1. The Role of Minimal Residual Disease in Multiple Myeloma
C. Ola Landgren, MD, PhD Chief, Multiple Myeloma Section Memorial Sloan Kettering Cancer Center New York, New York
Welcome to this program on the role of minimal residual disease (MRD) in multiple myeloma.
My name is Ola Landgren. I’m Chief of the Myeloma Section at Memorial Sloan Kettering Cancer Center in New York.

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3. Faculty Disclosure
C. Ola Landgren, MD, PhD, has disclosed that he has received consulting fees from Bristol-Myers Squibb, Celgene, and Onyx.
On this slide, I have provided my disclosures.

4. Agenda Why MRD Testing for Pts With Multiple Myeloma?
Assessing and Monitoring MRD Response to Therapy
Using MRD Results to Improve Outcomes for Myeloma Pts
MRD, minimal residual disease.
The agenda for this program has 3 components. The first one being why minimal residual disease (MRD) testing for patients with multiple myeloma is important.

5. Continued Improvement in OS in Pts With Multiple Myeloma
Ages 0-50 Yrs
100 75 50 25
2.5
7.5
5.0
10.0
Cumulative Percent Survival
OS, overall survival.
On this slide, you see patients diagnosed with multiple myeloma up to the age of 50 years in the United States. This comes from the SEER NCI registry. And as you can see, there’s a continued improvement in overall survival in this age group. The top line indicates that on average patients live longer than 10 years in this age group.
Yrs After Diagnosis
Kristinsson SY, et al. Leukemia. 2014;28:

6. Newer Myeloma Drugs Are Associated With Better Clinical Response
100
≥ VGPR OR 80 60
Response Rate of Pts Treated (%) 40
CRd, carfilzomib, lenalidomide, and low-dose dexamethasone; CTD, cyclophosphamide, thalidomide, and dexamethasone; Dex, dexamethasone; Len, lenalidomide; OR, overall response; Thal, thalidomide; RVD, lenalidomide, bortezomib, and dexamethasone; VAD, vincristine, doxorubicin, dexamethasone; VCD, bortezomib, cyclophosphamide, and dexamethasone; VGPR, very good partial response; VRDC, bortezomib, lenalidomide, dexamethasone, and cyclophosphamide.
Newer myeloma drugs are associated with better clinical response. As illustrated on this figure, you see on the x-axis from left to right combinations of drugs that have been given over the years as induction therapies for patients with multiple myeloma. On the left, VAD therapy, no longer used; dexamethasone; cyclophosphamide/thalidomide and dexamethasone; thalidomide and dexamethasone; bortezomib and cyclophosphamide and dexamethasone; lenalidomide/dexamethasone; bortezomib/thalidomide/ dexamethasone; bortezomib/lenalidomide/dexamethasone and cyclophosphamide; bortezomib/lenalidomide and dexamethasone; and carfilzomib/lenalidomide and dexamethasone.
And as you can see on this slide, several of these combinations of therapies provide 100% overall response. Overall response, as you probably know, means a partial response or better. But I do think, more importantly, this figure shows that from left to right, the proportion of patients that reach deeper response keeps on going up. And on the very right, the carfilzomib and lenalidomide and dexamethasone combination provides over 80% very good partial response, which translates into a 90% or more reduction of the M-spike. 20
VAD
Dex
CTD
Thal-Dex
VCD
Len-Dex
VTD
VRDC
CRd
RVD
Induction Regimen
Mailankody S, et al. Nat Rev Clin Oncol. 2015;[Epub ahead of print].

7. Clinical Response to MM Therapy Affects OS
CR vs nCR: P = .1 CR vs PR: P = .07 CR vs SD: P = .02
nCR vs PR vs SD: P = .9
1.0
0.8 CR
0.6
nCR
Probability of OS PR SD
0.4
CR, complete response; MM, multiple myeloma; nCR, near complete response; OS, overall survival; PD, progressive disease; PR, partial response; SD, stable disease.
Importantly, in multiple myeloma, several studies show that the clinical response to myeloma therapy impacts overall survival. As you can see in this slide by Lahuerta et al, published in the Journal of Clinical Oncology 2008, patients that reached a complete response had the longest overall survival.
0.2 PD 12 24 36 48 60 72 84 96
Mos
Lahuerta JJ, et al. J Clin Oncol. 2008;26:

8. Improved Response Needs Improved Measurements
Conventional microscopy (CR)[1]
Immunohistochemistry (sCR)[2]
sCR CR
nCR
1.0
P = .01
1.0
0.8
0.8
0.6
0.6
< 5% BM plasma cells
Cumulative Survival
Probability of OS
0.4
0.4
BM, bone marrow; CR, complete response; nCR, near complete response; sCR, stringent complete response.
Investigators have looked at factors beyond these regular measures. On the left, splitting between above or below 5% plasma cells, you can see that patients in complete response with less than 5% plasma cells, they have a better survival than those who have more than 5% plasma cells. And on the right, if you add the free light chains to the conventional complete response, which would make the patient either stringent complete response or not stringent complete response, you can also see that there is a difference in terms of overall survival among those patients who reach a stringent complete response.
0.2
0.2
≥ 5% BM plasma cells 2 4 6 8 10 12 14 1 2 3 4 5 6 7
Years Since Diagnosis
Yrs Since Transplantation
1. Chee CE, et al. Blood. 2009;114: Kapoor P, et al. J Clin Oncol. 2013;31:

9. IMWG Revised Uniform Response Criteria for MM
Negative IFE of serum and urine, disappearance of any soft tissue plasmacytomas, and < 5% PCs in bone marrow (2 consecutive assessments)
Stringent CR
CR plus normal FLC ratio and absence of clonal PCs by immunohistochemistry or flow cytometry (2 consecutive assessments)
Immunophenotypic CR
Stringent CR plus absence of clonal BM PCs with minimum 1 million PCs analyzed by flow cytometry > 4 colors
Molecular CR
CR plus negative allele-specific oligonucleotide PCR (sensitivity 10-5)
BM, bone marrow; CR, complete response; FLC, free light chain; IFE, immunofixation electrophoresis; IMWG, International Myeloma Working Group; MM, multiple myeloma; PC, plasma cell; PCR, polymerase chain reaction.
As you saw on the previous slides, depth of response correlates with overall survival, and there are also studies showing consistently that depth of response impacts progression-free survival. On this slide, you see the International Myeloma Working Group revised Uniform Response Criteria for multiple myeloma.
Complete response is a negative immunofixation of serum and urine, disappearance of any soft tissue plasmacytoma, and less than 5% plasma cells in the bone marrow at 2 consecutive assessments. The stringent complete response, which was shown on a previous slide, is defined as a complete response plus a normal free light chain ratio and absence of clonal plasma cells by immunohistochemistry or flow cytometry, also at 2 consecutive assessments. The immunophenotypic complete response is defined as stringent complete response plus absence of clonal bone marrow plasma cells with a minimum of 1 million plasma cells analyzed by flow cytometry with at least 4 colors. And a molecular complete response is complete response plus negative allele-specific oligonucleotide PCR, also called ASO PCR. And this is reported to have a sensitivity of 10-5.
Palumbo A, et al. J Clin Oncol. 2014;32:

10. MRD Status Is a Relative Measure
1 x 1012
At diagnosis
PR (50% reduction in M protein)
nCR (immunofixation positive only)
Number of Myeloma Cells
CR (immunofixation negative)
ASO-PCR, allele-specific oligonucleotide polymerase chain reaction; CR, complete response; MRD, minimal residual disease; nCR, near complete response; PR, partial response.
I think it’s important to point out that minimal residual disease, or MRD status, is not an arbitrary yes/no question in multiple myeloma. At least, not yet. It is a relative measure. Looking at the top of this slide at diagnosis, you see there are 1 x 1012 number of myeloma cells in the patient. At the partial response, there is a 50% reduction of the M-spike. Per criteria, near complete response is an immunofixation positive only, and you could also have a 90% or more reduction by the serum protein electrophoresis and fall into this category, and then it would be called very good partial response. A complete response would mean that both serum protein electrophoresis and the immunofixation have to be negative.
Now we’re going down in the red text, the nonquantitative ASO PCR of the bone marrow—that’s what we call MRD negative if you don’t pick up myeloma cells there. And if we pick it up, we call it MRD positive. We have further more sensitive technologies, quantitative ASO PCR of the bone marrow, high-quality flow cytometry of the bone marrow, and deep sequencing of the bone marrow. These technologies have more sensitive opportunities to detect residual cells. At this point, we don’t know if there could be patients that are cured or if we need to develop further sensitive technologies.
Nonquantitative ASO-PCR of bone marrow
1 x 108
1 x 106
Quantitative ASO-PCR of bone marrow High-quality flow cytometry of bone marrow Deep sequencing of bone marrow
MRD+
1 x 104
MRD-
Cure
Mailankody S, et al. Nat Rev Clin Oncol [Epub ahead of print]

11. Mos From MRD Assessment Mos From MRD Assessment
Relapse and OS of Pts in CR at Day 100 After HDT/ASCT, Based on FISH and MRD
100
100
Median: NR
Median: 83 mos 80 80 60 60
Progression (%)
Median:
23 mos
OS (%)
Median: 47 mos 40 40
Median: 21 mos
Median:
6 mos 20 20
ASCT, autologous stem cell transplantation; CR, complete response; FISH, fluorescence in situ hybridization; HDT, high-dose therapy; MRD, minimal residual disease; NR, not reached; OS, overall survival.
As I showed you before, patients that reach deep responses by conventional measures have better progression-free and overall survival. There have been studies focusing on patients that reach complete response, adding measures of MRD to see whether it’s possible to predict who’s going to have a longer or shorter both progression-free and overall survival. And here is a study from the Spanish study group showing that relapse and overall survival, meaning progression-free and overall survival, of patients at CR (complete response) Day 100 after an autologous stem cell transplant differs if you factor in MRD status as well as FISH markers. On the left, you see standard risk FISH with MRD negative on top and 83 months’ median progression-free survival. The high-risk FISH or MRD positive, either of these, that would translate into a median of 23 months. And for patients who have both high-risk FISH and who are MRD positive, they have a median of only 6 months’ progression-free survival. And on the right, you see the same pattern for overall survival with a median for the top group not reached, median 47 months for the middle group and 21 months for the high-risk group.
P < .001
P < .001 20 40 60 80
100 20 40 60 80
100
Mos From MRD Assessment
Mos From MRD Assessment
Standard-risk FISH + MRD negative (n = 58)
High-risk* FISH or MRD positive (n = 45)
High-risk* FISH + MRD positive (n = 7)
*t(4;14), t(14;16) and/or del(17p).
Paiva B, et al. Blood. 2012;119:

12. Impact of MRD-Negative Status on High-Risk and Standard-Risk MM Pts
1.0
P < .001
1.0
P < .001
0.8
0.8
0.6
0.6
PFS (Proportion)
OS (Proportion)
0.4
0.4
0.2
0.2
HR, high risk; MM, multiple myeloma; MRD, minimal residual disease; SR, standard risk.
Here is another slide by the Spanish study group, looking at the impact of MRD-negative status on high-risk and standard-risk myeloma patients. And as you can see, across the board—from left you have the progression-free survival and to the right you have overall survival—you see that those patients that are MRD negative with a standard-risk profile by cytogenetics have the best outcome, while MRD positivity with a high-risk cytogenetic profile corresponds with the worst outcome. 12 24 36 48 60 72 84 96 12 24 36 48 60 72 84 96
Mos Since MRD Assessment
Mos Since MRD Assessment
MRD negative/
standard risk
(n = 101)
MRD negative/
high risk*
(n = 19)
MRD positive/
standard risk
(n = 130)
MRD positive/
high risk*
(n = 22)
*t(4;14), t(14;16), and/or del(17p).
Rawstron A, et al. J Clin Oncol. 2013;31:

13. MRD Kinetics Discriminate Chemosensitive From Chemoresistant Pts
100 80 60 40 20
Median: 9 yrs
Progression (%)
Δ 6 yrs
Median: 5 yrs
MRD, minimal residual disease.
On this slide, we see how MRD status impacts the kinetic of the disease in terms of interpretation. You see MRD kinetics discriminate the chemosensitive from the chemoresistant patients. This graph shows time to progression in patients who are MRD negative and stay MRD negative, patients that are MRD positive that convert into MRD negativity, and patients that were MRD positive and stayed MRD positive. And the median is 9 years, 5 years, and 3 years.
P < .001
Median: 3 yrs
140
120
100 80 60 40 20
Mos From MRD Assessment
MRD-  MRD-
(n = 63)
MRD+  MRD-
(n = 84)
MRD+  MRD+
(n = 189)
Transplant-eligible pts included in the GEM2000 & GEM2005MENOS65 studies. Median follow-up: 5 yrs.
Paiva B, et al. Blood. 2010;116. Abstract Rawstron A, et al. J Clin Oncol. 2013;31:

14. Mos From MRD Assessment Mos From MRD Assessment
Pts in CR After HDT/ASCT With Persistent MRD* Have Similar Outcomes to Pts in nCR/PR
100 80 60 40 20
100 80 60 40 20
MRD- vs CR/MRD+: P < .001
MRD- vs CR/MRD+: P < .001
MRD- median OS: 12 yrs
MRD- median TTP: 5 yrs
Progression (%)
OS (%)
ASCT, autologous stem cell transplantation; CR, complete response; HDT, high-dose therapy; MRD, minimal residual disease; nCR, near complete response; PR, partial response; OS, overall survival; TTP, time to tumor progression.
Patients in complete response after a high-dose therapy with an autologous stem cell transplant with persistent MRD, they have similar outcomes to patients in near complete response or partial response. Here you see on the left progression-free survival and on the right overall survival. So the MRD-negative patients, they are the ones that are standing out.
140
120
100 80 60 40 20
140
120
100 80 60 40 20
Mos From MRD Assessment
Mos From MRD Assessment
MRD- (n = 213)
CR but MRD+ (n = 88)
nCR/MRD+ (n = 54)
PR (n = 131)
Transplant-eligible pts included in the GEM2000 & GEM2005MENOS65 studies. Median follow-up: 5 yrs.
*Measured by less sensitive 4-color flow.
Paiva B, et al. Blood. 2010;116. Abstract Rawstron A, et al. J Clin Oncol. 2013;31:

15. Agenda Why MRD Testing for Pts With Multiple Myeloma?
Assessing and Monitoring MRD Response to Therapy
Using MRD Results to Improve Outcomes for Myeloma Pts
MRD, minimal residual disease.
Now, we are entering the second part of this presentation: assessing and monitoring MRD response to therapy.

16. MRD of Bone Marrow by Flow Cytometry = Ruling Out Evidence of Abnormal Cells
MRD of bone marrow based on blind biopsies
Limitation on any test performed
Potential for false negatives
MRD, minimal residual disease.
As you probably know, minimal residual disease of the bone marrow by flow cytometry is a matter of ruling out evidence of abnormal residual cells that are there, based on a blind bone marrow biopsy with an aspirant. So that’s a limitation of any test that we do; whether we use cell-based or molecular-based tests, we are still relying on blind biopsies, so we don’t know to what degree that potentially impacts the results. If the disease is heterogeneous in the marrow, it could potentially give us false negative results.

17. Flow Cytometry-Based MRD Testing in Myeloma
Flow cytometry relies on differentiation of malignant from normal plasma cells based on cell-surface marker expression differences
Key standard markers: CD38, CD19, CD56, CD45, and CD138
Additional markers to consider: CD27, CD81, CD117
MRD, minimal residual disease.
Defining abnormal plasma cells in the bone marrow includes a lot of work that the flow lab has to do. Several markers are considered to be the standard markers, including CD38, 19, 56, 45, and 138. Also additional markers, as shown on this slide, CD117, 27, and 81, are included, and there are additional other markers that people have proposed to differentiate the normal from the abnormal cells.
Mailankody S, et al. Nat Rev Clin Oncol [Epub ahead of print]

18. 2-Tube 8-Color Flow Cytometry Panel: Targets
Fluorophore
Target Cell-Surface Marker in Tube 1
Target Cell-Surface Marker in Tube 2
FITC
CD38 PE
CD56
PerCP-Cy5.5
CD45
CD229
PC7
CD19
APC
CD117
clgk
APC-C750
CD81
clgλ
V450
CD138
BV510
CD27
On this slide, you see 2 sets of markers in two 8-color panels that have been proposed by the Spanish study group, and using this panel of markers, one would be able to rule out evidence of minimal residual disease in a manner that has been proven to be very, very solid. And this is acknowledged by the International Myeloma Working Group as a strategy to reach consensus for the purpose of MRD testing in multiple myeloma.
Mailankody S, et al. Nat Rev Clin Oncol. 2015;[Epub ahead of print].

19. Flow Cytometry Helps Predict Outcomes Posttransplant
Progression-Free Survival
Overall Survival
1.0
MRD+ not CR MRD+ CR MRD- not CR MRD- CR
1.0
0.8
0.6
0.4
0.2
0.8
0.6
Probability of PFS
X23 = P < .001
Probability of OS
MRD+ not CR MRD+ CR MRD- not CR MRD- CR
0.4
X23 = P = .0385
0.2
CR, complete response; MRD, minimal residual disease; OS, overall survival; PFS, progression-free survival.
Using flow cytometry in patients that are in complete response, vs not complete response, has been investigated by multiple investigators around the world. Here’s a study that was recently published by Dr. Rawstron, from Leeds in the UK, showing patients that were MRD positive by flow, not in CR, and then you can see MRD positive in complete response, MRD negative not in complete response, and the best category, obviously, MRD negative in complete response. And you can see on the left the progression-free survival and on the right overall survival. Again, a deep response translates into a better outcome, both in terms of progression-free and overall survival, here illustrated by flow as the tool for MRD detection. 12 24 36 48 60 72 84 96 12 24 36 48 60 72 84 96
Mos Since MRD Assessment
Mos Since MRD Assessment
Rawstron AC, et al. J Clin Oncol. 2013;31:

20. Molecular MRD = Looking for Abnormal Cell Content
Searching DNA from cells extracted through bone marrow aspirant
Research is ongoing into “liquid biopsies,” ie, detecting free-circulating DNA in the bloodstream
Varied technologies available for molecular MRD assay
More sensitive than standard flow cytometry, but more labor-intensive
MRD, minimal residual disease.
I also would like to emphasize that there are molecular tests available, and using a molecular test, one is looking for abnormal cell content. For example, one can look for DNA that can be extracted from cells that have been captured through a bone marrow aspirant and also there is ongoing research trying to identify free-circulating DNA in the bloodstream. At this point, that is not yet available for standard clinical care, but for the bone marrow there are technologies available allowing sequencing of the VDJ sequence for the determination of MRD positivity or MRD negativity. And those technologies are more sensitive than the standard flow cytometry test, but they also include more work, and all these factors have to be taken into account when deciding which platform to use.

21. Available Molecular MRD Assays
ASO-PCR for VDJ rearrangements
High throughput VDJ sequencing
High throughput exome sequencing
ASO-PCR, allele-specific oligonucleotide polymerase chain reaction; MRD, minimal residual disease; VDJ, variable diversity joining.
Available molecular MRD assays for myeloma at this point include ASO PCR for VDJ rearrangement, high-throughput VDJ sequencing, and high-throughput exome sequencing. The difference between 2 and 3 is that the VDJ sequencing is only focusing on the VDJ sequence, while the exome sequencing has no particular restriction of focus, so it includes the whole sequence for exome.

22. MRD by High-Throughput Sequencing Predicts Prognosis in Pts With CR
Quantitative; with amplification and sequencing of immunoglobulin gene segments using consensus primers for IGH-VDJH, IGH-DJH, and IGK
TTP (CR Pts)
100
MRD- MRD+ Threshold: 10-5
n = 26 80 60
TTP (%)
MRD stratifies the CR population into 2 groups with strikingly different prognoses
CR, complete response; IGH-DJH, immunoglobulin heavy-chain incomplete; IGH-VDJH, immunoglobulin heavy-chain locus complete; IGK, immunoglobulin κ locus; MRD, minimal residual disease; TTP, time to tumor progression.
In a recent study by the Spanish study group that has done most of the work at this point using flow cytometry, they also evaluated MRD status by high-throughput sequencing of the VDJ sequence, and they showed that it predicts prognosis in patients with complete response. Here, as you can see, MRD negative in orange and MRD positive in blue. And they were able to show it was a highly significant difference for progression-free survival. This assay is based on 3 primers: IgH for VDJH and IgH for DJH and Ig kappa. 40
n = 36 20
P = .0009 50
100
150
Mos
Martinez-Lopez J, et al. Blood. 2014;123:

23. Comparison of MRD Assessment Techniques in Pts With MM
Parameter
ASO-PCR
VDJ Sequencing
Exome or Genome Sequencing
Flow Cytometry
Universal assay
No (pt-specific primers)
Yes
Sensitivity*
1 in 105
Unknown
1 in 104 to 10†
Sample source
BM aspirate
BM aspirate or peripheral blood
Sample quality assessment No
Sampling error
Likely
Can be overcome by using peripheral blood
Clonal evolution
Not detected
Limited detection
Detectable
Turnaround time
Days
1 wk
Days to wks
Hrs
Interobserver variation
Substantial
Clinical benefit associated with MRD- status
Improvements in PFS and OS
ASO-PCR, allele-specific oligonucleotide polymerase chain reaction; BM, bone marrow; MM, multiple myeloma; MRD, minimal residual disease; OS, overall survival; PFS, progression-free survival; VDJ, variable diversity joining.
I already talked about different options and the fact that they have strengths and weaknesses and, as you can see on this slide, looking at different types of parameters on the left, such as the access to a universal assay, sensitivity, sample source, sample quality assessment, sampling error, possibility to study clonal evolution, the turnaround time, the interobserver variation, and the clinical benefit associated with MRD-negative status, you can see for each of these technologies how they are similar or how they differ.
I think that the VDJ sequencing, which is the second from the left, and flow cytometry are probably the 2 most interesting technologies at this point. You can see that both of them have universal assays. Flow cytometry and VDJ sequencing either have the same sensitivity if flow cytometry’s optimized or VDJ sequencing is more sensitive. There are differences for the quality assessment and also the flow has a limitation that it cannot study clonal evolution. Importantly, the turnaround time differs; flow is hours, while VDJ sequencing could take up to a week.
*Expressed as the minimum cell sample size required for detection of 1 clonal cell. †4-6 color flow cytometry (1 in 104); 8-10 color flow cytometry (1 in 105).
Mailankody S, et al. Nat Rev Clin Oncol. 2015;[Epub ahead of print].

24. Agenda Why MRD Testing for Pts With Multiple Myeloma?
Assessing and Monitoring MRD Response to Therapy
Using MRD Results to Improve Outcomes for Myeloma Pts
MRD, minimal residual disease.
Now I’m switching over to the third and last part of this presentation: using MRD status to improve outcomes for myeloma patients.

25. Survey of Flow Cytometry–Based MRD Testing in Myeloma
Surveyed directors of flow cytometry laboratories at 30 major US medical institutions with active myeloma programs
26 institutions responded
Number of myeloma specimens per yr for flow cytometry
> 100 specimens: 19 institutions
specimens: 2 institutions
NA: 5 institutions
Perform myeloma MRD
Yes: 11 institutions
No: 15 institutions
MRD, minimal residual disease; NA, not available.
We recently conducted a survey of flow cytometry–based MRD testing in multiple myeloma, and we reached out to 30 major United States medical institutions with an active myeloma program. Within a week, 26 institutions responded to us. We asked them many questions. Some of them are included on this and the following slides. The first question we asked was, “What’s the number of myeloma specimens you evaluate each year for flow cytometry for the purpose of MRD testing?” As you can see, 19 institutions said more than 100 specimens. Two institutions said specimens. And 5 institutions could not really provide a number. We further asked if they used this information to perform formal MRD testing on these patient samples, and as you can see, 11 institutions said yes and 15 institutions said no.
Flanders A, et al. Blood. 2013;122:

26. Survey of Flow Cytometry–Based MRD Testing in MM: Colors and Gating
Number of “colors” in MRD flow analysis?[1]
4 colors: 0 institutions
5 colors: 2 institutions
6-9 colors: 9 institutions
Gating strategies: how you determine which cells will be studied?[1]
SSC, CD45: 1 institution
SSC, CD45, CD38: 2 institutions
CD38, CD138: 2 institutions
SSC, CD38, CD138, CD45: 6 institutions
67%
92%
Precision/concordance as determined by EMN[2]
EMN, European Myeloma Network; MM, multiple myeloma; MRD, minimal residual disease.
In order to gather more information on the actual MRD analysis using flow, we asked what number of colors they used in their flow machines and also what gating strategies they had. As you can see, 2 institutions used 5 colors and 6-9 colors was used by 9 institutions. And I do think in 2015, using 6-9 colors is probably considered to be a standard.
Now, gating strategies, meaning how do you hone in on which cells you study? There was a study done in Europe by Rawstron et al in 2008, and they conducted gating with different types of markers. And they let 2 laboratories do the same type of approach, and then they compared the results to look for concordance. And as you can see, on the very right, the 2 middle ones had only a 67% concordance, determined by the European Myeloma Network. And they recommended the combination of side scatter, CD38, CD138, and CD45, which had a 92% concordance. And for this particular survey, only 6 of the institutions used this same gating strategy.
1. Flanders A, et al. Blood. 2013;122: Rawstron AC, et al. Haematologica. 2008;93:

27. Survey of Flow Cytometry–Based MRD Testing in MM: Antigens Studied
European Myeloma Network recommendations[1]
Which antigens do you study in myeloma MRD?[2]
Abnormal plasma cells: CD38+, CD138+, CD45 -, CD19-, CD56+, CD20+, CD27 dim or -, CD28+, CD81 dim or -, CD117+, monoclonal k/l
Recent studies show that up to 30% normal PCs are CD19- or CD45- and 15% normal PCs are CD56+
CD38
CD138
CD45
CD19
CD56
CD20
CD27
CD28
CD81
CD117
Antigen
CD38
CD138
CD45
CD19
CD56
CD20
Ic k/l
CD27
CD28
CD81
CD117
#Labs 11 10 9 5 3
MM, multiple myeloma; MRD, minimal residual disease; PCs, plasma cells
Looking further into detail of which markers to use, the European Myeloma Network has recommended, as you can see on top in red, the markers that you absolutely need to have in order to determine abnormal vs normal plasma cells. And in green, markers that they recommend that you have in your lab. In the lower part of this figure, you see how the 11 laboratories that answered that they do MRD testing in myeloma pan out with regard to these markers. You can see that for CD38, 138, 45, and 19, 11 out of the 11 laboratories have these markers. Ten out of the 11 have CD56. Now, for the green ones, you see that the numbers go from left to right from 9 all the way down to 3 laboratories using these markers. This is very important. For example, there are studies recently done showing that 30% of normal plasma cells are CD19 negative or CD45 negative. Also, there are studies showing that 15% of normal plasma cells are CD56 positive.
So if one does not have the correct markers, one may falsely look for cells, calling them abnormal, or missing abnormal cells, calling them normal. It takes a lot of markers, and also one has to evaluate a lot of events. .
1. Rawstron AC, et al. Haematologica. 2008;93: Flanders A, et al. Blood. 2013;122:

28. Survey of Flow Cytometry–Based MRD Testing in MM: Events
Institution
Events Acquired in MRD Testing, n
Abnormal Plasma Cells Needed to Call MRD, Maximum n
Maximum Possible Sensitivity, %
Antigens Studied, n 1
3,000,000-4,000,000 20
0.0005 12 2
1,800,000 50
0.003 9 3
1,000,000
0.005 8 4
500,000 25 6 5 NA
300, ,000
30-35
0.006 7
250, ,000
0.01 10
100,000
0.02
Variable 11 30
12-26
Do not perform MRD testing
27-30
Did not respond
MM, multiple myeloma; MRD, minimal residual disease; NA, not available.
As I mentioned to you, the number of events that have been acquired really impacts the opportunity to rule out MRD in the sample. When we asked these institutions how many events they acquire before they provide an answer to the clinician, as you can see there’s a huge range. Institution number 1 looks through 3-4 million cells in the bone marrow, and they say if 20 or more of the cells have features of abnormal cells, then we will call it MRD positive. That leads to a maximum possible sensitivity of %. If you go further down on the list, you see how the number of events acquired differs and goes all the way down to 100,000. Institution number 8, they review 100,000 cells of the bone marrow. They also use 20 as the cutoff, so if 20 or more are positive, they would call it MRD positive. This takes them to a maximum possible sensitivity of 0.02%. So as you can see, the maximum possible sensitivity varies with a factor greater than 100.
Flanders A, et al. Blood. 2013;122:

29. Flow Cytometry–Based MRD Testing: By the Numbers
53-yr-old female with myeloma
Abnormal plasma cells at diagnosis: CD19-, CD45-, CD38 dim, CD20-, CD56+, CD81-, CD27 dim
Now MRD workup posttherapy
100,000 cells
500,000 cells
1 million cells
3 million cells
MRD, minimal residual disease.
As you saw on the last slide, the detection rate varies a lot based on the number of events you would go through in the bone marrow. Let’s put this in clinical context. Here is a 53-year-old woman with myeloma. The abnormal plasma cells at diagnosis are standard-looking, CD19 negative, 45 negative, CD38dim, CD20 negative, 56 positive, CD81 negative, and CD27dim. Now, we actually took the bone marrow aspirants from this patient, and we ran it for MRD testing in our machine. And let’s look on the very right. We let the machine count 3 million cells, and then applying the features of abnormal, as you can see on the very right, there are 30 abnormal plasma cells that were detected in this patient. So using 20 as the cutoff, this patient would be deemed to be MRD positive.
What we did now is that we reset the computer and we let the computer only look at the first 1 million cells. Although we had 3 million cells evaluated, we only looked at the first million. Now you see, 12 plasma cells were found to be abnormal. This patient would now be deemed to be MRD negative. And then again, we reset the computer and we counted the first 500,000 cells. Now there are 6 abnormal plasma cells. And lastly, on the very left, we counted only 100,000 cells and there are no abnormal plasma cells. So this illustration of one single patient shows that the number of events that you count will impact how you determine whether the patient has or has no detectable MRD in the bone marrow.
No abnormal plasma cells
6 abnormal plasma cells
12 abnormal plasma cells
30 abnormal plasma cells
Stetler-Stevensen A, et al. Unpublished data.

30. Gating strategy to analyze plasma cells (CD138+, CD38+)
MRD Status in NDMM Treated by CRd: Advanced Flow Cytometry of Bone Marrow
Gating strategy to analyze plasma cells (CD138+, CD38+)
Analyze 3-4 x 106 bone marrow cells
8-color flow panel*
CD38
CD138
CD19
CD20
CD56
CD45
CD27
CD28
MRD-: < 20 abnormal plasma cells detected
CRd, carfilzomib, lenalidomide, low-dose dexamethasone; MRD, minimal residual disease; NDMM, newly diagnoses multiple myeloma.
We applied these same approaches in a clinical series that we have conducted recently at the NIH. This study focused on MRD status in newly diagnosed myeloma patients treated with carfilzomib, lenalidomide, and dexamethasone, and we used advanced flow cytometry of bone marrow aspirant that was developed by the Spanish study group. We analyzed 3-4 million bone marrow cells and we used two 8-color flow panels, the same as I showed you on the previous slides.
*European Myeloma Network Gating Criteria.
Korde N, et al. ASH Abstract 2105.

31. MRD Status in NDMM Treated by CRd: Results
Among 27 nCR/sCR* pts tested by advanced flow, all 27 are MRD negative
Pt #7: Pre CRd: Abnormal PCs CD19-, CD45 dim, CD56dim+ 25
MRD negative 20
Flow
positive after 8 cycles 15
Pts
Post CRd: Normal PCs CD19+, normal CD45, CD56-
Flow
negative after 8 cycles
CR, complete response; CRd, carfilzomib, lenalidomide, low-dose dexamethasone; MRD, minimal residual disease; NDMM, newly diagnoses multiple myeloma; nCR, near complete response; PC, plasma cell; PR, partial response; sCR, stringent complete response; SD, stable disease; VGPR, very good partial response.
Study results were presented at ASH 2014, the MRD status in newly diagnosed myeloma patients treated with the combination of carfilzomib, lenalidomide, and dexamethasone. Among 27 patients reaching a near complete response or a stringent complete response tested by advanced flow, all these patients were MRD negative. You also see that patients that were in the stable disease partial response or very good partial response that there are positive patients after 8 cycles.
And there is one patient who is in the very good partial response who is negative by flow. Why is that? We don’t have the final answer, but our interpretation is that the M-spike, which in this case was IgG kappa, was circulating in the bloodstream and remained detectable in the blood in the absence of cells that were not detectable by flow cytometry in the bone marrow. And there is scientific evidence that there is a reuptake of IgG immunoglobulin in the body after depletion of such releasing cells. So that’s the interpretation, but we need to look into this further. 10 5
CR/sCR
nCR
VGPR PR SD
*2 pts (1 sCR and 1 nCR) not available for interpretation.
Korde N, et al. ASH Abstract 2105.

32. Response Status in Pts With High-Risk Smoldering MM Treated by CRd
2.5
2.0
1.5
1.0
0.5
100 80 60 40 20
nCR or better
VGPR PR
Monoclonal protein 8 8 8 25 58 67 42 83 91
Mean Monoclonal Protein (g/dL)
Pts (%)
100 83 67 50 42
CRd, carfilzomib, lenalidomide, low-dose dexamethasone; MM, multiple myeloma; nCR, near complete response; PR, partial response; VGPR, very good partial response.
So, as I showed you on the previous slide, after we found prominent MRD-negative findings in newly diagnosed multiple myeloma patients treated with carfilzomib/lenalidomide and dexamethasone, we were motivated to carry out a pilot study, which I presented at ASH And as you can see on this slide, the average end spike at baseline was more than 2 g/dL. And as you can further see on the X axis, when you go from 1 all the way out to 8 combination cycles, you see that 100% of the patients reached a near complete response or better in this pilot study including 12 patients. 33 17 8 9
Baseline 1 2 3 4 5 6 7 8
Number of Treatment Cycles Administered
Best response and mean monoclonal protein levels, by cycle (cycles 1-6, N = 12; cycles 7-8, n = 11)
Landgren O, et al. ASH Abstract 4746.

33. MRD Status in Pts With High-Risk Smoldering MM Treated by CRd
Pt Number 1 2 3 4 5 6 7 8 9 10 11 12
After 8 cycles or achievement of a CR
Flow cytometry
Next-generation
sequencing
After 1 yr of extended dosing or achievement of a CR
CR, complete response; CRd, carfilzomib, lenalidomide, low-dose dexamethasone; MM, multiple myeloma; MRD, minimal residual disease.
Based on the finding that all the 12 out of 12 patients on this pilot study reached a near complete response or better after 8 combination cycles, and these are again the high-risk smoldering myeloma patients, we were of course interested in looking into MRD status. And as you can see on this slide, patient number 1 was positive for MRD both by flow cytometry and next-generation VDJ sequencing. Patient number 5 was negative for MRD status by flow cytometry but had positive uptake by the next-generation sequencing. And all these were after 8 cycles of complete response.
We, at this point, have only samples for a few individuals after 1 year of extended dosing with lenalidomide maintenance. And as you can see for patient number 2, the flow cytometry picked up detectable MRD status, while the next-generation sequencing is still negative. And for patients 3 and 4, the flow cytometry testing is negative at 1 year of maintenance. Key questions that remain to be answered with this study will be longitudinal monitoring of MRD status in high-risk smoldering myeloma and of course also to expand the sample size to see what these results pan out to be in the larger setting.
Positive for MRD Negative for MRD Data not available
MRD status in evaluable pts (N = 12) after 8 cycles and after 1 yr extended dosing with lenalidomide.
Landgren O, et al. ASH Abstract 4746.

34. Maintenance Therapy in MM: For Whom and How Long?
Addressing the question using MRD within clinical trials
Several potential drawbacks of maintenance therapy:
Unnecessary exposure to chemotherapy can lead to adverse events, increased costs, and potential secondary malignancies
Thus, a major step for researchers moving forward is to determine how long maintenance therapies should be administered
MRD+
Maintenance
HDT/ ASCT
Induction
Consolidation
Maintenance R
MRD-
No Maintenance
1st yr
2nd yr
3rd yr
Serial MRD monitoring
ASCT, autologous stem cell transplantation; HDT, high-dose therapy; MM, multiple myeloma; MRD, minimal residual disease.
One key clinical question regarding maintenance therapy is for whom and how long. So addressing the question in clinical trials, there are now efforts looking at monitoring of MRD in patients treated with maintenance or no maintenance if they’re MRD positive or MRD negative. These studies are ongoing, and at this point, we do not have the information available. But the studies are designed obviously to see whether we can stop therapy in MRD-negative patients or if we need to continue and for how long. And these results are urgently needed.
GEM2012MENOS65 (NCT ) & GEM2014MAIN
Kumar S. Clin Adv Hematol Oncol. 2014;12:

35. Change in MRD Status (%)
Impact of Thalidomide Maintenance on MRD Status in Pts With MM Post-ASCT 70 60 50 40 30 20 10
Thalidomide No maintenance
Change in MRD Status (%)
ASCT, autologous stem cell transplantation; MM, multiple myeloma; MRD, minimal residual disease.
Until we have the results from these on-going efforts based on newer drugs, the only information that we have available is based on studies that were based on older drugs. Here is one example, a study published by the British group, using thalidomide as maintenance. And if you look on the very left, you see in patients that were on thalidomide maintenance, and on no maintenance, that there is a difference in change of MRD status. There are patients that convert from MRD positive and they become MRD negative. And on the very right, you can see the patients that are no maintenance are going from MRD negative and become MRD positive.
So, there is clearly a change in both directions. Again, we need more data using more modern therapies with longer follow-up, and also importantly, we need to know if patients stay on maintenance, if the disease comes back if it is the same response as it would be in the absence of maintenance because ultimately we need to factor in the impact on overall survival.
Become MRD Negative
Remain MRD Negative
Remain MRD Positive
Become MRD Positive
Subset (n = 99) of trial pts randomized to thalidomide maintenance therapy or placebo after ASCT
Rawstron A, et al. J Clin Oncol. 2013;31:

36. MRD Status Affects Both PFS and OS
Study
Treatment Arms
Test Method
Outcomes (MRD- vs MRD+)
Paiva et al
(2008)
6 alternating cycles of VBMCP and VBAD, followed by HDT/ASCT (n = 577)
4-color flow cytometry
Median PFS: 71 vs 37 mos (P < .001) Median OS: not reached vs 89 mos (P = .02)
(2012)
6 cycles of VMP or VTP (n = 102)
Median PFS: not reached vs 35 mos (P < .02) Median OS: not significantly different
Korthals et al
Idarubicin or dexamethasone plus HDT/ASCT (n = 53)
ASO-PCR
Median EFS: 35 vs 20 mos (P = .001)
Median OS: 70 vs 45 mos (P = .04)
Rawstron et al
(2013)
CVAD or CTD plus HDT/ASCT (n = 378)
6-color flow cytometry
Median PFS: 28.6 vs 15.5 mos (P = .001)
Median OS: 80.6 vs 59.0 mos (P = .018)
Puig et al
(2014)
VBMCP or VBAD induction therapy plus HDT/ASCT or 6 cycles of VMP or VTP (n = 170)
VBMCP or VBAD induction therapy plus HDT/ASCT: median PFS: 54 vs 27 mos (P = .001); OS not significantly different 6 cycles of VMP or VTP: median PFS: not reached vs 31 mos (P = .029); OS not significantly different
Martinez- Lopez et al
VBMCP or VBAD induction therapy plus HDT/ASCT or 6 cycles of VMP or VTP (n = 133)
Next-generation VDJ sequencing
Median TTP: 80 vs 31 mos (P < .0001) Median OS: not reached vs 81 mos (P = .02)
ASCT, autologous stem cell transplantation; ASO-PCR, allele-specific oligonucleotide polymerase chain reaction; CTD, cyclophosphamide, thalidomide, and dexamethasone; CVAD, cyclophosphamide, vincristine, doxorubicin, and dexamethasone; EFS, event-free survival; HDT, high-dose therapy; MRD, minimal residual disease; OS, overall survival; PFS, progression-free survival; TTP, time to tumor progression; VBAD, vincristine, carmustine, doxorubicin, and dexamethasone; VBMCP, vincristine, carmustine, melphalan, cyclophosphamide, and prednisone; VDJ, variable diversity joining; VMP, bortezomib, melphalan, and prednisone; VTP; bortezomib, thalidomide, and prednisone.
Talking of progression-free and overall survival takes us back to the discussion of different types of technologies that one could use for MRD testing. Here is a summary of a couple of studies that have used flow, ASO-PCR, and also next-generation VDJ sequencing. Some of these results I have shown on the previous slides, and as you can see the median progression-free and overall survival is significantly better for patients that reach MRD negativity vs positivity, whichever platform that’s being used in this table.
Mailankody S, et al. Nat Rev Clin Oncol. 2015;[Epub ahead of print].

37. MRD and Drug Approval
“ we conclude that the introduction of well-validated protocols for flow cytometric MRD testing in multiple myeloma has the potential to pave the way for cell-based and molecular-based surrogate biomarkers as endpoints in clinical trials for multiple myeloma within the near future.”
MRD, minimal residual disease.
Beyond using MRD testing for the purpose of evaluating outcome in clinical trials, and ultimately to link it with clinical outcomes, one could envision that MRD could be used for drug approval. At this point, we are not yet there in myeloma, but there was a workshop at the FDA about a year ago. I was the lead author for this paper that was published in the American Journal of Hematology. And in the end of this paper, we wrote, “We conclude that the introduction of well-validated protocols for flow cytometric MRD testing in multiple myeloma has the potential to pave the way for cell-based and molecular-based surrogate biomarkers as endpoints in clinical trials for multiple myeloma within the near future.”
And I do think if we could generate data using these different MRD platforms and show that they are associated with clinical endpoints that would benefit patients because it would make drug development much shorter and would provide the patients with newer drugs much faster.
I think this is a major step forward. I also think that the impact from ongoing studies would be that we could implement MRD testing in the clinic on a day-to-day basis, and we could use it to guide therapy. We are not yet there, but I think we are moving there very fast, and I hope that we will be there in the near future.
Landgren O, et al. Am J Hematol. 2014;89:

38. A Few More Perspectives
MRD and myeloma biology?
MRD early vs late: does it matter?
MRD and maintenance: role?
MRD and high-risk SMM: even better?
MRD and drug approval: prime time?
MRD, minimal residual disease; SMM, smoldering multiple myeloma.
There are several questions that remain unanswered. We have touched on some of these today, but more work needs to be done. What about the role of MRD testing and the biology of myeloma? I showed you that high-risk disease has poor outcome, but in both high-risk and low-risk disease, MRD negative seems to do better compared to MRD positive. Does it matter if MRD happens early or if it happens later? That, we don’t know. What’s the role of MRD in maintenance? Can one use MRD negativity to guide if maintenance could be stopped or maybe could be restricted to a certain duration? Again, we don’t know the answer.
MRD and high-risk smoldering myeloma: Is that even better if a patient is started on therapy earlier on as high-risk smoldering on a clinical trial? At this point, high-risk smoldering myeloma in the standard setting is not recommended to be treated outside of trial, but if a patient on a trial reaches MRD negativity, is that even better? There is some data suggesting that. And lastly, as we already talked about, MRD as a potential surrogate endpoint for drug approval: Is it prime time? I think we are moving in that direction. We are not yet there.

39. Summary and Conclusions
In 2015, more and more myeloma pts achieve deeper responses to therapy
Deeper responses = better outcomes
In some pts, a deep response in not required for good outcome
In pts with poor outcomes, however, typically poor responses are observed
Highly effective therapies are becoming available for both newly diagnosed and relapsed/refractory myeloma
Clinicians need to better assess treatment response!
In conclusion, in 2015, more and more myeloma patients achieved deeper responses to therapy. Deeper responses translate into better clinical outcomes, both progression-free and overall survival. In some patients, a deep response is not required for a good outcome. However, in patients with poor outcomes, typically poor responses are observed. Highly effective therapies are becoming available for both newly diagnosed and relapsed and refractory myeloma patients. Clinicians need to better assess treatment responses.
I would like to thank you very much for your attention and for participating in this program on the role of minimal residual disease in multiple myeloma..

40. Go Online for More CCO Coverage of Multiple Myeloma
Multiple Myeloma ClinicalQuiz™ test your knowledge of optimal myeloma management with this interactive quiz game Interactive Case Challenges work through challenging patient cases and review the implications of emerging clinical trial data Text-Based Modules plus downloadable PowerPoint slides
clinicaloptions.com/oncology