Dickkopf-related protein 1 expression in bone marrow of multiple myeloma patients: correlation with bone disease and plasma cell malignancy type.

BACKGROUND: Previous studies have pointed out the role of dickkopf-related protein 1 (DKK 1) - Wnt inhibitor, which is essential for osteoblast functioning, in the development of osteolytic lesions in multiple myeloma (MM). AIM: To assess the DKK 1 expression displayed by myeloma cells in bone marrow trephine biopsies of patients with and without osteolytic lesions, and in different malignancy grades of the disease. METHODS: The expression level of DKK 1 was assessed immunohistochemically in bone marrow of 49 MM patients presented with and without osteolytic lesions (the 1st and the 2nd group, respectively). RESULTS: Levels of weak, moderate, and strong DKK 1 expression were distributed - as 43.33, 27.78 and 25.56%, and 63.91, 18.80, and 1.50%, respectively when evaluating the samples obtained from the 1st and the 2nd group. Statistically significant differences were found when the levels of DKK 1 expression in the 1st and the 2nd group were compared (χ2 = 51; df = 3; p < 0.001). CONCLUSIONS: DKK 1 contributes to the development of osteolytic lesions in MM. The present study provides morphological evidence that inhibition in Wnt signaling may lead to bone damage observed in the advanced stage of the disease.

Progressive multifocal encephalopathy in a patient with non-Hodgkin follicular lymphoma.

Progressive multifocal leukoencephalopathy (PML) is a rare and often fatal demyelinating disease of the central nervous system caused by John Cunningham virus (JCV). We present a case report of patient with non-Hodgkin follicular lymphoma, who developed PML after hematopoietic stem cell transplantation and rituximab-bendamustine therapy. JCV DNA was proven both in peripheral blood and cerebrospinal fluid. Patient with 4 years history of follicular lymphoma presented with progressing weakness in the right arm and leg and postural instability. Magnetic resonance imaging scans showed bilateral hyperintense lesions in the cerebellum and centrum semiovale consistent with findings in PML. JCV DNA was detected in patient peripheral blood and cerebrospinal fluid by real time polymerase chain reaction assay in CERBA laboratory (France). Human herpes simplex 6 and 7 DNA were also detected in peripheral blood by PCR. Patients condition rapidly deteriorated with exitus letalis after 3 months and 2 weeks from onset of symptoms. This case draws attention to risk for developing PML in patients with long-standing hematological malignancies.

Association of elevated vitamin B12 with oncohematological diseases in a cohort of 79,524 patients from Latvia.

AIM: Currently there are some large-scale studies of elevated total vitamin B12 in relation to diseases and their prognosis. Aim of this retrospective study was to determine association of increased B12 as an additional diagnostic marker of oncohematological diseases by a statistical analysis of clinical data of 79,524 patients. MATERIALS AND METHODS: Overall Latvian population representative data on B12  testing in 79,524 patients were obtained from laboratory database. The following exclusion criteria were applied: fluctuating B12 results within a three-month period, elevated (> 100 U/L) alanine transaminase or aspartate transaminase, hepatitis (HAV, HBV, and HCV) infection, reduced glomerular filtration rate (< 45 mL/min/1.73 m2). As a control group, individuals with normal B12 level and any oncologic diagnosis (solid cancer or hematological malignancies) were selected. RESULTS: After application of step-by-step exclusion filters, 1,373 patients were left with significantly increased level of plasma B12 (> 1,700 pg/mL). Odds ratios for oncohematological diseases in total and myeloid leukemia (including acute, chronic and unspecified) in patient group with elevated B12 were found to be 6.0 (95% CI 4.7-7.6; p < 0.0001) and 19.2 (95% CI 13.1-28.0; p <0.0001), respectively, as compared to the control group. CONCLUSION: Elevated total B12 could be considered as a potential marker for oncohematological disorders.

Prognostic value of the bone turnover markers in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is characterized by osteolytic bone disease resulting from increased osteoclast activity and reduced osteoblast function. AIM: The aim of our research was to determine connection between bone turnover markers and presence of bone lesions, their degree of severity, to monitor MM bone disease and to assess effectiveness of anti-myeloma treatment. MATERIALS AND METHODS: Serum samples and clinical data from 123 patients with newly diagnosed MM were collected at Riga East Clinical University Hospital (Riga, Latvia) from June 2014 to June 2016. Bone lesions detected by radiography, CT scans, MRI, and PET/CT were divided into degrees from 0 to 3 (0 - no bone involvement, 1 - ≤ 3 bone lesions, 2 - ≥ 3 bone lesions, 3 - fracture). Staging was performed applying Durie/Salmon (DS) and International Staging System classifications. Progressive disease was defined as development of one or more new bone lesions. The levels of bone metabolic markers ß-isomerized C-terminal telopeptide of collagen type I (ß-CTX) and bone-specific alkaline phosphatase (bALP) were monitored regularly in the year. RESULTS: Bone lesions were found in 86 (69%) patients. From these 6 (4%) patients had 1st degree, 11 (9%) had 2nd degree and 69 (56%) had 3rd degree bone lesions. Level of the bone resorption marker ß-CTX in the control group was 0.41 ng/ml, which is lower than in MM patients (p < 0.001). Spearman correlation coefficient analysis found a positive and statistically significant correlation (rs = 0.51, p < 0.001) between bone lesions degree and ß-CTX levels. Mean ß-CTX for patients without bone lesions was 0.72 ng/ml (SD = 0.64), but for patients with 3rd degree bone lesions it was 1.34 ng/ml (SD = 0.65) difference being 38% (p < 0.001). In patients who responded to therapy after 6 months of treatment reduction of ß-CTX was found compared to baseline values (M = -0.65). In contrast, in patients who did not respond to therapy, there was a statistically significant (p < 0.001) increase in ß-CTX values after six months of treatment compared to baseline values (M = 0.42). Exact cutoff value of ß-CTX is 0.79. When analyzing mean bALP, no significant difference between MM patients and control group was found. ANOVA statistical analysis showed no statistically significant differences in bALP levels at different degrees of bone lesions (p = 0.95) in MM patients. Analysis of bALP suitability as MM diagnostic marker using receiver operating characteristics curve showed that bALP is not applicable for clinical diagnosis of MM (AUC 0.5, p > 0.05). However, ß-CTX was found to be an excellent diagnostic marker for MM (AUC 0.91; 95% confidence interval, 0.88-0.94; p < 0.001). CONCLUSIONS: Patients with MM and bone lesions have increased value of bone resorption marker ß-CTX. There is a correlation between bone resorption marker and degree of bone lesions. Changes in ß-CTX levels may be used to monitor the effectiveness of myeloma treatment.

Generic imatinib in the treatment of chronic myeloid leukemia: two years' experience in Latvia.

BACKGROUND: Imatinib is tyrosine kinase inhibitor (TKI) and as a targeted anti-cancer agent has significantly changed chronic myeloid leukemia (CML) prognosis and patient survival. Currently TKI is the main therapy in CML Philadelphia chromosome-positive (Ph-positive) cases. When generics of imatinib appeared in the pharmaceuticals market, reimbursement policies in many countries switched to using generics or encouraged use of generic imatinib to lower the expenses. Cost savings were substantial; however, for doctors and CML patients the efficacy, safety and quality of generic imatinib were an issue of concern. OBJECTIVE: Since the global number of CML patients, who in the future will have to switch from original imatinib to generic imatinib, is high, the aim of study was to monitor, whether during 24 months of generic imatinib usage patients maintain the achieved major molecular response (MMR) or whether the treatment results are inferior. METHODS: We conducted a retrospective study, which included CML patients, who were above 18 years of age and who until May 2013 had used at least for 2 years (24 months) the original imatinib, and following that used at least for 24 months one of the generic imatinib medicines. In 2013, before switching to generic imatinib, all patients had reached MMR in accordance with European LeukemiaNet (ELN) Guidelines. Every three months blood count, BCR-ABL fusion gene (BCR-ABL), biochemical analysis and side effect were monitored. RESULTS: Our study proved that CML patients, who had achieved MMR by original imatinib therapy, retained MMR during 24 months of generic imatinib therapy. Nobody was switched to second line generation TKI. During observation period neither haematological, nor non-hematological toxicity was found. CONCLUSION: Our study proved that CML patients, who had achieved MMR by original imatinib therapy, retained MMR during 24 months of generic imatinib therapy. This demonstrates that generic imatinib is not inferior to original imatinib. As to expenses, the annual costs of generic imatinib are lower by 96%, which is a significant benefit to health-care financing.

Treatment and outcome of 2904 CML patients from the EUTOS population-based registry.

The European Treatment and Outcome Study (EUTOS) population-based registry includes data of all adult patients newly diagnosed with Philadelphia chromosome-positive and/or BCR-ABL1+ chronic myeloid leukemia (CML) in 20 predefined countries and regions of Europe. Registration time ranged from 12 to 60 months between January 2008 and December 2013. Median age was 55 years and median observation time was 29 months. Eighty percent of patients were treated first line with imatinib, and 17% with a second-generation tyrosine kinase inhibitor, mostly according to European LeukemiaNet recommendations. After 12 months, complete cytogenetic remission (CCyR) and major molecular response (MMR) were achieved in 57% and 41% of patients, respectively. Patients with high EUTOS risk scores achieved CCyR and MMR significantly later than patients with low EUTOS risk. Probabilities of overall survival (OS) and progression-free survival for all patients at 12, 24 and 30 months was 97%, 94% and 92%, and 95%, 92% and 90%, respectively. The new EUTOS long-term survival score was validated: the OS of patients differed significantly between the three risk groups. The probability of dying in remission was 1% after 24 months. The current management of patients with tyrosine kinase inhibitors resulted in responses and outcomes in the range reported from clinical trials. These data from a large population-based, patient sample provide a solid benchmark for the evaluation of new treatment policies.

The EUTOS population-based registry: incidence and clinical characteristics of 2904 CML patients in 20 European Countries.

This population-based registry was designed to provide robust and updated information on the characteristics and the epidemiology of chronic myeloid leukemia (CML). All cases of newly diagnosed Philadelphia positive, BCR-ABL1+ CML that occurred in a sample of 92.5 million adults living in 20 European countries, were registered over a median period of 39 months. 94.3% of the 2904 CML patients were diagnosed in chronic phase (CP). Median age was 56 years. 55.5% of patients had comorbidities, mainly cardiovascular (41.9%). High-risk patients were 24.7% by Sokal, 10.8% by EURO, and 11.8% by EUTOS risk scores. The raw incidence increased with age from 0.39/100,000/year in people 20-29 years old to 1.52 in those >70 years old, and showed a maximum of 1.39 in Italy and a minimum of 0.69 in Poland (all countries together: 0.99). The proportion of Sokal and Euro score high-risk patients seen in many countries indicates that trial patients were not a positive selection. Thus from a clinical point of view the results of most trials can be generalized to most countries. The incidences observed among European countries did not differ substantially. The estimated number of new CML cases per year in Europe is about 6370.

Peripheral blood lymphocyte phenotype of ZAP-70⁺ and ZAP-70⁻ patients with B-cell chronic lymphocytic leukaemia.

BACKGROUND: Up to now, the immune status of chronic lymphocytic leukemia (CLL) patients in association with the expression of zeta-chain-associated protein kinase 70 (ZAP-70) in leukemic cells has not been evaluated. AIM: The aim of this work was the study of the peripheral blood (PB) T-lymphocyte phenotypes in ZAP-70-positive (ZAP-70(+)) and ZAP-70-negative (ZAP-70(-)) untreated patients with CLL. MATERIALS AND METHODS: ZAP-70-, CD25-, CD3-, CD4-, and CD8-positive lymphocytes were enumerated by flow cytometry in PB of 120 untreated CLL patients. CD8+, CD3+CD4+ and CD3+CD25+ cells were counted for the non-leukemic lymphocytes. RESULTS: The patients were distributed into two groups: the ZAP-70(+) group of high CLL progression (n = 61), and the ZAP-70(-) group of low CLL progression (n = 59). In the ZAP-70(+) group, the ratio CD4/CD8 (0.33 ± 0.62; p = 0.001) and the numbers of the CD3+ (34.8 ± 8.1%; p = 0.01), CD3+CD4+ (24.4% ± 4.8; p = 0.001), and CD3+CD25+ (6.2 ± 0.91%; p = 0.001) lymphocytes were reduced and the percentage of the CD8+ cells (73.1 ± 4.6%; p = 0.0001) was above the norm. In the ZAP-70(-) group, the number of the CD3+CD4+ cells (36.9 ± 6.1%; p = 0.001) was within the norm, but the numbers of the CD8+ (11.3 ± 1.1%; p = 0.0001) and CD3+ (41.2 ± 5.3%; p = 0.05) lymphocytes were reduced; the ratio CD4/CD8 (3.26 ± 0.88; p = 0.001) and the percentage of the CD3+CD25+ cells (27.1 ± 3.4%; p = 0.0001) were above the norm. CONCLUSIONS: Our data show that the increased CD4/CD8 ratio, caused by the reduced number of the CD8+ lymphocytes, and the increased number of CD3+CD25+ cells are characteristic for the ZAP-70(-) group (slow progressing) of untreated CLL patients. In ZAP-70(+) patients, the CD4/CD8 ratio was significantly below the norm indicating an active disease process. Results of our study contribute to identification of CLL patients with different prognosis in routine diagnostic/prognostic procedures.

Identifying the stage of new CLL patients using TK, ZAP-70, CD38 levels.

UNLABELLED: Serum thymidine kinase (TK), zeta-associated protein of 70 kDa (ZAP-70) and CD38 levels have been shown to be correlated with survival in chronic lymphocytic leukaemia (CLL). AIM: To investigate the possible correlations between TK, ZAP-70 and CD38 levels as prognostic markers in new diagnosed Rai stages of CLL patients. METHODS: 120 CLL patients were enrolled. ELISA was used to measure serum TK level, flow cytomerty - to determine ZAP-70 and CD38 expression applying ZAP-70 Kit and monoclonal antibody to CD38, respectively. RESULTS: Significantly higher levels of TK were found in the high progression group of CLL patients that corresponded to stage II (Rai classification). An elevated level of TK, CD38 and ZAP-70 together was also found in the II stage. The coefficient of correlation between CD38 and ZAP-70 is reliable (p < 0.001). There is also a correlation between the level of TK and the disease stage (p < 0.05). Other parameters do not show this correlation. CONCLUSION: The determination of TK, ZAP-70 and CD38 together allows patients susceptible to a possible stage of the disease, to be identified. Estimation of the factors at an early stage of the disease may allow an earlier commencement of treatment.

Progressive multifocal leukoencephalopathy following fludarabine treatment in a chronic lymphocytic leukemia patient.

UNLABELLED: Progressive multifocal leukoencephalopathy (PML) is a neurological disease caused by infection of the central nervous system (CNS) with the JC polyomavirus (JCV). JCV is endemic and infects a large proportion (70-90%) of healthy individuals worldwide, but infection is latent. JCV reactivation may occur, if the immune function is compromised. AIM: To present a PML case in a CLL patient after a long course of disease and treatment with fludarabine. JCV virus infection in this patient was proven both in brain biopsy material and blood. METHODS: Patient with a nine-year history of CLL was hospitalized with the weakness in the right leg and left hand, tremors, speech difficulties. An MRI diagnosed infiltrative glial tumor of the left hemisphere, proliferating predominantly in the frontal lobe, more in the gyrus frontalis superior region. CNS tumor biopsy performed. RESULTS: Morphology and immunoprofile of the lesion consistent with progressive multifocal leukoencephalopathy. The material from biopsy was diagnosed as positive for JCV DNA. JCV and HHV-7 genomic sequences were found in patient's PBL DNA sample. In a plasma DNA sample, only genomic sequences were detected. CONCLUSION: The present case draws attention to the fact that the use of fludarabine and its combinations in CLL therapy increases the risk of JCV infection reactivation and development of serious complications like PML.

Human retrovirus type 5 sequences in non-Hodgkin's lymphoma of T cell origin.

DNA of a recently described fifth exogenous retrovirus (HRV-5) has been found in blood samples from patients with autoimmune diseases and lymphoma. We analyzed HRV-5 sequence in DNA extracted from whole blood of 17 patients with T cell non-Hodgkin's lymphoma (NHL) and 186 patients with hematological malignancies other than NHL, using a sensitive PCR technique. While all samples of patients with hematological malignancies other than NHL were negative, 2 of the 17 patients with T cell NHL were HRV-5 DNA positive. Both HRV-5-positive patients had T cell NHL of high-grade malignancy (stage IV) and diffuse distribution of the lymphoma, including infiltration of bone marrow or lung and pleura. The difference in HRV-5 DNA detection frequency between NHL and control groups is significant (p value of 0.0004 judged by the Fisher exact test). These data, together with our previous finding of HRV-5 DNA in three B cell NHL cases, are compatible with an association between HRV-5 and NHL, of both T cell and B cell origin.

Human retrovirus 5 sequences in peripheral blood cells of patients with B-cell non-Hodgkin's lymphoma.

A recently described sequence from a probable 5th human exogenous retrovirus, HRV-5, is related to type A, B and D retroviruses. It was initially detected in a salivary gland biopsy from a patient with Sjögren's syndrome, but it is not consistently associated with this disease. We searched for the HRV-5 sequence in DNA extracted from whole blood of 300 blood donors, 81 patients with hematological malignancy and 21 patients with neurological disease using PCR. While samples from none of the blood donors and the neurological patients became positive, 3 of the 81 patients with hematological malignancy were HRV-5 DNA positive. All 3 had B-cell non-Hodgkin's lymphoma of low grade. The difference in frequency between NHL and controls is statistically significant. HRV-5 DNA was found in DNA from whole blood and in plastic-adherent cells but not in tumor cell DNA. Thus, monocytes/macrophages may be preferred targets for HRV-5. Our result, together with a previous finding of HRV-5 DNA in 2 NHL cases, is compatible with an association between HRV-5 and NHL, whether causal or not.