Minimal Residual Disease

1. Minimal Residual Disease in
Chronic lymphocytic leukemia

2. Background
• Chronic lymphocytic leukemia (CLL) is an incurable
disease with a heterogeneous clinical course
• In the last decades, the aim of therapy for patients with
CLL has shifted from palliation to disease eradication
• Particularly for younger patients who account for almost a
third of the entire population with this disease
• Accurate quantitation of posttreatment residual
disease burden in chronic lymphocytic leukemia (CLL)
is prognostically relevant
• Achievement of complete remission (CR) is associated
with superior progression-free (PFS) and overall
survival (OS) in first-line and relapsed/refractory CLL
Thompson et al , Blood, 21 Jan 2016 X Vol 127(3)

3. Background
• Most patients who achieve CR have persistent, low-
level disease, which is not detected with routine
imaging and laboratory tests
• But can be demonstrated in bone marrow (BM) and/or
blood with more sensitive flow cytometry (FLC) and
molecular methods, so-called minimal residual
disease (MRD).
• These residual cells ultimately are responsible for clinical
relapse
• The timing of relapse depends on the quantity of residual
disease and kinetics of residual leukemia cell division.
Thompson et al , Blood, 21 Jan 2016 X Vol 127(3)

4. Minimal Residual Disease (MRD)
Patients will be defined as having a clinical
remission in the absence of MRD when they have
blood or marrow with less than one CLL cell per
10 000 leukocytes
Hallek, BLOOD, 15 June 2008
CLL: chronic lymphocytic leukemia

5. Schematic representation to illustrate the paradigm of the deeper the
response, the longer the (progression-free) survival (filled lines)
Tam CS, et al., Blood 2008

6. Increasing complete remissions in CLL
Increasing percentages of CR and MRD-CR after first-line treatment in CLL patients. The results
presented are after the administration of: chlorambucil; fludarabine(for which only clinical responses
are shown); alemtuzumab; the FC combination; FCR; FCM; RFCM(for which MRD-cases as assessed by
high-sensitivity methods are shown)
Percentages of MRD+ cases or those without MRD evaluation are depicted in blue; MRD-cases are in
green. Ghia,P, hematology, 2012

7. Clinical studies assessing MRD in CLL
Ghia,P, hematology, 2012

8. MRD: How to detect it
• MRD can be assessed by distinct approaches
• Based on PCR
• Based on flow cytometry
• For both approaches, different methods exist that
allow to reach strikingly different levels of sensitivity
(above or below the threshold of negativity of 10-4
Hallek, BLOOD, 15 June 2008

9. Timeline for development of MRD assays in
CLL
Ghia,P, hematology, 2012

10. Characteristics of an ideal MRD test
• In CLL, an ideal MRD assay should have the following
characteristics:
• Quantitative, with specific levels predictive of time-to-
clinical events
• Standardized methodology and interpretation of laboratory
data, to allow comparison between laboratories and clinical
trials
• Broadly applicable, regardless of pretreatment biological
characteristics
• High throughput, with rapid turnaround time; and relatively
noninvasive, technically simple, with objective and easy to
interpret results
MRD testing in CLL is potentially more complex and challenging
than for other leukemias, because current methodology focuses
on sampling low-level disease from blood or BM, which is likely
limiting
Thompson et al , Blood, 21 Jan 2016 X Vol 127(3)

11. Methodology for MRD detection
Method Sensitivity Quantitative
(Range)
Standardized Sample
Requirement
Influences on
Assay
performance
FLC CD5/19
kappa/lamda
10-2 No No Live leukocytes Number of
benign B cells
Quantitative
4-color FLC
10-4,10-5 Yes to 10-4 Yes ≥107 live
leukocytes*
Number of
available
leukocytes;
sensitivity
reduced in
paucicellular
specimens
Consensus
IGHV PCR
10-3 No Yes ≈106 total
leukocytes†
Number of
benign B cells,
clone
Thompson et al , Blood, 21 Jan 2016 X Vol 127(3)
Immunoglobulin heavy chain variable: IGHV, Complementarity determining region 3: CDR3
Real-time quantitative PCR: RQ-PCR

12. Methodology for MRD detection
Method Sensitivity Quantitative
(Range)
Standardized Sample
Requirement
Influences on
Assay
performance
ASO IGHV
RQ-PCR
10-5 Yes, to 10-4 Yes ≈106 total
leukocytes†
Clone (CDR3
region)
Nested ASO
IGHV PCR
10-6 No No ≈106 total
leukocytes†
Clone (CDR3
region)
Consensus
IGHV PCR
using
HTS
10-6 Yes, to 10-5 no 1 mg DNA for
baseline
sample,
6-60 mg for
follow-up†
Specific
sequences
may amplify
poorly in
mutated CLL
Thompson et al , Blood, 21 Jan 2016 X Vol 127(3)
Immunoglobulin heavy chain variable: IGHV, Complementarity determining region 3: CDR3
Allele-specific oligonucleotide: ASO, Real-time quantitative PCR: RQ-PCR
High-throughput sequencing: HTS

13. Consensus MRD protocol
Rawstron et al. Leukemia 2007

14. Importance Of The Sample Type
Blood generally can be used for making this assessment
except during the period within 3 months of completing
therapy, particularly for patients treated with
alemtuzumab, rituximab, and other antibodies targeting
CLL
Rawstron et al. Leukemia 2007

15. Importance Of The Sample Type
Rawstron et al. Leukemia 2007

16. Pros and cons
Rawstron et al. Leukemia 2007

17. CLL MRD Flow Cytometry
Rawstron et al. Leukemia 2007

18. Is it meaningful to reach MRD negativity?
Modified from Moreno et al, Best Pract 2010

19. Is it meaningful to reach MRD negativity?

20. Clinical data using MRD analysis

21. MRD assessment following combination
chemotherapy and CIT in first-line treatment
Studies where treatment-na¨ıve patients treated with combination
chemotherapy or CIT, where MRD analysis with a sensitivity of≥10-4 was
performed
• Achieving MRD-negative status was an independent predictor of
PFS after treatment with combination chemotherapy and both
low-intensity and moderate intensity CIT regimens
• Level of MRD was significantly associated with OS in all series
where it was reported.
Thompson et al , Blood, 21 Jan 2016 X Vol 127(3) CIT: Chemoimmunotherapy

22. MRD assessment postsalvage treatment and in treatment of
high-risk populations
Results of posttreatment MRD assessment and correlation with
PFS and survival in patients treated for relapsed/refractory or
high-risk CLL
MRD negative remission was associated with superior PFS after
FCR treatment, in patients with TP53 deletion treated with
alemtuzumab plus high-dose methylprednisolone, and with
salvage alemtuzumab monotherapy, independent of clinical
response category
Thompson et al , Blood, 21 Jan 2016 X Vol 127(3)

23. MRD assessment post–allogenic stem cell transplant
Clinical significance of posttreatment MRD analysis as
determined by a method with sensitivity of at least 10-4, after
first-line combination chemotherapy or chemoimmunotherapy
Thompson et al , Blood, 21 Jan 2016 X Vol 127(3)
Studies showed that MRD-negative status at 12months
post–allogenic stem cell transplant (allo-SCT) was
associated with superior event-free survival

24. Use of MRD results to guide
treatment decisions

25. Consolidation treatment (CT)
• Treats low-level residual disease
• Expected to otherwise produce clinical relapse
• Treatment at a time of lower disease burden could
reduce likelihood of developing drug resistance
Thompson et al , Blood, 21 Jan 2016 X Vol 127(3)

26. Consolidation treatment (CT)
• Consolidation with alemtuzumab after CIT
• Alemtuzumab consolidation after fludarabine and rituximab
improved response:
• 61% of patients in PR achieved CR (50% MRD negative)
• 43% of patients with MRD-positive CR became MRD negative
• But, Infection-related mortality was unacceptably high
• No PFS or OS benefit was seen
Consolidation with alemtuzumab after CIT is not
recommended
Thompson et al , Blood, 21 Jan 2016 X Vol 127(3) CIT: Chemoimmunotherapy

27. Consolidation treatment (CT)
• Lenalidomide consolidation after CIT with pentostatin,
fludarabine, and cyclophosphamide
• Improved responses in 24% of patients with measurable
disease
• 4 became MRD negative
• Time to next treatment appeared prolonged relative to
historic data
• Many patients required dose reduction, particularly due to
hematologic toxicity
The Cancer and Leukemia Group B 10404 study will
further clarify the utility of lenalidomide consolidation
after fludarabine-based CIT
Thompson et al , Blood, 21 Jan 2016 X Vol 127(3) CIT: Chemoimmunotherapy

28. Consolidation treatment (CT)
• Toxicity of any consolidation therapy must be balanced
against the benefit of improving depth of response
• Availability of potent novel therapies with more
favorable toxicity profiles
• May lead to reexamination of the feasibility of MRD-directed
consolidation after CIT
• Venetoclax and obinutuzumab
• Can induce MRD-negative CR
• Would likely be well tolerated as consolidation therapy.
• Other novel therapies, to be tested in this setting
• Immune checkpoint inhibitor mAbs
• Cellular therapy
Thompson et al , Blood, 21 Jan 2016 X Vol 127(3)

29. MRD-guided therapy based on
interim-analysis of MRD levels
No published study has prospectively used interim
MRD levels to guide therapeutic decisions

30. GCLLSGCLL8 study
• MRD analysis was performed in blood after 3 cycles of
FC/FCR;
• All patients subsequently received 6 cycles, regardless
of interim MRD results
• Those achieving MRD-negative status had similar PFS,
regardless of whether 3 or 6 cycles were required to
achieve MRD-negative remission
Böttcher et al. JCO 2012
Fludarabine and cyclophosphamide, Rituximab: FCR
Fludarabine and cyclophosphamide :FC

31. Eradication of bone marrow minimal residual
disease may prompt early treatment
discontinuation in CLL
by Paolo Strati, Michael J. Keating, Susan M. O'Brien, Jan Burger,
Alessandra Ferrajoli, Nitin Jain, Francesco Paolo Tambaro, Zeev Estrov,
Jeffrey Jorgensen, Pramoda Challagundla, Stefan H. Faderl, and William G.
Wierda
Blood
Volume 123(24):3727-3732
June 12, 2014

32. Survival by MRD status
Strati P et al, Blood Volume 123(24):3727-3732, June 12, 2014

33. Landmark analysis at MRD final assessment for
survival by MRD status
Strati P et al, Blood Volume 123(24):3727-3732, June 12, 2014

34. PFS and OS according to MRD/therapy groups
Strati P et al, Blood Volume 123(24):3727-3732, June 12, 2014

35. All data suggests
Early treatment cessation in patients who achieve MRD-
negative status after 3 cycles of FCR may be feasible
without compromising long-term disease control, but
this requires prospective study
Thompson et al , Blood, 21 Jan 2016 X Vol 127(3)

36. Use of MRD analysis as a clinical trial
end point
• Very long follow-up is required to determine whether
PFS after receiving novel agents or regimens is
superior to existing CIT regimens
• This may significantly delay new drug development
• Strong correlation between achieving MRD-negative
status and survival outcomes,
• MRD status is receiving consideration from regulatory
agencies as a potentially meaningful end point for clinical
trials.
• Major caveat
• MRD assessment cannot generally be used to assess the
efficacy of novel regimens such as ibrutinib as monotherapy
or in combination with rituximab
• These regimens rarely achieve MRD-negative status and are given
continuously until disease progression
Strati P et al, Blood Volume 123(24):3727-3732, June 12, 2014

37. Summary
• Existing treatment paradigms in CLL are undergoing significant
change
• Development of rational combinations and sequencing with novel
therapies will increase the number of patients achieving such
remissions
• The availability of sensitive and specific methods to quantify residual
disease may allow individualized therapy in the future.
• Patients who achieve early MRD-negative status may have treatment
de-escalated to limit toxicity
• Those initially failing to achieve MRD-negative remission could
receive consolidation or maintenance therapy with non–cross-
resistant agents
• Highly sensitive and specific MRD detection methodology must be
available
• Multicolor FLC is the current standard, more sensitive methods such
as IGHV-HTS may ultimately prove to have superior predictive power;
however, this will need to be determined prospectively.

38. Thank You