Bone Marrow Transplantation (2015), 1–11 © 2015 Macmillan Publishers Limited All rights reserved 0268-3369/15 www.nature.com/bmt

REVIEW

Minimal residual disease testing after stem cell transplantation for multiple myeloma AM Sherrod1, P Hari2, CA Mosse3, RC Walker4 and RF Cornell1 Increased use of novel agents and autologous stem cell transplantation has led to a significant improvement in PFS and overall survival in patients with multiple myeloma. Despite improved treatment strategies, most patients eventually relapse due to persistent low levels of disease in the bone marrow. Increasingly sensitive methods to measure or detect such disease have been evaluated, including multi-parametric flow cytometry, PCR, next-generation sequencing and imaging modalities. The following literature review examines current methods for detecting and monitoring minimal or measurable residual disease (MRD) in the post-transplant setting. Improved methods for detecting MRD will refine the current definitions of remission and could guide treatment approaches. Bone Marrow Transplantation advance online publication, 20 July 2015; doi:10.1038/bmt.2015.164

INTRODUCTION Multiple myeloma (MM) is a clinically and genetically heterogeneous plasma cell disorder, accounting for more than 24 000 new cancer diagnoses and 11 000 deaths annually in the United States.1,2 Over the past few decades, overall survival (OS) has improved from an age-adjusted 5-year relative survival of 35.6% in 1998–2001 to 44% in 2006–2009.3 This is largely due to increased use of autologous stem cell transplantation (ASCT) and the introduction of novel agents, such as immunomodulatory agents (i.e., thalidomide, lenalinomide and pomalidomide) and proteasome inhibitors (i.e., bortezomib and carfilzomib). Updated International Myeloma Working Group (IMWG) uniform response criteria were published in 2006 to more accurately classify treatment responses in the modern era of myeloma therapies with proteasome inhibitors and immunomodulatory agents.4 Historically, attainment of CR in MM was uncommon with conventional therapies such as melphalan and prednisone (MP) or vincristine, doxorubicin, and dexamethasone (VAD). Although these regimens were active, treatment was often not sufficient to eliminate overt disease detectable by conventional biochemical and pathological techniques (i.e., protein electrophoresis, immune fixation and immunohistochemistry). In the current era, in the treatment-naïve patient, modern combination therapies (i.e., bortezomib, lenalidomide, and dexamethasone) yield overall response rates (ORR) of nearly 100% and CR rates of 30%–50%.5,6 There is extensive evidence that attainment of CR either in the pre- or post-ASCT period predicts improved PFS and OS.7–9 A stringent CR (sCR), a deeper response, is achievable in the current therapeutic era and should be the goal in a subset of patients with MM. Despite deep responses, most CRs are not sustained and the vast majority of patients eventually relapse. The timing of relapse can be quite heterogeneous with significant variations in PFS and

OS for this subgroup. Most relapses are attributed to persistent low levels of disease in the bone marrow (BM). This highlights the need for better methods to detect minimal or measurable residual disease (MRD) in those with traditional CR or sCR. Increasingly sensitive laboratory techniques to more readily detect residual disease in the BM have been evaluated. These include multiparametric flow cytometry (MFC), PCR and next-generation sequencing (NGS) (Figure 1). Advanced imaging modalities such magnetic resonance imaging (MRI) and positron emission tomography–computed tomography (PET/CT) have also been investigated to detect anatomical locations of persistent or residual MM. Here we review the literature describing the current methods for detecting and monitoring MRD. These technologies have implications following consolidative ASCT when the probability of achieving CR and MRD negativity is the highest. In order to better define responses and treatment approaches, MRD techniques must be validated and further compared for reliability and applicability to practice. CURRENT IMWG CRITERIA: GOING BEYOND TRADITIONAL CR The definition of CR has evolved over time as therapies have improved. Before the advent of ASCT, a rare CR was defined as a > 75% reduction in myeloma paraprotein. With the introduction of high-dose chemotherapy and ASCT, the definition of CR required a BM study with o5% plasma cells, clonality unspecified, as well as the complete absence of serum and urine paraprotein, as assessed by immunofixation (IFE). IFE has a sensitivity to detect 100–150 mg/L of protein in the serum or urine, and, together with serum-free light chains (SFLC), can identify the vast majority of patients with a secretory plasma cell neoplasia.10–13 In 2006, the IMWG defined sCR as a deeper response.4 This response level

1 Division of Hematology and Oncology, Department of Internal Medicine, Vanderbilt University Medical Center, Nashville, TN, USA; 2Division of Hematology and Oncology, Department of Internal Medicine, Medical College of Wisconsin, Milwaukee, WI, USA; 3Department of Pathology and Laboratory Medicine, Vanderbilt University Medical Center, Nashville, TN, USA and 4Department of Clinical Radiology and Radiological Sciences, Vanderbilt University Medical Center, Nashville, TN, USA. Correspondence: Dr RF Cornell, Division of Hematology and Oncology, Department of Internal Medicine, Vanderbilt University Medical Center, 2220 Pierce Avenue, 777 PRB, 1301 Medical Center Drive, Nashville, TN 37232-6307, USA. E-mail: [email protected] Received 8 March 2015; revised 28 May 2015; accepted 2 June 2015

Minimal residual disease testing AM Sherrod et al

2 1012

At diagnosis

150-500mg/L

CR

1.2 mg/L (k) 1.7 mg/L (I)

sCR

10-4 to -5

MFC

10-5 to -6

ASO-PCR

10-6

NGS

Number of MM cells

Figure 1.

Minimal residual disease modality and sensitivity of detection.

requires meeting standard CR parameters in addition to normalization of the SFLC ratio (SFLCR) and the absence of clonal plasma cells in the BM by immunohistochemistry or two- to four-color flow cytometry. Sensitivity for this IFE assay is 1.2 mg/L for κ and 1.7 mg/L for λ.14 NOVEL BIOCHEMICAL TECHNIQUES FOR SEROLOGICAL MONITORING OF MM Serum-free light chains SFLC immunoassays with calculated SFLCR are widely used for screening, detection and monitoring of plasma cell gammopathies. Compared with absolute FLC concentration, the use of the SFLCR is a more sensitive marker of monoclonal FLC production, because the nonclonal, uninvolved FLC is incorporated into the calculation. Normalization of the SFLCR has been associated with superior outcomes following treatment.6 Several studies have compared IFE and SFLC assays in the detection of monoclonal gammopathies. More than 50% of patients with abnormal proteins detected by IFE have normal SFLC values; therefore, IFE is the preferred diagnostic test.15,16 Kapoor et al.6 evaluated the prognostic impact of SFLCR normalization in 443 patients following ASCT. Median time to progression (TTP) for patients achieving sCR was significantly longer compared with conventional CR or near CR (50 vs 20 vs 19 months, respectively). Median OS for patients who achieved sCR (n = 144) was significantly longer compared with conventional CR (n = 37) or near CR (n = 91) (not reached (NR) vs 81 vs 60 months, respectively, Po0.001). By multivariable analysis, post-ASCT sCR was an independent prognostic factor for survival (hazard ratio (HR): 0.44, 95% confidence interval (CI): 0.25–0.80, P = 0.008).6 In addition, suppressed levels of the clonal or involved FLC (iFLC) below the level of uninvolved FLC is associated with a better prognosis. Smeltzer et al.17 evaluated FLC in 697 patients following ASCT. Patients were considered to have ‘suppressed’ FLC if the iFLC fell below the value of the uninvolved FLC within 12 months of ASCT. Median TTP was 36 vs 19 months (P o0.001), and median OS was 6.5 vs 4.7 years (P = 0.001), favoring those with a suppressed FLC. Duration of FLC suppression was significantly associated with superior outcomes. These observed improvements in clinical outcomes were independent of CR attainment.17 Cornell et al.18 reported similar findings when they evaluated FLC in 50 patients following allogeneic stem cell transplantation. Patients with sustained iFLC suppression had improved OS compared with those that lacked evidence of iFLC suppression at 1-year post allogeneic stem cell transplantation (P = 0.03). By multivariate analysis, lack of iFLC suppression at 1-year post Bone Marrow Transplantation (2015), 1 – 11

allogeneic stem cell transplantation was associated with significantly increased mortality (HR: 7.01, 95% CI: 1.21–40.8, P = 0.03). Patients with iFLC recovery before 1 year had increased relapse risk compared with those patients that exhibited sustained iFLC suppression (HR: 10.4, 95% CI: 2.95–36.6, P o 0.001).18 FLC assay has limited utility in the setting of renal insufficiency. The κ-light chains are produced at double the rate of the λ-light chain. However, renal clearance of the λ-light chain is slower because of its dimeric structure. Therefore, in those patients with normal renal function, the concentration of κ-light chain is reduced compared with the λ-light chain, with a median κ/λ ratio of 0.58. In the setting of renal insufficiency, the reticuloendothelial system is increasingly used for FLC metabolism. As the reticuloendothelial system is not influenced by molecular weight, the half-lives of κ and λ are nearly equivalent. This results in a relative increase in κ-light chain concentration and abnormal ratio independent of their rate of production.19 In the setting of chronic inflammation, polyclonal immunoglobulins and FLC are elevated, resulting in abnormal FLC ratios and complicating the interpretation of disease response. It is also unclear whether the FLC ratio and difference in light chains (ΔFLC), which is a surrogate of oligoclonal reconstitution, are the most predictive measures of prognosis. Oligoclonal bands. In the setting of MM, oligoclonal bands are defined as the appearance of a serum monoclonal protein different from that observed at diagnosis and are thought to be secondary to a robust immune response. There is evidence to indicate that the emergence of oligoclonal bands following treatment with novel therapies and ASCT is associated with improved outcome. Although initially described as transient, there is growing evidence that an oligoclonal response can last for years.20 Oligoclonal bands were first reported in 1998, when Zent et al.21 found that 10% of patients had oligoclonal bands with isotype switch following ASCT. Patients with oligoclonal reconstitution and electrophoretic bands had significantly higher CR rates (67% vs 37%, P = 0.001), event-free survival (P = 0.004) and OS (P o 0.001).21 de Larrea et al.22 reported that in a study of 34 patients who reached CR following ASCT, 41% of patients had oligoclonal bands. Interestingly, 32.4% of these patients had an abnormal SFLCR. This calls into question the prognostic significance of the SFLCR in the current IMWG response criteria, as it does not distinguish between patients with and without oligoclonal bands, who are negative for their original protein.22 © 2015 Macmillan Publishers Limited

Minimal residual disease testing AM Sherrod et al

3 Table 1.

28

Antigens used in detection of aberrant plasma cells

Antigen

Normal expression profile (percentage expression on normal plasma cells)

Abnormal expression profile

Percentage of myeloma cases with abnormal expression

CD19 CD56 CD117 CD20 CD28 CD27 CD81 CD200

Positive (470%) Negative (o 15%) Negative (0%) Negative (0%) Negative/weak ( o15%) Strongly positive (100%) Positive (10%) Weakly positive

Negative Strongly positive Positive Positive Strongly positive Weak or negative Weak or negative Strongly positive

95% 75% 30% 30% 15%–45% 40%–50% Not published Not published

Heavy chain/light chain (HevyLite immunoassay) The added utility of the SFLCR has prompted interest in the analysis of κ/λ ratios for intact immunoglobulin (Ig) subsets (IgGκ, IgGλ, IgAκ and IgAλ heavy chain/light chain (HLC) immunoassays). HLC immunoassays produce ratios of tumor-produced (involved) Ig to polyclonal (uninvolved) Ig concentrations (for example, IgGκ/ IgGλ). Preliminary evaluations suggest that HLC κ/λ ratios are useful for both diagnosis and disease monitoring in MM. Of particular interest is the ability of the HLC assays to quantify suppression of the uninvolved polyclonal Ig of the same isotype as the tumor. Tovar et al.23 investigated the prognostic impact of serum HLC in 37 myeloma patients in CR following ASCT. Increased IgAκ/IgAλ and IgMκ/IgMλ ratios of the uninvolved isotype in patients with CR were associated with longer PFS (P = 0.006 and 0.01, respectively), with a statistical trend toward OS in the IgAκ/IgAλ group (P = 0.068). Bradwell et al.24 examined the prognostic significance of HLC subsets compared with monoclonal protein quantification by SPEP, SFLC and cytogenetic markers in 339 patients. Abnormal IgGκ/IgGλ and IgAκ/IgAλ ratios present in the respective tumor isotypes at clinical presentation were predictive of shorter PFS (HR: 1.9, P o0.001), predominantly owing to the suppression of the uninvolved (polyclonal) Ig of the same isotype as the tumor (HR: 1.8, P = 0.002). HLC ratios were independently prognostic (P = 0.001), suggesting that HLC ratios may have a role in clinical management of MM.24 Overall, a relatively higher HLC ratio of the uninvolved immunoglobulin is predictive of significantly longer PFS and OS in patients with MM in CR. Importantly, HLC ratios provide a composite measure of both tumor immunoglobulin production and immunoparesis rather than solely a measure of the malignant clone and MRD. This parameter is also a surrogate marker of robust immune recovery following ASCT. However, this assay is ineffective for determining the disease status of light chain-only secretors. Further studies investigating potential role of HLC measurement in MM are anticipated. PROS AND CONS FOR NOVEL BIOCHEMICAL TECHNIQUES One advantage of the aforementioned biochemical techniques is their ease of applicability. These tests are performed on the serum or urine and require neither an invasive procedure, such as BM biopsy, nor advanced imaging as with MRI or PET/CT scan. Results are rapid and highly reproducible with relatively low expense. These tests can be applied to any patient with secretory disease, including those with extramedullary myeloma, and reflect global plasma cell secretory activity in the patient. The SFLC assay is particularly useful in patients with light-chain myeloma or oligosecretory MM for disease monitoring. These tests may mitigate the need for serial BM biopsies in patients with myeloma. Concomitantly, they reveal the quality of immune reconstitution, in particular after ASCT. Furthermore, the SFLC assay can be performed rapidly, owing to automation, and the interpretation of results remains objective. © 2015 Macmillan Publishers Limited

However, these tests are not without some limitations. Test results can, in some cases, be a reflection of treatment-related immunosuppression rather than decreased disease burden. They are not applicable in patients with non-secretory disease. In many cases of advanced relapsed myeloma, the plasma cell clone becomes non-secretory. In such cases, serologic disease monitoring is challenging. Without adequate clinical and imaging follow-up, complacency can occur. NOVEL METHODS TO MEASURE MRD Although multiple studies have shown that attainment of CR either pre- or post ASCT predicts for prolonged PFS and OS, caution should be exercised in the interpretation of these findings. It is still unclear whether the benefits to those in CR are due to disease biology, innate patient characteristics, treatment effect or a combination of these factors. Notably, a subset of patients who are not in CR and with persistent residual paraprotein burden following ASCT may maintain long remissions without upgrading their response to CR. In addition, a correlation between depth of response and clinical outcome is not always seen.25,26 It is apparent, however, that attaining a CR state is extremely important for those with higher risk myeloma, as determined by cytogenetics, who are at risk of early progression after ASCT. Finally, the relationship between depth of response and survival offers an excellent surrogate endpoint with which to evaluate and compare therapeutic strategies.27 In order to further define this relationship, newer MRD techniques have been developed. Multi-parametric flow cytometry MFC is a quantitative MRD technique that allows for the discrimination between myelomatous plasma cells and normal plasma cells in BM samples based on differential antigen expression (Table 1).28 This technique is applicable in 90% of patients and the sensitivity is sufficient to detect 1 atypical plasma cell in 10 000 to 100 000 total plasma cells (10 − 4 to 10 − 5) in the BM.28–31 Antibody panel design is an essential and difficult part of MFC testing for MRD in patients with MM. Identification of appropriate antigens, panel testing for optimal monoclonal antibody and fluorochrome combinations, and validation in clinical trials must take place before a standard panel can be established. Several six-color and eight-color panels have been explored. These panels have been validated using the expression of CD27, CD56, CD 81 and CD117 in combination with CD19, CD38, CD45 and CD138.32,33 The European Myeloma network and International Clinical Cytometry Society and European Society for Clinical Cell Analysis Myeloma MRD Consensus Committee recommend that laboratories adopt one of these validated panels for use in MRD testing33–35 (Tables 2 and 3). Multiple studies using MFC have shown effective outcome discrimination using this MRD technique (Table 4). Two large prospective studies showed an OS benefit for MRD-negative Bone Marrow Transplantation (2015), 1 – 11

Minimal residual disease testing AM Sherrod et al

4

(MRD − ) patients using MFC. Paiva et al.36 analyzed 295 MM patients treated with induction therapy followed by ASCT. MRD − status post ASCT predicted significantly superior PFS (71 vs 37 months, Po 0.001) and OS (NR vs 89 months, P = 0.002) when compared with MRD-positive (MRD+) patients. In the subgroup of patients who achieved CR, MRD − predicted improved 5-year PFS (71 vs 37 months, P o0.01) and OS (87 vs 59%, P = 0.09). When the prognostic importance of MRD − pre-ASCT was assessed, MRD − was associated with significantly improved PFS (P o 0.001), with a Table 2.

Recommended six-color flow cytometry panel

Fluorophore

Target cell-surface marker in tube 1

Target cell-surface marker in tube 2a

FITC PE PerCP-Cy5.5 PE-Cy7 APC APC-Cy7

CD27 CD81 CD19 CD38 CD138 CD45

CD56 CD117 CD19 CD38 CD138 CD45

Abbreviations: APC = allophycocyanin; APC-Cy7 = allophycocyanin-cyanine 7; FITC = fluorescein isothiocyanate; PE = phycoerythrin; PerCP-Cy5.5 = peridinin chlorophyll protein-cyanine 5.5; PE-Cy7 = phycoerythrin-cyanine 7. Reprinted with permission from Mailankody et al.35 aTube 2 is complementary if further demonstration of clonality is needed among cells with phenotypic deviation are identified in Tube 1.

Table 3.

Recommended eight-color flow cytometry panel

Fluorophore

Target cell-surface marker in tube 1

Target cell-surface marker in tube 2a

FITC PE PerCP-Cy5.5 PE-Cy7 APC APC-Cy750 V450 BV510

CD38 CD56 CD45 CD19 CD117 CD81 CD138 CD27

CD38 CD56 CD229 CD19 cIgk cIgλ CD138 CD27

Abbreviations: APC = allophycocyanin; APC-Cy7 = allophycocyanin-cyanine 7; FITC = fluorescein isothiocyanate; PE = phycoerythrin; PerCP-Cy5.5 = peridinin chlorophyll protein-cyanine 5.5; PE-Cy7 = phycoerythrin-cyanine 7. Reprinted with permission from Mailankody et al.35 aTube 2 is complementary if further demonstration of clonality is needed among cells with phenotypic deviation are identified in Tube 1.

Table 4.

trend toward significance for OS (P = 0.06). MRD status at day 100 post ASCT was identified as the most important independent prognostic factor for PFS and OS.36 Rawstron et al.32 assessed MRD using MFC in transplant-eligible patients pre-ASCT (n = 378), at day 100 post ASCT (n = 397), and in transplant-ineligible patients following induction (n = 245). MRD − at day 100 post ASCT predicted improved PFS (28.6 vs 15.5 months, P o0.001) and OS (80.6 vs 59 months, P = 0.018). In patients who achieved CR, MRD − predicted improved PFS (P o 0.001) and OS (P = 0.039) when compared with patients who were MRD − /IFE+ or MRD+. Achieving MRD − was predictive of outcomes in patients with both favorable and adverse cytogenetic profiles. Twenty-eight percent of MRD+ patients who received maintenance thalidomide became MRD − , suggesting maintenance thalidomide improves MRD status in some cases. MRD status after induction therapy in transplant-ineligible patients was not predictive of outcome (PFS, P = 0.10).32 Using MFC, there is conflicting information on the impact of MRD − status and survival outcomes in high-risk disease. The Spanish PETHEMA group, using the GEM2000 protocol, has shown improved PFS and OS in patients achieving MRD − status for both high- and standard-risk disease.36 Researches in the United Kingdom conducted the MRC Myeloma IX trial, which demonstrated the rate of MRD − achievement was similar for both high- and standard-risk groups (60% and 62%). However, when measured post ASCT, patients with high-risk disease had inferior PFS and OS regardless of the MRD status compared with standard risk disease (P o0.01). Additional research is required to better understand the impact of MRD status within subgroups.32 Overall, MFC appears to be both sensitive and applicable with prognostic implications. One limitation of MFC is the need to obtain a BM specimen of sufficient quality. A poor quality sample will affect the flow cytometry results.35 In addition, the absence of a current standardized protocol for MFC in myeloma results in variability between laboratories. Roschewski et al.37 pointed out the vast heterogeneity among academic institutions with regard to MRD testing, noting major differences in antibody panels used to distinguish abnormal from normal plasma cells and major discrepancies in the cutoffs for MRD positivity.37 Allele-specific oligonucleotide PCR PCR techniques are already employed for other hematological malignancies including CML, ALL and acute promyelocytic leukemia for establishing prognosis, monitoring disease burden and guiding therapy. In MM, PCR with allele-specific oligonucleotides (ASO-PCR) is the most sensitive approach in the detection of malignant plasma cells, reaching a sensitivity up to 10 − 5. The clinical value of qualitative approaches has been modest owing to

Multi-parametric flow cytometry studies N

Study Paiva et al.

36

Rawstron et al.32

Population

Treatment regimen

MRD threshold −4

295

Post-ASCT

VBMCP/VBAD

0.01% (⩽10

)

102

Non-transplant eligible

VMP, VTP

0.01% (⩽10 − 4)

378

Pre-ASCT

CTD, CVAD

0.01% (⩽ 10 −4)

397

Post ASCT group

CTD, CVAD

0.01% (⩽10 − 4)

245

Non-transplant eligible

MP, aCTD

0.01% (⩽ 10 −4)

Outcomes (MRD − /MRD+) PFS 71 vs 37 months, Po0.001 OS NR vs 89 months, P = 0.002 PFS NR vs 35 months, P = 0.02 Trend toward improved OS PFS 44.2 months, Po0.001; OS not significant PFS 29 vs 16 months, Po0.001 OS 81 vs 59 months, P = 0.018 PFS 34 vs 14 months, P = 0.007 Trend toward improved OS

Abbreviations: aCTD = attenuated CTD; ASCT = autologous stem cell transplantation; CTD = cyclophosphamide, thalidomide, dexamethasone; CVAD = cyclophosphamide, vincristine, doxorubicin, dexamethasone; MP = melphalan, prednisone; MRD = minimal or measurable residual disease; VBMCP/VBAD = carmustine, melphalan, cyclophosphamide, prednisone/vincristine, carmustine, doxorubicin, dexamethasone.

Bone Marrow Transplantation (2015), 1 – 11

© 2015 Macmillan Publishers Limited

Minimal residual disease testing AM Sherrod et al

5 Table 5.

ASO-PCR studies

Study

N

Population

Treatment regimen

MRD threshold

Outcomes (MRD − /MRD+)

Bakkus et al.42 Putkonen et al.43

67 37

VAD Not reported

0.015% 0.01% (⩽10 − 4)

PFS 64 vs 16 months, P = 0.001 PFS 70 vs 19 months, P = 0.003 OS NR

Ladetto et al.41

31

Post-ASCT CR/nCR Post-ASCT or alloHCT CR/VGPR Post-ASCT

VAD induction VTD consolidation

0.0001% (⩽10 − 6)

PFS 100% vs 57% at 42 months, Po0.001

Abbreviations: alloHCT = allogeneic hematopoietic cell transplantation; ASCT = autologous stem cell transplantation; ASO = allelic-specific oligonucleotide; MRD = minimal or measurable residual disease; nCR = near CR; NR = not reached; VAD = vincristine, doxorubicin, dexamethasone; VGPR = very good partial response; VTD = bortezomib, thalidomide, dexamethasone.

the high sensitivity of the assay, as most cases remain positive despite heterogeneous outcomes.38 As an alternative, ASO realtime quantitative PCR provides an accurate quantification of residual disease, thus overcoming the problem. Several reports using this technique have been published, showing effective outcome discrimination in the transplant setting29,39–43 (Table 5). Limitations of ASO-PCR include expense, time consumption and lack of widespread availability. Turnaround time can be weeks, as the process requires PCR amplification of IGHV-J, IGHD-J and IGKDEL; clonal gene rearrangement identification; DNA sequencing of clonal genes; and design of a patient-specific ASO primer complementary to the corresponding clone at diagnosis. The patient-specific primer is then used in follow-up assessments for MRD. In essence, a unique primer is developed for each patient based on the antibody generated by that patient’s myeloma clone. That primer must be validated as a clinical laboratory reagent and used only for the patient from whom it was derived. Mutations in the clonal antibody gene during tumor evolution can effectively invalidate any result from ASO-PCR testing. Owing to these limitations, successful ASO-PCR occurs in 42%–86% of patients, thus hampering its use in clinical practice. Comparison of MFC and ASO-PCR Comparisons of MRD techniques are vital to determine the relative applicability, sensitivity and prognostic value of each test, facilitating the optimal design for prospective clinical trials. There have been multiple studies comparing these techniques (Table 6).40,44,45 In three consecutive trials, Puig et al.46 analyzed differences in ASO-PCR and MFC in 170 patients that had achieved at least a PR following treatment. MRD negativity was defined as ⩽ 10 − 4. PCR was applicable in only 42% of cases, owing to lack of clonality, unsuccessful sequencing or suboptimal ASO-PCR performance. In contrast, MFC was applicable in 490% of myeloma cases. There was significant correlation between MRD detection by both techniques (r = 0.881). Both techniques identified significantly different PFS in those obtaining MRD − (defined as o 10 − 4) compared with MRD+ disease in patients who were intensively treated (PCR: 54 vs 27 months, P = 0.001; MFC: 45 vs 27 months, P = 0.02) and non-intensively treated (PCR: NR vs 31 months, P = 0.029; MFC: NR vs 27 months P = 0.002). In patients who achieved CR, MRD − had superior outcomes compared with MRD +, with improved PFS (PCR: 49 vs 26 months, P = 0.001; MFC: 45 vs 25 months, P = 0.001) and OS (PCR: NR vs 60 months, P = 0.008; MFC: 72 vs 45 months, P = 0.014). Further comparative analyses of MPC- and ASO-PCR-based techniques indicated that although ASO-PCR has the potential to be more sensitive (measuring up to 10 − 6 cells), newer generation six- and eight-color MFC approaches may have sensitivities approaching or surpassing ASO-PCR.46 Moreover, the applicability of MFC to 490% of myeloma cases, compared with the 42%–86% typically reported for ASO-PCR, favors MFC as the more practical method for MRD assessment. © 2015 Macmillan Publishers Limited

Next-generation sequencing. NGS has been explored as an alternative strategy for detection of MRD in a number of lymphoid malignancies. This technique allows parallel sequencing of the unique clonal immunoglobulin heavy chain (IGH) present in every mature B cell and plasma cell. Sequencing is performed at diagnosis to determine the unique IGH rearrangement expressed by the malignant clone. In follow-up studies, this unique sequence can be identified among 105–106 amplified IGHs. Sensitivity for IGH-based NGS detection of MRD has been reported at ⩽ 10 − 6 cells. Martinez-Lopez et al.45 investigated the prognostic value of MRD detection in MM patients using a sequencing-based platform in BM samples from 133 MM patients who achieved ⩾ VGPR after frontline therapy. NGS was conducted by amplification of genomic DNA using locus-specific primer sets for complete IGH (IGH-VDJH), incomplete IGH (IGH-DJH) and the immunoglobulin κ locus (IGK). A high-frequency myeloma clone that was suitable for tracking in follow-up analyses was identified in 90% of patients. MRD negativity was defined as an MRD level of ⩽ 10 − 5.45 Concordance between NGS and MFC or ASO-PCR was 83% and 85%, respectively. Patients who were MRD − by sequencing had a significantly longer time to TTP (80 vs 31 months; P o 0.001) and increased OS (NR vs 81 months; P = 0.02), compared with patients who were MRD+. When stratifying patients by decreasing levels of MRD, the respective median TTP were 27 months (MRD ⩾ 10 − 3), 48 months (MRD 10 − 3 to 10 − 5) and 80 months (MRD o 10 − 5) (P from 0.003–0.0001). Thus, an increase in TTP was observed in patients with reduced MRD. Ninety-two percent of VGPR patients were MRD+. In patients obtaining CR, TTP was significantly longer for MRD − compared with MRD+ patients (131 vs 35 months; P o0.001). By multivariate analysis, MRD − status by deep sequencing was the single variable that achieved statistical significance (HR: 8.6, P = 0.012).45 Of note, the sequencing platform used in this study, LymphoSIGHT, is commercially available and, therefore, easily applicable in all clinical settings. IMAGING Imaging techniques have been investigated as a means to monitor treatment response and predict outcome. Whole-body X-ray (WBXR) has historically been the gold standard imaging modality, but this technique has significant limitations (Figure 2). WBXR is not adequate for the detection of early lytic lesions or in monitoring treatment response. New or enlarging bone lesions detected by WBXR generally signify progression. However, lytic lesions rarely show evidence of healing on X-ray. In this respect, WBXR is of questionable benefit and is not routinely recommended in follow-up evaluations.47 In recent years, more sensitive and specific imaging techniques have been investigated to detect focal bone lesions that may be harboring viable monoclonal plasma cells. Recently, revised IMWG criteria have incorporated these newer imaging modalities for the detection of bone lesions consistent with MM. A diagnosis of MM requires evidence of osteolytic bone lesions by CT and/or Bone Marrow Transplantation (2015), 1 – 11

Minimal residual disease testing AM Sherrod et al

6 Table 6.

Studies comparing MFC and PCR

Study

N

Population

Treatment regimen

MRD threshold

PCR outcomes (MRD − /MRD+)

MFC outcomes (MRD − /MRD+)

Sarasquete et al.44

32

CR post-ASCT

VBMCP/VBAD

0.01% (⩽10 − 4)

PFS 34 vs 15 months, P = 0.04 PFS 68% vs 28%; P = 0.001 PFS 54 vs 27 months, P = 0.001

PFS 27 vs 10 months, P = 0.05 PFS 75% vs 25%, P = 0.002 PFS 45 vs 27 months, P = 0.02

PFS: 61 vs 36 months, P = 0.001 OS NR vs 66 months, P = 0.03

PFS 67 vs 42 months, P = 0.005 OS NR vs 69 months, P = 0.004

Martinez-Sanchez et al.40 Puig et al.46

Martinez-Lopez et al.45

53

−4

Post-ASCT

VBMCP/VBAD

0.01% (⩽10

)

170

Post-ASCT; Nontransplant

0.01% (⩽10 − 4)

133

4VGPR

VBMCP/VBAD, T D or VTD; VMP or VTP in elderly VBMCP/VBAD, TD or VTD; VMP or VTP in elderly

0.1% (⩽10 − 3)

Abbreviations: ASCT = autologous stem cell transplantation; MFC = multi-parametric flow cytometry; MRD = minimal or measurable residual disease; NR = not reached; TD = thalidomide, dexamethasone; VBMCP/VBAD = carmustine, melphalan, cyclophosphamide, prednisone/vincristine, carmustine, doxorubicin, dexamethasone; VGPR = very good partial response; VMP = bortezomib, melphalan, prednisone; VTD = bortezomib, thalidomide, dexamethasone; VTP = bortezomib, thalidomide, prednisone.

Figure 2. False-negative WBXR. Focal osteolytic lesions of MM are seen well with PET/CT, compared with skeletal survey. WBXR lateral view of lumbar spine (far left) demonstrates no evidence of tumor. PET/CT (second from left) reveals diffuse myeloma marrow infiltration (shorter arrows) and focal lytic bone lesions (longer arrows) of L3 and L4. Axial PET/CT fused images are to the far right with corresponding CT-only images second from the right. (Reprinted from permission from Walker et al.66).

18F-fluorodeoxyglucose-PET (FDG-PET), or the identification of more than one focal lesion, ⩾ 5 mm in diameter, by MRI.48 These imaging modalities have also been explored in defining response to treatment and impact on prognosis; however, they have not yet been incorporated into the IMWG consensus response guidelines.49–51 Positron emission tomography–computed tomography FDG-PET/CT is a functional imaging modality that can show focal osteolysis (CT), focal or diffuse areas of abnormal FDG uptake or CT Bone Marrow Transplantation (2015), 1 – 11

soft tissue masses (intra- or extramedullary). Although FDG-PET/CT has low spatial resolution (6–8 mm), the combined CT component allows for a direct anatomic correlation of focal FDG uptake and provides high-resolution bone images that are superior to X-ray in detecting lytic bone lesions. In addition to the detection of hypermetabolic tumor, effective suppression of FDG activity early in the course of therapy has been linked to superior patient outcome in myeloma.52–54 Bartel et al.55 investigated the prognostic role of complete FDG suppression on PET/CT imaging in MM patients treated with induction therapy pre-ASCT. Among those with o 100% © 2015 Macmillan Publishers Limited

Minimal residual disease testing AM Sherrod et al

7 suppression of FDG uptake in focal lesions and extramedullary disease at 30 months post ASCT, complete metabolic response of focal lesions and metastatic spread before transplantation was associated with superior event-free survival (92% vs 71%, P o0.001) and OS (89% vs 63%, (P o 0.001). By multivariate analysis, normalization of PET findings before transplantation was associated with improved event-free survival (HR: 0.51, P = 0.038) and OS (HR: 0.41, P = 0.017).55 In 2011, Zamagni et al.56 demonstrated that PET/CT was useful in evaluating response to therapy and had prognostic significance post ASCT. In their analysis of 192 patients treated with induction chemotherapy followed by ASCT, persistent FDG-avid lesions were predictive of shorter PFS (47% vs 32%, P = 0.017) and OS (79% vs 66%, P = 0.018) at 48 months. By multivariate analysis, incomplete FDG suppression was strongly associated with inferior PFS (HR: 1.89, P = 0.030) and OS (HR: 3.9, P = 0.032). In 23% of patients who achieved a CR, persistent FDG-avid lesions were associated with inferior PFS (30% vs 61%, P = 0.019) at 4 years. These data emphasize the necessity of residual disease evaluation, not only in the BM but also outside of the BM using the most sensitive, appropriate and available imaging modality.56 Magnetic resonance imaging. MRI has also been explored as a sensitive method for the detection of bone disease. MRI can detect marrow infiltration and epidural extension of myeloma cells, rather than just bony destruction. In a comparison of axial MRI and WBXR, Lecouvet et al.51 found that MRI more frequently detected lytic lesions. However, WBXR was deemed superior, because it demonstrated more appendicular lesions.51 When whole-body examination was performed, including the appendicular skeleton, MRI had a higher detection rate than WBXR.57 A comparison of axial MRI versus whole body MRI revealed that

a

Baseline - diffuse and focal

~ 10% of patients had lesions exclusively outside of the axial skeleton.58 A systematic review of imaging techniques in the diagnosis of myeloma bone disease compared MRI with WBXR and/or CT. Although MRI detected more lesions in the axial skeleton compared with WBXR, WBXR detected more lesions in the ribs compared with MRI.59 Imaging comparisons. CT, PET/CT and MRI have been compared in multiple studies. Baur-Melnyk et al.60 found that MRI revealed more extensive disease in half of the patients evaluated when compared with whole-body CT. MRI detected focal plasmacytomas within the marrow before the development of osteolytic changes that were visible by CT or X-ray.60–62 Bannas et al.63 prospectively evaluated 31 patients following ASCT with imaging techniques compared with standard laboratory response criteria. PET/CT had a sensitivity of 50% and a specificity of 85%, and MRI had a sensitivity of 80% and a specificity of 38%. False positives on MRI were most often associated with persistent nonviable lesions.63 Derlin et al.64 found that remission status determined by PET/CT (not MRI) was significantly correlated with standard response criteria. Compared with PET/CT, MRI is superior for the detection of diffuse bone disease, central nervous sytem and spinal cord imaging, and characterizing pathological fractures (Figure 3). MRI is limited by increased imaging time, has a relatively limited field of view and focal lesions can remain persistently positive for years after therapy (Figure 4). PET/CT is optimal for measurement of extramedullary disease, requires less imaging time and metabolic changes seen on PET/CT offer an earlier response evaluation. PET/CT is limited by increased radiation exposure and suboptimal characterization of the central nervous sytem lesions and diffuse bone involvement. Furthermore, the SUV cutoff to define active

b

c

CR (baseline + 240 d)

Relapse (baseline + 610 d)

Figure 3. Sagittal views of short tau-inversion recovery-weighted MRI series demonstrating baseline diffuse and focal disease, treatment response, and relapse. (a) At time of diagnosis, diffuse tumor infiltration and focal lesions are seen (arrows). (b) Complete response is seen 240 days later, with normal MRI appearance. (c) Relapse is seen 610 days after diagnosis, with slight increase in diffuse, heterogeneous marrow signal, recurrent focal lesion at L4 (bottom arrow) and new focal lesion at L2 (top arrow). (Reprinted with permission from Walker et al.66). © 2015 Macmillan Publishers Limited

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8

a

b

c

d

e

f

Figure 4. Baseline whole-body anterior MRI (a and b) and PET/CT (c) images of patient newly diagnosed with MM. MRI was performed using three-dimensional maximum-intensity projection with diffusion weighting and restriction on apparent diffusion coefficient. All three images reveal severe diffuse and focal marrow infiltration. Treatment response seen on whole-body anterior MRI (c and e) vs PET/CT (f). Improvement is seen on MRI, with some persistent abnormalities and normalization on PET. By tumor markers, patient was in remission, correlating best with PET image. (Reprinted with permission from Walker67).

disease has not been validated prospectively. False-positive results can occur with PET at the site of BM biopsy, spinal surgery or other procedures (Table 7).65 In conclusion, although skeletal survey remains the standard of care, more sensitive imaging techniques have established significant value in assessing extramedullary disease response following therapy. PET/CT appears to be superior for re-evaluating myeloma after induction and ASCT, and has prognostic value in diagnosing persistent hypermetabolic activity following ASCT. However, at this time it is unclear whether therapy directed at such sites of persistent hypermetabolic activity can improve prognosis in the subgroup with persistent PET/CT positivity. Thus, PET/CT has a role in detection of extramedullary residual disease and may be complementary to MRD detection in marrow samples. In order for this to be of routine clinical use, standardized definitions for PET/CT imaging, thresholds for PET positivity and standardized protocols for semi-quantitative SUV are needed. In addition, future validation of the optimal timing for PET/CT evaluations after treatment interventions is necessary.66,67 PRACTICAL QUESTIONS The importance of MRD measurement remains a critical question in the management of MM. MRD measurement has proved useful in other hematological malignancies, such as ALL, acute promyelocytic leukemia and CML, and has the potential to be useful for MM. The incorporation of modern therapies and ASCT into the initial management of newly diagnosed myeloma has resulted in deeper levels of remission and more durable responses. Based on Bone Marrow Transplantation (2015), 1 – 11

this, some have suggested that the myeloma response criteria be updated to include MRD status.35 Although multiple MRD techniques have demonstrated an improvement in PFS, the impact on OS is less well defined. Given the toxicity and morbidity associated with the intense therapies that are required to achieve a state of MRD negativity, it is imperative that we standardize an optimal MRD testing technique that consistently translates to improved OS. Two large studies that used MFC have shown precisely this; however, other studies have used ASO-PCR and NGS for MRD detection. MFC is relatively inexpensive, with a sensitivity of 10 − 4 to 10 − 5, applicability of 490% and flow cytometry labs are widely available to most patients. In contrast, ASO-PCR and NGS are more labor intensive, more costly and less widely available (Table 8). The optimal timing for MRD testing remains unclear. It remains to be determined whether lack of MRD negativity at certain time points indicates a need for additional therapy or if, conversely, an MRD − state suggests that no additional therapy is needed. Additional studies are required to determine whether a patient that is MRD − before ASCT would benefit from the transplant. Some patients achieve durable long-term responses with detectable disease in a PR status. We question whether there are similar patient populations where achieving MRD − is not as important. No data are available to suggest that MRD negativity pre-ASCT or in the nontransplant eligible population translates into an OS benefit. This may be because of the timing of testing or may suggest that treatment goals should be different in the nontransplant eligible population. Larger MRD studies have not uniformly incorporated three-drug novel agent regimens into © 2015 Macmillan Publishers Limited

Minimal residual disease testing AM Sherrod et al

9 Table 7.

Comparison of imaging techniques for multiple myeloma Skeletal CT

Radiation exposure Sensitivity for bone disease

Low (3.5 mSv) Sensitive for focal bone lesions, but cannot differentiate active from inactive focal lesions. DXA best to assess bone density and changes in density Compression CT is best for all, but cannot assess the fractures age Detect spinal cord or Not trustworthy CNS compromise Metabolic activity of Not useful lesions EMD Visible at times Diffuse marrow involvement

Not useful

Response evaluation

Not useful

Imaging time

Fastest

Cost False positive

Least expensive Benign bone cysts and erosive changes from arthritis can mimic lytic bone lesions Limited

Occult infection

MRI

PET CT

None MRI is most sensitive but can be slow to demonstrate early response to treatment

High (7 mSv) PET/CT is best for assessment of treatment response, early treatment response and for detection of extramedullary disease Good because of CT component

MRI best to detect acute/subacute MRI best Some usefulness with DCE MRI MRI is good if the EMD is within the field of view Most sensitive, but limited with chemotherapy rebound or marrow stimulation Slow response to treatment (up to years) Slowest – up to several hours for a thorough exam (whole-body MRI is faster, but not yet established in the evaluation of MM) Most expensive Benign bone cysts and erosive changes can be difficult to distinguish, especially if small ( o5 mm) Very good if in the field of view

Poor negative predictive value for subtle disease PET/CT most useful PET/CT most useful Moderate utility Shows earliest response to treatment About 40 min

Common from inflammatory etiologies Most useful

Abbreviations: CNS = central nervous sytem; CT = computed tomography; DCE = dynamic contrast-enhanced; DXA = dual-energy X-ray absorptiometry; EMD = extramedullary disease; MM = multiple myeloma; MRI = magnetic resonance imaging; PET = positron emission tomography.

Table 8.

Comparison of novel methods of MRD measurement MFC −4

ASO-PCR −5

−5

NGS −6

Sensitivity Limitations

10 to 10 (higher with eight-color) Cell number, background

Clonal identification Testing duration Standardization

Somatic hypermutations can affect target site Rapid Easily done for MM

10 to 10 Technical applicability of color assay Clonal aberrant phenotypes can emerge Weeks Not uniform, ongoing

Bioinformatics expertise Applicability

Not needed 90%

Not needed 42%–86%

10 − 6 Cell number Results can be affected by subclone identification 5–10 days Mostly service basis (EUROCLONALITY, EUROMRD, lymphoSIGHT) Needed 80%–91%

Abbreviations: ASO = allelic-specific oligonucleotide; MFC = multi-parametric flow cytometry; MM = multiple myeloma; MRD = minimal or measurable residual disease; NGS = next-generation sequencing.

their induction protocols. The newer proteasome inhibitor– immunomodulatory agent combinations will likely result in significant proportions of MRD − status pre-ASCT. It is possible that MRD negativity may emerge as a valid and early surrogate endpoint for extended response times and survival. On a cautionary note, we remind the reader that MM is a heterogeneous chronic disease with wide variability in the risk of progression. Increasing treatment toxicity with the sole aim of deepening remission may not be a ‘one-size-fits-all’ approach for all patients. RECOMMENDATIONS As the treatment paradigm for MM continues to evolve and as the depth and duration of responses continue to improve, more sensitive measures of disease evaluation, including newer biochemical markers and imaging modalities to measure clonal © 2015 Macmillan Publishers Limited

plasma cell burden, should be integrated into the response algorithm. Future standardized MRD testing techniques have the potential to improve interim disease evaluation, risk stratification and, ultimately, treatment decisions. As MRD research develops, there are two areas where testing becomes important. The first is the decision to proceed with upfront ASCT versus delayed ASCT following a set number of therapeutic cycles. Currently, the role of upfront ASCT is being studied in the era of novel agent triplets (IFM DFCI study, NCT01208662). In this study, after RVd (lenalidomide, bortezomib and dexamethasone) induction, patients are randomized to upfront ASCT followed by two additional cycles of RVd versus an additional five cycles of consolidation RVd with the option of a delayed ASCT at relapse. As more sophisticated MRD techniques and induction therapies have become available, a responseadapted ASCT strategy may be more relevant in the modern era. As opposed to a fixed number of RVd cycles, MRD status could Bone Marrow Transplantation (2015), 1 – 11

Minimal residual disease testing AM Sherrod et al

10 identify patients who benefit from upfront ASCT as a responsedeepening strategy. A reasonable modern study design would be to randomize patients who achieve MRD − status to upfront ASCT versus delayed ASCT. In parallel, patients not achieving MRD − status with limited induction cycles could be randomized to many strategies such as immediate ASCT versus further cycles of induction until the achievement of an MRD response followed by re-randomization to ASCT or not. Response-based treatment assignments will define the depth of response required for sustained benefit, avoid overtreatment of those who have achieved maximal benefit and clarify whether the modality that induces an MRD − state matters beyond the response level itself. The answers to these questions are of particular interest in the United States, as increasing numbers of patients will become MRD − with additional cycles of chemotherapy. Similarly, determination of MRD status during maintenance therapy may elucidate the optimal duration of therapy for MM after ASCT. CONFLICT OF INTEREST The authors declare no conflict of interest.

REFERENCES 1 Child JA, Morgan GJ, Davies FE, Owen RG, Bell SE, Hawkins K et al. High-dose chemotherapy with hematopoietic stem-cell rescue for multiple myeloma. N Engl J Med 2003; 348: 1875–1883. 2 Siegel R, Naishadham D, Jemal A. Cancer statistics. CA Cancer J Clin 2013; 63: 11–39. 3 Pulte D, Redaniel MT, Lowry L, Bird J, Jeffreys M. Age disparities in survival from lymphoma and myeloma: a comparison between US and England. Br J Haematol 2014; 165: 824–831. 4 Durie BG, Harousseau JL, Miguel JS, Blade J, Barlogie B, Anderson K et al. International uniform response criteria for multiple myeloma. Leukemia 2006; 20: 1467–1473. 5 Richardson PG, Weller E, Lonial S, Jakubowiak AJ, Jagannath S, Raje NS et al. Lenalidomide, bortezomib, and dexamethasone combination therapy in patients with newly diagnosed multiple myeloma. Blood 2010;. 116: 679–686. 6 Kapoor P, Kumar SK, Dispenzieri A, Lacy MQ, Buadi F, Dingli D et al. Importance of achieving stringent complete response after autologous stem-cell transplantation in multiple myeloma. J Clin Oncol 2013; 31: 4529–4535. 7 van de Velde HJK, Liu X, Chen G, Cakana A, Deraedt W, Bayssas M. Complete response correlates with long-term survival and progression-free survival in high-dose therapy in multiple myeloma. Haematologica 2007; 92: 1399–1406. 8 Lahuerta JJ, Mateos MV, Martínez-López J, Rosiñol L, Sureda A, de la Rubia J et al. Influence of pre- and post-transplantation responses on outcome of patients with multiple myeloma: sequential improvement of response and achievement of complete response are associated with longer survival. J Clin Oncol 2008; 26: 5775–5782. 9 Gay F, Larocca A, Wijermans P, Cavallo F, Rossi D, Schaafsma R et al. Complete response correlates with long-term progression-free and overall survival in elderly myeloma treated with novel agents: analysis of 1175 patients. Blood 2011; 117: 3025–3031. 10 Dispenzieri A, Kyle R, Merlini G, Miguel JS, Ludwig H, Hajek R et al. International Myeloma Working Group guidelines for serum-free light chain analysis in multiple myeloma and related disorders. Leukemia 2008; 23: 215–224. 11 Katzmann JA, Dispenzieri A, Kyle RA, Snyder MR, Plevak MF, Larson DR et al. Elimination of the need for urine studies in the screening algorithm for monoclonal gammopathies by using serum immunofixation and free light chain assays. Mayo Clin Proc 2006; 81: 1575–1578. 12 Bradwell AR, Carr-Smith HD, Mead GP, Tang LX, Showell PJ, Drayson MT et al. Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine. Clin Chem 2001; 47: 673–680. 13 Malpas J, Bergsagel D, Kyle R, Anderson K.Myeloma Biology and Management, 3rd edn Elsevier: Oxford, 2004. 14 Bradwell A, Carr-Smith H, Mead G, Tang L, Showell P, Drayson M et al. Highly sensitive automated immunoassay for immunoglobulin free light chains in serum and urine. Clin Chem 2001; 47: 673–680. 15 Wood PB, McElroy YG, Stone MJ. Comparison of serum immunofixation electrophoresis and free light chain assays in the detection of monoclonal gammopathies. Clin Lymphoma Myeloma Leuk 2010; 10: 278–280.

Bone Marrow Transplantation (2015), 1 – 11

16 Wolff F, Thiry C, Willems D. Assessment of the analytical performance and the sensitivity of serum free light chains immunoassay in patients with monoclonal gammopathy. Clin Biochem 2007; 40: 351–354. 17 Smeltzer J, Gertz M, Rajkumar SV, Dispenzieri A, Lacy M, Buadi F et al. Suppression of involved immunoglobulin free light chain post therapy and survival outcomes following autologous stem cell transplantation for myeloma. Biol Blood Marrow Transplant 2013; 19: S156. 18 Cornell RF, Dogra S, Brazauskas R, Goodman S, Jagasia MH, Kassim AA et al. Sustained suppression of involved free light chain predicts long term outcomes in multiple myeloma after allogeneic hematopoietic stem cell transplantation: a multi-institutional study. BMT Tandem Meeting 2014; 20: S74. 19 Hutchison CA, Harding S, Hewins P, Mead GP, Townsend J, Bradwell AR et al. Quantitative assessment of serum and urinary polyclonal free light chains in patients with chronic kidney disease. Clin J Am Soc Nephrol 2008; 3: 1684–1690. 20 Wadhera RK, Kyle RA, Larson DR, Dispenzieri A, Kumar S, Lazarus HM et al. Incidence, clinical course, and prognosis of secondary monoclonal gammopathy of undetermined significance in patients with multiple myeloma. Blood 2011; 118: 2985–2987. 21 Zent CS, Wilson CS, Tricot G, Jagannath S, Siegel D, Desikan KR et al. Oligoclonal protein bands and ig isotype switching in multiple myeloma treated with highdose therapy and hematopoietic cell transplantation. Blood 1998; 91: 3518–3523. 22 de Larrea CF, Cibeira MT, Elena M, Arostegui JI, Rosiñol L, Rovira M et al. Abnormal serum free light chain ratio in patients with multiple myeloma in complete remission has strong association with the presence of oligoclonal bands: implications for stringent complete remission definition. Blood 2009; 114: 4954–4956. 23 Tovar N, Fernández de Larrea C, Elena M, Cibeira MT, Aróstegui JI, Rosiñol L et al. Prognostic impact of serum immunoglobulin heavy/light chain ratio in patients with multiple myeloma in complete remission after autologous stem cell transplantation. Biol Blood Marrow Transplant 2012; 18: 1076–1079. 24 Bradwell A, Harding S, Fourrier N, Mathiot C, Attal M, Moreau P et al. Prognostic utility of intact immunoglobulin Ig′κ/Ig′λ ratios in multiple myeloma patients. Leukemia 2013; 27: 202–207. 25 Barlogie B, Tricot G, Anaissie E, Shaughnessy J, Rasmussen E, van Rhee F et al. Thalidomide and hematopoietic-cell transplantation for multiple myeloma. N Engl J Med 2006; 354: 1021–1030. 26 Cavo M, Tosi P, Zamagni E, Cellini C, Tacchetti P, Patriarca F et al. Prospective, randomized study of single compared with double autologous stem-cell transplantation for multiple myeloma: Bologna 96 Clinical Study. J Clin Oncol 2007; 25: 2434–2441. 27 van Rhee F, Giralt S, Barlogie B. The future of autologous stem cell transplantation in myeloma. Blood 2014; 124: 328–333. 28 Rawstron AC, Orfao A, Beksac M, Bezdickova L, Brooimans RA, Bumbea H et al. Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders. Haematologica 2008; 93: 431–438. 29 Korthals M, Sehnke N, Kronenwett R, Schroeder T, Strapatsas T, Kobbe G et al. Molecular monitoring of minimal residual disease in the peripheral blood of patients with multiple myeloma. Biol Blood Marrow Transplant 2013; 19: 1109–1115. 30 Lioznov M, Badbaran A, Fehse B, Bacher U, Zander AR, Kroger NM. Monitoring of minimal residual disease in multiple myeloma after allo-SCT: flow cytometry vs PCR-based techniques. Bone Marrow Transplant 2008; 41: 913–916. 31 Mason KD, Juneja S. Go with the flow for monitoring response in myeloma with minimal residual disease. Leuk Lymphoma 2008; 49: 177–178. 32 Rawstron AC, Child JA, de Tute RM, Davies FE, Gregory WM, Bell SE et al. Minimal residual disease assessed by multiparameter flow cytometry in multiple myeloma: impact on outcome in the Medical Research Council Myeloma IX Study. J Clin Oncol 2013; 31: 2540–2547. 33 van Dongen JJ, Lhermitte L, Böttcher S, Almeida J, van der Velden VH, Flores-Montero J et al. EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia 2012; 26: 1908–1975. 34 Landgren O, Gormley N, Turley D, Owen RG, Rawstron A, Paiva B et al. Flow cytometry detection of minimal residual disease in multiple myeloma: lessons learned at FDA-NCI roundtable symposium. Am J Hematol 2014; 89: 1159–1160. 35 Mailankody S, Korde N, Lesokhin AM, Lendvai N, Hassoun H, Stetler-Stevenson M et al. Minimal residual disease in multiple myeloma: bringing the bench to the bedside. Nat Rev Clin Oncol 2015; 12: 286–295. 36 Paiva B, Vidriales M-B, Cerveró J, Mateo G, Pérez JJ, Montalbán MA et al. Multiparameter flow cytometric remission is the most relevant prognostic factor for multiple myeloma patients who undergo autologous stem cell transplantation. Blood 2008; 112: 4017–4023. 37 Roschewski M, Stetler-Stevenson M, Yuan C, Mailankody S, Korde N, Landgren O. Minimal residual disease: what are the minimum requirements? J Clin Oncol 2014; 32: 475–476.

© 2015 Macmillan Publishers Limited

Minimal residual disease testing AM Sherrod et al

11 38 Martinelli G, Terragna C, Zamagni E, Ronconi S, Tosi P, Lemoli R et al. Polymerase chain reaction-based detection of minimal residual disease in multiple myeloma patients receiving allogeneic stem cell transplantation. Haematologica 2000; 85: 930–934. 39 Galimberti S, Benedetti E, Morabito F, Papineschi F, Callea V, Fazzi R et al. Prognostic role of minimal residual disease in multiple myeloma patients after non-myeloablative allogeneic transplantation. Leuk Res 2005; 29: 961–966. 40 Martínez-Sánchez P, Montejano L, Sarasquete ME, García-Sanz R, Fernández-Redondo E, Ayala R et al. Evaluation of minimal residual disease in multiple myeloma patients by fluorescent-polymerase chain reaction: the prognostic impact of achieving molecular response. Br J Haematol 2008; 142: 766–774. 41 Ladetto M, Pagliano G, Ferrero S, Cavallo F, Drandi D, Santo L et al. Major tumor shrinking and persistent molecular remissions after consolidation with bortezomib, thalidomide, and dexamethasone in patients with autografted myeloma. J Clin Oncol 2010; 28: 2077–2084. 42 Bakkus MHC, Bouko Y, Samson D, Apperley JF, Thielemans K, Camp BV et al. Post-transplantation tumour load in bone marrow, as assessed by quantitative ASO-PCR, is a prognostic parameter in multiple myeloma. Br J Haematol 2004; 126: 665–674. 43 Putkonen M, Kairisto V, Juvonen V, Pelliniemi T-T, Rauhala A, Itälä-Remes M et al. Depth of response assessed by quantitative ASO-PCR predicts the outcome after stem cell transplantation in multiple myeloma. Eur J Haematol 2010; 85: 416–423. 44 Sarasquete M, Garcia-Sanz R, Gonzalez D, Martinez J, Mateo G, Martinez P et al. Minimal residual disease monitoring in multiple myeloma: a comparison between allelic-specific oligonucleotide real-time quantitative polymerase chain reaction and flow cytometry. Haematologica 2005; 90: 1365–1372. 45 Martinez-Lopez J, Lahuerta JJ, Pepin F, González M, Barrio S, Ayala R et al. Prognostic value of deep sequencing method for minimal residual disease detection in multiple myeloma. Blood 2014; 123: 3073–3079. 46 Puig N, Sarasquete ME, Balanzategui A, Martinez J, Paiva B, Garcia H et al. Critical evaluation of ASO RQ-PCR for minimal residual disease evaluation in multiple myeloma. A comparative analysis with flow cytometry. Leukemia 2014; 28: 391–397. 47 Caers J, Withofs N, Hillengass J, Simoni P, Zamagni E, Hustinx R et al. The role of positron emission tomography-computer tomography and magnetic resonance imaging in diagnosis and follow up of multiple myeloma. Haematologica 2014; 99: 629–637. 48 Rajkumar SV, Dimopoulos MA, Palumbo A, Blade J, Merlini G, Mateos M-V et al. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma. Lancet Oncol 2014; 15: e538–e548. 49 Zamagni E, Nanni C, Patriarca F, Englaro E, Castellucci P, Geatti O et al. A prospective comparison of 18 F-fluorodeoxyglucose positron emission tomography-computed tomography, magnetic resonance imaging and whole-body planar radiographs in the assessment of bone disease in newly diagnosed multiple myeloma. Haematologica 2007; 92: 50–55. 50 Kröpil P, Fenk R, Fritz L, Blondin D, Kobbe G, Mödder U et al. Comparison of whole-body 64-slice multidetector computed tomography and conventional radiography in staging of multiple myeloma. Eur Radiol 2008; 18: 51–58. 51 Lecouvet FE, Malghem J, Michaux L, Maldague B, Ferrant A, Michaux JL et al. Skeletal survey in advanced multiple myeloma: radiographic bersus MR imaging survey. Br J Haematol 1999; 106: 35–39. 52 Spaepen K, Stroobants S, Dupont P, Van Steenweghen S, Thomas J, Vandenberghe P et al. Prognostic value of positron emission tomography (PET)

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with fluorine-18 fluorodeoxyglucose ([18 F]FDG) after first-line chemotherapy in non-hodgkin’s lymphoma: is [18 F]FDG-PET a valid alternative to conventional diagnostic methods? J Clin Oncol 2001; 19: 414–419. Allal AS, Dulguerov P, Allaoua M, Haenggeli C-A, El Ghazi EA, Lehmann W et al. Standardized uptake value of 2-[18 F] fluoro-2-deoxy-d-glucose in predicting outcome in head and neck carcinomas treated by radiotherapy with or without chemotherapy. J Clin Oncol 2002; 20: 1398–1404. Maisey NR, Webb A, Flux GD, Padhani A, Cunningham DC, Ott RJ et al. FDG–PET in the prediction of survival of patients with cancer of the pancreas: a pilot study. Br J Cancer 2000; 83: 287–293. Bartel TB, Haessler J, Brown TLY, Shaughnessy JD, van Rhee F, Anaissie E et al. F18-fluorodeoxyglucose positron emission tomography in the context of other imaging techniques and prognostic factors in multiple myeloma. Blood 2009; 114: 2068–2076. Zamagni E, Patriarca F, Nanni C, Zannetti B, Englaro E, Pezzi A et al. Prognostic relevance of 18- F FDG PET/CT in newly diagnosed multiple myeloma patients treated with up-front autologous transplantation. Blood 2011; 118: 5989–5995. Ghanem N, Lohrmann C, Engelhardt M, Pache G, Uhl M, Saueressig U et al. Whole-body MRI in the detection of bone marrow infiltration in patients with plasma cell neoplasms in comparison to the radiological skeletal survey. Eur Radiol 2006; 16: 1005–1014. Baur-Melnyk A, Buhmann S, Dürr HR, Reiser M. Role of MRI for the diagnosis and prognosis of multiple myeloma. Eur J Radiol 2005; 55: 56–63. Regelink JC, Minnema MC, Terpos E, Kamphuis MH, Raijmakers PG, Pieters – van den Bos IC et al. Comparison of modern and conventional imaging techniques in establishing multiple myeloma-related bone disease: a systematic review. Br J Haematol 2013; 162: 50–61. Baur-Melnyk A, Buhmann S, Becker C, Schoenberg SO, Lang N, Bartl R et al. Whole-body MRI versus whole-body MDCT for staging of multiple myeloma. AJR Am J Roentgenol 2008; 190: 1097–1104. Walker R, Barlogie B, Haessler J, Tricot G, Anaissie E, Shaughnessy JD et al. Magnetic resonance imaging in multiple myeloma: diagnostic and clinical implications. J Clin Oncol 2007; 25: 1121–1128. Walker RC, Brown TL, Jones-Jackson LB, De Blanche L, Bartel T. Imaging of multiple myeloma and related plasma cell dyscrasias. J Nucl Med 2012; 53: 1091–1101. Bannas P, Hentschel H, Bley T, Treszl A, Eulenburg C, Derlin T et al. Diagnostic performance of whole-body MRI for the detection of persistent or relapsing disease in multiple myeloma after stem cell transplantation. Eur Radiol 2012; 22: 2007–2012. Derlin T, Peldschus K, Münster S, Bannas P, Herrmann J, Stübig T et al. Comparative diagnostic performance of 18 F-FDG PET/CT versus whole-body MRI for determination of remission status in multiple myeloma after stem cell transplantation. Eur Radiol 2013; 23: 570–578. Mesguich C, Fardanesh R, Tanenbaum L, Chari A, Jagannath S, Kostakoglu L. State of the art imaging of multiple myeloma: Comparative review of FDG PET/CT imaging in various clinical settings. Eur J Radiol 2014; 83: 2203–2223. Walker R, Barlogie B, Haessler J, Tricot G, Anaissie E, Shaughnessy JD Jr et al. Magnetic resonance imaging in multiple myeloma: diagnostic and clinical implications. J Clin Oncol 2007; 25: 1121–1128. Walker RC, Jones-Jackson LB, Rasmussen E, Miceli M, Angtuaco EJC, Van Rhee F et al. PET and PET/CT imaging in multiple myeloma, solitary plasmacytoma, MGUS, and other plasma cell dyscrasias. Positron Emission Tomography. Springer: London, 2006, pp 283–302.

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