Polymorphisms and haplotypes in the multidrug resistance 1 gene (MDR1/ABCB1) and risk of multiple myeloma.

MDR1(ABCB1) gene encodes for P-glycoprotein (P-gp, MDR1, ABCB1), an ATP-binding cassette superfamily member involved in the transport of xenobiotics. Here, we investigated whether common MDR1 single nucleotide polymorphisms (1236C>T, 2677G>A/T and 3435C>T) affect predisposition to multiple myeloma. Genotyping was performed in 111 myeloma patients and 96 controls by PCR-based assays. Haplotypes were inferred using PHASE algorithm. We found comparable allele and genotype frequencies among myeloma patients and controls. Moreover, patient and control groups did not differ regarding MDR1 haplotype distribution (p=0.18). In conclusion, our results do not support major influence of MDR1 variants on the risk of myeloma in Caucasians.

Richter's syndrome in the brain first manifested as an ischaemic stroke.

Isolated central nervous system involvement in Richter's syndrome (RS) is extremely rare and only 6 such cases have been described, so far. We report a 60-year-old woman with B-cell chronic lymphocytic leukemia (B-CLL) heavily pretreated with cladribine based regimens and rituximab in whom RS in the brain was first manifested as a stroke. Initial cranial computed tomography (CT) revealed a hypodense area in the right parietal lobe showing no contrast enhancement. The follow-up CT done after 2 months showed an irregular, slightly hyperdense tumor surrounded by oedema with mass effect and midline shift. However, cerebrospinal fluid (CSF) examinations revealed no pathological changes. Neurosurgical operation was performed and the diagnosis of diffuse large B-cell lymphoma (DLBCL) has been established on the basis of histological and immunological investigation of the tumor. The pattern of immunoglobulin heavy chain (IgH) gene rearrangement in the patients' bone marrow aspirate and brain tumor was identical and suggested that both tumors originated from the same B-cell progenitors. The patient was then treated with brain irradiation (2000 cGy) and complete remission as assessed by MRI was achieved. Significant neurological improvement was observed and no clinical progression was stated 3 months after radiotherapy.

Rituximab followed by cladribine in the treatment of heavily pretreated patients with indolent lymphoid malignancies.

The purpose of this study was to determine the efficacy and toxicity of combined therapy consisting of rituximab (RIT), an anti-CD20 monoclonal antibody, and cladribine (2-chlorodeoxyadenosine, 2-CdA) (RC regimen) in patients with refractory or relapsed indolent lymphoproliferative disorders. Twenty six CD20 antigen positive patients, 15 with B-cell chronic lymphocytic leukemia (B-CLL) and 11 with low grade non-Hodgin's lymphoma (LG-NHL) were enrolled to the study. Fourteen patients (53.8%) had refractory disease, the other 12 (46.2%) were recurrent after prior chemotherapy. RC regimen consisted of RIT at a dose of 375 mg/m2 in 6 h infusion on day 1 and 2-CdA at a dose of 0.12 mg/kg, in 2 h infusion, given on days 2-6. The RC courses were repeated at 4 week intervals or longer if severe myelosuppression occurred. Seventy eight cycles of RC with median of 3 cycles per patient were administered (range 1-5 cycles). Four patients (15.4%) (95% CI 1.5-29.3%), 1 with B-CLL and 3 with LG-NHL, achieved a complete response (CR). Fourteen patients (53,8%) (95%CI 34.6-72.9%), including 10 with B-CLL and 4 with LG-NHL, had a partial response (PR). Overall response rate (OR) was 69.2% (95%CI 51.4-86.9%) in the whole group, from 63.6% (95% CI 35.2 92.0%) in LG-NHL to 73.3% (95%CI 50.1-95.7%) in B-CLL patients. Twelve of 18 patients with CR/PR are still in remission, with the median follow up 10 (7-28 months). The median failure-free survival (FFS) of responders was 6.5 months. Hypersensitivity to RIT was the major toxicity of RC regimen, and occurred in 9 patients (34.6%), mostly only during the first infusion of RIT. Severe neutropenia (grade III) was seen in 3 patients (11.5%). Anemia and thrombocytopenia associated with RC treatment were observed in 5 (19.2%) and 2 patients (7.7%), respectively. Four episodes (15.4%) of grade III-IV infections were observed. There was no treatment related mortality. During the follow-up six patients (23.1%) died from the disease progression. In conclusion, the combination of RIT and 2-CdA is an effective and well tolerated treatment, even for heavily pre-treated patients, and the results seem to be better than in patients previously treated in our institution with 2-CdA alone. This regimen can be considered as an alternative treatment of CD-20 positive indolent lymphoproliferative disorders.

Multiple myeloma in a patient with systemic lupus erythematosus, myasthenia gravis and non-familial diffuse palmoplantar keratoderma.

The coexistence of autoimmune diseases and malignancies including lymphoproliferative diseases is often reported in the literature. Here we report an unusual case with two autoimmune diseases--myasthenia gravis (MG) and systemic lupus erythematosus (SLE) associated with unique palmoplantar keratoderma (PK) which preceded the development of multiple myeloma (MM) for twenty and seven years respectively. MG associated with non-malignant thymoma developed in 1981 and was successfully treated with thymectomy and physostigmine. Thirteen years later SLE was diagnosed and until now it is also accompanied by skin lesions corresponding to non-familial, diffuse palmoplantar keratoderma which is resistant to treatment. In 2001 the patient revealed inguinal and abdominal lymphadenopathy first diagnosed as extramedullary plasmacytoma and then as multiple myeloma on the basis of bone marrow infiltration and monoclonal gammopathy. Therapy with VAD regimen achieved complete remission of the MM and significant improvement of the skin changes lasting for six months. We failed to collect sufficient numbers of CD 34+ cells for peripheral blood stem cell transplantation. Now the malignancy is in partial remission after CHOP therapy and the skin lesions have returned to their initial status. To our knowledge, this is the first case to be reported with coexistence of these four diseases.

Circulating VEGF and its soluble receptors sVEGFR-1 and sVEGFR-2 in patients with acute leukemia.

Angiogenesis plays an important role in the pathogenesis of acute leukemia, and vascular endothelial growth factor (VEGF) is a crucial, positive regulator of this process. The biological activity of VEGF is mediated by two different receptor tyrosine kinases: VEGFR-2 and VEGFR-1. The soluble form of VEGFR-1 is likely to be a negative regulator of VEGF availability, but the physiological role of sVEGFR-2 is still unclear. The plasma levels of sVEGFR-1 and sVEGFR-2 in patients with acute leukemia have not been investigated. We measured the plasma concentrations of VEGF and its two soluble receptors in 39 AML and 15 ALL patients as well as in the control group, using the ELISA assay. We also correlated the plasma levels of these proteins with disease status and known prognostic factors. The sVEGFR-1 level was significantly higher in patients with AML and ALL than in the healthy subjects (p < 0.002 and p < 0.03 respectively). The sVEGFR-2 level was significantly higher in AML patients compared with the control group (p < 0.03). The VEGF levels in AML and ALL patients and in healthy subjects did not differ significantly. The sVEGFR-1 level was higher in AML patients with > 50% of blasts in the bone marrow (BM), WBC > 20 G/L and elevated LDH level, than in the group with BM blasts < 50% (p < 0.01), WBC < 20 G/L (p < 0.02) and a normal LDH level (p < 0.05). Positive correlations between sVEGFR-1 level and WBC (p < 0.02),% of BM blasts (p < 0.05), the absolute blast count in peripheral blood (ABC) (p < 0.009) and LDH (p < 0.000001) were found. The sVEGFR-1/VEGF ratio (R1) was calculated, and a positive correlation between R1 and ABC in AML (p < 0.03) was determined. A higher (above median) sVEGFR-1/VEGF ratio correlated with a lower CR rate and a shorter survival (p < 0.03 and p = 0.0007 respectively). In conclusion, the plasma concentration of sVEGFR-1 is higher in leukemia patients than in healthy subjects and correlates with tumour burden and poor prognosis. The sVEGFR-1/VEGF ratio may be of greater prognostic value than VEGF alone. Further investigation is recommended to better determine their function.

High serum level of endostatin in multiple myeloma at diagnosis but not in the plateau phase after treatment.

We investigated the serum concentration of endostatin in 84 patients with multiple myeloma (MM) and in 13 healthy controls. The level of measured anti-angiogenic agent was correlated with the phase and stage of the disease, and most importantly with clinical and laboratory parameters depicting the disease activity (haemoglobin, creatinine, albumins, calcium, M-component, C-reactive protein, beta2-microglobulin, lactate dehydrogenase, stage of bone disease) as well as serum levels of pro-angiogenic cytokines such as vascular endothelial growth factor, hepatocyte growth factor, fibroblast growth factor and transforming growth factor-beta. The median serum level of endostatin in MM patients was 58 ng/ml and was statistically significantly higher than in the control group (median, 40 ng/ml; p=0.015). MM patients in phase I (at diagnosis) had higher levels of endostatin (median, 69 ng/ml) than those in phase II (plateau phase after treatment) (median, 49 pg/ml; p=0.044). We did not find any statistical correlation between the level of endostatin and stage of MM according to the Durie and Salmon system. The serum concentration of endostatin in MM patients with a normal level of albumins was significantly higher than in others with hypoalbuminaemia (median, 62 ng/ml versus 39 ng/ml; p=0.033). Also, patients with a normal value of lactate dehydrogenase had a higher concentration of endostatin than those with values >425 U/l (median, 70 ng/ml versus 39 ng/ml; p=0.019). We did not show any statistical correlation between the concentration of endostatin and level of haemoglobin, creatinine, calcium, C-reactive protein, beta2-microglobulin and stage of bone disease. We failed to find positive or negative correlations between the level of endostatin and vascular endothelial growth factor, hepatocyte growth factor, fibroblast growth factor and transforming growth factor-beta. The concentration of endostatin did not influence the probability of survival in MM patients in our study. In conclusion, our data indicate that endostatin has a higher level in MM patients than in healthy controls. Highest values were stated in active phases of the disease (at presentation and in progression). Different clinical and laboratory parameters generally do not influence the concentration of endostatin (except albumins and lactate dehydrogenase).

Coexistence of chronic lymphocytic leukemia and essential thrombocythemia.

The association of chronic lymphocytic leukemia (CLL) with essential thrombocythemia (ET) is an extremely rare event and until now 3 patients with such coexistence have been reported in the literature. We report a 77-year-old white woman in whom these two disorders were diagnosed concomitantly on the basis of peripheral blood count and cytology, bone marrow cytology and histology, immunophenotyping, as well as exclusion criteria. The diagnosis of ET was also supported by spontaneous in-vitro erythroid colony growth and by evaluation of thrombopoietin (TPO) serum level. Interphase FISH analysis allowed to detect 13q14.3 deletion in 98% of lymphocytes nuclei. In contrast this aberration was not observed in the megakaryocytes. The results of PCR analysis of IgG gene rearrangement showed distinct bands characteristic for monoclonal lymphoid population in bone marrow, peripheral blood and inguinal lymph node. The patient was started on hydroxyurea 1 g/day and normalization of the platelet count was achieved. Possible etiopathogenic relationships between both disorders and differential diagnosis of ET and reactive thrombocytosis (RT) are discussed.

Chronic myelomonocytic leukemia coexisting with B-cell chronic lymphocytic leukemia.

Coexistence of B-cell chronic lymphocytic leukemia (B-CLL) and chronic myelomonocytic leukemia (CMML) is an unusal event, and to our knowledge, only four such cases have been reported in the literature. We report a 68-year-old white woman in whom these two diseases were diagnosed concomitantly. The diagnosis was made on the basis of peripheral blood count, morphology and immunophenotyping, and bone marrow cytology and histology. Interphase FISH analysis detected a 13q14.3 deletion in lymphocytes nuclei and no such abnormality in monocytes nuclei. The PCR analysis of IgH gene rearrangement in the bone marrow, as well as the peripheral blood lymphocytes, showed two different monoclonal IgH configurations as the result of biallelic clonal rearrangement of IgH genes suggesting an origin of lymphocytes from B-cell progenitors. The patient was originally treated with prednisone 1 mg/kg/day because of progressive significant thrombocytopenia, without improvement. Subsequently, she received one course of cladribine (2-CdA). Significant reduction of lymphocytes in the peripheral blood was observed. However, rapid increase of monocytes was seen shortly after the 2-CdA treatment. Subsequently, she received hydroxyurea (1.5 g/day) without hematological improvement. The patient died in January 2003, three months after diagnosis because of progression of both leukemias and associated pneumonia. Possible etiopathogenic relationship between both disorders is discussed.

Aggressive primary plasma cell leukemia with skin manifestations, trisomy 8 and molecular oligoclonal features.

Plasma cell leukemia (PCL) is a very rare variant of multiple myeloma (MM) occurring in about 2% of newly diagnosed patients. Plasma cell leukemia may develop during the course of MM (secondary PCL) or it can occur without any prior sign of MM (primary PCL). We report a case of aggressive primary PCL with unusual clinical, cytogenetic and molecular features. A 36-year-old male patient was first seen because of fever and bone pain. On the skin of his chest, back, abdomen, and palpebras, there were nodular infiltrations resembling urticaria. White blood cell count was 10.8 x 10(9)/l with 41% plasmacytes. Bone marrow aspiration was hypercellular, 93.5% of cells were atypical plasmacytes and plasmablasts. The cytogenetic analysis of G-banded chromosomes in bone marrow cells yielded the trisomy 8. The skin biopsy specimen showed intensive infiltrates of uninucleated blastic cells similar to those found in the bone marrow. Immunophenotyping of bone marrow and skin neoplastic cells showed CD45+, CD45Ro+, CD68+, CD38+ and cytoplasmic kappa light chain +. The neoplastic cells stained negatively for lambda light chain, CD3, CD20, CD30, EMA, CD15, CD34, CD56 and factor VIII. The pattern of IgL genes rearrangement in the bone marrow aspirate, peripheral blood mononuclear cells, and skin specimens was examined by PCR analysis. All studied specimens showed three different IgK gene configurations suggesting that the neoplastic cells originated as a result of oligoclonal lymphoproliferation process. The patient received two courses of VAD (vincristine, doxorubicin, dexamethasone) without improvement and three courses of CHOP with only temporary stabilization of the disease. He died 5 months after the diagnosis of PCL because of disease progression and pneumonia.

Acute lymphoblastic leukemia in adult first manifested as severe aplastic anemia--role of molecular analysis in correct diagnosis.

Aplastic anemia (AA) may sometimes precede the diagnosis of acute lymphoblastic leukemia (ALL) in children. Such presentation of ALL is externally rare in adults and until now only few such cases have been reported. We present a 40-year-old male with ALL common type, which developed 14 months after the diagnosis of severe AA, successfully treated with corticosteroids. ALL was treated with standard induction chemotherapy but remission has not been achieved. The patient died 6 weeks after the diagnosis of ALL because of central nervous system bleeding. The pattern of IgH gene rearrangement analyzed with PCR method in bone marrow from the period of AA diagnosis and in peripheral blood mononuclear cells from ALL diagnosis showed two different monoclonal IgH configurations as the results of biallelic monoclonal rearrangement of IgH genes. The observed bands in both specimens were identical and indicated that leukemic cells originated from B-cell progenitor were also present in the bone marrow when AA was diagnosed. We suggest that molecular analysis of monoclonality in patients with AA may be important for proper selection of the rare cases of ALL first presenting as marrow aplasia.

Lymphoplasmacytic lymphoma with monoclonal gammopathy-related pseudo-Gaucher cell infiltration in bone marrow and spleen--diagnostic and therapeutic dilemmas.

Gaucher-like cells have occasionally been described in various haematological malignancies including Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma (MM) and chronic myelogenous leukaemia (CML). A special type of this phenomenon is crystal-storing histocytosis or the so-called pseudo-pseudo Gaucher cells (PPGC) in which crystalline protein storage in macrophages is induced by paraproteinemia. Here we describe a 54-year-old man with an initial suspicion of Gaucher disease and monoclonal IgA gammopathy in whom a correct diagnosis of lymphoplasmacytic lymphoma (LPL) with massive infiltration of bone marrow and spleen by PPGC was confirmed by immunological, ultrastructural and molecular characterisation. The activity of leukocyte beta-glucocerebrosidase was only slightly elevated (7.3 nmol/mg protein/1 h) which ruled out the diagnosis of classic Gaucher's disease. The patient received two courses of CHOP without improvement and anti-CD20 monoclonal antibody (rituximab) with only temporary stabilisation. Subsequently, he underwent splenectomy because of prolonged severe pancytopenia and a suspicion of hypersplenism. After splenectomy significant haematological improvement was observed. Following anti-CD20 therapy, changes in immunoprofile and morphology of tumour cells were evident. Before treatment the population of LPL was more divergent, with expression of LCA, CD20, CD38 and CD138. However, after the treatment, there were more mature plasma cells which no longer expressed CD20 antigen-this picture was more consistent with the diagnosis of plasma cell myeloma. Similarly, in the spleen there were no CD-20-positive cells evident. Finally, the patient received two courses of VAD vincristine, doxorubicin, dexamethasone) with further haematological improvement but complete response was not achieved.