Focus on cellular MRD...Focus on cellular MRD •Clonality assessment: •CD19/5/K/L assessment is rapid, globally available and usually sufficient if >1% CLL •Not quantitative and

1. Adapted from: Ghia P. Hematology 2012; 2012:97–104; 2. Hillmen P, et al. J Clin Oncol 2007; 25:5616–5623; 3. Catovsky D, et al. Lancet 2007; 370:230–239; 4. Eichhorst BF, et al. Blood 2009; 114:3382–3391; 5. Eichhorst BF, et al. Blood 2006; 107:885–891; 6. Fischer K, et al. J Clin Oncol 2012; 30:3209–3216; 7. Hallek M, et al. Lancet 2010; 376:1164–1174; 8. Böttcher S, et al. J Clin Oncol 2012; 30:980–988; 9. Bosch F, et al. Clin Cancer Res 2008; 14:155–161; 10. Bosch F, et al. J Clin Oncol 2009; 27:4578–4584; 11. Seymour J, et al. Lancet Oncol 2017; 18:230-240.

Focus on cellular MRD

Andy C. Rawstron HMDS Leeds, UK

Cellular MRD analysis: clonality vs. phenotype

One dot ≈ one cell

One-colour (marker): CD19

There are 10% B-cells

Three colours

(CD19/κ/λ):


90% are either κ or λ

=> polyclonal

10% have no κ or λ

=> progenitors



Three colours

(CD19/CD5/CD20):

There are 10% B-cells:

85% are CD5+/-CD20++

10% are CD5-CD20+/-

5% are CD5+CD20+(wk)

Five colours

(CD19/CD5/CD20/κ/λ):


85% are CD5+/-CD20++

either κ or λ

=> polyclonal

10% are CD5-CD20+/-

no κ or λ

=> progenitors


Five colours

(CD19/CD5/CD20/κ/λ):


5% are CD5+CD20+(wk)

monoclonal

=> 0.5% neoplastic


Clonality assessments are not quantitative not reproducible MRD

CLL cells Normal B cells

CD19+ κ/λ

Consensus IgH-PCR

0.1% 5% 1.5

0.01% 5% 1.5

0.1% 0.1% 3.4

0.01% 0.1% 1.7

0.01% 0% >10

_ _

? _

_ _

+ +

+ +

_

?

_

+

+

Clonality assessment: poor specificity & sensitivity + detection limit depends on polyclonal background

Not detected

CLL

cel

ls %

of

leu

cocy

tes

0.01

0.1

1

10

100

0.0001 0.001 0.01 0.1 1 10 100 1000 10000

Kappa:Lambda ratio

>40% of polyclonal are MRDPOS

>5% of “monoclonal” are MRDNEG

Rawstron et al, Leukemia 2013 PMID 23041722


• Clonality assessment: • CD19/5/K/L assessment is rapid, globally available and usually sufficient if >1% CLL

• Not quantitative and less informative than disease-specific quantification

Flow Cytometry MRD Detection

• Develop assess for direct quantification of CLL cells

• Tested 50 combinations of 27 markers to identify expression patterns that discriminate CLL cells from normal B-cells • in both peripheral blood and bone

marrow • lowest inter-laboratory variability • lowest false positive rate

Plots are gated to show CD19+ B-lymphocytes CLL cells Normal B-cells

CLL cells

Mature Normal B-cells

B-progenitors Plasmablasts Plasma cells

Contamination




• Multi-parameter (≥6 marker) flow cytometry: • Rapid, globally available, quantitative measurement of typical CLL >0.01%

• The techniques for assessing MRD have undergone a critical evaluation and have become well standardized. Six-color flow cytometry (MRD flow), allele-specific oligonucleotide PCR, or high-throughput sequencing using the ClonoSEQ assay are reliably sensitive down to a level of <1 CLL cell in 10 000 leukocytes.

• Refinement and harmonization of these technologies has established that a typical flow cytometry–based assay comprises a core panel of 6 markers (ie, CD19, CD20, CD5, CD43, CD79b, and CD81).

• As such, patients will be defined as having undetectable MRD (MRD-neg) remission if they have blood or marrow with <1 CLL cell per 10 000 leukocytes.

• The blood generally can be used for making this assessment because the marrow will have detectable CLL when it is also found in the peripheral blood. However, there are therapies that preferentially clear the blood but not the marrow (such as monoclonal antibodies); therefore, it may be important to confirm that the marrow aspirate also is MRD-neg when the blood is found to be MRD-neg.

Minimal Residual Disease in CLL – iwCLL Guidelines 2018

Hallek M, et al. Blood 2008; 111:5446–5456. iwCLL, International Workshop on CLL; PCR, polymerase chain reaction.

• EMA CLL: “If MRD is not detectable in PB, it is mandatory to confirm MRD status in the BM”.

• EMA CLL: “MRD response rate is defined as the proportion of patients in the ITT population in whom a clinical complete response (CR) and undetectable MRD status in bone marrow is achieved”

• EMA myeloma: “It is recommended to use two different methods within the same trial”.

• FDA: “The sensitivity of the MRD assay should be at least 10-fold below the clinical decision-making threshold (the definition of MRD). For example, if MRD positive or negative is defined as detection of greater or less than 1x10-5 cells, respectively, then the assay should be optimized and validated to have an analytical sensitivity of at least 1x10-6”.

Minimal Residual Disease in CLL – EMA/FDA considerations

https://www.ema.europa.eu/en/documents/scientific-guideline/evaluation-anticancer-medicinal-products-man-appendix-4-condition-specific-guidance-rev2_en.pdf

https://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM623333.pdf

https://www.ema.europa.eu/en/documents/scientific-guideline/draft-guideline-use-minimal-residual-disease-clinical-endpoint-multiple-myeloma-studies_en.pdf

MRD Analysis Requires a Quantitative Method That Is Not Influenced by the Polyclonal Background with Prospective Validation

• Flow cytometry: Multiparameter assessment of CLL phenotype that is not clonality based

• PCR: Real-time quantitative ASO-PCR using patient-specific primers, not consensus primers

• High-throughput sequencing: Amplification of all B-cell sequences and enumeration of CLL-specific immunoglobulin gene

•

Development of ‘MRD’ as a regulatory endpoint:

Identify MRD endpoint in clinical trials

Develop assay

Standardisation of assay

Apply standardised assay prospectively

Apply to regulatory action

1995

2007

2012

2002

Flow Cytometry MRD Detection

Rawstron AC, et al. Leukemia 2016; 30:929-936; Rawstron AC, et al. Leukemia 2013; 27:142–149; Rawstron AC, et al. Leukemia 2007; 21:956–964.

Parameters Tubes Detection limit

Cells required for 0.01% – LoD

6 (4-colour) 4 0.005% 4–20 million

8 (6-colour) 2 0.001% 2–10 million

10 (8-colour) 1 0.001% 1–5 million

V450 V500 FITC PE PerCP-Cy5.5 PE-Cy7 APC APC-

H7

CD 5

CD 3

CD 81

CD 79b

CD 22

CD 19

CD 43

CD 20

Mary Sartor Sydney 10-colour flow assay

Much better reagents became available

Requests to include: CD45, CD38, CD160, CD200, CD305, ROR1

LoD, limit of detection. .

Rawstron AC, et al. Leukemia 2016; 30:929–936.

Antigen Typical expression (% positive vs control)

Control population in normal peripheral blood

Minimum relative fluorescence

intensity (preferred)

Positive Negative

CD5 Positive (>20%) CD3+ T-cells

CD19+ B-cells

>30 (>65)

CD20 Weak CD19+ B-cells

CD3+ T-cells

>10 (>20)

CD43 Positive (>20%) CD3+ T-cells

CD20+ B-cells

>15 (>40)

CD79b Weak CD20+ B-cells

CD3+ T-cells

>15 (>30)

CD81 Weak CD3+ T-cells

Granulocytes >12 (>20)

Weak indicates ≤20% reduction in fluorescence intensity relative to the median expression observed with a reference population of polyclonal B-cells using the same antibody. The minimum relative fluorescence intensity would provide separation of CLL cells from normal B-cells in >95% of cases with a preferred relative fluorescence intensity being the level at which 99% of cases have optimal separation of CLL cells from normal B-cells.

Requires ≥6 markers to achieve 0.01% –

available to most labs

The core panel must meet these 6 specifications, but

is flexible thereafter

Identifying a suitable reagent CLL cells

Normal B-cells Normal T-cells

Optimal

Acceptable

Kits that meet the ERIC specification are available

A = non-leucocytes (CD43-CD19-) B = mononuclear leucocytes C = CD19+ mononuclear leucocytes D = CD19-CD5+ mononuclear leucocytes

T-cells = B and D Granulocytes = Not A and not B CLL cells = B and C and E and F and G NormB = B and C and not (E and F and G)

ERIC specification for the detection of CLL cells in a normal background

• The minimum required antibody panel would comprise CD19 to identify B-cells and CLL cells, in combination with CD5, CD43, CD79b, CD81 and either CD20 or CD22 to discriminate CLL cells from normal B-cells. Other gating markers should be considered if CD19 alone would not be adequate to separate CLL cells from other leucocytes in the treatment setting, e.g. if an anti-CD19 therapeutic antibody was involved.

CD20 much better than CD22 for discriminating CLL There are no mature B-cells during anti-CD20 therapy no benefit to including both CD20 and CD22

8-CLR vs core markers: CD19, CD5, CD20, CD43, CD79b, CD81, +/- CD3 and CD22

Rawstron AC, et al. Leukemia 2016; 30:929–936. Rawstron AC, et al. Blood 2001; 98:29–35.


• CD3 may be informative if only 500,000 events or fewer have been acquired and the result is within 0.5log of the 0.010% threshold ( MRD threshold)

• CD3 is uninformative for results that are above 2x the limit of quantitation (50 events) or below the limit of detection (20 events).

• For example if 500,000 events are acquired and there are fewer than 20 events (<0.0040% CLL) or more than 100 CLL-phenotype events (>0.020% CLL), the inclusion of CD3 would have no significant impact on the results but if there are 20-100 CLL-phenotype events then the inclusion of CD3 may adjust the result below the 0.010% threshold (e.g. a result of 0.012% without analysis of CD3 may equate to a result of 0.0080% when CD3 is included in the analysis).


• CD3 may be informative if only 500,000 events or fewer have been acquired and the result is within 0.5log of the 0.010% threshold ( MRD threshold)

• CD3 is uninformative for results that are above 2x the limit of quantitation (50 events) or below the limit of detection (20 events).

• For example if 500,000 events are acquired and there are fewer than 20 events (<0.0040% CLL) or more than 100 CLL-phenotype events (>0.020% CLL), the inclusion of CD3 would have no significant impact on the results but if there are 20-100 CLL-phenotype events then the inclusion of CD3 may adjust the result below the 0.010% threshold (e.g. a result of 0.012% without analysis of CD3 may equate to a result of 0.0080% when CD3 is included in the analysis).

• Any benefit of CD3 is further reduced when additional CLL-specific markers such as ROR1 are included in the analysis


• It is acceptable to include any additional markers to the core panel if they do not compromise the separation of CLL cells from normal B-cells provided by the core panel markers.

Flow Cytometry for MRD detection

Rawstron AC, et al. Leukemia 2016; 30:929-936;

Rawstron AC, et al. Leukemia 2013; 27:142–149;

Rawstron AC, et al. Leukemia 2007; 21:956–964.

Leeds current: CD19 gating CD19 BB700 ERIC core markers ROR1 (+ HLADR)


• Ideally, a sample prior to treatment or with detectable disease should be evaluated to determine if the core panel is sufficient, i.e. >95% of CLL cells can be separated from normal B-cells by two or more markers from the core panel.



0

0.5

1

1.5

2

2.5

3

Baseline M1 M6

Expression relative to screening

0

0.5

1

1.5

2

2.5

3

Baseline M1 M6


0

0.5

1

1.5

2

2.5

3

Baseline M1 M6


0

0.5

1

1.5

2

2.5

3

Baseline M1 M6


CD5 CD43 ROR1 CD200



• In addition it is preferable to evaluate a sample ~ 1 month after starting inhibitor therapy to determine if there are changes in expression profile that may affect residual disease detection





• ERIC recommended core markers CD19, CD5, CD20, CD43, CD79b, CD81 • Additional markers useful with inhibitor treatments, particularly ROR1


• The use of statements such as MRD-positive or MRD-negative can be poorly informative because the threshold for MRD detection is not fixed. Therefore the report should indicate the limit of quantification, (defined as 100* 50 / total number of cells analysed) for cases with detectable residual disease, and the limit of detection for cases with no detectable disease (defined as 100* 20/ total number of cells analysed, assuming the laboratory has demonstrated an appropriate limit of blank).

Guidelines on the validation of cell-based assays

• Sensitivity • Either: “lowest signal detectable above

background” • Or: “true positive / true positive + false

negative”

• Limit of Blank (LOB) = highest signal in the absence of measurand, calculated as mean (blank) + 1.645 SD (95% of negative values are below this limit)

• Limit of Detection (LOD) = level at which 95% of samples with low level of measurand are detected above the limit of blank, calculated as LOB + 1.645 SD

• Limit of Quantitation (LoQ) = lowest level of measurand that can be reliably detected and whose total error (bias + Imprecision) meets a desired criterion for accuracy (clinical utility)

Clinical and Laboratory Standards Institute (CLSI) has published the guideline EP17, Protocols for Determination of Limits of Detection and Limits of Quantitation. UKAS ISO15189 guidelines


• The minimum population size for reproducible detection of CLL cells in a multiparameter analysis has been demonstrated to be 20 events 3,4. The LoD can be defined as 20/total leucocytes, i.e. a LoD of 0.010% requires acquisition of at least 200,000 leucocytes.

• The minimum population size for reproducible quantification of CLL cells in multiparameter analysis has been demonstrated to be 50 events 3,4. The LoQ can be defined as 50/total leucocytes, i.e. a LoQ of 0.010% requires acquisition of 500,000 leucocytes.

• Each laboratory should confirm that they achieve the same limit of detection by testing at least twenty normal samples and demonstrating a limit of blank below ten (<10) events. More extensive testing would be required to validate a lower LoD/LoQ

Case 8

Case 8 Limit of detection = 0.016% (20/123101) Limit of quantitation = 0.041% (50/123101)


• Examples:

• If there are 80 CLL events detected in a total of 1 million total cells, the report would state “CLL cells = 0.0080% (limit of quantitation 0.0050%)”.

• If CLL cells are not detectable the report should state “CLL cells not detected (limit of detection 0.0020%).

• If CLL cells are detected at a level intermediate to the limit of detection and quantitation, e.g. 40 CLL events in 1 million total cells, the report should state “CLL cells detected below the quantitative range (0.0020 – 0.0050%).





• ERIC recommended core markers CD19, CD5, CD20, CD43, CD79b, CD81 • Additional markers useful with inhibitor treatments, particularly ROR1

• Detection limit is dependent on the number of cells (leucocytes) analysed

Documents

Focus on cellular MRD...Focus on cellular MRD •Clonality assessment: •CD19/5/K/L assessment is rapid, globally available and usually sufficient if >1% CLL •Not quantitative and