Aneta Mikulášová

Mutational Spectrum, Copy Number Changes, and Outcome: Results of a Sequencing Study of Patients With Newly Diagnosed Myeloma

PURPOSE At the molecular level, myeloma is characterized by copy number abnormalities and recurrent translocations into the immunoglobulin heavy chain locus. Novel methods, such as massively parallel sequencing, have begun to describe the pattern of tumor-acquired mutations, but their clinical relevance has yet to be established. METHODS We performed whole-exome sequencing for 463 patients who presented with myeloma and were enrolled onto the National Cancer Research Institute Myeloma XI trial, for whom complete molecular cytogenetic and clinical outcome data were available. RESULTS We identified 15 significantly mutated genes: IRF4, KRAS, NRAS, MAX, HIST1H1E, RB1, EGR1, TP53, TRAF3, FAM46C, DIS3, BRAF, LTB, CYLD, and FGFR3. The mutational spectrum is dominated by mutations in the RAS (43%) and nuclear factor-κB (17%) pathways, but although they are prognostically neutral, they could be targeted therapeutically. Mutations in CCND1 and DNA repair pathway alterations (TP53, ATM, ATR, and ZNFHX4 mutations) are associated with a negative impact on survival. In contrast, those in IRF4 and EGR1 are associated with a favorable overall survival. We combined these novel mutation risk factors with the recurrent molecular adverse features and international staging system to generate an international staging system mutation score that can identify a high-risk population of patients who experience relapse and die prematurely. CONCLUSION We have refined our understanding of genetic events in myeloma and identified clinically relevant mutations that may be used to better stratify patients at presentation.

The spectrum of somatic mutations in monoclonal gammopathy of undetermined significance indicates a less complex genomic landscape than that in multiple myeloma

Monoclonal gammopathy of undetermined significance is a pre-malignant precursor of multiple myeloma with a 1% risk of progression per year. Although targeted analyses have shown the presence of specific genetic abnormalities such as IGH translocations, RB1 deletion, 1q gain, hyperdiploidy or RAS gene mutations, little is known about the molecular mechanism of malignant transformation. We performed whole exome sequencing together with comparative genomic hybridization plus single nucleotide polymorphism array analysis in 33 flow-cytometry-separated abnormal plasma cell samples from patients with monoclonal gammopathy of undetermined significance to describe somatic gene mutations and chromosome changes at the genome-wide level. Non-synonymous mutations and copy-number alterations were present in 97.0% and in 60.6% of cases, respectively. Importantly, the number of somatic mutations was significantly lower in monoclonal gammopathy of undetermined significance than in myeloma (P<10−4) and we identified six genes that were significantly mutated in myeloma (KRAS, NRAS, DIS3, HIST1H1E, EGR1 and LTB) within the monoclonal gammopathy of undetermined significance dataset. We also found a positive correlation with increasing chromosome changes and somatic gene mutations. IGH translocations, comprising t(4;14), t(11;14), t(14;16) and t(14;20), were present in 27.3% of cases and in a similar frequency to myeloma, consistent with the primary lesion hypothesis. MYC translocations and TP53 deletions or mutations were not detected in samples from patients with monoclonal gammopathy of undetermined significance, indicating that they may be drivers of progression to myeloma. Data from this study show that monoclonal gammopathy of undetermined significance is genetically similar to myeloma, however overall genetic abnormalities are present at significantly lower levels in monoclonal gammopathy of undetermined significant than in myeloma.

Gain(1)(q21) is an Unfavorable Genetic Prognostic Factor for Patients With Relapsed Multiple Myeloma Treated With Thalidomide but Not for Those Treated With Bortezomib

UNLABELLED Chromosomal aberrations are important prognostic factors in multiple myeloma diagnosis. We evaluated the effect common high-risk chromosomal aberrations in a cohort of 102 patients with relapsed disease treated with bortezomib or thalidomide. Our results showed that patients treated with thalidomide with a gain(1)(q21) had inferior survival compared with the bortezomib group. Therefore, bortezomib-based regiments are more effective for patients with relapsed multiple myeloma with an incidence of gain in the gain(1)(q21). BACKGROUND Prognostic impact of specific chromosomal aberrations in patients with relapsed multiple myeloma (MM) treated with the novel agents is briefly described. PATIENTS AND METHODS We analyzed the prognostic value of an extended panel of chromosomal aberrations [del(13)(q14), del(17)(p13), t(4;14)(p16;q32), gain(1)(q21), and hyperdiploidy] by using the technique of interphase fluorescence in situ hybridization in a cohort of 102 patients with relapsed MM treated with thalidomide- or bortezomib-based protocols. RESULTS The gain(1)(q21) had a negative impact on overall survival for patients with MM treated with thalidomide (15.7 vs. 41.3 months; P = .004). Moreover, we confirmed the negative impact of the cumulative effect of 2 or more cytogenetic changes that occur simultaneously on the overall survival in the thalidomide group (20.3 months vs. not yet reached; P = .039). We did not find any significant impact of the aberrations studied on overall survival in the bortezomib cohort of patients. CONCLUSION We conclude that bortezomib-based protocols are able to partially overcome the negative prognostic impact of the tested chromosomal abnormalities in patients with relapsed MM.

Genomewide profiling of copy-number alteration in monoclonal gammopathy of undetermined significance.

Monoclonal gammopathy of undetermined significance (MGUS) is a benign condition with an approximate 1% annual risk of symptomatic plasma cell disorder development, mostly to multiple myeloma (MM). We performed genomewide screening of copy‐number alterations (CNAs) in 90 MGUS and 33 MM patients using high‐density DNA microarrays. We identified CNAs in a smaller proportion of MGUS (65.6%) than in MM (100.0%, P = 1.31 × 10−5) and showed median number of CNAs is lower in MGUS (3, range 0–22) than in MM (13, range 4–38, P = 1.82 × 10−10). In the MGUS cohort, the most frequent losses were located at 1p (5.6%), 6q (6.7%), 13q (30.0%), 14q (14.4%), 16q (8.9%), 21q (5.6%), and gains at 1q (23.3%), 2p (6.7%), 6p (13.3%), and Xq (7.8%). Hyperdiploidy was detected in 38.9% of MGUS cases, and the most frequent whole chromosome gains were 3 (25.6%), 5 (23.3%), 9 (37.8%), 15 (23.3%), and 19 (32.2%). We also identified CNAs such as 1p, 6q, 8p, 12p, 13q, 16q losses, 1q gain and hypodiploidy, which are potentially associated with an adverse prognosis in MGUS. In summary, we showed that MGUS is similar to MM in that it is a genetically heterogeneous disorder, but overall cytogenetic instability is lower than in MM, which confirms that genetic abnormalities play important role in monoclonal gammopathies.

Analysis of B-Cell Subpopulations in Monoclonal Gammopathies

BACKGROUND Multiple myeloma (MM) is characterized by accumulation of pathological plasma cells (PCs) in bone marrow (BM) as a result of deregulation of B-cell development. To clarify its pathophysiology it is necessary to investigate in detail the developmental stages of B-cells. MATERIALS AND METHODS Enumeration of total CD19-positive (CD19(+)) cells and their subpopulations together with PCs was done in peripheral blood (PB) and BM of newly diagnosed monoclonal gammopathy patients and control subjects. Representation of subsets was compared among groups and relationships between subset percentage and cytogenetic/biochemical findings were analyzed. RESULTS A lower number of total CD19(+) cells was found in MM, particularly in advanced stages of disease. Reduction of naive (P < .01) and transitional B-cells (P < .05) and increase of switched memory and switched CD27(-) B-cells and germinal center founder cells were detected in PB of MM compared with controls (P < .01). Similar results were found in BM. β2 microglobulin level in MM positively correlated with the number of PCs and negatively with percentage of naive B-cells (P < .05). CONCLUSION Our results provided a detailed phenotypic profile and enumeration of B and PC subpopulations in monoclonal gammopathy patients. A reduced number of B-cells and particularly a differentiation shift to more numerous antigen-stimulated forms was observed in MM. This might indicate a potential source of myeloma-initiating cells in one of these subpopulations.

A first Czech analysis of 1887 cases with monoclonal gammopathy of undetermined significance

Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant condition with a risk of malignant conversion.

Incidence of cytogenetic aberrations in two B lineage subpopulations in multiple myeloma patients analyzed by combination of whole-genome profiling and FISH.

Multiple myeloma (MM) is an incurable malignant disease of the terminal developmental stage of B-lymphocytes. While genetic heterogeneity of MM is widely described, little is known about its genetic basis as well as primary damage during plasma cells (PC) development. In this study, we focused on genome-wide screening of DNA copy number changes using oligonucleotide-based array-CGH together with I-FISH of the IgH locus rearrangements in pair samples of bone marrow B-cells (CD19+) and CD138+ PC from newly diagnosed MM patients. The IgH disruption was found in 8.9% (4/45) of CD19+ samples and in 57.8% (26/45) of CD138+ samples. The genomic profiling using array-CGH identified copy number alterations (CNAs) in 10% (2/20) of CD19+ samples in regions known to be important for MM pathogenesis. In contrast, we found CNAs in 100% (16/16) of CD138+ samples. Most common chromosomal abnormalities were trisomies of odd-numbered chromosomes (3, 5, 7, 9, 11, 15, 19 and 21), gain 1q, gain Xq and monosomy of chromosome 13. We did not find any correlation between incidence of CNAs in CD19+ and CD138+ cells. In conclusion, effective utilization of FISH and array-CGH can identify genetic lesions in premalignant stages leading to better understanding and characterization of MM. Keywords: Multiple myeloma; CD19+; CD138+; cytogenetics; array-CGH; FISH.