regimen. However, complete remission cannot be achieved in 10%-30% of patients, or the disease recurs after the first chemotherapy cycle, on what account the salvage chemotherapy is administered. The initial BEP chemotherapy was applied in patients with gonadal nonseminomatous tumors B2 clinical stage after the left side inguinal orchiectomy. Due to manifested resistance, further chemotherapy was given according to VIP and one cycle by VEIP regimen. Upon inserting the urinary catheter because of total urinary retention, the phlegmonous inflammation and subsequently the necrosis of preputial and corpus skin of the penis were observed. The necrectomy with autotransplant was performed after demarcation. The skin necrosis of penis is very rare post chemotherapy complication in the stage of evident peripheral pancytopenia that may be the result of toxic effects of the used cytostatics or development of local infection with resulting necrosis.

[Bcl-2 and Bax protein interaction in B-lymphocytes of peripheral blood in patients with chronic lymphocytic leukemia].

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a neoplastic disease characterized by the accumulation of morphologically mature monoclonal CD5+ B cells in the early phase (G0/G1) of the cell cycle. The accumulation of neoplastically transformed B-lymphocytes (CLL cells) is primarily the consequence of apoptosis blocking in these cells. Bcl-2 proteins are well-known modulators of this process. Some of these proteins are anti-apoptotic while the others are pro-apoptotic. All contain at least one of the four conserved regions called the Bcl-2 homologous domains (BH1-BH4). Evidence indicates that Bcl-2 and Bax form homo- and heterodimers. The anti-apoptotic effect of Bcl-2 protein is based on its ability to bind Bax protein in the heterodimer form, and thus to block the forming of Bax/Bax proapoptotic homodimers. The ratio of Bcl-2/Bax represents the cell autonomous rheostat which determinates the type of the cell reaction to an apoptotic stimulus. METHODS: The aim of this study was to determine the level of interaction between these two proteins in CLL cells using the co-immunoprecipition method. The study included the analysis of 20 peripheral blood specimens from 20 patients with CLL, and 20 peripheral blood specimens from healthy persons, who were in the control group. Specimens were precipitated with the monoclonal antibody for Bcl-2 protein, and immunoblotted with the palyclonal antibady for Bax protein (IP: Bcl-2/WB:Bax). At the same time, specimens were precipitated with the polyclonal antibody for Bax protein, and immunoblotted with the monoclonal antibody for Bcl-2 protein (IP: Bax/WB:Bcl-2). The intensity of Bcl-2 and Bax protein's binding compared to the control samples of the peripheral blood from healthy persons, was increased in CLL cells. RESULTS: IP: Bax/WB: Bcl-2 showed a high level of "free" Bcl-2 protein which was not bound in the heterodimer form to Bax protein. Simultaneously IP: Bcl-2/WB: Bax showed that a higher quantity of Bax protein was bound in the heterodimer form to Bcl-2 protein as opposed to the quantity of pro-apoptotic Bax protein potentialy bound in the homodimer form. CONCLUSION: Further studies involving larger groups of patients are necessary to explore the potential significance of the Bcl-2/Bax protein ratio as a prognositc parameter in the CLL treatment.

[Use of high-dosage chemotherapy with autologous hematopoietic stem cell transplantation as a first-line therapy for the patients with poor-prognosis testicular tumors].

BACKGROUND: High-dose chemotherapy followed by hematopoietic stem cell support can be used as a first-line treatment in patients with germ-cell tumor (GCT) with poor prognosis. Long-term survival rate is attained in 50% of these patients. The aim of this paper was to present the experience at the Department of Hematology, Military Medical Academy, with high-dose cytostatic therapy as first-line chemotherapy in GCT patients with poor prognosis. METHODS: Between 1997 and 2003, five patients with high-risk germ-cell tumors were treated with high-dosage chemotherapy followed by an autologous stem cell transplantation. All the patients were with non-seminomatous germ-cell tumors with mixed histology, and one was with extragonadal retroperitoneal germ-cell tumor. RESULTS: The follow-up period ranged from 8 to 33 months. Three patients achieved complete remision, two patients only partial remision, and one was not followed-up. One patient was with residual tumor resection, using retroperitoneal lymphadenectomy, after autologous stem cell transplantation. All the patients were treated according to standard protocols. CONCLUSION: Early high-dose chemotherapy associated with hematopoietic stem cell support as a first-line treatment in the patients with germ-cell tumor with a poor prognosis, represented an efficient treatment modality.

[Expression of the Bcl-2 family of proteins in peripheral blood B-lymphocytes in patients with chronic lymphocytic leukemia]. / Ekspresija Bcl-2 familije proteina u limfocitima B periferne krvi bolesnika sa hronicnom limfocitnom leukemijom.

Chronic lymphocytic leukemia (CLL) is a neoplastic disease characterized by the accumulation of morphologically mature monoclonal CD 5+ B cells in the early phase (G0/G1) of the cell cycle. It is considered that the accumulation of neoplastically transformed lymphocytes B (CLL cells) is primarily the consequence of the disturbance, i.e., blockade of these cells' apoptosis process. Apoptosis is the specific process of programmed cell death regulated by numerous extracellular and intracellular mechanisms. The Bcl-2 proteins are well-known modulators of this process. Some of these proteins (such as Bcl-2, and Bcl-XI) are anti-apoptotic, while others (such as Bad or Bax) are pro-apoptotic. Our study included the analysis of 20 peripheral blood specimens from 20 patients with CLL, and 20 peripheral blood specimens of healthy persons who represented the control group. Using Western blotting analysis, we quantitatively examined the protein expression of Bcl-2 family (Bcl-2, Bax, Bad, and Bcl-XI). The level of Bcl-2 (p = 3.68 x 10(-10)), Bax (p = 0.019), and Bad (p = 0.073) proteins expression was significantly increased in all the analyzed peripheral blood samples of patients, while the level of Bcl-XI protein (p = 0.75) did not significantly differ in peripheral blood samples of patients, compared to the controls. The results of this study showed that the increased level of expression of Bcl-2, Bax, and Bad protein represented the most striking feature of CLL cells. Moreover, the variations in the expression of only one protein of the Bcl-2 family could not represent the prognostic parameter in the treatment of this disease.

[Effect of chlorambucil on B-lymphocytes in the peripheral blood of patients with chronic lymphocytic leukemia--study of cellular ultrastructure]. / Dejstvo hlorambucila na limfocite B periferne krvi bolesnika sa hronicnom limfocitnom leukemijom--ispitivanje ultrastrukture celija.

B type Chronic Lymphocytic Leukemia (B-CLL) is a malignant disease characterized by the progressive accumulation of morphologically mature, but immunologically dysfunctional CD 5+ lymphocytes in the blood, bone marrow and lymphatic organs in the early phase of the cell cycle. B-CLL is an example of human malignancy caused by alternations in the pathways of programmed cell death--apoptosis. Recent investigations showed a probable role of apoptosis as a prognostic parameter in B-CLL patients. Since the introduction of chlorambucil in the therapy in 1952, besides all the achievements in modern oncology, chlorambucil remained the most common antineoplastic agent in the treatment of CLL. Numerous experimental studies, both in vitro and in vivo, showed the capability of antineoplastic agents to induce the process of apoptosis of neoplastically transformed cells. In this study the effect of chlorambucil on B lymphocytes was monitored in 16 samples of peripheral blood taken from B-CLL diagnosed patients. According to the investigations performed in this study by ultrastructure analysis of B-CLL cells, it was concluded that chlorambucil either induced apoptosis in B-CLL cells, or activated cell response to the stress.