Academic literature on the topic 'Intraclonal heterogeneity'

AbstractMonoclonal gammopathy of undetermined significance (MGUS) can transform to multiple myeloma (MM). In myeloma, mutated VHgenes with sequence homogeneity reveal a postfollicular origin. Previously, some MGUS cases showed mutated VH genes with intraclonal variation, indicating an earlier stage of arrest. We investigated progression from 2 of 2 MGUS to MM, in which VH genes confirmed clonal evolution. In one MGUS case, intraclonal heterogeneity was evident, and transformation to myeloma occurred rapidly with apparent homogeneity in the emergent clone. However, residual MGUS-derived sequences were detectable at this time. Heterogeneity in MGUS does not associate with benign disease, but it indicates an origin from a tumorigenic cell, most likely surface immunoglobulin+, undergoing somatic mutation. The remaining case displayed intraclonal homogeneity at the MGUS stage, conceivably resulting from a self-cloning outgrowth from MGUS with heterogeneity. Transformation can occur at either MGUS stage, but it involves a single cell in which somatic mutation is then silent.

Abstract Multiple myeloma (MM) pathogenesis has been explained for many years by the cancer biology dogma introduced by Peter Nowell: first, a single plasma cell would be immortalized by an error in the immunoglobulin genes rearrangement process; then, a progressive stepwise acquisition of somatic cell mutations would induce a sequential selection and domination by the fittest clone. In line with this idea of “myeloma stability”, SNP arrays studies in diagnostic-relapse paired samples have revealed the presence of common clonal characteristics. Biologically, the M-protein remains usually constant across MM evolution and further, the variable domain of the rearranged immunoglobulin heavy chain genes (or CDR3 region) has been used as a patient-specific myeloma fingerprint in minimal residual disease (MRD) studies. However, massive genome studies with Next Generation Sequencing (NGS) have challenged this concept, showing a significant intraclonal heterogeneity at diagnosis with the possible presence of several clonal progenitors or tumor-initiating cells. In this study, we have characterized and compared the CDR3 region in 52-paired samples from 26 MM patients aiming: 1) to assess mono-clonality in MM evolution through the analysis of the CDR3 sequence and, 2) to validate ASO RQ-PCR approaches for MRD in MM, based on the constancy and specificity of the CDR3 region. Samples were obtained at diagnosis and progression (19 pairs) or at 2 different timepoints of progressive disease (7 pairs). Median time between sampling was 2 years. M-protein subtype remained stable in all pairs but 1, associated with a light-chain escape phenomenon. All samples proceeded from bone marrow (BM) except for 2 pairs, composed by BM and extramedullary disease (spleen and testes). Two major cytogenetic changes were identified: increased 13q14 deletion (from 7 to 54%) in 1 pair and increased 17p (p53) deletion (from 5 to 87%) in a further one. Treatments administered between sampling included most of the current approaches used in MM (data not shown). Genomic DNA isolation, PCR amplification and sequencing were performed following conventional methods. Germline VH, DH and JH segments were identified by comparison with public databases. CDR3 region was first identified in all samples and then compared between the two samples in the 26 pairs: the sequence of nucleotides was constantly identical in each pair, including those associated with major cytogenetic changes, a light-chain escape, extramedullar vs. BM infiltration and relapsed (and therefore, treatment selected) vs. refractory disease. Therefore, we can first conclude that the main tumor clone in MM retains a specific signature across all stages of disease evolution that allows the identification of samples as evolutionary related. This major clone signature is not modified by clinical or biological changes in the disease nor under different treatment pressures and would thus identify disease relapse and progression. Our results have also a clear impact on the validity of molecular MRD techniques. The high rate of complete responses (up to 50-60%) currently achieved in MM has prompted the use of new techniques for disease assessment. Today, ASO RQ-PCR, based on the use of specific primers and probes complementary of the VDJH rearrangement, continues to be the most sensitive approach. One pitfall of this technique would be the potential instability of PCR targets over time, which would induce false negative results. In B-cell precursor ALL, this is estimated to happen in 30-40% of cases but has not been deeply evaluated in MM yet. With the present study, we can also conclude that the junction region of the VDJH rearrangement in MM constantly identifies the myeloma cells responsible for relapse and therefore can be used as a reliable target for MRD assessment by ASO RQ-PCR and more recently, by NGS methods. If the CDR3 region remains stable, the novel concept of clonal tiding in MM should not be interpreted as a poly- or oligoclonal but subclonal. In MM, tides can be subclonal, but the ocean remains monoclonal. Disclosures No relevant conflicts of interest to declare.

Abstract Background: DNA methylation in AML/MDS plays a major role in the pathogenesis of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The major genes involved in DNA methylation in AML/MDS are IDH1 and 2, TET2 and DNMT3A. Mutations in IDH1/2 result in the production of an aberrant metabolite, 2-hydroxyglutarate, which acts as a competitive inhibitor of a-ketoglutarate and inhibits TET2 oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Mutations in TET2 or IDH1/2 are associated with reduced levels of 5hmC and genomic hypermethylation. TET2 mutations and IDH1/IDH2 mutations are believed to be mutually exclusive. In addition, DNMT3A as a DNA methyltransferase enzyme is commonly mutated in AML/MDS and its mutation is believed to lead to hypomethylation. Understanding the interaction between these genes may influence therapy with IDH1/2 inhibitors. Toward better understanding of interaction between these genes, we analyzed the mutation profile of these genes in patients with AML/MDS. Methods: A total of 1182 bone marrow (BM) aspirate samples were tested by the commercially available TruSight Myeloid Next Generation Sequencing Panel (Illumina, San Diego, CA). We extracted DNA from bone marrow aspirate using the QIAamp DNA Mini Kit. This NGS panel covers hot spot mutations in 54 genes. The average depth of sequencing was 10,000X. Results: IDH1/2 mutations were detected in 201 of the 1182 (17%). IDH1 was detected in 87 (7.4%) and IDH2 was detected in 120 (10.1%). This included 6 patients who had mutations in both IDH1 and IDH2. Variant (mutant) allele frequency (VAF) was significantly higher (P=0.01) in IDH2 as compared to IDH1 (median of 43.35% vs 35.0%, respectively). Thirteen patients (6.5%) had mutant VAF >50% suggesting homozygosity, 11 of which had IDH2 mutation. Two of the 6 patients with both IDH1 and IDH2 mutations had VAF <20%, raising the possibility of two independent clones. TET2 mutations were detected in 15 (7.5%) of the patients with IDH1/2 mutations. There was significant difference (P=0.03) in VAF between IDH1/2 and TET2. Nine of these patients showed comparable VAF while the other 6 patients showed completely different VAF, suggesting subclonal heterogeneity. In addition, 58 (29%) patients showed mutations in IDH1/2 and DNMT3A. While there was no significant difference in VAF between IDH1/2 and DNMT3A, VAF in IDH1/2 was >50% in 6 of these patient and in DNMT3A in 3 patients. Twenty four patients had TP53 mutation, of which 16 had IDH1 mutation and 8 had IDH2 mutation, which is disproportional with the prevalence of IDH1 mutation. There was no statistically significant difference in VAF between TP53 and IDH1/2, but 4 of these patients had both DNMT3 and IDH2 mutations and one had both IDH1 and IDH2 mutations. None of the patients with TP53 mutation had TET2 mutation. Conclusions: IDH2 mutations may coexist with IDH1 and TET2 mutations. This co-mutation appears to be in the same clone in some patients and in a separate clone in others. The presence of VAF>50% in 6.5% of patients, which suggests homozygosity, along with co-presence of IDH1 and IDH2 and TET2 mutations suggests possible dosage effects in the biology of MDS/AML. The high rate (29%) of co-presence of DNMT3A with IDH/1/2 mutations also suggests cooperation between the two mechanisms in influencing DNA methylation and leukemogenesis. The relatively high incidence of TP53 mutation in IDH1 patients suggests that IDH1 mutation might be associated with more aggressive disease than IDH2. This data suggests that there is interaction and significant interclonal and intraclonal heterogeneity in DNA methylation genes in AML/MDS. Complete profiling of these genes is necessary for better understanding and proper prediction of clinical behavior particularly when patients treated with DNA methylation inhibitors. Figure Figure. Disclosures Ma: Neogenomics Laboratories: Employment. Thangavelu:Neogenomics Laboratories: Employment. De Dios:Neogenomics Laboratories: Employment. Funari:Neogenomics Laboratories: Employment. Albitar:Neogenomics Laboratories: Employment, Equity Ownership.

Abstract Introduction: Recent data suggest that MDS evolves by accumulating mutations. Early mutations may involve genes that require additional mutations prior to clinical manifestation as MDS. We explored if mutant allele burden and the relative mutation of one gene to another gene could provide information on the interclonal and intraclonal progression of MDS using next generation sequencing (NGS) in patients with early MDS. Methods: NGS data was generated from 96 patients diagnosed with MDS with marrow blast count <5% using a targeted sequencing covering mutations in the following genes: TET2, SF3B1, ASXL1, DNMT3A, SRSF2, RUNX1, NRAS, ZRSR2, EZH2, ETV6, TP53, CBL, NPM1, JAK2, U2AF1, IDH1, KRAS, IDH2, FLT3, PTPN11, SETBP1, and BCOR. The average depth of sequencing was 10,000X. Differences in mutant allele frequency between two genes in the same sample were considered significant if they were >10%. A difference of 10% to 20% was considered mild, 20%-30% moderate, and >30% severe. A heat map reflecting these differences in mutant allele frequency was generated. Results: In this group of early MDS patients, 63 patients (66%) had more than one gene mutated and 38 (40%) had a significant (>10%) difference in allele frequency. The median number of genes mutated was 2 (range 1 to 5). Difference in mutant allele frequency was severe in 15 patients (16%), intermediate in 15 patients (16%), and mild in 13 patients (14%). TET2 was the most commonly mutated gene (43 patients, 45%) and was rarely the sole mutation with most cases exhibiting a mutation in a second gene (39 patients, 91%). The mutant allele burden was highest in TET2 in 26 of these 39 patients (67%), reflecting early event in the tumorigenic process. Of the 13 cases with TET2 mutation and allele burden less than the companion gene, 6 had a mutation in SF3B1, 3 had significant cytogenetic abnormalities (monosomy 5, del(7q), and trisomy 8), 2 had a mutation in SRSF2, 1 had a mutation in ZRSR2 and 1 had a mutation in ASXL1, which suggests that these abnormalities might be the initiating event. A second TET mutation (biallelic mutation) was detected in 16 of the 39 patients. SF3B1 was the most common gene having a solitary mutation (10% of all patients), although mutation in SF3B1 was detected in 27 patients (26% of all patients). All solitary SF3B1 mutations were associated with normal karyotypes, except for one patient with del(11q). JAK2 was mutated with SF3B1 in two cases diagnosed as RARS-T (refractory anemia with ring sideroblasts and thrombocytosis). In one case, the JAK2 and SF3B1 mutation allele frequencies were similar, but in the other, the JAK2 mutant allele frequency was 23% higher, suggesting that a myeloproliferative neoplasm was the initiating process. ASXL1 was mutated in 14 cases, 13 of which had additional mutations. DNMT3A gene was mutated in 18 cases, 5 of which were solitary; two of these five showed cytogenetic abnormalities. TP53 was mutated in 13 cases, but except for one case, all had either mutation in another gene or a cytogenetic abnormality. Conclusion: These data suggest that in patients with clinically confirmed early MDS, TET2 mutations are most likely the initiating oncogenic event, but mutations in other genes or cytogenetic abnormalities most likely lead to clinically confirmed MDS. In contrast, patients with SF3B1 mutation can have clinical disease without additional mutations. Our data suggest that SRSF2, ZRSR2, and ASXL1 may initiate mutagenesis in patients with MDS. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract Intraclonal subpopulations of circulating chronic lymphocytic leukemia (CLL) cells with different proliferative histories and reciprocal surface expression of CXCR4 and CD5 have been observed in the peripheral blood of CLL patients and named proliferative (PF), intermediate (IF), and resting (RF) cellular fractions. Here, we found that these intraclonal circulating fractions share persistent DNA methylation signatures largely associated with the mutation status of the immunoglobulin heavy chain locus (IGHV) and their origins from distinct stages of differentiation of antigen-experienced B cells. Increased leukemic birth rate, however, showed a very limited impact on DNA methylation of circulating CLL fractions independent of IGHV mutation status. Additionally, DNA methylation heterogeneity increased as leukemic cells advanced from PF to RF in the peripheral blood. This frequently co-occurred with heterochromatin hypomethylation and hypermethylation of Polycomb-repressed regions in the PF, suggesting accumulation of longevity-associated epigenetic features in recently born cells. On the other hand, transcriptional differences between paired intraclonal fractions confirmed their proliferative experience and further supported a linear advancement from PF to RF in the peripheral blood. Several of these differentially expressed genes showed unique associations with clinical outcome not evident in the bulk clone, supporting the pathological and therapeutic relevance of studying intraclonal CLL fractions. We conclude that independent methylation and transcriptional landscapes reflect both preexisting cell-of-origin fingerprints and more recently acquired hallmarks associated with the life cycle of circulating CLL cells.

Understanding the biology of Multiple Myeloma (MM) is of primary importance in the struggle to achieve a cure for this yet incurable neoplasm. A better knowledge of the mechanism underlying the development of MM can guide us in the development of new treatment strategies. Studies both on solid and haematological tumours have shown that cancer comprises a collection of related but subtly different clones, a feature that has been termed “intra-clonal heterogeneity”. This intra-clonal heterogeneity is likely, from a “Darwinian” natural selection perspective, to be the essential substrate for cancer evolution, disease progression and relapse. In this context the critical mechanism for tumour progression is competition between individual clones (and cancer stem cells) for the same microenvironmental “niche”, combined with the process of adaptation and natural selection. The Darwinian behavioural characteristics of cancer stem cells are applicable to MM. The knowledge that intra-clonal heterogeneity is an important feature of tumours’ biology has changed our way to addressing cancer, now considered as a composite mixture of clones and not as a linear evolving disease. In this variable therapeutic landscape it is important for clinicians and researchers to consider the impact that evolutionary biology and intra-clonal heterogeneity have on the treatment of myeloma and the emergence of treatment resistance. It is clear that if we want to effectively cure myeloma it is of primarily importance to understand disease biology and evolution. Only by doing so will we be able to effectively use all of the new tools we have at our disposal to cure myeloma and to use treatment in the most effective way possible. The aim of the present research project was to investigate at different levels the presence of intra-clonal heterogeneity in MM patients, and to evaluate the impact of treatment on clonal evolution and on patients’ outcomes.

Multiple myeloma (MM) is a largely incurable haematological malignancy characterised by the aberrant proliferation of malignant plasma cells (PCs) in the bone marrow (BM). Next generation sequencing (NGS) studies have shown that MM patients display complex mutational landscapes involving intraclonal genetic heterogeneity. While intraclonal heterogeneity is now an established feature of MM, the genomic changes and tumour evolution associated with the transformation from the asymptomatic disease stages of Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smouldering Multiple Myeloma (SMM) to MM remains unknown. This thesis presents a unique assessment of the genomic architecture and subclonal evolution associated with the natural history of disease transformation, with the analyses of a rare cohort of paired BM samples from patients when first diagnosed with MGUS or SMM, who later went on to develop MM (n = 10). Whole exome sequencing (WES) and bioinformatic analyses identified that clonal heterogeneity was present at the asymptomatic MGUS/SMM stages of disease, with a changing spectrum of acquired mutations associated with transition to MM. Subclonality was observed at MGUS/SMM, with the presence of between 5 to 11 subclones. The progression to MM was characterised by a prevailing model of subclonal evolution defined by clonal stability, where the transformed PC subclones of MM were already present at the MGUS/SMM stage. RNA sequencing (RNAseq) revealed that the patterns of expressed genes at MGUS/SMM to MM were found to be relatively homogeneous. Moreover, RNAseq revealed that mutant genes identified by WES were generally not expressed, expressed at low levels, with most genes showing wild-type expression. Analysis of the methylome was carried out using whole genome bisulphite sequencing (WGBS). Significant hypomethylation was observed in PCs recovered at all disease stages (MGUS, SMM and MM) compared to normal PCs. Interestingly, the degree of hypomethylation observed at MGUS was maintained with progression to SMM and MM stages. In addition, the phenomenon of RNA editing in SP140, a recurrently mutated gene in human MM patients, was investigated in the 5TGM1 MM PC line. A high impact C>T (ie. U) RNA editing change was identified in exon 2 of Sp140, resulting in an early STOP codon, which was hypothesised to result in the formation of truncated Sp140 protein that may contribute to MM pathogenesis. In mouse cell lines, Sp140 RNA editing was not restricted to the 5TGM1 cell line, but editing was not observed in any human MM PC lines. CRISPR-Cas9 mediated mutation of the mouse Apobec1 and Apobec3 genes, showed that neither of these cytidine deaminases were responsible for this RNA editing phenomenon. These studies show that MGUS/SMM patients, that progress in a short time frame, appear to be sufficiently genetically complex to be on the threshold of transformation to MM. Furthermore, the intrinsic genomic complexity of MM is present at the asymptomatic stages of disease (MGUS and SMM), suggesting that extrinsic factors from the tumour microenvironment play an important role in mediating progression. Indeed, these studies suggest that early intervention at MGUS/SMM may be possible to prevent progression and result in durable cure for patients.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2018