Human Adrenocortical Carcinoma (NCI-H295R) Cell Line as an In Vitro Cell Culture Model for Assessing the Impact of Iron on Steroidogenesis.

The aim of this in vitro study was to examine the dose-dependent effects of iron as a potential endocrine disruptor in relation to the release of sexual steroid hormones by a human adrenocortical carcinoma (NCI-H295R) cell line. The cells were exposed to different concentrations (3.90, 62.50, 250, 500, 1000 µM) of FeSO4.7H2O and compared with the control group (culture medium without FeSO4.7H2O). Cell viability was measured by the metabolic activity assay. Quantification of sexual steroid production was performed by enzyme-linked immunosorbent assay. Following 48 h culture of the cells in the presence of FeSO4.7H2O, significantly (P < 0.001) increased production of progesterone was observed at the lowest concentration (3.90 µM) of FeSO4.7H2O, whereas the lowest release of progesterone by NCIH295R cells was noted after addition of 1000 µM of FeSO4.7H2O, which did not elicit cytotoxic action (P > 0.05). Testosterone production was substantially increased at the concentrations ≤ 62.50 µM of FeSO4.7H2O. Lower levels of testosterone were recorded in the groups with higher concentrations (≥ 250 µM) of FeSO4.7H2O (P > 0.05). The presented data suggest that iron has no endocrine disruptive effect on the release of sexual steroid hormones, but its toxicity may be reflected at other points of the steroidogenesis pathway.

The quality of life following allogeneic hematopoietic stem cell transplantation - a multicenter retrospective study.

UNLABELLED: Although allogeneic haematopoietic stem cell transplantation (allo-HSCT) offers a unique curative potential, it may be connected with high treatment-related morbidity and mortality. Besides many organ complications, allo-HSCT may significantly affect quality of life (QOL). PATIENTS AND METHODS: Between January 2011 and December 2012, five hundred and ninety patients (pts) from 6 transplant centers in the Czech Republic filled in the questionnaire for the quantitative measurement of QOL using Functional Assessment of Cancer Therapy-General (FACT-G) version 4. Study cohort characteristics were as follows: 325 males, 340 pts received myeloablative conditioning, 383 pts received PBPC, representation of diagnoses; acute leukemia (n=270), bone marrow failure (n=36), chronic myeloid leukemia (n=74), myelodysplastic/myeloproliferative syndrom (n=110), lymphoproliferative disease (n=93). The median age at allo-HSCT was 43 years (range: 1.7 - 71.0), the median time from allo-HSCT to questionnaire completing was 3.8 years (range: - 0.2 - 21.6). The earliest allo-HSCT was performed in November 1989, the last in September 2012. In this retrospective study, we investigated the impact of various factors on the QOL after allo-HSCT: age, gender, diagnosis, type of conditioning, time from diagnosis to allo-HSCT, disease stage, graft type, donor type, time from allo-HSCT to questionnaire completing, GVHD, relapse. Only data from patients who were more than 3 months after allo-HSCT were used for the multivariate analysis. The overall results of the total FACT-G score (median=85.0; range: 29-108) as well as the results of each specific dimension - PWB (median=23.0; range: 5-28), SWB (median=24.0; range: 7-28), EWB (median= 19.0; range: 4-24), FWB (mean=21.0; range: 2-28) showed a value in the highest quartile of the possible evaluation. In multivariate analysis, an inferior QOL score was reported for patients with aGVHD (p=0.002), cGVHD (p<0.001), QOL decreased with increasing age (p=0.048) and increased with time elapsed since allo-HSCT (p<0.001).Allogeneic HSCT represents an important intervention into the overall integrity of the organism. In particular, the development of GVHD can cause very serious organ, but also mental problems which can significantly reduce the QOL. The QOL is steadily increasing with increasing interval from allo-HSCT but improvement and disappearance of these complications may take many years, and sometimes these effects may probably persist permanently.

Quantitative monitoring of WT1 expression in peripheral blood before and after allogeneic stem cell transplantation for acute myeloid leukemia - a useful tool for early detection of minimal residual disease.

Overexpressed Wilms tumor gene 1 (WT1) has been found in a majority of patients with acute myeloid leukemia (AML). The aim of this study was to confirm the applicability of WT1 expression measurement as a marker of minimal residual disease (MRD). The expression of WT1 gene was measured by real-time polymerase chain reaction in peripheral blood (PB) according to European Leukemia Net (ELN) recommendations. The WT1 expression was related to the expression of a reference gene Abelson (ABL) and the results were calculated as a number of WT1 copies related to 104 copies of ABL gene. The upper normal limit of WT1 expression was set at 50 copies of WT1 to 104 copies of ABL. Morphological, flow cytometry and chimerism examinations were evaluated according to standard protocols.A total of 51 AML patients with overexpressed WT1 gene were analyzed. The median follow-up after transplantation was 14 (2-72) months. WT1 expression levels exceeding the upper normal limit were considered as a sign of impending hematological relapse, in accord with morphological, flow cytometry and chimerism data, as well as with the expression of the specific fusion genes. Moreover, in 7 patients the rise of WT1 expression preceded all other standard methods. Patients with high WT1 expression before allogeneic hematopoietic stem cell transplantation (allo-HSCT) had significantly worse outcome than patients with low WT1 level. Examination of WT1 expression in PB of patients with AML is a useful tool for MRD monitoring. Moreover, the WT1 gene expression before stem cell transplantation seems to be of prognostic significance.

[Treatment of patients with relapsed/refractory Hodgkin lymphoma]. / Lécba pacientu s relabovaným/refraktérním Hodgkinovým lymfomem.

BACKGROUNDS: This retrospective study evaluated treatment outcomes in patients undergoing autologous stem cell transplantation (ASCT) for relapsed/refractory Hodgkin lymphoma (HL). PATIENTS AND METHODS: Overall, 194 HL patients treated with ASCT between 2000 and 2009 were analyzed. Survival was calculated using Kaplan-Meier method and differences in survival between subgroups with log-rank test. RESULTS: Best responses observed after ASCT: 124 complete and 35 partial remissions, 2 patients with stable disease and 33 relapses/progressions. During a median follow-up of 44 months, seventy patients after ASCT progressed/relapsed. Thirty-seven patients received salvage chemotherapy only with or without radiotherapy, 25 underwent allogeneic stem cell transplantation (SCT), 4 the second ASCT and 4 refused treatment. 5-year overall survival after ASCT was 71% and progression-free survival 54%. Median survival of the 70 patients relapsing after ASCT was 16.9 months. Median survival in patients after allogeneic SCT was 31.8 months and 12.4 months in patients treated with other modalities (p = 0.21). Overall mortality was 26.3% (51/194 patients): 13.4% progressions/relapses of HL and 12.9% non-relapse mortality. CONCLUSION: Efficacy of ASCT was confirmed in 54% progression-free survivors. Median survival after ASCT failure is relatively short. There is a slightly longer overall survival after allogeneic SCT, although not statistically significant when compared to other approaches.

The effect of allogeneic stem cell transplantation on high risk chronic lymphocytic leukaemia: a single centre retrospective analysis.

In recent years, many studies have confirmed that allogeneic stem cell transplantation (allo-SCT) can provide long-term disease control and possible cure in selected patients with chronic lymphocytic leukaemia (CLL), including those with a biologically highly unfavourable risk profile. A retrospective analysis of allo-SCT in 30 patients with CLL whose risk profile was unfavourable and who were treated in the years 2000-2009 was performed. The aim was to compare the results of allo-SCT by prognostic factors and conditioning type and evaluate the results of unrelated transplantation. The median age was 54 years. Donors were 8 HLA-matched siblings and 22 unrelated volunteers, 11 of whom were mismatched. Eighteen patients were treated with reduced intensity conditioning. Twelve patients received myeloablative conditioning. Estimated overall survival (OS) at 3 years was 78%, progression-free survival (PFS) 71%, relapse incidence 10% and non-relapse mortality (NRM) 16%, respectively, with a median follow-up of 35 months. According to molecular/cytogenetic characteristics, OS and PFS for the high risk group (17p- or 11q-) were 89 and 77%, respectively, not significantly different from those with standard risk. Graft-versus-host disease (GVHD) was associated with greater toxicity; significantly higher NRM for patients with aGVHD (p = 0.04) and worse PFS for patients with cGVHD (p = 0.04). Our results for the refractory disease group (77% responses) indicate that chemoresistance may be overcome by the GVL effect. Transplants from unrelated donors may be considered comparable to those from related donors.

[The current role of haematopoietic stem cell transplantation in the treatment of lymphomas--review]. / Soucasné postavení transplantace krvetvorných bunek v lécbe lymfomu--prehled.

Despite the undeniable progress in first-line treatment, a significant proportion of patients with lymphoproliferative disease still relapse. Transplant strategy is one of the most effective options for such patients. Autologous stem cell transplantation has for many years been the standard position in the treatment of chemosensitive relapse of diffuse large B cell lymphoma, Hodgkin's lymphoma and follicular lymphoma. Recently, autologous stem cell transplantation has had significant importance in first-line therapy of some T-cell lymphoma and mantle cell lymphoma as well. It is possible that this situation will change after the evaluation of clinical protocols using rituximab, either in first or second-line therapy or as part of the conditioning regimen and maintenance therapy. In recent years, there has also been a significant shift to the use of allogeneic transplantation, particularly through the introduction of so-called reduced intensity (RIC-alo). Many studies have demonstrated a significant reduction in mortality using RIC-alo, while preserving the GVL effect. In recent years, significant attention has also been focused on the identification of prognostic factors that could lead to earlier intensive therapy, including RIC-alo, particularly in mantle cell lymphoma, some T-cell lymphoma and Hodgkin's lymphoma. However, relapses remain a major problem of treatment failure and more accurate identification of patients suitable for early transplant strategy will be required.

The results of allogeneic transplants in patients with malignant lymphoma-a retrospective analysis of data from the Czech National Registry.

We processed data of 79 patients (pts) with malignant lymphoma from the National Registry of haematopoietic stem cell transplants conducted between 1997 and 2006. The haematopoietic stem cell donor in 48 pts was an HLA matched relative, and in 30 pts an unrelated volunteer. Sixty (77%) pts were transplanted with reduced intensity conditioning (RIC), eleven (23%) pts with myeloablative conditioning (MC). Acute graft-versus-host disease (aGVHD) was recorded in 26 (33%) pts. Chronic GVHD was diagnosed in 19 (36%) of the 53 assessable pts. Transplant-related mortality (TRM) in the first 100 days, 1 year and 3 years for the whole group was 26%, 33% and 33%. Twenty (26%) of the pts relapsed. During the median follow-up of 26 months the overall survival (OS) was 44%, the progression free survival (PFS) was 54% and cumulative incidence of relapse was 45%. Pts with chemoresistant disease had significantly worse results (OS at 3 years 22% vs. 56%, p=0.002). We did not find any correlation between the incidence of GVHD and the frequency of relapse. Similarly, we did not observe any difference in survival between patients following MC vs. RIC. Survival of pts transplanted from related donors did not differ statistically from unrelated donors. Key words: Lymphoma, allogeneic, transplantation, GVHD.

Chemical, instrumental and sensory characteristics of cooked pork ham.

Instrumental, chemical and sensory parameters of cooked pork ham were evaluated. Principal component analysis was carried out on the basis of the instrumental variables related to colour and texture. The four PCs account for almost 94% of the total variance in the data set. The PCA only separated 3 hams with a(∗)>10. Hardness was correlated with non-collagen muscle protein (P⩽0.01), gumminess (P⩽0.01) and ash (P⩽0.05). Sensory evaluated tenderness showed positive significant correlation with L(∗) (P⩽0.01). The most important colour parameter seems to be a(∗), which was negatively correlated with sensory evaluated parameter colour (P⩽0.01). The PCA performed on all parameters (sensory, chemical and textural) discriminated two groups of hams differing in non-collagen muscle protein content and hardness.

[High dose therapy with autologous stem cell transplantation in patients with Hodgkin's lymphoma: long-term follow-up in patients treated in one center]. / Vysokodávkovaná terapie s autologní transplantací krvetvorných bunek u nemocných s Hodgkinovým lymfomem: dlouhodobé sledování nemocných lécených v jednom centru.

BACKGROUND: Majority of patients with Hodgkin's Lymphoma (HL) can be cured by first line therapy. The high dose therapy (HDT) with autologous stem cell transplantation (ASCT) is the option which can be used in the situation when the conventional therapy failed. METHODS AND RESULTS. Beginning 1994 till 2005 84 pts with HL who did not respond the conventional chemotherapy underwent 105 HDT procedures with ASCT. The median age at the time of HDT was 30.5 years. The reason for salvage therapy followed by HDT with ASCT was the failure to achieve 1st complete remission-- CR (n 16) or the subsequent relapse or progression (n 68). The disease status at the time of HDT after conventional salvage chemotherapy was assessed as chemosensitive in 65 pts (77.4%) and chemoresistant in 19 pts (22.6%). The most frequent HDT regimen used was BEAM (82 HDT), 22 pts entered into the tandem HDT program. Bone marrow only was used as the source of progenitor cells in 4 ASCT, peripheral blood progenitor cells (PBPC) only were used in 85 ASCT and the combination of both in 16 ASCT. The disease status after the HDT with ASCT was assessed (77 pts were qualifiable) as CR in 39 pts (50.6%), PR in 31 (40.3%) and as stable disease or progression in 7 pts (9.1%). Treatment related mortality in HDT with PBPC was 3.9%. The median follow up is 5.3 years. The five year probability of event free survival (EFS) is 43.1% and overall survival 53.2%. The EFS and OS probability respectively for the chemosensitive patients was 48.6% and 62.9% respectively. The status at HDT and the results after it have prognostic significance. There were observed 39 deaths and 26 of them were caused by disease progression. Secondary tumor was observed in 5 pts and in all of them it caused the death. CONCLUSIONS: The HDT with ASCT allows the long-term survival without disease progression in about a half of the patients with reasonable toxicity.

[Monitoring markers of bone turnover in multiple myeloma]. / Monitorace markeru kostní prestavby u mnohocetného myelomu.

PURPOSE: To assess the efficacy of bone turnover markers monitoring for prediction of overall survival and event-free survival in patients with multiple myeloma. To determine the value of initial measurement for differential diagnosis and evaluation of myeloma stage and activity. METHODS AND RESULTS: We evaluated 79 consecutive, previously untreated patients with myeloma and 24 with monoclonal gammopathy of undetermined significance. Urine excretion rates of pyridinoline (PYR) and deoxypyridinoline (DPYR) as markers of bone resorption and serum osteocalcin (OC) as marker of bone formation were measured at diagnosis and further repeatedly during the course of disease. High-performance liquid chromatography (HPLC) was used to measure urinary excretion rates of PYR and DPYR, serum OC levels was analysed using RIA method. Significant correlation was found between PYR and DPYR levels (p < 0.001) and between DPYR and OC (p < 0.05). Significantly higher urine PYR and DPYR (p < 0.001 and p < 0.01, respectively), but not serum OC, were found in myeloma comparing MGUS. In patients with MM PYR and DPYR levels were significantly higher in stage III vs. I and II (p < 0.01), the correlation with disease activity was not found. The grade of bone involvement according to RTG (osteolysis, osteoporosis, absence of bone lesions) was not reliable as a prognostic factor in our study. Univariate overall survival analysis showed prognostic significance for initial PYR (p < 0.05) but not for DPYR and/or OC. Rapid decrease of PYR (after 1 month of chemotherapy) was associated with the shortest median survival (608 days), however, significant difference was observed only comparing the patients in which PYR decreased 12 months after start of chemotherapy. In the univariate analysis increase of DPYR was the only variable significant for event-free survival (p < 0.05), increase of PYR tend to be significant (p < 0.1). CONCLUSIONS: We confirmed possible contribution of pyridinium cross-links for differential diagnosis of MM and MGUS and the correlation with advanced stage of MM. Initial measurement of PYR and monitoring of both PYR and DPYR during the course of myeloma could be helpful for prediction of overall survival and event-free survival. According to our experience OC is not useful for diagnosis, assessment of disease stage and activity and prediction of survival of patients with MM.

[Effect of treatment with recombinant erythropoietin in patients with multiple myeloma and kidney failure]. / Efekt lécby rekombinantním erytropoetinem u pacientu s mnohocetným myelomem a selháním ledvin.

The efficacy of recombinant human erythropoietin (r-HuEPO) in patients with multiple myeloma (MM) has been confirmed in several clinical trials. We report our experience of r-HuEPO treatment in 5 myeloma patients with renal failure. The therapy with r-HuEPO (Eprex, Janssen-Cilag or Recormon, Boehringer, Mannheim) was started after 4-8 months from diagnosis, the drug was administered intravenously (in one patient subcutaneously after cessation of hemodialysis treatment), two or three times weekly. The initial doses were 4-12,000 units/week (mean 8,400). In all patients good response during the first month of therapy was observed. Median Hb and hematocrit increased from 70 g/l and 20.8% to 87 g/l and 26% after 1 month and to 105 g/l and 30.3% after 4-6 months, respectively. The need for blood transfusion decreased significantly--from 2.72 TU/month to 0.13 TU/month. WHO performance status and patients self-assessment of quality of live improved substantially after r-HuEPO. No serious adverse events, including hypertension and/or thromboembolic events were observed. In accordance with some previous reports we conclude r-HuEPO is effective and safe treatment in patients with MM and renal failure. Moreover, lower doses of growth factor could be effective in this particular group of patients.