Whole Exome Sequencing of Aberrant Plasma Cells in a Patient with Multiple Myeloma Minimal Residual Disease.

Multiple myeloma is a plasma cell dyscrasia. It is the second most common hematological malignancy which is characterized by proliferation of clonal plasma cells producing harmful monoclonal immunoglobulin. Despite treatment modalities greatly evolved during the last decade, small amount of aberrant residual cells reside in patients after therapy and can cause relapse of the disease. Characterization of the residual, resistant clones can help to reveal important therapeutic targets for application of effective and precious treatment. We use CD38, CD45, CD56 and CD19 sorted aberrant plasma cells to perform next generation sequencing of their exome. Among the 213 genes in which at least one variant was present, the most interesting was found gene NRAS, one of the most often mutated gene in multiple myeloma, and homologs of 88 gene panel previously used for multiple myeloma sequencing among which was a gene previously identified as gene meaningful in bortezomib resistance. Nevertheless, the results of next generation exome sequencing need to be interpreted with caution, since they rely on bioinformatical analysis, which is still being optimized. The results of next generation sequencing will also have to be confirmed by Sanger sequencing. Final results supported by larger cohort of patients will be published soon.Key words: multiple myeloma - minimal residual disease - exome - next generation sequencing.

[The preparation of anticancer vaccine for patients with multiple myeloma on the base of monoclonal immunoglobulin loaded dendritic cells]. / Príprava protinádorové vakcíny pro pacienty s mnohocetným myelomem na bázi dendritických bunek nalozených monoklonálním imunoglobulinem.

BACKGROUND: On June 2006, phase II clinical trial focused on anticancer vaccination of multiple myeloma patients, was started. On September 2007, the immune and clinical response evaluation of first four patients was finished.The anticancer vaccine contained dendritic cells loaded with monoclonal immunoglobulin produced by myeloma cells. METHODS AND PATIENTS: Within the frame of phase II clinical trial were vaccinated four myeloma patients with stable disease. It was administered six vaccines for each patient, monthly. The dendritic cells were cultured from the patient's peripheral blood mononuclear cells and loaded with autologous monoclonal immunoglobulin under the good manufacturing practice conditions. After the safety and quality control, the satisfactory vaccine was administered to the patient. The functional characteristic of dendritic cells was evaluated using flow cytometry, the immune response was evaluated using ELISpot. The clinical response was monitored using monoclonal immunoglobulin concentration in patient's sera. RESULTS AND CONCLUSION: The immune response detected using ELISpot was observed in 3/4 patients. The monoclonal immunoglobulin concentration was changeable for all twelve months, but never exceeded the range of 25% for minimal clinical response achievement. During the vaccination, no significant toxicities or negative side-effects were observed. The clinical trial is going on with vaccination other patients with multiple myeloma.

[Selective depletion of alloreactive T cells and study of anti-tumor activity of specific T cell clones in patients with leukemia]. / Selektivní deplece aloreaktivních T lymfocytu a studium protinádorové aktivity specifických klonu T lymfocytu u pacientu s leuikémií.

BACKGROUND: Graft-versus-host disease (GVHD) is a severe complication of allogeneic transplantation of hematopoietic stem cells. Donor T cells play a major role in GVHD leading to the host tissue damage, mainly the skin, liver, and gastrointestinal tract. A selective depletion using an anti-CD25 immunotoxin can eliminate harmful alloreactive T cells while preserving other donor T cells with antileukemic and antiinfectious reactivity. PATIENTS AND METHODS: We performed 15 mixed lymphocyte reactions with clinical specimens from 12 patients with various types of leukemia (7x AML, 3x ALL, 1x CML, 1x CLL) and PBMC from 15 healthy volunteers from Transfusive station FN Brno Bohunice. RESULTS: In our experiments we have demonstrated, that antileukemic (GVL) effect of donor, especially CD4+ T cells was well preserved (7.46%), while unfavourable alloreactive (GVH) reaction of donor T cells was completely removed. The graft-versus-host (GVH) reactivation of donor cells was negligible ever after repeated stimulation with irradiated patient's PBMC. CONCLUSION: We have shown that anti-CD25 immunotoxin (IT), RFT5-SMPT-dgA, launched against alpha chain for human interleukin 2 (IL-2), led to long-term selective depletion of alloreactive donor T cell clones while their antileukemic activity was well preserved. Base on our results the clinical phase I/II study was designed. This study was initiated in year 2007 in three clinical centers in Czech Republic.

Cytogenetic and immunological changes after dermal exposure to polycyclic aromatic hydrocarbons and UV radiation.

Goeckerman's therapy (GT), which combines exposure to coal tar (polycyclic aromatic hydrocarbons - PAHs) and UV radiation (UV) is often used as the first option for treatment of psoriasis. However, PAHs and UV represent mutagenic, carcinogenic and immunotoxic agents. Therefore GT can represent a health risk for the patients. The group under observation consisted of thirty patients undergoing GT. Before and after the treatment, blood samples were collected and chromosomal aberrations and selected immunological markers were determined. The relationships between chromosomal aberrations and immunological markers and the extent (duration) of exposure to GT were evaluated. The Psoriasis Area and Severity Index (PASI) score confirmed the high efficacy of GT. However, significantly elevated levels of chromosomal aberrations of peripheral lymphocytes were also found after the therapy (p<0.001). The levels of chromosomal abnormalities correlated to the extent and the total duration of exposure to PAHs (r = 0.682, p<0.01 and r = 0.605, p<0.05). After the therapy, significantly decreased levels of IgE, IgM isotypes of immunoglobulin, alpha(2)-macroglobulin and transferrin together with beta(2)-microglobulin were found. From the immunological markers listed above only the decreased level of alpha(2)-macroglobulin correlated to the extent of exposure to PAHs (r = -0.568, p<0.05). No correlation was found between chromosomal aberrations, significantly changed immunological markers and the duration of UV exposure. Our study revealed that GT has a significant impact on both genetic and immunological parameters of psoriatic patients. The results indicate that GT could increase genotoxic risk and modulates immunity of treated patients.

[Comparison of standard prognostic factors with the deletion of 13q14 detected by interphase fluorescence in situ hybridization on separated and unseparated bone marrow cells in multiple myeloma]. / Porovnání standardních prognostických faktoru u pacientu s mnohocetným myelomem s delecí 13q14 stanovenou metodou interfázní fluorescencní in situ hybridizace na separovaných a neseparovaných bunkách kostní direne.

BACKGROUND: Cytogenetic abnormalities of chromosome 13 are emerging as important prognostic factors in multiple myeloma and have been associated with poor prognosis. METHODS AND RESULTS: The occurrence of 13q14 deletion and other standard laboratory parameters were determined in 40 patients with multiple myeloma. We found that interphase fluorescence in situ hybridization using a locus specific probe for RB1 gene on immunomagnetically selected myeloma cells was more sensitive than non selected cells. The 13q14 deletion was found in 10 of 40 (25.0%) of bone marrow samples without cell selection and in 25 of 40 (62.5%) of samples with CD138+ enriched myeloma cells. Negative correlation was found between albumin and the 13q14 deletion in separated (p = 0.003) as well as in cells without selection (p = 0.010). No significant correlation was found in overall survival of separated and unseparated cells (p = 0.830; p = 0.260) and a similar result was obtained for treatment response after transplantation of separated cells (p = 0.520) or non-separated cells (0.190). CONCLUSIONS: Our results confirm that immunomagnetic selection of CD138+ cells increases the probability of detection of the 13q14 deletion in bone marrow samples. The correlation was found between albumin and the 13q14 deletion in both of type of cells.

Lack of renal changes in stainless steel welders exposed to chromium and nickel.

Biochemical markers of kidney damage were examined in 52 male stainless steel welders (manual metal arc welding) exposed to chromium and nickel. No difference was found in the mean urinary excretion of total proteins, albumin, protein 1, transferrin, retinol-binding protein, lactate dehydrogenase, lysozyme, or beta-N-acetylglucosaminidase in a comparison with matched referents. Beta 2-microglobulin was slightly increased in those welders with a urinary chromium concentration of greater than 64.5 nmol.mmol-1 creatinine. The prevalences of abnormal values did not differ from those observed in the reference group. No correlation was found between the concentrations of chromium or nickel in urine and that of proteins or enzymes. No consistent or clinically significant renal impairment was revealed among the stainless steel welders exposed to a chromium air concentration slightly above the current threshold limit value of the American Conference of Governmental Industrial Hygienists for water-soluble hexavalent chromium compounds (50 micrograms.m-3).

Possible genotoxic risk of combined exposure to pharmaceutical coal tar and UV-B radiation.

Goeckerman's therapy of psoriasis combines exposure to pharmaceutical coal tar and UV-B radiation. In the pilot study (15 patients had been diagnosed with psoriasis, the average time period in hospital therapy was 24 days, the average age of the patients was 29 years, 47% of them were smokers) a level of genotoxic risk from therapy was evaluated by using chromosomal aberration of peripheral lymphocytes. The study suggested the presence of an increased genotoxic risk from the therapy. The PASI scores (Psoriasis Area and Severity Index) were monitored.

[Breastfeeding support in nine Czech maternity hospitals 1998-1999]. / Péce o kojení v devíti ceských porodnicích v letech 1998-1999.

OBJECTIVE: To find out the state of breastfeeding support in prenatal and perinatal care and infant feeding in first 6 months in 6 big cities of Czech Republic in 1998-1999. DESIGN: Cross-sectional multicentre descriptive study of 6 medical schools. SETTING: Centre of Preventive Medicine, 3rd Medical Faculty, Charles University, Prague. METHODS: In the first year of the study (1998), 1104 mothers were interviewed by neonatologists at the discharge from 9 maternity hospitals. After 6 months, 1019 mothers of the same sample were interviewed again by students and staff members of 6 medical schools. Obtained data were analyzed in EpiInfo 6 programme using ANOVA and x2 test. RESULTS: 29 percent of mothers participated in prenatal classes. 70 percent of newborns were put to the breast within two hours after birth. 44 percent of mothers reported problems with breastfeeding in the hospital. 77 percent of mothers appreciated the assistance with breastfeeding in the hospital. Mothers with higher education, living with partner or husband, mothers who participated in prenatal classes, mothers after vaginal labour, mothers whose children were put to the breast within two hours after birth and mothers who did not have problems with breastfeeding in the hospital were breastfeeding fully for significantly longer period. At the discharge from the hospital, 93.5 percent of newborns were fully breastfed. At the end of the 6th month, 23.1 percent of infants were fully breastfed and 29.9 percent of infants were breastfed while receiving complementary food. CONCLUSION: Breastfeeding rate at the end of the 6th month of age of infants increased apparently in mothers from 6 big cities of Czech Republic in comparison with national data from 1991.

[New findings in monitoring health status of welders and grinders of stainless steel]. / Novejsí zkusenosti se sledováním zdravotního stavu svárecu a brusicu nerezových ocelí.

During the occupational preventive care check up we investigated a group of 20 stainless steel welders and grinders in the factory producing technology for chemical industry. Results have been compared with 21 healthy persons--blood donors. In the group exposed there have not been discovered any marked deviations in either their health status, or in the CBC and biochemical screening results, though there had been found substantially increased chromium and nickel concentrations, mainly in grinders. Also the levels of polycyclic aromatic hydrocarbons were in some cases even higher than on the busy city crossing. The chromosome aberrations investigations proved to be very sensitive and confirmed that employees of the followed up factory are exposed to an increased genotoxic risk.

[Biological and medical monitoring of persons with occupational exposure to fibrous materials]. / K biologicko-medicínskému monitorování osob profesionálne exponovaných vláknitým materiálum.

Health status indicators in 3 groups of workers professionally exposed to fibrous materials and 1 control group have been followed. In spite of the fact that asbestos as a compound of friction elements has been forbidden and replaced by non-asbestos materials, quite important changes in peripheral lymphocytes aberrations we found in persons working with these new materials.

[Biological monitoring of occupational exposure in welders of stainless steel. Immunologic methods]. / Biologické monitorování pracovní expozice u svárecu nerezových ocelí. Imunologické metody.

Biological monitoring of occupational exposure to toxic compounds enables an early detection of adverse health effects. Apart from the standard biological monitoring methods of occupational exposure represented by metabolites and enzymes activities analysis also immunological detection methods seem to be promising. The changes of immunological system are demonstrable extraordinary sensitive markers of load of organism. The process of welding belongs between important sources of pollution of working environment with heavy metals and other harmful compounds including polycyclic aromatic hydrocarbons. According to IARC classification the welding fumes are included into group 2B and are supposed as possible human carcinogens. Numbers of different epidemiological studies have proved relationship between welding and serious health disorders namely malignancies. We recorded a number of changes in immunological parameters when compared with those in control group in our study. The results of analysis were statistically evaluated. The statistically significant reduction of number of phagocytosis capable cells was found in the group of welders when compared with the control group. The levels of neopterin, beta-2-microglobulin and IL-1 beta have indirectly monitored cell component of immunological response. In the case of these markers a statistically significant increases were found in the group of welders. We found statistically significant IgM decrease and statistically significant IgA increase in humoral responses with the same group when compared with control group.

[Health status of persons occupationally exposed to chromium, nickel, manganese and polycyclic aromatic hydrocarbons]. / Zdravotní stav osob profesionálne exponovaných chromu, niklu, manganu a polycyklickým aromatickým uhlovodíkum.

Occupational environment monitoring and biological-medical monitoring of persons professionally exposed to welding fumes have been performed. Chromium, manganese and polycyclic aromatic hydrocarbons in welding fumes represents an important health risk. Pollutant concentrations found in metal welding fumes represented only fractions of those acceptable ones. Polycyclic aromatic hydrocarbons have been reached the concentration found in a busy road crossing in Hradec Králové (compared with these as in Czech Republic no maximum acceptable levels for PAHs having been declared). Family, personal and occupational history have been taken. Health state including total haematological count, biochemical and cytogenetical changes of 19 stainless steel welders were checked-up. The level of mercapturates in urine were examined as well. The data were statistically compared with those of non exposed (control group). No changes witnessing the above mentioned risk factors influence on the haematological, biochemical and cytogenetical findings were ever proved. In conclusion, our results did not confirm an increased professional risk in this group of welders.

The chromosomal aberrations investigation in children permanently living in the lead polluted area.

The cytogenetic analysis of peripheral blood lymphocytes in 28 children permanently living in the lead polluted area and in 30 children from the control region was carried out. In the lead exposed group 0-6% of aberrated cells (AB.C.) with the average value of 1.94% of AB.C. were found. The most common findings were represented by the structural aberrations of the chromatid type, in lesser degree also the isochromatid breaks. In the control group 0-3% of AB.C. with average 1.37% was found, which corresponds to the spontaneous level of the chromosomal aberrations 1.2-2% of AB.C. in the unladen population. The differences in the numbers of the aberrated cells between the exposed and control groups are not statistically important. Nevertheless, in the exposed area a certain shift of the AB.C. frequency towards higher values (4.5-6% of AB.C.) is being noticed.

Dose-related stress reaction in male rats chronically exposed to lead acetate.

Male Wistar rats were given 0.5% lead acetate and natrium acetate equimolar to 2% lead acetate for 3 months, 1 and 2% lead acetate for 2 months. Noradrenaline (NA) in hypothalamus and striatum corticosterone, thyroxine and 3,5,3'-triiodothyronine in serum and catecholamines (CA) in adrenals were determined at one month's interval. Results were accomplished by body and organ weights. All the changes depend on lead exposure level and time of exposure. At the three doses, lead exposure produced a significant decrease of NA in hypothalamus and striatum, increase of CA in adrenals and increase of kidney weight. Significant changes of all hormones, liver, spleen and kidney weight were observed in rats given 2% lead acetate. The stress effects of acetate was excluded by the lack of biochemical, organ or body weight effects of sodium acetate. Our results suggest a nonspecific stress reaction in rats activating hypothalamo-pituitary-thyroid-adrenal axis.

[Chromosome aberrations in peripheral blood lymphocytes in workers occupationally exposed to styrene]. / Vysetrení chromozómových aberací v lymfocytech periferní krve zamestnancu profesionálne exponovaných styrénu.

We performed the investigation of the number of chromosomal aberrations in the peripheral blood lymphocytes of 13 women--workers occupationally exposed to styrene. Our set consisted exclusively of women in the age span from 23 to 54 years. Nine of the workers were smokers, 4 of them did not smoke. The control group was represented by 6 women working in the offices of the same factory. The clinical investigation of both the groups of women was performed at the Clinic of occupational diseases. The common physical check-up was combined with the hematological and biochemical tests including the assessment of the mandelinic acid level in the urine. Also the styrene concentration in the working place was measured by the help of the Regional hygiene center. The average working day's concentration of styrene was found to be 225 +/- 89 mg.m-3 ranging from 83 to 366 mg.m-3. From hte total number of 1220 cytogenetically investigated cells in the group of higher risk in 31 of them (2.54% AB.C.) the chromosomal aberration were found. In 27 cases (2.21%) the structural aberrations were involved; mostly the chromatid breaks and four times the chromosomal breaks were present. Four cells were laden by the numeric aberrations (type of 4n). Moreover, there was also checked the number of gaps (total of 9 gaps; 6 of chromatid, 3 chromosomal ones) and the number of satellite association of chromosomes (total of 87). The number of chromosomal aberrations in the exposed group is statistically significantly higher when compared with the control group (1.17% of AB.C.). Due to the restricted volume of the set our results cannot be taken as a confirmation of the relevant factory to be a working place with the higher exposition to genotoxic agents. Nevertheless, concerning the upper limit of spontaneous aberrations in the unladen population reaching maximally 2% we must consider our results to witness the increased occupational hazard.

The time course of biochemical and histological changes following carbon tetrachloride-induced liver damage in rats of both sexes.

A detailed analysis is presented of the time changes in the development of liver damage 6, 12, 24, 48 and 72 hours after i.p. administration of carbon tetrachloride [CCl4] in a dose of 0.75 ml, i.e. 1 200 mg/kg body weight to rats of both sexes. The severity of liver damage was assessed from the histological and biochemical changes of AST, ALT, alkaline phosphatase and GMT serum activity. From our experiments it follows that in male rats the level of transaminases increases earlier than in female rats, as early as 6 h after the administration of CCl4, reaching a maximum 12 h later. These changes prevail for a longer time period, the level of transaminases remaining increased even 72 h after CCl4 administration. In female rats the biochemical changes occur later reaching the maximum elevation of AST and ALT 24 h after CCl4 administration. The values slowly return to normal after 48 h, and after 72 h the levels of transaminases are identical with the control group. The above given biochemical results are in good agreement with the histological findings demonstrating a higher regenerative activity in female rats. This finding was also proved by specific liver DNA activity assay.

Effect of realimentation after several days' pure carbohydrate intake on DNA synthesis in regenerating rat liver.

The authors studied the effect of realimentation after several days' isolated glucose or fructose intake on DNA synthesis in liver regenerating after partial hepatectomy (PH) (65-70%) or after carbon tetrachloride (CCl4) poisoning 1.5 ml/kg. Two days before PH or the administration of CCl4 and two days after, the experimental rats were given glucose (50% solution) of fructose (50% solution) as the only source of energy. Rats with PH were then fed for one day on a standard laboratory diet (25 cal% protein) or a high protein diet (81 cal% protein). Rats with CCl4 liver damage were fed for one day on the standard laboratory diet only. In the rats given glucose, liver DNA synthesis and the total amount of these nucleic acids in the liver 48 hours after CCl4 administration was lower than in the controls or the rats given fructose. In all the experimental groups (PH and CCl4), stimulation of liver DNA synthesis was observed after one day's realimentation. The total DNA content of the liver of rats with PH rose markedly during realimentation. The experiments indicate that the regenerative activity of damaged liver can be influenced by the nutritional regimen.

Effect of realimentation and triiodothyronine on DNA synthesis in intact and partially resected rat liver.

Whereas DNA synthesis in the liver of rats with permanent access to food fell markedly between the 72nd and 96th hour after partial hepatectomy, the decrease in the same post-operative interval in rats realimented after 3 days' starvation was less pronounced. The difference between the values in the control and the experimental group 96 hours after partial hepatectomy was statistically significant for p less than 0.01). In rats given glucose after the operation, 24 hours' realimentation caused a nonsignificant increase in DNA synthesis. The difference between the values 96 h after partial hepatectomy in this group and in the group fed the whole time on the standard diet was statistically significant for p less than 0.001. The administration of triiodothyronine (200 microgram/kg i.g.) to the above groups of rats at the outset of realimentation did not affect the development of changes in liver DNA synthesis. It likewise had no effect on the outcome of the realimentation of rats with an intact, unresected liver fed 3 days on plain glucose. In the intact control rats fed the whole time on the standard laboratory diet, triiodothyronine produced a distinct increase in liver DNA synthesis (p less than 0.01).