Mutational characterisation in multiple myeloma (MM) currently relies on bone marrow (BM) biopsy, which fails to capture the putative spatial and genetic heterogeneity of this multifocal disease. Analysis of plasma (PL)-derived circulating free tumour DNA (ctDNA) as an adjunct to BM biopsy, for mutational characterisation and tracking disease progression, was evaluated. Paired BM MM cell DNA and ctDNA from 33 relapsed/refractory (RR) and 15 newly diagnosed (ND) patients were analysed for KRAS, NRAS, BRAF and TP53 mutations using the OnTarget Mutation Detection (OMD) platform. OMD detected 128 mutations (PL=31, BM=59, both=38) indicating the presence of PL mutations (54%). A higher frequency of PL-only mutations was detected in RR patients than ND (27.2% vs 6.6%, respectively), authenticating the existence of spatial and genetic heterogeneity in advanced disease. Activating RAS mutations were more highly prevalent than previously described with 69% harboring at least one RAS mutation. Sequential ctDNA quantitation with droplet digital PCR through longitudinal PL tracking of specific clones in seven patients demonstrated changes in fractional abundance of certain clones reflective of the disease status. We conclude that ctDNA analysis as an adjunct to BM biopsy represents a noninvasive and holistic strategy for improved mutational characterisation and therapeutic monitoring of MM.
We demonstrate a unique parameter for biomolecule separation that results from the nonlinear response of long, charged polymers to electrophoretic fields and apply it to extraction and concentration of nucleic acids from samples that perform poorly under conventional methods. Our method is based on superposition of synchronous, time-varying electrophoretic fields, which can generate net drift of charged molecules even when the time-averaged molecule displacement generated by each field individually is zero. Such drift can only occur for molecules, such as DNA, whose motive response to electrophoretic fields is nonlinear. Consequently, we are able to concentrate DNA while rejecting high concentrations of contaminants. We demonstrate one application of this method by extracting DNA from challenging samples originating in the Athabasca oil sands.concentration ͉ DNA ͉ electrophoresis ͉ purification ͉ SCODA M ethods for separating different molecular species are the cornerstone of analytic techniques in molecular biology. Of these, nucleic acid extraction from complex sources is a molecular separation problem of great importance to current challenges in genomics, metagenomics, forensics, biodefense, food and water safety, and clinical molecular diagnostics.The ubiquitous column-and bead-based nucleic acid extraction methods that dominate the field of nucleic acid extraction use selective chemical affinity between nucleic acids and ion exchange or similar resins and beads to capture target molecules. Although these methods often involve mechanical steps including filtration and centrifugation, they are relatively inexpensive and work well in a variety of samples. Their inadequacy lies in the fact that the separations are based on chemical affinity and therefore perform poorly in the presence of contaminant molecules that either have similar chemical properties to nucleic acids or foul the capture matrix (1). Precipitation methods are often used after column or bead extractions to remove contaminants that carry through; however, this further reduces yield of the methods, particularly in cases with low target concentrations.This weakness of existing methods is a critical problem for DNA extraction from environmental samples. For example, humic acids, a family of contaminants abundant in soil, coextract with DNA in phenol-based separations owing to their solubility in the aqueous phase (1) and partly carry through column-and bead-based methods. The situation worsens with a low starting concentration of nucleic acids because the dual challenge must then be faced of concentrating few nucleic acids while rejecting large amounts of contaminants. A universal, simple, and highly selective method to concentrate DNA from contaminated and low-abundance sources would be very desirable.We have found a unique solution to this problem in the physics of electrophoresis. It has long been known that nucleic acid molecules, because of their exceptionally long contour lengths and high linear charge density, exhibit complex electrophoretic...
Forensic crime scene sample analysis, by its nature, often deals with samples in which there are low amounts of nucleic acids, on substrates that often lead to inhibition of subsequent enzymatic reactions such as PCR amplification for STR profiling. Common substrates include denim from blue jeans, which yields indigo dye as a PCR inhibitor, and soil, which yields humic substances as inhibitors. These inhibitors frequently co-extract with nucleic acids in standard column or bead-based preps, leading to frequent failure of STR profiling. We present a novel instrument for DNA purification of forensic samples that is capable of highly effective concentration of nucleic acids from soil particulates, fabric, and other complex samples including solid components. The novel concentration process, known as SCODA, is inherently selective for long charged polymers such as DNA, and therefore is able to effectively reject known contaminants. We present an automated sample preparation instrument based on this process, and preliminary results based on mock forensic samples.
In the field of molecular analysis of cancer, there exists a need for a clinical device that can automate protocols for immunohistochemical and in situ hybridization diagnostic staining on tissue microarrays. The Tissue Microarray Antibody Spotter (TMAS) has been developed to provide fundamental improvements over current histological staining techniques by enabling precision application of reagents to individual biopsies within a tissue microarray. This allows for multiplexed reactions on a single slide and promises to significantly reduce costs associated with immunohistochemistry and in situ hybridization based assays. Additionally, because TMAS allows for testing of different biomarkers on each element of a tissue array, a complete cancer profile can be obtained from a single TMA slide. Ultimately this may lead to costeffective, faster and more accurate diagnosis of the patient.
Background: Multiple myeloma (MM) currently relies on bone marrow (BM) biopsy for mutational characterisation, which does not capture the putative spatial and genetic heterogeneity of this multi-focal disease. Circulating cell-free tumour DNA (ctDNA) is a powerful non-invasive biomarker, which is being utilised to monitor tumor dynamics in a number of cancers. In this study, analysis of peripheral blood plasma (PL) - derived ctDNA and contemporaneously sourced BM MM cells was undertaken to determine if ctDNA can be utilised as an adjunct to BM biopsy for mutational characterisation and non-invasive therapeutic monitoring of disease progression. Methods: Blood (30ml) from MM patients was collected into Streck Cell-Free DNA BCT tubes and DNA extracted using the QIAamp circulating nucleic acid kit (Qiagen). BM aspirates from MM patients were CD138 enriched and subject to DNA extraction (Qiagen). Paired BM MM DNA and PL samples from 54 patients (New Diagnosis (ND) = 17; Relapsed/ Refractory (RR) = 35 and monoclonal gammopathy of undetermined significance (MGUS) =2] were analysed in the high-sensitive OnTargetTM mutation detection platform (OMD, Boreal Genomics) that included 96-mutations in the KRAS, NRAS, CTNNB1, EGFR, PIK3CA, TP53, FOXL2, GNAS and BRAF genes. OMD findings were validated with ddPCR (Biorad QX200 droplet digital PCR system). Sequential ctDNA quantitation with ddPCR through longitudinal PL tracking of specific clones in two patients for the monitoring of disease was also performed. Results: OMD detected a total of 141 mutations (BM and PL=42; BM-only=63 and PL only=36) with the presence of mutations in the PL (55.3%) authenticating the existence of ctDNA harbouring mutations. The detection of PL-only mutations (25.5%) signified the existence of mutant clones predominantly or exclusively, distant to the BM biopsy site. RR patients had a higher number of PL-only mutations compared to ND (34 vs 2 mutations), with none detected in MGUS, denoting that spatial and genetic heterogeneity is more evident in patients with advanced disease. Activating RAS mutations were highly prevalent in 34/54 patients (63%) harboring at least one RAS mutation. Specifically, KRAS mutations were found in 26/54 patients (48%), with 17/35 RR (48.5%), 9/17 ND (53%) and 1/2 MGUS patients harboring at least 1 KRAS mutation, indicating that mutations in this gene occur early in MM pathogenesis. NRAS mutations were found to be present at a higher frequency in RR patients compared to ND patients (45.7% vs 35.2%). BRAF mutations were equally distributed amongst RR and ND patients, in contrast, TP53 mutations were found exclusively in RR patients. Additionally, in 2 RR patients, a total of 5 PIK3CA mutations (E542K, E545K, H1047R, C420R, E81K) were present, 4 of which were found in PL-only, denoting the presence of these mutations distant to the BM biopsy site. Two ND patients had 1 GNAS mutation each (R201H and R201C). Preliminary validation of the OMD findings was performed in 12 patients using ddPCR with 90.9% concordance between the two platforms. Furthermore, 4/13 mutations (30.7%) that were negative in OMD were detected by ddPCR, indicating a higher sensitivity for ddPCR. Sequential ddPCR of ctDNA for Patient #1 with advanced relapsed disease for previously identified TP53 R273H and KRAS G12D mutations showed that the fractional abundance of KRAS G12D rapidly increased coincident with relapse of the disease, while that of TP53 273H did not change significantly over time. For Patient #2, relapsed disease was associated with the reappearance of mutant KRAS G12V and KRAS G12D clones that had been present at diagnosis in the BM and the emergence of 2 new clones NRAS G13D and NRAS Q61K. Conclusions This study provides the groundwork for utilisation of PL-ctDNA as an adjunct to BM biopsy for comprehensive mutational characterisation and as a non-invasive biomarker for therapeutic monitoring in MM patients. Importantly, a higher frequency of RAS mutations and presence of mutations in PIK3CA and GNAS, which have not been previously reported in MM, provides evidence of a more complex mutational landscape in MM than previously shown with BM whole exome sequencing studies. Furthermore, sequential PL tracking of specific mutant clones in two patients enabled monitoring of tumor dynamics. In conclusion, incorporating PL ctDNA evaluation may represent a significant advance in attempts to personalize future MM treatment strategies. Disclosures Mai: Boreal Genomics: Employment. Walsh:Boreal Genomics: Employment. Broemeling:Boreal Genomics: Employment. Marziali:Boreal Genomics: Employment. Wiggin:Boreal Genomics: Employment.
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