Combination of Flow Cytometry and Functional Imaging for Monitoring of Residual Disease in Myeloma

Abstract

Introduction The iliac crest is the usual sampling site for minimal residual disease (MRD) monitoring in Multiple Myeloma (MM). However, the disease distribution in the bone marrow (BM) is often heterogeneous. Functional imaging can be used to complement MRD detection at a single site, thereby accounting for asymmetrically distributed disease. Diffusion weighted MRI with background suppression (DWIBS) is a novel functional imaging method that can detect disease in a higher proportion of newly diagnosed MM (NDMM) patients than 18F-fluorodeoxyglucose positron emission tomography (PET), as it is independent of the tumor metabolism. Yet, its performance for monitoring of residual disease has not been described. The aims of this study were 1) to compare DWIBS to PET for the detection of residual disease in patients achieving complete remission (CR), and 2) to test whether DWIBS and PET could complement MRD flow cytometry with a sensitivity of 1x10-5. To address these aims, we investigated 168 NDMM and 33 relapsed patients for whom DWIBS, PET, and MRD were available at the onset of CR during first-line and salvage therapy, respectively. Methods All patients signed written consent in accordance with the Declaration of Helsinki. Residual focal lesions (FLs) were defined as well delineated focal intensities above the surrounding BM background. For DWIBS FLs were considered if restriction could be confirmed on ADC maps. 8-color MRD flow cytometry with a limit of detection of 1x10-5 was available for 83 NDMM and all 33 salvage therapy patients. The Kaplan-Meier method was used for survival analyses. PFS time was measured from onset of CR to relapse or death from any cause or censored at the date of last contact. Paired-end whole exome sequencing of CD138-enriched MM cells was performed on an Illumina HiSeq 2500. Mutations were called from BWA aligned sequencing reads using MuTect. Subclonal reconstruction was done using SciClone. Results Compared to PET, DWIBS detected more CR patients with residual FLs (21% vs. 6%), and the concordance between PET and DWIBS was low. Only 6 of the DWIBS-positive patients also presented with FLs in PET. Yet, 5 patients had PET+/DWIBS- FLs, suggesting that the two techniques are complementary. Both, DWIBS+ and PET+ FLs negatively impacted PFS (p<0.05). For 83 patients MRD data were available. Combining MRD and imaging, residual disease was detectable in 53 patients (64%). The best outcome was seen for 30 double negative (MRD-/Imaging-) patients (3 events with a median follow-up of 3.6 years), the worst outcome was seen for 10 double positive (MRD+/Imaging+) patients (median PFS: 2.1 years). Only 4 of 86 patients were MRD-/Imaging+, indicating that residual FLs are rare in MRD-negative NDMM patients at a sensitivity of 1x10-5. A heterogeneous disease distribution is a common feature of late-stage patients. To test if this increased heterogeneity confounded MRD, we investigated a set of 33 heavily pretreated patients who achieved CR during salvage therapy. Combining MRD and imaging data, we detected residual disease in 25 patients (76%). Of note, the proportion of patients, who were MRD-negative but had residual FLs on functional imaging was significantly higher compared to NDMM (8/16 vs 4/34 patients, p=0.01). At the same time, 10 patients (30%) were MRD+ but Imaging-, supporting the idea that a combined MRD/Imaging approach can improve detection of residual disease and should be used in late-stage patients. To obtain insights in the underlying biology, we performed longitudinal multi-region sequencing of a subset of these CR patients. Our findings support the concept of persistence and progression of multiple spatially separated clones in the BM irrespective of being in an MRD-negative CR. Thereby, focal residual disease could be shown to contribute to relapse. Conclusion DWIBS is a promising tool for detection of residual disease and complements PET. The combination of MRD diagnostics and functional imaging improves prediction of outcome, with double-negativity and double positivity defining groups with excellent and dismal PFS, respectively. Prospective trials using this information to tailor therapy are warranted. From a biological perspective, this study highlights the confounding effects of spatial heterogeneity and limited dissemination of clones within the BM on MRD diagnostics. This may especially be true for patients achieving deep responses during salvage therapies. Disclosures Roy Choudhury: University of Arkansas for Medical Sciences: Employment, Research Funding. Epstein:University of Arkansas for Medical Sciences: Employment. Barlogie:International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; Millenium: Consultancy, Research Funding; Multiple Myeloma Research Foundation: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; Celgene: Consultancy, Research Funding; Dana Farber Cancer Institute: Other: travel stipend. Davies:Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Research Funding.

The Impact of Tumor Heterogeneity on Diagnostics and Novel Therapeutic Strategies in Multiple Myeloma

“…In the large FL, we identified a clone, which shared a “KRASGly12Val” with one of the baseline clones but presented with 65 new missense mutations that were not detectable at baseline despite deep multi-region sequencing. In the iliac crest sample, we found an additional resistant clone, which was also not detectable at baseline, and presented with another site-unique clonal KRAS mutation [ 49 ]. This observation highlights that even in MRD-negative patients, multiple spatially separated clones can survive.…”

“…Our group and others have shown that functional whole-body imaging can be used to monitor residual disease in MM and improve conventional MRD diagnostics [ 49 , 55 ]. In our study, 24% of first-line MM patients presented with residual FLs in DWIBS and/or PET-CT at the onset of CR, and these lesions were associated with short progression-free survival [ 49 ]. Although DWIBS is more sensitive than PET-CT in this context, the two techniques are complementary because some residual FLs are only detectable in PET-CT [ 49 ].…”

“…In our study, 24% of first-line MM patients presented with residual FLs in DWIBS and/or PET-CT at the onset of CR, and these lesions were associated with short progression-free survival [ 49 ]. Although DWIBS is more sensitive than PET-CT in this context, the two techniques are complementary because some residual FLs are only detectable in PET-CT [ 49 ]. Combining flow cytometry for MRD detection and functional imaging, we demonstrated that only 4 of 83 MRD-negative patients, who achieved CR during first-line treatment, still presented with residual FLs [ 49 ].…”

Challenges and opportunities in the assessment of measurable residual disease in multiple myeloma

“…This limitation can be, at least partially, mitigated by the concomitant use of functional imaging with fluoro-2-deoxyglucose positron emission tomography (FDG-PET). The presence of focal areas of FDG uptake postinitial therapy has been associated with higher risk of relapse (Zamagni et al, 2011;Moreau et al, 2017;Rasche et al, 2019), including among patients who were "MRD negative" in bone marrow assessment Rasche et al, 2019) with sensitivity of 10 À4 to 10 À5 . Therefore, the combination of undetectable tumour cells in marrow by NGS or NGF and lack of FDG uptake in marrow or extramedullary sites will identify patients with the best outcomes post-initial therapy (Rasche et al, 2019) and should be required for experimental approaches with therapy discontinuation.…”

“…The presence of focal areas of FDG uptake postinitial therapy has been associated with higher risk of relapse (Zamagni et al, 2011;Moreau et al, 2017;Rasche et al, 2019), including among patients who were "MRD negative" in bone marrow assessment Rasche et al, 2019) with sensitivity of 10 À4 to 10 À5 . Therefore, the combination of undetectable tumour cells in marrow by NGS or NGF and lack of FDG uptake in marrow or extramedullary sites will identify patients with the best outcomes post-initial therapy (Rasche et al, 2019) and should be required for experimental approaches with therapy discontinuation. Can be performed locally, few centres have implemented Sample shipped to central laboratory from any site Required sample 2 9 10 7 nucleated cells Up to 20 µg of DNA (~3 9 10 6 nucleated cells) Can be performed in stored samples No Yes Limit of detection 2 9 10 À6 (20 events) 6Á8 9 10 À7 Limit of quantification 5 9 10 À6 (50 events) 1Á8 9 10 À6 FDA cleared No Yes FDA, US Food and Drug Administration; MM, multiple myeloma; MRD, minimal residual disease…”