The Development and Pilot Testing of a New MR Imaging Protocol to Quantify Myeloma Disease Burden and Bone Loss

* Osteotronix' fineSA® (Fine Structural Analysis, FSA) technology extracts microstructural information from Magnetic Resonance Imaging (MRI) data sets, as a correlate of trabecular wall thickness, to indicate bone remodelling. The FSA metric has been shown to correlate tightly with gold standard bone density measurements in rats \[Evans et al, 2014\] and human cadaveric spine specimens \[Rafferty et al, 2016\]. * In this study, we had collected data during the novel MR protocol at both baseline and follow-up time points. However, we were unable to complete analysis of the FSA metrics, because of disruptions due to COVID-19, therefore the results have not been possible to report.

Time frame: At baseline

Population: \- In this study, we had collected data during the novel MR protocol at both baseline and follow-up time points. However, we were unable to complete analysis of the FSA metrics as the company we contracted to undertake this was dissolved, an alternative is not available and there is no remaining budget. Therefore the results will never be possible to report.

* This was intended as a novel end-point produced by OCMR scientists, in which high-resolution 3D imaging of the spine and pelvis are analysed for the extent of vertebral collapse. * Unfortunately, we were unable to collect data for the total spinal hole volume and total spine collapse volume at the point of novel magnetic resonance (MR) scan, due to technical challenges.

Time frame: At baseline

Population: \- Unfortunately, we were unable to collect data for the total spinal hole volume and total spine collapse volume at the point of novel MR scan, due to technical challenges.

* This was intended as a novel end-point produced by OCMR scientists, in which high-resolution 3D imaging of the spine and pelvis are analysed for lytic lesions (holes). * Unfortunately, we were unable to collect data for the total spinal hole volume and total spine collapse volume at the point of novel MR scan, due to technical challenges.

Time frame: At baseline

Population: \- Unfortunately, we were unable to collect data for the total spinal hole volume and total spine collapse volume at the point of novel MR scan, due to technical challenges.

Primary Objective 1: To assess whether the novel magnetic resonance (MR) protocol and exploratory bone biomarkers can improve quantification of tumour burden in patients with new or relapsed myeloma at baseline assessment, compared to paraprotein levels alone. This particular section analysed the correlation between the Apparent Diffusion Coefficient (ADC) measurements (from the Diffusion Weighted Magnetic Resonance Imaging (DW-MRI) component of the sequences) of lytic bone lesions, with standard clinical correlates of tumour burden (serum paraprotein, and serum paraprotein-associated immunoglobulin level). The measurement of ADC from DW-MRI is further described by Messiou et. al. \[1\] \[1\] Messiou, Christina, et al. Guidelines for acquisition, interpretation, and reporting of whole-body MRI in myeloma: myeloma response assessment and diagnosis system (MY-RADS). Radiology 291.1 (2019): 5-13.

Time frame: At baseline

Population: Subset of Group 1a and 1b, on active chemotherapy, with lytic bone lesions identified on baseline novel MR scan, amenable to ADC measurement, and a paired baseline serum paraprotein, or paraprotein-associated immunoglobulin, level available (where measurement was unsuccessful the number analysed is lower than the overall number analysed).~Group 1c, 2 and 3 participants did not have a lytic bone lesion identified on novel MR scan, and were therefore not included in the analysis.

Primary Objective 1: To assess whether the novel Magnetic Resonance (MR) protocol and exploratory bone biomarkers can improve quantification of tumour burden in patients with new or relapsed myeloma at baseline assessment, compared to paraprotein levels alone. This section examined correlation between baseline bone biomarkers and baseline serum paraprotein in a pooled cohort of patients from Groups 1 and 2, using Spearman's Rank Correlation Coefficients.

Time frame: At baseline

Population: Pooled cohort of patients from Groups 1 and 2 who had measured: i) available baseline serum paraprotein; ii) by baseline bone biomarkers.

Primary Objective 1: To assess whether the novel MR protocol and exploratory bone biomarkers can improve quantification of tumour burden in patients with new or relapsed myeloma at baseline assessment, compared to paraprotein levels alone. Participants' baseline novel MR scan was analysed by an expert radiologist, and pattern of disease was qualitatively classified using the MY-RADS (Myeloma Response Assessment and Diagnosis System) imaging recommendations, described in Figure 2 by Messiou et. al. \[1\]. This particular section analysed whether standard clinical correlate of tumour burden (serum paraprotein) differed by radiological pattern of disease (e.g., normal, focal, diffuse). \[1\] Messiou, Christina, et al. Guidelines for acquisition, interpretation, and reporting of whole-body MRI in myeloma: myeloma response assessment and diagnosis system (MY-RADS). Radiology 291.1 (2019): 5-13.

Time frame: At baseline

Population: Population is all participants who had a novel MR scan at baseline with: i) available MY-RADS pattern of disease assessment; ii) available baseline serum paraprotein.

Primary Outcome 2: To assess whether the novel magnetic resonance (MR) protocol and exploratory bone turnover markers can improve quantification of bone loss in patients with myeloma (new, relapsed, smouldering) and Monoclonal Gammopathy Of Uncertain Significance (MGUS) at baseline assessment, compared to Dual-energy X-ray Absorptiometry (DXA) and established bone turnover markers alone. In this particular section, in a pooled cohort of participants from Groups 1 and 2, Spearman's rank correlation coefficients were calculated between all baseline bone turnover biomarkers and: 1. Baseline novel MR Apparent Diffusion Coefficient (ADC) measurements; 2. Baseline DXA (Dual-energy X-ray Absorptiometry) BMD (Bone Mineral Density) at lumbar spine (L1-4); 2\) Baseline DXA (Dual-energy X-ray Absorptiometry) BMD (Bone Mineral Density) at femoral neck.

Time frame: At baseline

Population: Pooled population of participants from Groups 1 and 2. Where the numbers analysed are lower than the overall number of participants analysed, this reflects either failure of paired measurements for bone turnover marker and DXA and ADC, or that ADC measurements were not indicated because baseline novel MR scan did not show a lytic lesion.

Primary Outcome 2: To assess whether the novel magnetic resonance (MR) protocol and exploratory bone turnover markers can improve quantification of bone loss in patients with myeloma (new, relapsed, smouldering) and Monoclonal Gammopathy Of Uncertain Significance (MGUS) at baseline assessment, compared to Dual-energy X-ray Absorptiometry (DXA) and established bone turnover markers alone. In this particular section, Spearman's rank correlation coefficient was performed to assess correlations between all pairs of bone turnover markers, measured at baseline in a pooled cohort of participants from Groups 1 and 2: 1. P1NP (Procollagen Type 1 N-terminal Propeptide); 2. CTX-1 (Collagen Cross-Linked C-Telopeptide Type I); 3. ALP (Alkaline Phosphatase); 4. DKK1 (Dickkopf WNT Signaling Pathway Inhibitor 1); 5. Sclerostin; 6. Ratio of RANKL (Receptor Activator of Nuclear Factor Kappa-Β Ligand) to OPG (Osteoprotegerin).

Time frame: At baseline

Population: Pooled population of participants from Groups 1 and 2 who had bone biomarkers measured at baseline.

Primary Outcome 2: To assess whether the novel magnetic resonance (MR) protocol and exploratory bone turnover markers can improve quantification of bone loss in patients with myeloma (new, relapsed, smouldering) and Monoclonal Gammopathy Of Uncertain Significance (MGUS) at baseline assessment, compared to Dual-energy X-ray Absorptiometry (DXA) and established bone turnover markers alone. This particular section analysed the inter-group difference in baseline serum ALP (Alkaline Phosphatase) bone turnover marker levels, in patients from Groups 1a (new myeloma), 1b (relapsed myeloma), 1c (smouldering myeloma) and 2 (MGUS).

Time frame: At baseline

Population: All participants recruited at baseline from Groups 1 and 2 who had successful measurement of baseline serum ALP.

Primary Outcome 2: To assess whether the novel magnetic resonance (MR) protocol and exploratory bone turnover markers can improve quantification of bone loss in patients with myeloma (new, relapsed, smouldering) and Monoclonal Gammopathy Of Uncertain Significance (MGUS) at baseline assessment, compared to Dual-energy X-ray Absorptiometry (DXA) and established bone turnover markers alone. This particular section analysed the inter-group difference in baseline serum CTX-1 (Collagen Cross-Linked C-Telopeptide Type I) bone turnover marker levels, in patients from Groups 1a (new myeloma), 1b (relapsed myeloma), 1c (smouldering myeloma) and 2 (MGUS).

Time frame: At baseline

Population: All participants recruited at baseline from Groups 1 and 2 who had successful measurement of baseline serum CTX-1.

Primary Outcome 2: To assess whether the novel magnetic resonance (MR) protocol and exploratory bone turnover markers can improve quantification of bone loss in patients with myeloma (new, relapsed, smouldering) and Monoclonal Gammopathy Of Uncertain Significance (MGUS) at baseline assessment, compared to Dual-energy X-ray Absorptiometry (DXA) and established bone turnover markers alone. This particular section analysed the inter-group difference in baseline serum DKK1 (Dickkopf WNT Signaling Pathway Inhibitor 1) bone turnover marker levels, in patients from Groups 1a (new myeloma), 1b (relapsed myeloma), 1c (smouldering myeloma) and 2 (MGUS).

Time frame: At baseline

Population: All participants recruited at baseline from Groups 1 and 2 who had successful measurement of baseline serum DKK1.

Primary Outcome 2: To assess whether the novel magnetic resonance (MR) protocol and exploratory bone turnover markers can improve quantification of bone loss in patients with myeloma (new, relapsed, smouldering) and Monoclonal Gammopathy Of Uncertain Significance (MGUS) at baseline assessment, compared to Dual-energy X-ray Absorptiometry (DXA) and established bone turnover markers alone. This particular section analysed the inter-group difference in baseline serum P1NP (Procollagen Type 1 N-terminal Propeptide) bone turnover marker levels, in patients from Groups 1a (new myeloma), 1b (relapsed myeloma), 1c (smouldering myeloma) and 2 (MGUS).

Time frame: At baseline

Population: All participants recruited at baseline from Groups 1 and 2 who had successful measurement of baseline serum P1NP.

Primary Outcome 2: To assess whether the novel magnetic resonance (MR) protocol and exploratory bone turnover markers can improve quantification of bone loss in patients with myeloma (new, relapsed, smouldering) and Monoclonal Gammopathy Of Uncertain Significance (MGUS) at baseline assessment, compared to Dual-energy X-ray Absorptiometry (DXA) and established bone turnover markers alone. This particular section analysed the inter-group difference in baseline ratio between RANKL (Receptor Activator of Nuclear Factor Kappa-Β Ligand) and OPG (Osteoprotegerin) \[calculated as RANKL (pg/L) divided by OPG (pg/L)\] bone turnover marker levels, in patients from Groups 1a (new myeloma), 1b (relapsed myeloma), 1c (smouldering myeloma) and 2 (MGUS).

Time frame: At baseline

Population: All participants recruited at baseline from Groups 1 and 2 who had successful measurement of both baseline serum RANKL and baseline serum OPG.

Primary Outcome 2: To assess whether the novel magnetic resonance (MR) protocol and exploratory bone turnover markers can improve quantification of bone loss in patients with myeloma (new, relapsed, smouldering) and Monoclonal Gammopathy Of Uncertain Significance (MGUS) at baseline assessment, compared to Dual-energy X-ray Absorptiometry (DXA) and established bone turnover markers alone. This particular section analysed the inter-group difference in baseline serum sclerostin bone turnover marker levels, in patients from Groups 1a (new myeloma), 1b (relapsed myeloma), 1c (smouldering myeloma) and 2 (MGUS).

Time frame: At baseline

Population: All participants recruited at baseline from Groups 1 and 2 who had successful measurement of baseline serum sclerostin.

Secondary Objective 3: To assess how quality of life compares between groups and longitudinally in patients with myeloma, Monoclonal Gammopathy of Undetermined Significance (MGUS), and healthy volunteers. The EuroQol 5-Dimension (EQ-5D) assess the mobility, self-care, usual activities, pain/discomfort, anxiety and depression on a 5-point scale, in which a lower score represents better quality of life (1 = 'no problems', 5 = maximum problems, for each domain). The second part of the EQ-5D assess health on a scale where 100 is the best health and 0 is the worst health.

Time frame: At baseline and six months

Population: All participants from Groups 1, 2 and 3 were invited to complete EQ5D questionnaires at baseline and 6-month follow-up visits.~The total number of patients analysed for each EQ5D question varied slightly depending on how many responses were received from the questionnaires.

Secondary Objective 1: To assess whether the novel Magnetic Resonance (MR) protocol can improve detection of longitudinal changes in tumour burden in patients with new or relapsed myeloma during therapy, compared to the International Myeloma Working Group (IMWG) Response Group classification alone. This section compared two indicators of therapy response: 1. IMWG Response Group classification \[1\], based on % change in serum paraprotein 2. % change in Apparent Diffusion Coefficient (ADC) measurements in participants where there was a lytic bone lesion identified on both baseline and follow-up novel MR scan amenable to ADC measurement \[2\]. Ref: 1. https://www.myeloma.org/resource-library/international-myeloma-working-group-imwg-uniform-response-criteria-multiple 2. Messiou, Christina, et al. Guidelines for acquisition, interpretation, and reporting of whole-body MRI in myeloma: myeloma response assessment and diagnosis system (MY-RADS). Radiology 291.1 (2019): 5-13

Time frame: Comparison between baseline and follow-up at 6month

Population: Participants who had successfully measured paired serum paraprotein (amenable to IMWG Response Group classification) and novel MR scans (with a lytic bone lesion amenable to ADC measurement, with a successfully calculated % change in ADC) at baseline and 6-month follow-up.

Secondary Objective 1: To assess whether the novel Magnetic Resonance (MR) protocol can improve detection of longitudinal changes in tumour burden in patients with new or relapsed myeloma during therapy, compared to the International Myeloma Working Group (IMWG) Response Group classification alone. This section compared two indicators of therapy response: 1. % change in Apparent Diffusion Coefficient (ADC) measurements in participants where there was a lytic bone lesion identified on both baseline and follow-up novel MR scan amenable to ADC measurement \[1\]. 2. MY-RADS RAC (Myeloma Response Assessment and Diagnosis System Response Assessment Classification) based on expert radiologist interpretation of paired novel MR imaging, guided by Messiou et. al. criteria \[1\]. Ref: \[1\] Messiou, Christina, et al. Guidelines for acquisition, interpretation, and reporting of whole-body MRI in myeloma: myeloma response assessment and diagnosis system (MY-RADS). Radiology 291.1 (2019): 5-13

Time frame: Comparison between baseline and follow-up at 6 month

Population: Participants with new or relapsed myeloma who had successfully measured paired novel MR scans at baseline and 6-month follow-up, with a successfully graded MY-RADS RAC, and a lytic bone lesion also a lytic bone lesion amenable to longitudinal calculation of % change in ADC.

Secondary Objective 1: To assess whether the novel Magnetic Resonance (MR) protocol can improve detection of longitudinal changes in tumour burden in patients with new or relapsed myeloma during therapy, compared to the International Myeloma Working Group (IMWG) Response Group classification alone. This section compared two indicators of therapy response: 1. IMWG Response Group classification \[1\], based on % change in serum paraprotein 2. MY-RADS RAC (Myeloma Response Assessment and Diagnosis System Response Assessment Classification) based on expert radiologist interpretation of paired novel MR imaging, guided by Messiou et. al. criteria \[2\]. Ref: 1. https://www.myeloma.org/resource-library/international-myeloma-working-group-imwg-uniform-response-criteria-multiple 2. Messiou, Christina, et al. Guidelines for acquisition, interpretation, and reporting of whole-body MRI in myeloma: myeloma response assessment and diagnosis system (MY-RADS). Radiology 291.1 (2019): 5-13.

Time frame: Comparison between baseline and follow-up at 6 months.

Population: Participants from Group 1 who had successfully measured paired novel MR scans and serum paraproteins, between baseline and 6-month follow-up, and with a successfully graded MY-RADS RAC, and IMWG Response Group classifications.

Secondary Objective 2: To assess whether the novel Magnetic Resonance (MR) protocol and exploratory bone biomarkers can improve detection of longitudinal changes in bone loss in patients with myeloma (new, relapsed, smouldering) and Monoclonal Gammopathy of Undetermined Significance (MGUS) during therapy, compared to Dual-energy X-ray Absorptiometry (DXA) and established bone biomarkers alone. This section examined whether longitudinal change in bone turnover markers differed by chemotherapy responders vs non-responders. * The % change in bone biomarker measurements was expressed as a ratio of follow-up / baseline of paired measurements * Participants were classified by International Myeloma Working Group (IMWG) Response Group classification, as responder (partial response, very good partial response or complete response) or non-responder (stable, progressive or relapse).

Time frame: Comparison between baseline and follow-up at 6month

Population: Pooled cohort of participants from Groups 1 and 2 who had a IMWG response classification (responder vs non-responder) and paired serum bone turnover markers measured at baseline and follow-up.

Secondary Objective 2: To assess whether the novel Magnetic Resonance (MR) protocol and exploratory bone biomarkers can improve detection of longitudinal changes in bone loss in patients with myeloma (new, relapsed, smouldering) and Monoclonal Gammopathy of Undetermined Significance (MGUS) during therapy, compared to Dual-energy X-ray Absorptiometry (DXA) and established bone biomarkers alone. This particular section examined the correlation between longitudinal changes in bone turnover markers between one another (calculated as a ratio of follow-up measurement divided by baseline measurement). Spearman's rank correlation was performed for the longitudinal % change between different biomarkers, to assess the relationship between longitudinal changes in these measures.

Time frame: Comparison between baseline and follow-up at 6month

Population: Pooled population of participants from Groups 1 and 2 who had paired measurements of follow-up and baseline bone turnover markers.~For some rows, the total number of participants analysed are less than the overall number of participants analysed, due to assay failure of some bone biomarkers variably on individual samples.

Secondary Objective 2: To assess whether the novel Magnetic Resonance (MR) protocol and exploratory bone biomarkers can improve detection of longitudinal changes in bone loss in patients with myeloma (new, relapsed, smouldering) and Monoclonal Gammopathy of Undetermined Significance (MGUS) during therapy, compared to Dual-energy X-ray Absorptiometry (DXA) and established bone biomarkers alone. This section examined the correlation (using Spearman's Rank Correlation Coefficient) between longitudinal changes (expressed as a ratio of follow-up / baseline of paired measurements) in bone turnover markers and: 1. Longitudinal changes in DXA Bone Mineral Density (BMD) at lumbar spine (L1-4) and femoral neck; 2. Longitudinal changes in novel MR Apparent Diffusion Coefficient (ADC) measurements.

Time frame: Comparison between baseline and follow-up at 6month

Population: Participants from Groups 1 and 2 were analysed who had a paired bone turnover marker measurements available.~Where the number analysed for an outcome measure is lower than the overall number of participants analysed, this is due to failure of measurement of either DXA BMD or novel MR ADC, or because ADC measurement was not indicated due to lack of a lytic bone lesion identified on MR.

This questionnaire assesses the experience of the Novel Magnetic Resonance (MR) and Dual-energy X-ray Absorptiometry (DXA) scans. Answers were recorded on a 5 point Likert scale where the lower number represents a better outcome. Scale descriptors: Q1 (Overall Experience): 1 (Very comfortable)/ 2 (Comfortable)/ 3 (Neither comfortable or uncomfortable)/ 4 (Uncomfortable)/ 5 (Very uncomfortable) Q2 (Adverse Effects): 1 (YES) / 0 (NO) Q3 (Length of Time): 0 (Too short) / 1 (Just right) / 2 (Too long) Q4 (Pain/Discomfort): 1 (No Increase)/ 2 (Mild Increase)/ 3 (Moderate Increase)/ 4 (High Increase)/ 5 (Severe Increase) Q5 (Likely to Reparticipate): 0 (Extremely Unlikely) / 1 (Unlikely) / 2 (Neither Likely or Unlikely) / 3 (Likely) / 4 (Extremely Likely) Q6 (How similar to expectations): 1 (YES) / 0 (NO) Q7 (Comfort with Staff): 1 (YES) / 0 (NO)

Time frame: At baseline and six months

Population: Baseline: all participants from Groups 1,2,3 were invited for novel MR scan, and only participants from Groups 1 and 2 were invited for DXA Follow-up: only participants from Groups 1 and 2 were invited for a novel MR scan and also a DXA.

We first addressed an important practical limitation of the study design. Patients with new myeloma usually commenced chemotherapy imminently after their diagnosis, typically a few months before they were able to consent for the present study. Therefore, most patients had already started chemotherapy at the point of baseline novel Magnetic Resonance (MR) scan, and had received variable durations of chemotherapy at baseline and follow-up scans. We hypothesised that this may be a potential confounding factor, as more recent chemotherapy may reduce tumour cellularity and therefore increase diffusion \[and therefore Apparent Diffusion Coefficient (ADC) measurements\] at lytic bone lesions. In this particular section, we analysed the difference between baseline ADC measurements in patients with new and relapsed myeloma, grouped by time elapsed since chemotherapy (ongoing, recent or never chemotherapy).

Time frame: At baseline

Population: Analysis included patients from Groups 1a and 1b who had a novel MR scan at baseline, with a lytic bone lesion identified amenable to ADC measurement.

We first addressed an important practical limitation of the study design. Patients with new myeloma usually commenced chemotherapy imminently after their diagnosis, typically a few months before they were able to consent for the present study. Therefore, most patients had already started chemotherapy at the point of baseline novel Magnetic Resonance (MR) scan, and had received variable durations of chemotherapy at baseline and follow-up scans. We hypothesised that this may be a potential confounding factor, as more recent chemotherapy may reduce tumour cellularity and therefore increase diffusion \[and therefore Apparent Diffusion Coefficient (ADC) measurements\] at lytic bone lesions. In this particular section, we analysed the difference between follow-up ADC measurements in patients with new and relapsed myeloma, grouped by time elapsed since chemotherapy (ongoing, recent or never chemotherapy).

Time frame: At follow-up

Population: Analysis included patients from Groups 1a and 1b who had a novel MR scan at follow-up, with a lytic bone lesion identified amenable to ADC measurement.