MRD techniques: differences between NGS and NGFcme-utilities.com/mailshotcme/Material for Websites...May 04, 2018 · Other factors affecting MRD sensitivity • Number of cells analyzed:

MRD techniques: differences between

NGS and NGF

Jill CORREUniversity Cancer Institute

Toulouse, FRANCE

Paris , 2018-05-04

No disclosure

Diagnosis(1012 cells)

(1010 cells)

(0 cell)

CR≥ 2 log

reductionof tumor

load

?

With current treatment, many patients reach CR. However, most of them relapse, reflecting a persistent disease

that cannot be detected by conventional methods.

Now we have definitively a clearer idea with MRD, even if

there is still more to learn.

Amongst patients achieving (s)CR, those having MRD negative have best clinical outcome (PFS and OS), whatever the technique

Flores-Monteiro et al, Leukemia 2017(PETHEMA GEM2012)

Avet-Loiseau et al, ASH 2015(IFM/DFCI2009)

P-value : p<0.0001

Negative (<10-6)

PositivePositive

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Pat

ient

s w

ithou

t pro

gres

sion

(%)

06

1218

2430

3642

48

p

MRD is a precision medecine tool that could help to adjust and personalize management of MM

HOW TO ASSESS MRD ?

Technical aspects

• Until now, response was evaluated on indirect immuno-biochemical markers. However :

- Non-secretory MM- Are all MM cells always able to secrete M protein during all disease course ?- Ig half-life may induce delay between response measure and reality- Metabolism of FLCs by the kidneys may impact urine assessment

• Perfect technique : Evaluate directly the tumoral compartment Stable during disease course Informative for all patients Highly sensitive Highly specific Reproducible Easy to perform in routine Quick Limited sample quantity Transportable sample Proven clinical utility Not expensive

• SENSITIVITY :

Probability of being tested positive when disease present prevent false negative

for instance : 1 tumoral cell in 1 000 000 cells analyzed = 10-6

Sampling quality is a crucial determinant of the sensitivity

• SPECIFICITY:

Probability of being tested negative when disease absent prevent false positive

Technical aspects

• Cellular-based : Next Generation Flow (NGF)

• Molecular-based : Next Generation Sequencing (NGS)

Two biological methods should be used


• Molecular-based : Next Generation Sequencing (NGS)


Normal plasma cells

Normal plasma cellsLess typical (<30%)

MM cells(all combinations are

possible)

CD38 + bright + low (80%)CD138 + +CD19 + - - (96%)CD45 + -/low - (73%)CD27 + low -/low (40-68%)CD81 + -/low (55%)CD56 - + + (60-75%)

CD117 - + (30-32%)cIg/ polyclonal clonal

Multiparametric Flow Cytometry (MFC)

Adapted from Flores-Montero et al, Cytometry Part B 2016

A panel is needed to confidently distinguish MM cells from normal plasma cells.

From Mailankody et al. Nat Rev Clin Oncol 2015

NGF Improved technology and consensus guidelines for standardization

of flow-based MRD

Stetler-Stevenson et al, Cytometry Part B 2016, Flores-Monteiro et al, Leukemia 2017

* Multiclone CD38 (not blocked by anti-CD38 MoAbs)

• Optimal panel: current Euro-Flow NGF method

• Bulk lysis sample processing: to consistently measure 5-10.106 cells per tube

• Novel analysis strategies: software algorithms whichautomatically detect MM cells to remove subjectivity

NGF

Flores-Monteiro et al, Leukemia 2017

All Plasma cells = 0.005% of all BM cells

MM cells = 127 cells = 0.001% of all BM cells

Others BM cells MM cellsNormal plasma cells


• Molecular-based : Next Generation Sequencing (NGS) or Deep Sequencing


Adaptive © 2017

3221144321 25544321 3

B and T Cells Undergo V(D)J Recombination to Generate Unique DNA Sequences

V(D)J recombination is undergone by B and T cells to increase diversity in the immune receptor repertoire

14

Additional diversity is introduced at junctions

Unique DNA sequences can be used to identify each T or B cell

V D J

Potential IgH diversity: ~1011

3 3

Abbas AK, et al. Cell and Molec Immuno. Philadelphia, PA: Elsevier. 2015:176-177. The clonoSEQ Assay is currently available as a CLIA-certified laboratory service. clonoSEQ is not currently FDA cleared by the FDA. clonoSEQ test results should only be used taking into account all available clinical information and should not be used as the sole determinant of patient management.

NGSClonal V(D)J rearrangement as a molecular marker of MM for one patient

MRD negative

MRD positive

DIAGNOSIS = CALIBRATIONclonotype identification

(patient specific)

FOLLOW UP = MRD evaluation residual clonotype

33 2 333 2 3

33 2 333 2 3

33 2 333 2 3

33 2 333 2 3

NGS• All possible rearrangements are amplified by highly multiplexed V and J

consensus primers (IgH (VDJ, DJ), IgL, IgL)

Multiplex Primers

V D J

V D J

• A second round of PCR adds sequences (barcodes and adaptors)

• The library is ready for sequencing

Myeloma

NGS

MRD

Myeloma

NGS

Myeloma

NGS

AMPLIFY HOUSEKEEPING GENES

NT = Total Number of Leukocytes

Myeloma

NGS



Myeloma

NGS



Myeloma

NGS



NGS vs NGF

• Sensitivity 10-6

• 2.106 cells (12µg of DNA)• Can be delayed (stored sample)• 10 days – 2 weeks• Baseline sample required• Applicable in 97% pts• Only two platforms, very few institutions• Not affected by mAb-based

treatment• …• …• Etc …

• Sensitivity 10-6

• if 20.106 cells• Fresh samples (<24-48h)• Few days• Not mandatory• Applicable in almost 100% pts• Many labs for MFC, less for NGF• Additional information about

immune recovery• …• …• Etc …

Use the most sensitive technique available

P<0.001

P=0.06

P=0.30

0

25

50

75

100

0 12 24 36 48 60

Time since MRD assessment

>=10-4

[10-5;10-4[ 1

[10-6;10-5[

<10-6

Avet-Loiseau et al, ASH 2015

Prog

ress

ion

free

surv

ival

<10-6

10-5 to 10-6

10-4 to 10-5

≥10-4

The highest sensitivity is the most discriminant for NGS…

Paiva et al, ASH 2017**

… and for NGF

A common pitfall for NGS and NGF : aspirate hemodilution

• Frequent +++ in MM

• Not a problem for genomic assessment since plasma cells are sorted, but for MRD assessement, can induce false negative results

• Recommandations * : - use the first aspiration- draw 2mL of bone marrow (never more than 4-5mL)- collect multiple aspirations with the needle redirected for each aspirate

* Kumar et al, Lancet Oncol 2016**

A common pitfall for NGS and NGF : aspirate hemodilution

- NGS : additional analysis required = smear from tube used for MRDHemodiluted sample if megakaryocytes absent and erythroblasts < 5%

• Is extent of hemodilution assessable ?

- NGF : global bone marrow cell analysis can be done with the panelHemodiluted sample if erythroblasts, mast cells… do not reach the normal BM range

Flores-Montero et al, Leukemia 2017

YES, assessable but more complicated than it soundsand we need to standardize it

• « Patchy » disease i.e. heterogeneous distribution of MM cells within the BM imaging-guided biopsy ?

• Extramedullary disease : - relapse +++- Paiva et al, ASH 2017 : 3% of patients MRD-negative relapsed

all with extramedullary disease

Other factors affecting MRD sensitivity

• Number of cells analyzed:

- NGF : 20.106 cells(specificity : 20 MM cells must be detected to identify MRD)

- NGS : 2.106 cells (specificity : clonotype defined by the presence of ≥ 2 identical sequencing reads)

Requested to reach10-6 sensitivity

• « Patchy » disease i.e. heterogeneous distribution of MM cells within the BM imaging-guided biopsy ?

• Extramedullary disease : - relapse +++- Paiva et al, ASH 2017 : 3% of patients with MRD negative

Other factors affecting MRD sensitivity

• Number of cells analyzed:

- NGF : 20.106 cells(specificity : 20 MM cells must be detected to identify MRD)

- NGS : 2.106 cells (specificity : clonotype defined by the presence of ≥ 2 identical sequencing reads)

Requested to reach10-6 sensitivity

Kumar et al, Lancet Oncol 2016**

IMWG response criteria and MRD assessment in MM

Towards standardized reporting of resultsfor biological MRD

May be reported :

- Technique used

- Nb of cells analyzed

- Level of achievied sensitivity

- Extent of hemodilution

- … ?

Conclusions

• Use the most sensitive available technique

• NGS and NGF are equivalent, if 10-6 sensitivity is reached

• Need to prospectively compare both in clinical trials

• Experience is MANDATORY +++

• Should be performed in accredited labs, with an accredited technique

• Quality sample is of crucial importance : MRD results must be taken into account only if sample is representative of BM

Paiva et al, Blood 2014

• Implication of MRD in patient managment has to be established

Questions

• Depth of response and biology are independent prognostic factors :

- What is the impact of MRD according to risk and molecular subgroups ?

- Does MRD negativity is needed for all patients to achieve long term survival ?

• MRD in blood ?

- Advantages of liquid biopsy patchy distribution / extramedullary disease / hemodilution much less invasive

- Feasible by NGS with circulating tumoral DNA

- Enough sensitive ?

Need comparative prospective studies between BM and blood

Questions

Many thanks to all my colleaguesUnité Génomique du Myélome / Equipe 13 Pharmacogénomique du Myélome

Hervé Avet-LoiseauLudovic MartinetBettina CoudercAlice CleynenMichel AttalMurielle RousselBenjamin HébraudSabrina MaheoLaure BuissonNadège Carrié-ConstantinMarie-Véronique JoubertDo Souto Ferreira Laura

Céline MazzottiAudrey BeaufilsSandy BernouOlivier BoulbesJean-Michel HerreraBruno La CollaStéphanie LaffaureEmmanuelle Le TrionnaireLaurine MartinCharlotte ThéralMarine CuisinierCharlotte Avet-Loiseau

corre.jill@iuct‐oncopole.fr

Comparing NGS and NGF in 31 patients

Flores-Montero et al, Leukemia 2017

• Applicability 100% NGF vs 87% for NGS* (*with kit 2.0, almost 100%)

• Around 30% of discrepancy in low MRD levels(<10-4)

Most cases : MRD NGF+ and NGS- HSM ?

Deserves further investigations.

Oligoclonality and NGS ?• MM is a clonal disease originating from the transformation process of a single

plasma cell : myeloma cells are traditionally thought to have one clonal Iggene sequence that remains stable throughout the course of the disease.

• Oligoclonality (multiple clonotypes) at diagnosis in around 15% MM patients- 2% (11/620) with unrelated Ig sequences indicating the presence of two

independent myelomas(8/11: sample available at diagnosis and post-treatment 4/8 : relative frequencies dramatically different)

- 13% (79/620) with related Ig sequences indicating that both myeloma clones are derived from a common ancestor that arose after the pro B cell stage when VDJ recombination is completed (evolution after the MM malignant lesions occur) :

- Either somatic hypermutation process and differed by only a few bases (around 4 mutations in the CDR3 region)

- Either same VDJ sequence associated with two distinct isotypes

• 3.4% (7/206) of patients MRD+ display clonal evolution as soon as 6 months after treatment, sometimes suggesting differential sensitivity

Munshi et al, ASH 2016

NGS limitations ?

• Some sequences are known to occur naturally at high frequency in healthy populations = Monoclonal B-cell lymphocytosis (7% of healthy subject over 45 yrs) :

may theorically lead to false positive

In practice we check if each sequence is suitable for subsequent MRD tracking : the rearrangement uniqueness affects

- its trackable feature- the sensitivity of the test(meaning that if the sequence is not so specific, more cells are requested).

• Lack of standardization ? NGS almost approved by FDA

Phenotype of MM propagating cells and NGF ?

• More closely resembles B cells than plasma cells ?

• Lack of data to make definite conclusion

Other techniques ?

• NGS has supplanted ASO-qPCR because has overcome some of its limitations especially the low applicability+++, requiring patients specific primers, time consuming, less sensitive and specific.

• dPCR : highly sensitive and less expansive but same pb than ASO-PCR for design primer and undetectable MRD if mutations occur in CDR3 region.

• WES/WGS for MRD : technically feasible but not evaluated. More adapted to detect genomic heterogeneity and clonal evolution.

• Hevylite for MRD : increased IgAk/IgAl and IgMk/IgMl of the uninvolved istotype have been associated with longer PFS

- Reflect the degree of immune plasma cell recovery after ASCT which couldehance the capacity to control the disease for longer

- Reflect a functionnal consequence of MRD negativity- Most data must be collected in CR patients- Could be a complement to NGF/NGS

• Stringent CR : may be dropped, since more sensitive flow become common place (ratio may not be so important in sCR definition)

MRD techniques: differences between NGS and NGFcme-utilities.com/mailshotcme/Material for Websites...May 04, 2018 · Other factors affecting MRD sensitivity • Number of cells analyzed:

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