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.

Biomarkers in Immunoglobulin Light Chain Amyloidosis.

Immunoglobulin light chain amyloidosis (AL amyloidosis - ALA) is a monoclonal gammopathy characterized by presence of aberrant plasma cells producing amyloidogenic immunoglobulin light chains. This leads to formation of amyloid fibrils in various organs and tissues, mainly in heart and kidney, and causes their dysfunction. As amyloid depositing in target organs is irreversible, there is a big effort to identify biomarker that could help to distinguish ALA from other monoclonal gammopathies in the early stages of disease, when amyloid deposits are not fatal yet. High throughput technologies bring new opportunities to modern cancer research as they enable to study disease within its complexity. Sophisticated methods such as next generation sequencing, gene expression profiling and circulating microRNA profiling are new approaches to study aberrant plasma cells from patients with light chain amyloidosis and related diseases. While generally known mutation in multiple myeloma patients (KRAS, NRAS, MYC, TP53) were not found in ALA, number of mutated genes is comparable. Transcriptome of ALA patients proves to be more similar to monoclonal gammopathy of undetermined significance patients, moreover level of circulating microRNA, that are known to correlate with heart damage, is increased in ALA patients, where heart damage in ALA typical symptom.Key words: amyloidosis - plasma cell - genome - transcriptome - microRNA.

Diagnostic Tools of Waldenströms Macroglobulinemia - Best Possibilities for Non-invasive and Long-term Disease Monitoring.

Waldenströms macroglobulinemia (WM) is a B-cell malignancy characterized by high level of monoclonal immunoglobulin M (IgM) paraprotein in blood serum and associated with the bone marrow infiltration by malignant cells with lymphoplasmacytic differentiation. WM remains incurable advances in therapy. Most of WM cases are associated with a somatic point mutation L265P in MYD88. Significantly higher risk of progression from the IgM monoclonal gammopathy of undetermined significance (IgM MGUS) to WM for patients with mutated MYD88 gene suggests that this mutation is an early oncogenic event and plays a central role in development of malignant clones. The second, most prevalent mutation in WM is found in the CXCR4 gene and is often associated with drug resistance and aggressive disease presentation. Therefore, detection of these mutations (MYD88L265P and CXCR4S338X) could be useful diagnostic and prognostic tool for the patients with WM. While detection of these mutations in bone marrow sample is common, the aim of our study was to compare sensitivity of detection of mutation from different cell fraction from peripheral blood and bone marrow. The results show possibility to describe MYD88 and CXCR4 mutation status even from peripheral blood sample (sensitivity for MYD88L265P was 100%, for CXCR4S338X 91%), which significantly facilitate material collection. Moreover, comparable detection sensitivity of these mutations in bone marrow and peripheral blood samples examined before and during the therapy offers a promising tool for more routine diagnostic and monitoring of disease progression.Key words: Waldenström macroglobulinemia - hematology - neoplasms - lymphoma - mutation - MYD88 - CXCR4.

Biobanking strategy and sample preprocessing for integrative research in monoclonal gammopathies.

AIMS: Some types of monoclonal gammopathies are typified by a very limited availability of aberrant cells. Modern research use high throughput technologies and an integrated approach for detailed characterisation of abnormal cells. This strategy requires relatively high amounts of starting material which cannot be obtained from every diagnosis without causing inconvenience to the patient. The aim of this methodological paper is to reflect our long experience with laboratory work and describe the best protocols for sample collection, sorting and further preprocessing in terms of the available number of cells and intended downstream application in monoclonal gammopathies research. Potential pitfalls are also discussed. METHODS: Comparison and optimisation of freezing and sorting protocols for plasma cells in monoclonal gammopathies, followed by testing of various nucleic acid isolation and amplification techniques to establish a guideline for sample processing in haemato-oncology research. RESULTS: We show the average numbers of aberrant cells that can be obtained from various monoclonal gammopathies (monoclonal gammopathy of undetermined significance/light chain amyloidosis/multiple myeloma (MM)/MM circulating plasma cells/ minimal residual disease MM-10 123/22 846/305 501/68 641/4000 aberrant plasma cells of 48/30/10/16/37×106 bone marrow mononuclear cells) and the expected yield of nucleic acids provided from multiple isolation kits (DNA/RNA yield from 1 to 200×103 cells was 2.14-427/0.12-123 ng). CONCLUSIONS: Tested kits for parallel isolation deliver outputs comparable with kits specialised for just one type of molecule. We also present our positive experience with the whole genome amplification method, which can serve as a very powerful tool to gain complex information from a very small cell population.

[Ovarian remnant syndrome]. / Syndrom ovariálního zbytku.

OBJECTIVE: We report a case of the patient who 4 years after bilateral salpingo-oophorectomy had adenocarcinoma of ovary. DESIGN: Case report. SETTING: Department of Obstetrics and Gynecology, Pardubická krajská nemocnice, a.s., Pardubice. SUBJECT AND METHOD: A case report of the patient diagnosed and treated at our department. CONCLUSION: The risk of ovarian remnant after operation is complication that is frequently related to a history of endometriosis, PID and multiple pelvic surgery. These complication is called ovarian remnant syndrome. Also an ovarian adenocarcinoma can develop in ovarian remnant. Effective therapy of ovarian remnant may usually be achieved by an operation demanding a wide approach to retroperitoneum.

[Experience of the Toxicology Information Center in Prague with pesticides]. / Zkusenosti toxikologického informacního strediska v Praze s pesticidy.

The Toxicological Information Centre replied in 1988 and 1989 some 5500 inquiries. This number comprised 5-7% concerning treatment of acute intoxications with pesticides. More than half of them concerned children who ingested pesticides. In adults in half of inquiries possible intoxication after ingestion was involved, in half intoxication was a result of inhalation or percutaneous intoxication. 10.5% and 12.5% of the inquiries concerning pesticide intoxications in adults were attempted suicide. The number of inquiries regarding occupational diseases during spraying was small (some 5 and 3 cases). The inquiries were divided by groups of pesticides into herbicides, insecticides, rodenticides, fungicides and others. From the analysis ensued that children were intoxicated most frequently by preparations against harmful animals (rodenticides, insecticides), in particular rodents and ants. In adults the stratification of intoxications into groups was less marked.

[Acute toxic liver lesion through industrial noxious substances and drugs in higher than therapeutic doses]. / Akutní toxické poskození jater prumyslovými skodlivinami a léky ve vyssích nez lecebných dávkách

From the accessible material an attempt was made to estimate the frequency of acute toxic disturbances of liver and its relation to all intoxications together with special regard to the composition of noxious substances on the conditions of this country. From 3700 cases of all acute intoxications or serious attempts of intoxication 54 cases were discovered to have marked hepatic disturbance (about 1.5%). The noxious substance causing hepatic disturbance in the overwhelming majority of cases was tetrachlormethan (and chemically related substances) or new herbicide drugs of the dipyridine compounds. The last mentioned group is highly lethal and the more important as it is a relatively new substance to which health control and practice is less accustomed as to a potential hepatotoxic drug than to tetrachlormethan. It is pointed further to a less frequent potential intoxication by phenylbutazone and its derivatives especially in children and to the acute intoxication by INH when high single doses of this drug are used.