A genomic approach towards an understanding of clonal evolution and disease progression in Multiple Myeloma

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, 201

Low temperature features in the heat capacity of unary metals and intermetallics for the example of bulk aluminum and Al3_3Sc

We explore the competition and coupling of vibrational and electronic contributions to the heat capacity of Al and Al$_3$Sc at temperatures below 50 K combining experimental calorimetry with highly converged finite temperature density functional theory calculations. We find that semilocal exchange correlation functionals accurately describe the rich feature set observed for these temperatures, including electron-phonon coupling. Using different representations of the heat capacity, we are therefore able to identify and explain deviations from the Debye behaviour in the low-temperature limit and in the temperature regime 30 - 50 K as well as the reduction of these features due to the addition of Sc.Comment: 10 pages, 6 figures in total, paper submitted to Physical Review

Comparative Prospective Study Reporting Intraoperative Parameters, Pedicle Screw Perforation, and Radiation Exposure in Navigation-Guided versus Non-navigated Fluoroscopy-Assisted Minimal Invasive Transforaminal Lumbar Interbody Fusion

Study DesignProspective cohort study.PurposeTo compare intraoperative parameters, radiation exposure, and pedicle screw perforation rate in navigation-guided versus non-navigated fluoroscopy-assisted minimal invasive transforaminal lumbar interbody fusion (MIS TLIF).Overview of LiteratureThe poor reliability of fluoroscopy-guided instrumentation and growing concerns about radiation exposure have led to the development of navigation-guided instrumentation techniques in MIS TLIF. The literature evaluating the efficacy of navigation-guided MIS TLIF is scant.MethodsEighty-seven patients underwent navigation- or fluoroscopy-guided MIS TLIF for symptomatic lumbar/lumbosacral spondylolisthesis. Demographics, intraoperative parameters (surgical time, blood loss), and radiation exposure (sec/mGy/Gy.cm2 noted from C-arm for comparison only) were recorded. Computed tomography was performed in patients in the navigation and non-navigation groups at postoperative 12 months and reviewed by an independent observer to assess the accuracy of screw placement, perforation incidence, location, grade (Mirza), and critical versus non-critical neurological implications.ResultsTwenty-seven patients (male/female, 11/16; L4–L5/L5–S1, 9/18) were operated with navigation-guided MIS TLIF, whereas 60 (male/female, 25/35; L4–L5/L5–S1, 26/34) with conventional fluoroscopy-guided MIS TILF. The use of navigation resulted in reduced fluoroscopy usage (dose area product, 0.47 Gy.cm2 versus 2.93 Gy.cm2), radiation exposure (1.68 mGy versus 10.97 mGy), and fluoroscopy time (46.5 seconds versus 119.08 seconds), with p-values of <0.001. Furthermore, 96.29% (104/108) of pedicle screws in the navigation group were accurately placed (grade 0) (4 breaches, all grade I) compared with 91.67% (220/240) in the non-navigation group (20 breaches, 16 grade I+4 grade II; p=0.114). None of the breaches resulted in a corresponding neurological deficit or required revision.ConclusionsNavigation guidance in MIS TLIF reduced radiation exposure, but the perforation status was not statistically different than that for the fluoroscopy-based technique. Thus, navigation in nondeformity cases is useful for significantly reducing the radiation exposure, but its ability to reduce pedicle screw perforation in nondeformity cases remains to be proven

Minimally Invasive Microscope-Assisted Stand-Alone Transarticular Screw Fixation without Gallie Supplementation in the Management of Mobile Atlantoaxial Instability

Study Design Retrospective study. Purpose To evaluate the clinico-radiological efficacy of stand-alone minimally invasive transarticular screw (MIS-TAS) fixation without supplemental Gallie fixation in the management of mobile C1–C2 instability. Overview of Literature Data evaluating the efficacy and feasibility of MIS-TAS in the literature is scanty. Methods Patients with mobile atlantoaxial instability and >2 years follow-up were included and managed by stand-alone TAS fixation using the Magerl technique and morselized allograft without additional fixation. Patient demographics and intra-operative parameters were noted. Clinical parameters (Visual Analog Scale [VAS] and Oswestry Disability Index [ODI]), neurology (modified Japanese Orthopaedic Association [mJOA]), and radiological factors (anterior atlanto-dens interval and space available for cord) were evaluated pre and postoperatively. Computed tomography (CT) was performed in patients who did not show interspinous fusion on X-ray at 1 year, to verify intra-articular fusion. Statistical analysis was performed using IBM SPSS ver. 20.0 (IBM Corp., Armonk, NY, USA); the Student t-test and analysis of variance were used to assess statistical significance (p <0.05). Results A total of 82 consecutive cases (three males, one female; mean age, 36.26±5.78 years) were evaluated. In total, 163 TASs were placed. Significant improvement was noticed in clinical (mean preoperative VAS=7.2±2.19, postoperative VAS=3.3±1.12; mean preoperative ODI=78.3±4.83, postoperative ODI=34.05±3.26) and neurological features (mean preoperative mJOA=14.73±2.68, postoperative mJOA=17.5±2.21). Radiological evidence of fusion was noted in 97.5% cases at final follow-up. Seventeen patients were found to have no interspinous fusions upon X-rays, but CT revealed facet fusion in all patients except in two. Inadvertent vertebral artery injury was noted in three cases. Conclusions Stand-alone TAS fixation with morselized allograft provides excellent radiological and clinical outcomes. The addition of a supplementary tension band and structural graft are not essential. This provides the opportunity to avoid the complications associated with graft harvesting and wiring

Genetic subtypes of smoldering multiple myeloma are associated with distinct pathogenic phenotypes and clinical outcomes

Smoldering multiple myeloma (SMM) is a precursor condition of multiple myeloma (MM) with significant heterogeneity in disease progression. Existing clinical models of progression risk do not fully capture this heterogeneity. Here we integrate 42 genetic alterations from 214 SMM patients using unsupervised binary matrix factorization (BMF) clustering and identify six distinct genetic subtypes. These subtypes are differentially associated with established MM-related RNA signatures, oncogenic and immune transcriptional profiles, and evolving clinical biomarkers. Three genetic subtypes are associated with increased risk of progression to active MM in both the primary and validation cohorts, indicating they can be used to better predict high and low-risk patients within the currently used clinical risk stratification models

Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view about chromatin structure has emerged, including its interrelationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded novel mechanistic and evolutionary insights about the functional landscape of the human genome. Together, these studies are defining a path forward to pursue a more-comprehensive characterisation of human genome function