CRISPR in Research and Treatment of Multiple Myeloma.

In the recent years, there was a remarkable advance in research and clinical implementation of the genome editing technologies. The most remarkable was a discovery of the bacterial adaptive immune system called CRISPR and its rapid transformation into a robust and broadly applicable technology that completely revolutionized both basic and applied biomedical research. Implementation of CRISPR makes genome modification easier, faster and significantly cheaper compare to any other currently available technology. It also offers a tremendous potential for desiging novel research approaches and future treatment options for various genetic diseases including multiple myeloma. The hightroughput use of CRISPR in pooled screen formats promises faster identification and validation of valuable drug targets together with revealing high-confidence biomarkers and unknown resistance mechanisms. This can provide clinicians with new diagnostic and prognostic tolls and ultimately allow more accurate patient stratification for personalised treatment with better eficacy. In this review, we summarize current knowledge about the CRISPR technology and focus especially on its impact in exploring gene functions, screening for novel drug targets, diagnostic markers and genes involved in resistance to commonly used drug in the treatment of multiple myeloma. Finally, we also highlight a potential future use of CRISPR in actual clinical practise.Key words: multiple myeloma - CRISPR - therapeutics.

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.

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.

Beyond the marrow: insights from comprehensive next-generation sequencing of extramedullary multiple myeloma tumors.

Extramedullary multiple myeloma (EMM) is an aggressive form of multiple myeloma (MM). This study represents the most comprehensive next-generation sequencing analysis of EMM tumors (N = 14) to date, uncovering key molecular features and describing the tumor microenvironment. We observed the co-occurrence of 1q21 gain/amplification and MAPK pathway mutations in 79% of EMM samples, suggesting that these are crucial mutational events in EMM development. We also demonstrated that patients with mutated KRAS and 1q21 gain/amplification at the time of diagnosis have a significantly higher risk of EMM development (HR = 2.4, p = 0.011) using data from a large CoMMpass dataset. We identified downregulation of CXCR4 and enhanced cell proliferation, along with reduced expression of therapeutic targets (CD38, SLAMF7, GPRC5D, FCRH5), potentially explaining diminished efficacy of immunotherapy. Conversely, we identified significantly upregulated EZH2 and CD70 as potential future therapeutic options. For the first time, we report on the tumor microenvironment of EMM, revealing CD8+ T cells and NK cells as predominant immune effector cells using single-cell sequencing. Finally, this is the first longitudinal study in EMM revealing the molecular changes from the time of diagnosis to EMM relapse.

Current applications of multiparameter flow cytometry in plasma cell disorders.

This corrects the article DOI: 10.1038/bcj.2017.90.

Mutations in LZTR1 drive human disease by dysregulating RAS ubiquitination.

The leucine zipper-like transcriptional regulator 1 (LZTR1) protein, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, is implicated in human disease, yet its mechanism of action remains unknown. We found that Lztr1 haploinsufficiency in mice recapitulates Noonan syndrome phenotypes, whereas LZTR1 loss in Schwann cells drives dedifferentiation and proliferation. By trapping LZTR1 complexes from intact mammalian cells, we identified the guanosine triphosphatase RAS as a substrate for the LZTR1-CUL3 complex. Ubiquitome analysis showed that loss of Lztr1 abrogated Ras ubiquitination at lysine-170. LZTR1-mediated ubiquitination inhibited RAS signaling by attenuating its association with the membrane. Disease-associated LZTR1 mutations disrupted either LZTR1-CUL3 complex formation or its interaction with RAS proteins. RAS regulation by LZTR1-mediated ubiquitination provides an explanation for the role of LZTR1 in human disease.

Current applications of multiparameter flow cytometry in plasma cell disorders.

Multiparameter flow cytometry (MFC) has become standard in the management of patients with plasma cell (PC) dyscrasias, and could be considered mandatory in specific areas of routine clinical practice. It plays a significant role during the differential diagnostic work-up because of its fast and conclusive readout of PC clonality, and simultaneously provides prognostic information in most monoclonal gammopathies. Recent advances in the treatment and outcomes of multiple myeloma led to the implementation of new response criteria, including minimal residual disease (MRD) status as one of the most relevant clinical endpoints with the potential to act as surrogate for survival. Recent technical progress led to the development of next-generation flow (NGF) cytometry that represents a validated, highly sensitive, cost-effective and widely available technique for standardized MRD evaluation, which also could be used for the detection of circulating tumor cells. Here we review current applications of MFC and NGF in most PC disorders including the less frequent solitary plasmocytoma, light-chain amyloidosis or Waldenström macroglobulinemia.