minimal residual disease - leukemia

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Perspectives

Has MRD monitoring superseded other prognostic factors in adult ALL?

Monika Bruggemann,1 Thorsten Raff,1 and Michael Kneba1

1Department of Hematology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany

Significant improvements have been

made in the treatment of acute lympho-

blastic leukemia (ALL) during the past

2 decades, and measurement of submi-

croscopic (minimal) levels of residual dis-

ease (MRD) is increasingly used to moni-

tor treatment efficacy. For a better

comparability of MRD data, there are on-

going efforts to standardize MRD quantifi-

cation using real-time quantitative PCR of

clonal immunoglobulin and T-cell recep-

tor gene rearrangements, real-time

quantitative-based detection of fusion

gene transcripts or breakpoints, and mul-

tiparameter flow cytometric immunophe-

notyping. Several studies have demon-

strated that MRD assessment in childhood

and adultALL significantly correlates with

clinical outcome. MRD detection is par-

ticularly useful for evaluation of treat-

ment response, but also for early assess-

ment of an impending relapse. Therefore,

MRD has gained a prominent position in

many ALL treatment studies as a tool for

tailoring therapy with growing evidence

that MRD supersedes most conventional

stratification criteria at least for Ph-

negative ALL. Most study protocols on

adult ALL follow a 2-step approach with a

first classic pretherapeutic and a second

MRD-based risk stratification. Here we

discuss whether and how MRD is ready to

be used as main decisive marker and

whether pretherapeutic factors and MRD

are really competing or complementary

tools to individualize treatment. (Blood.

2012;120(23):4470-4481)

Introduction

Treatment outcome in acute lymphoblastic leukemia (ALL) pa-

tients depends on a combination of multiple factors, such as

properties of the leukemic cells (eg, proliferative capacity, suscep-

tibility to drugs, and other escape mechanisms), host factors

(eg, general fitness and concomitant diseases, treatment compli-

ance, host pharmacodynamics and pharmacogenetics), and treat-

ment given to eradicate the disease. Many of intrinsic leukemia cell

and host factors have already been elucidated with immunologic

and molecular methods and are ongoing translated into providing

prognostic information (Table 1). Based on retrospective analyses

of large cohorts of patients, conventional pretherapeutic risk

criteria, including age, elevated white blood cell count at diagnosis,

adverse immunophenotypic features, and cytogenetic as well as

molecular aberrations provide the basis for upfront risk stratifica-

tion in current treatment protocols. It has to be acknowledged,

however, that these advances in our understanding of ALL biology

in the past have only to a limited extent been accompanied by

improved survival of adult ALL patients with relapse still being the

main clinical problem. The source of these relapses is the persis-

tence of minimal residual disease (MRD) that is undetectable by

standard diagnostic techniques. Several studies have shown that

detection of MRD in childhood and adult ALL is an independent

risk parameter of high clinical relevance, both in de novo and

relapsed ALL as well as in ALL patients undergoing stem cell

transplantation (SCT).1-14 Consequently, an increasing number oftreatment protocols use MRD as a tool for treatment stratification.

In addition, postremission MRD monitoring is also used to predict

an impending relapse and to start preemptive salvage treatment in

time. Therefore, MRD is not only a prognostic factor but chal-

lenges the traditional concept of defining remission and relapse.

Prerequisite for application of MRD for treatment tailoring is an

adequate, sensitive, and standardized MRD methodology. The

focus of this Perspective is therefore to highlight pros and cons of

the different MRD techniques, to present the published experience

of MRD analysis and MRD-guided treatment in adult ALL, and to

discuss the value of MRD in different clinical settings compared

with other prognostic factors.

Does methodology of MRD detection matter?Pros and cons of different techniques

MRD assessment relies on the identification of specific molecularor immunophenotypic markers on the leukemia cells. Flow cytom-

etry (FCM) is applied to detect combinations of cell markers that

are present on the leukemic but not on normal bone marrow cells.

PCR is used to detect leukemia-specific fusion transcripts

(eg, BCR-ABL) or clone specific immunoglobulin (Ig) or T-cell

receptor (TCR) genes.

Each of these techniques has its own strengths and limitations,

which are summarized in Table 2 and are described in more detail

within the next 3 sections.

Multiparameter flow cytometry

MRD measurement by FCM is based on the detection of leukemia-

associated immunophenotypes that can be used to distinguish themfrom normal hematopoietic cells. Leukemia-associated immunophe-

notypes usually describe a subpopulation of cells of a given lineage

at a particular differentiation stage with aberrant molecular expres-

sion patterns, asynchrony, and/or profound overexpression or

underexpression of molecules.15 Therefore, unlike molecular meth-

ods, FCM needs the identification of a cluster of events for

definition of MRD positivity. Using 4-color flow cytometry,

Submitted June 9, 2012; accepted September 6, 2012. Prepublished online as

BloodFirst Edition paper, October 2, 2012; DOI 10.1182/blood-2012-06-379040.

2012 by The American Society of Hematology

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leukemia-associated immunophenotypes can be identified in 90%

of B-precursor and 95% of all T-ALL patients reaching detection

limits of 103-104.5,16-22

The major advantage of FCM is its rapidity, which allows

reporting of quantitative results within 1 day. This is specifically

useful when MRD results are needed quickly to guide therapy. In

addition, FCM allows the simultaneous assessment of cell qualities

requisite for emerging targeted therapies in ALL.23,24 A potential

pitfall of the method results from similarities between leukemic

lymphoblasts and nonmalignant lymphoid precursors in various

phases of regeneration or chemotherapy-induced alterations that

may lead to false positivity.18,25 In addition, phenotypic shifts

frequently occur in leukemic cells during induction among others

because of steroid-induced gene expression modulation.23-28 In

times of targeted therapies (eg, anti-CD19, anti-CD20, anti-CD22,

or anti-CD33 treatment), also completely unknown marker shifts

may occur potentially influencing detectability of residual leuke-

mic cells. Therefore, interpretation of MRD FCM data requires a

deep understanding of the expression patterns of benign hemato-

gones and leukemic cells within different treatment phases andshould be restricted to reference laboratories, even if FCM is

broadly available in many hematolgoy centers.16

In addition, careful standardization of experimental setup and

interpretation is needed to obtain comparable MRD results in

multicenter studies, which is one topic of international study

groups.29-33 Additional efforts focus on improving the method and

its sensitivity through usage of more colors ( 8), inclusion of new

markers, possibly more specific for leukemic cells, and develop-

ment of new software for fast and easy automated data analysis.

PCR analysis of Ig and TCR gene rearrangements

Ig and TCR gene rearrangements represent fingerprint-like DNA

regions of individual lymphoid cells and its descendants, making

them attractive targets for clone specific PCR. Prerequisite is the

molecular characterization of leukemia-specific Ig/TCR gene rear-

rangements for each individual patient, which is possible for the

majority ( 95%) of B- and T-lineage ALL. Quantification of these

target genes is nowadays performed with quantitative real-time

quantitative (RQ)PCR that allows for a higher degree of automa-tion and sample throughput without the need of post-PCR manipu-

lation of samples.34-37 Similarly to rearranged immune genes, also

some leukemia specific translocations (in particular MLL rearrange-

ments) can be used as targets for DNA-based quantitative RQ-PCR

analysis after the rearrangement break point at the DNA level has

been characterized.38,39 Sensitivity is determined exactly for each

assay and generally reaches 1:104-1:105, which is 0.5-1.0 log

higher than for published FCM-based MRD assays.19,21,40

Right from the start, DNA-based MRD diagnostics has been

highly standardized through the efforts of the EuroMRD Group.

Guidelines are published for determination of linearity, sensitiv-

ity, specificity, and reproducibility for each clone-specific

assay.36 Usage of DNA as analytical sample allows long

shipping times, sample storage, and retrospective cumulative

PCR analyses of sample batches. Nevertheless, there are some

caveats, including clonal evolution of Ig/TCR genes, which may

lead to false negativity. It is therefore recommended to use at

least 2 independent targets. On the other hand, false-positive

MRD results cannot be completely excluded as massive regen-

eration of normal lymphoid progenitors might lead to low levels

of nonspecific amplification.41,42 In addition, the method needs

the time-consuming initial characterization of the leukemic

Ig/TCR rearrangements with a panel of different PCR and

sequencing reactions. However, recent advances in sequencing

technology (next-generation sequencing) will probably acceler-

ate this process in the near future and even add a new molecular

technology for MRD assessment.43

Table 1. Pretherapeutic factors associated with outcome in adult ALL81-83

Factor Category Prognostic impact Potential impact on targeted therapy

Age Worse outcome with advancing age84,85

White blood cell count at diagnosis B: 30 109/L (B) High WBC associated with poor prognosis84,85

T: 100 109/L (T)

Immunophenotype CD20 expression Conflicting data concerning prognosis74,75 Monoclonal antibodies

T versus B Independent prognostic significance (T-ALL with

better prognosis) mainly in early studies

84,85

Monoclonal antibodies

Bispecific T-cell engager nelarabineCytogenetics t(9;22)/ BCR-ABL Poor prognosis86,87 TKI

t(4;11)/MLL-AF4 Poor prognosis86,87

t(8;14)

Hypodiploidy*

Near triploidy

Complex karyotype

t(1;19) Conflicting data concerning prognosis88,89

High hyperdiploidy Better prognosis86

del(9p)

Specific molecular alterations JAKmutations Emerging significance of poor prognosis90 JAK inhibitors

IKZFDeletions/sequence

mutations

Emerging significance of poor prognosis91,92

CRLF2 overexpression Emerging significance (mainly childhood ALL) of

poor prognosis92

CRLF antibodies

ERG/BAALC expression Conflicting data concerning prognosis93,94

NOTCH1mutations Conflicting data concerning prognosis95,96 NOTCH1 targeting

indicates not applicable; CRLF2, cytokine receptor-like factor 2; ERG, v-ets erythroblastosis virus E26 oncogene homolog (avian); BAALC, brain and acute leukemia,

cytoplasmic; IKZF, IKAROS family zinc finger; JAK, Janus kinase; TKI, tyrosine kinase inhibitors; and WBC, white blood cell count.

* 44 chromosomes/leukemic cell.

5 abnormalities.

50 chromosomes/leukemic cell.

MRDAS MAIN PROGNOSTIC FACTOR INADULT ALL 4471BLOOD, 29 NOVEMBER 2012 VOLUME 120, NUMBER 23



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Ta

ble2.Featuresoftechniquescurrentlyem

ployedforMRDdetectioninALL

G

eneralstatements

Pros

Cons

Flo

wcytometry

Aberrantantigene

expression

ApplicableforalmostallALLpatients

Immunophenotypicshiftsofleukemiccells

Sensitivitydependsontechnology(numberofcolors)

andoncellinput

Availabilityofmethodologyinmanylaboratories

Expandedandalteredprecursor-B-cellcompartmentduring

regeneration

Rapid

Lowcellularityduring/afterinduction

Quantitative

Relativelyhighcosts(dependsoncellinp

ut,numberof

markers/colorsandulteriorcytometeru

se)

Additionalinformationonbenigncells

Limitedsensitivity/applicabilityusing3-to

4-colorflowcytometry

Additionalinformationonmalignantcells

6-colorflowcytometry:extensiveknow

ledgeandexperiencefor

sensitiveandstandardizedanalysisne

eded

Identificationandmonitoringoftreatmenttargetspossible

Nosampleasservationandretrospective

analysispossible(on-site

availabilityofexpertoperatorsnecessa

ry)

Growings

tandardization(mainlythroughoutEurope)

DN

A-basedRQ-PCR

TargetsmainlyIga

ndTCRgenerearrangementsbut

alsoMLLgenerearrangementsorSIL-TAL-deletions

ApplicableforalmostallALLpatients

Time-consumingmarkercharacterization

Highsens

itivity

Potentialinstabilityoftargets(clonalevolutionphenomena,therefore

needforpreferably2targets/patient)

Highdegr

eeofstandardization

Extensiveknowledgeandexperienceneeded

Accepted

anduniformlyuseddefinitionofquantitativerangea

nd

sensitiv

ity

Relativelyexpensive

Well-establishedstratificationtoolinvariousclinicalprotocols

Mostpublisheddataforevidence-basedtreatmentdecisions

Stabilityo

fDNA(multicentersetting,shipmenttime)

Possibility

ofsamplestorageandretrospective/batchanalysis

Qu

antitativeRT-PCRoffusion

transcripts

TargetsmainlyBC

R-ABLtranscripts(35%ofadult

B-cellprecursor

ALL)

Highsens

itivity

Usefulonlyinaminorityofpatients

Unequivocallinkwithleukemic/preleukemicclone

InstabilityofRNA

Stabilityo

ftargetduringcourseoftreatment

UncertainquantitationbecauseofunknownnumberofRNA

transcripts/cell(potentialdifferencesdu

ringcourseoftreatment)

Fast

Falsepositivityresultingfromcross-conta

mination

Relatively

cheap

Standardizationnecessary

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PCR analysis of fusion transcripts

Leukemia-specific gene fusions represent ideal targets for MRD

detection because they are linked to the oncogenic process andare therefore highly specific. Only few translocations (mainly

MLL rearrangements38 and SIL-TAL1 translocations) are ana-

lyzed on DNA level, the broadest routine clinical use is the

quantification of aberrant BCR-ABL mRNA transcripts in

Philadelphia (Ph)positive ALL.

Advantages of quantitative reverse transcriptase-PCR (RT-PCR)

to evaluate fusion transcripts in Ph-positive ALL include the high

sensitivity of up to 106 because of presence of multiple specific

mRNA-transcripts per cell. Furthermore, these transcripts allow

the usage of the same primer/probe-combinations for quantita-

tive RT-PCR of many patients, thus keeping the costs for the

individual analyses low. In addition, there is a broad experience

in BCR-ABL PCR assays because it is widely used for MRD

monitoring in CML. However, methodologies are not fully

comparable for both entities (dominance of minor break-point

transcript in Ph-positive ALL compared with major breakpoint

cluster region in CML, differences in relevant MRD thresholds

with higher sensitivities being required in Ph-positive ALL).

A crucial point for BCR-ABL PCR is the sample quality, which

is adversely influenced by long transport times resulting in

degradation of RNA. In addition, lack of standardization of RNA

extraction, cDNA synthesis, selection of housekeeping genes, and

variation in the way quantitative RT-PCR analyses are carried out

lead to substantial differences in results and carry the risk of false

negativity. In addition, false positivity cannot be ruled out

(eg, because of cross-contamination) and is observed in external

quality controls even among experienced laboratories. There areongoing efforts to standardize methodology and interpretation to

allow a better comparability of PCR results within different clinical

trials.44

Proposal for a uniform description of MRD results

An important step toward a better comparability of MRD data are

the standardized and uniform description of MRD results. Irrespec-

tive of the methodology used a testing result may be (1) negative,

(2) positive without being quantifiable because of the rarity of the

event, and (3) positive at a quantifiable level (Figure 1).

Concerning Ig/TCR-PCR, these results are defined by the

EuroMRD Group based on the general performance, linearity,

reproducibility, and background of the individual assay.36 In

addition, for BCR-ABL and FCM MRD, there are ongoing

international efforts to define generally accepted guidelines for

standardized analysis and interpretation of results.30,33,44

Based on these efforts, also a uniform MRD terminologyreferring to terms that are already established for cytomorphology

with definitions of complete MRD response, MRD persistence, and

MRD relapse seems feasible16 (Figure 1).

MRD for assessment of remission and relapsecompared with other prognostic factors

The 2 major applications of MRD in adult ALL are (1) the

assessment of response to initial therapy for definition of MRD-

based risk groups and (2) the subsequent monitoring of remission

patients to detect MRD reoccurrence and allow a preemptive

salvage treatment.

MRD for initial remission assessment

The most significant application of MRD for de novo ALL is the

sensitive assessment of treatment efficacy in patients reaching a

complete morphological remission, thereby refining initial risk

stratification. Compared with childhood ALL with reports on

several thousand patients,2,45 MRD in adult ALL has been studied

less extensively. Nevertheless, also in adult ALL large studies have

shown that initial MRD kinetics is highly predictive for outcome

(Table 3).

Ph-negative ALL. Several studies reported on the independent

prognostic value of MRD in adult Ph-negative ALL performingeither Ig/TCR-quantitative RQ-PCR1,3,9,46,47 or FCM12,22,48 (Table

3). Concordantly, different European study groups demonstrated

that MRD persistence measured at different time points 1-6 months

after initial diagnosis is associated with a poor prognosis.1,3,9,22,46-48

Bruggemann et al3 and Vidriales et al22 additionally identified a

small subset of patients with a very rapid tumor clearance

(low-level/undetectable MRD after 2 weeks of therapy) and an

excellent prognosis. The presently largest MRD study on adult

ALL was recently published by Gokbuget et al and analyzed MRD

in Ph-negative patients with standard risk (SR, n 434) and high

risk (HR, n 146) features.46 A complete MRD response after

induction 2 and/or consolidation 1 was associated with a compa-

rable clinical benefit irrespective of pretherapeutic risk factors.

MRD was the only parameter with significant prognostic impact in

Figure 1. Proposals for definition of MRD terms in

ALL.Data fr om Bruggemann et al16 with permission.




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multivariate analysis. The biologic differences between SR and

HR patients were reflected by the significant different propor-

tion of patients reaching a complete MRD response between SR

and HR patients with 20% point lower rates of MRD

negativity in HR patients.

Whether or not rare factors, such as t(4;11), may retain their

prognostic significance is hard to state as these patients are diluted

within the whole population. Cimino et al analyzed a limitednumber of adult MLL-AF4positive patients demonstrating the

prognostic impact of MRD also for this rare biologic entity. 49

Ph-positive ALL. In Ph-positive ALL, most published studies

focus on detection and quantification of BCR-ABL transcripts. In

the pre-tyrosine kinase inhibitors (TKI) era, the level of MRD after

induction and/or consolidation treatment turned out to be a

powerful indicator of prognosis, although data differed concerning

the discriminative value of different time points for MRD assess-

ment10,50 (Table 3). Introduction of TKI substantially changed

treatment outcome and MRD kinetics. In a study by Lee et al, MRD

assessment after induction chemotherapy did not retain its prognos-

tic significance when followed by imatinib treatment.51 Conversely,

a reduction of BCR-ABL transcript levels of at least 3 log after the

first 4-week imatinib therapy was identified as the most powerful

predictor of a better disease free survival (DFS) and overall

survival (OS) rate after SCT (4-year DFS 82.1% vs 41.7%,

P .009; 4-year OS 82.3 vs 48.6, P .007). These findings

indicate that poor response to chemotherapy may be compensated

by subsequent administration of imatinib. Leguay et al presented

data of the GRAAL AFR03 study that imatinib combined with

high-dose chemotherapy improved molecular remission rate before

transplantation and led to an improved outcome.52 In contrast,

Yanada et al failed to establish an association between an early

MRD response to imatinib combined chemotherapy and outcome

and concluded that relapse risk may depend on factors unrelated to

initial treatment response53 (eg, outgrowth of preexisting subclones

with resistance mutations54

).

Postremission MRD monitoring

A second important application of MRD is postremission monitor-

ing of patients reaching complete MRD response for early detec-

tion of an impending relapse.

Ph-negative ALL. Raff at al55 were the first to demonstrate

within the German Multicenter ALL Study Group (GMALL) trials

that molecular relapse defined as reconversion to quantifiable

molecular MRD positivity was followed by a clinical relapse after a

median time of 4.1 month in Ph-negative ALL. Remarkably,

low-level, nonquantifiable MRD was not necessarily related to a

subsequent relapse confirming this MRD value as a sort of gray

area.16

A recent update of the Raff et al study55

by Gokbuget et al46

confirmed the close correlation between conversion to quantifiable

MRD positivity and subsequent relapse: in 34 patients with

conversion to quantifiable MRD positivity (MRD relapse,

n 13 during first year of treatment, n 21 subsequently), the

probability of continuous complete remission (CCR) was 21%

9% at 5 years. If patients with MRD-based SCT in first CR

were excluded the probability of CCR was only 0% (5% 5%

at 3 years).

Ph-positive ALL. In Ph-positive ALL, several studies pub-

lished already in the 1990s showed a significant relationship

between conversion to MRD positivity and subsequent relapse in

the pre-TKI era.10,56-59 Yanada et al confirmed these findings also

for Ph-positive patients being treated with imatinib-combined

chemotherapy.53 In a prospective study on 100 adult patients with

Ph-positive ALL, 29 showed an MRD elevation in CR. Of these,

12 of 13 who had not undergone allogeneic (allo) SCT experienced

a relapse, whereas only 3 of 16 patients who underwent allo-SCT

relapsed. The authors concluded that an increase in MRD is

predictive of a subsequent relapse, but such patients can be

successfully treated with allo-SCT.

MRD-based treatment intervention

Treatment stratification according to initial MRD response

The objective of measuring MRD response to initial therapy is to

adjust treatment with the ultimate goal to improve outcome of

MRD-HR patients and to reduce toxicity in MRD-LR patients

without worsening their prognosis.

Several clinical trials on adult ALL implemented MRD into

their treatment stratification16: The PETHEMAALL-AR-03 trial12,60

focuses on HR Ph-negative ALL and passes on SCT in first

complete remission in case of standard cytologic response ( 10%

blasts in BM on day 14) and MRD 5 104 after early

consolidation. In contrast, patients with a slow cytologic responseand/or MRD 5 104 after early consolidation receive allo-

SCT further on. Preliminary results indicate that the prognosis of

HR patients with adequate response to induction and adequate

clearance of MRD is not worsened by avoiding allo-SCT. The

combined MRD level after induction and consolidation therapy

was the main prognostic factor for CR, DFS, and OS, although a

part of MRD-LR patients received a MRD-based de-escalated

therapy.

Within the GMALL 07/03 trial, patients with persistent MRD

104 after induction (day 71) and/or first consolidation (week

16) were allocated to the MRD-HR group61 and qualified for

allo-SCT. Gokbuget et al recently showed the first results of

MRD-based treatment intensification in these patients: 120 of

504 evaluated patients (24%) were allocated to the MRD-HR

group (89 SR and 31 HR patients defined by conventional

criteria).46 In 47% of these patients, SCT was realized in first

CR, with the SCT rate being significantly higher in HR

compared with SR patients (71% vs 39%, P .002). The

probability of CCR after 5 years was significantly higher for

patients receiving an MRD-directed SCT in first CR compared

with those without SCT in first CR (66% 7% vs 12% 5%,

P .0001). This also translated into a better OS at 5 years (54%

8% vs 33% 7%, P .06).

Bassan et al described an MRD-oriented therapy for all

t(4;11)/t(9,22) ALL patients within the Northern Italy Leukemia

Group.1 MRD-positive patients (defined as MRD 104 before

induction-consolidation cycle 6 and MRD positivity before cycle8) were allocated to allogeneic or autologous SCT, whereas

MRD-negative patients received standard maintenance regardless

of classic risk factors. Four-year DFS was 76% in MRD-LR

patients versus only 24% in the MRD-HR group despite of

treatment intensification.

The first phase 2 clinical study prospectively analyzing an

MRD-based targeted therapy administered blinatumomab mono-

therapy in patients with MRD failure or MRD reappearance

(defined as quantifiable MRD 1 104 after end of first

consolidation).62 Sixteen of 20 evaluable patients (15 patients with

MRD failure, 5 patients with MRD relapse) became MRD negative

after 1 cycle of blinatumomab treatment. Twelve of the 16 MRD

responders had never achieved MRD negativity before. MRD

negativity translated into an ongoing hematologic remission in all




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16 MRD-negative patients within a median observation time of

405 days (1-year relapse free survival probability 78%). One

patient was censored because of withdrawal of informed consent

during second treatment cycle, 8 of these patients consecutively

underwent allo-SCT without any treatment related mortality, and

7 patients did not receive any further consolidation treatment,

indicating the possibility of an improved outcome with this

MRD-guided treatment, even if data can only be compared withhistorical controls.

Preemptive treatment in case of MRD recurrence

MRD assessment is also used for preemptive treatment interven-

tion in case of MRD recurrence. In the GMALL 07/03 trial, salvage

treatment was intended to be started at time of reoccurrence of

quantifiable MRD. In a recent publication,46 10 of 34 patients with

MRD relapse underwent MRD triggered SCT in ongoing first CR.

Three-year probability of CCR was 80% 18% compared with

only 5% 5% in patients without transplantation. Other study

groups also perform postremission MRD monitoring for selected

patient subgroups, with some of them drawing clinical conse-

quences in case of high-level MRD.16

Besides SCT, also targetedtherapies are investigated to improve outcome in patients with an

MRD relapse.

In Ph-positive ALL, postremission MRD monitoring is mainly

used to tailor treatment after SCT. Wassmann et al investigated the

effect of imatinib to decrease the relapse probability in case of

reconversion to MRD positivity after SCT.63 BCR-ABL transcripts

became undetectable by both quantitative and nested RT-PCR in

15 of 29 (52%) patients. This was associated with a sustained

remission, whereas MRD persistence 6-10 weeks after start of

imatinib treatment correlated with an almost certain relapse.

Proposal for selection of sampling timepoints and methodology

Optimal sampling time points and sampling frequency have to be

defined according to the individual protocol depending on the

treatment protocol and the stratification aim. In de novo Ph-

negative ALL, postinduction MRD assessment (after 2-4 months of

treatment) is considered to have the most important role for

evaluation of initial treatment response and MRD-based risk-

stratification. An MRD assessment during induction (after

2 weeks of treatment) additionally identifies patients with a rapid

tumor clearance and a particular good outcome. Concerning

postremission monitoring of MRD for early relapse identification,

the GMALL proposes 3-monthly intervals for a total of 3 years asthe majority of clinical relapses occur within this time64 and reconver-

sion to MRD-positivity precedesa clinical relapse with a median time of

4.1 months between first quantifiable MRD positivity and relapse.

In Ph-positive ALL, the value of MRD for initial remission

assessment is more limited in the era of TKI, whereas MRD

assessment is frequently used for postremission monitoring. How-

ever, compared with Ph-negative ALL, relapse kinetics seem to be

more rapid with median time between MRD elevation and relapse

of only 2-3 months with53 or without59 application of TKI.

Therefore, sampling frequency is recommended to be higher than

in Ph-negative ALL.

Comparisons of MRD results obtained by different methodolo-

gies show that different techniques cannot be considered fully

interchangeable. The main difference between FCM and Ig/TCR-

PCR seems to be sensitivity,19,21,40 with both methods quantifying

single signals/leukemic cell. In contrast, BCR-ABL-PCR measures

multiple transcripts/cell with copy numbers potentially varying

during the course of treatment. As there is evidence of multilineage

involvement of Ph-positive cells, target cells also may not be fully

concordant to other MRD techniques. Therefore, choice of the

MRD method in a particular protocol should be guided by the

question to be answered, the experience gained in former MRDtrials, the available technical expertise, the logistics, and whether or

not treatment intervention is planned according to MRD results.

Can pretherapeutic risk assessment beskipped in adult ALL?

The topic of this Perspective is to debate whether MRD superseded

other risk factors. To answer this question, it is essential to

illuminate the value of MRD within different clinical settings but

also to discuss pretherapeutic factors that partly changed their

meaning from prognostic to predictive classifiers.

Classic risk factors versus MRD: the conventional concept of

risk-oriented therapy

During the past 3 decades, treatment of adult ALL patients

composed an induction/consolidation chemotherapy followed ei-

ther by additional consolidation cycles and maintenance treatment

(partly supported by auto SCT) or allo-SCT. Although there was

also a discussion on optimization of chemotherapy elements, the

main therapeutic decision was whether or not to apply allo-SCT,

which on the one hand leads to a reduced relapse rate but on the

other hand is accompanied by severe side effects and a consider-

able treatment-related mortality. Data exist for both treatment

paradigms: Collaboration from the United Kingdom Medical

Research Council and the Eastern Cooperative Oncology Group

demonstrated the superiority of allo-SCT at least in younger

patients.65 On the other hand, pediatric-inspired chemotherapy

regimens show favorable outcomes in adolescents restraining

indication of allo-SCT.66-68 Most protocols struck a balance be-

tween both approaches generally recommending SCT for patients

with clinical risk factors and a high risk of postchemotherapy

relapse. Risk assignment is conventionally based on indirect

measures of leukemic burden and chemosensitivity, such as

chromosome, molecular, and immunophenotypic analyses at diag-

nosis (Table 1; Figure 2A). Compared with all these pretherapeutic

factors, MRD directly measures chemosensitivity for individual

patients and integrates different host-, leukemia-, and treatment-

related components of treatment outcome.Indeed, most published data recognize MRD as most important

independent prognostic factor in adult ALL that supersedes all

other risk factors, at least for Ph-negative ALL. Prospective studies

on MRD-oriented therapy also indicate that MRD-based treatment

is safely possible, allo-SCT may be avoided in MRD-LR Ph-

negative patients and seems to be particularly active in MRD-HR

patients. In addition, MRD measurement helps to prevent the

problem of stratifying patients with newly identified genetic risk

factors that are too rare to allow a reasonable assignment to a

special risk group.

However, available MRD data presented here are not fully

consistent. Optimal sampling time points and MRD thresholds

seem to differ between ALL subgroups, in particular in Ph-positive

compared with Ph-negative ALL. In addition, multivariate analyses




9/13

in published studies partially retain prognostic factors other than

MRD as independent variables (interestingly, the old-fashioned

factor elevated white blood cell count at diagnosis emerges

repeatedly). As an additional restriction, as true also for all other

prognostic factors, MRD assessment is not realizable in all patients

because of technical limitations (eg, lack of identification of

leukemia specific markers, missing follow-up samples). Depending

on the minimum technical requirements, the MRD methodology,

and the adherence to the protocol, MRD-based stratification using

stringent criteria seems to be feasible in 80%-90% of patients.2,69

It also must be admitted that no published study on adult ALL

performed a randomized comparison between action and no action

on MRD but only compared the results of MRD-based treatment to

historical outcome data or to groups of patients that did not receive

an MRD guided therapy for whatever reasons. Therefore, as

already started in childhood ALL, also in adult ALL randomized

trials have to be done to confirm these encouraging data. MRD

excellently qualifies for such an approach because it not only serves

as marker for initial treatment stratification but also allows for an

ongoing monitoring (Figure 2B). Thereby MRD can serve as

safety net enabling early reintensification in case of MRD-basedtreatment de-escalation.

Targeted therapies: prognostic factors become predictive

Increased understanding of molecular mechanisms of cancer and

availability of drugs targeting them is changing the meaning of

particular leukemic markers from solely prognostic toward predic-

tive factors (Table 1). In adult ALL, BCR-ABL positivity can serve

as a model for this transformation: Formerly, the detection of the Ph

chromosome during diagnostic workup of adult ALL prognosti-

cated an extremely poor outcome with remission rates being at least

10% lower than in Ph-negative ALL and with a median survival of

only 8 months.70 The sole curative option was an allo-SCT. but even in

this setting Ph positivity formed the risk group with the poorest

outcome, making Ph positivity a poorly tractable therapeutic prob-

lem.71 However, things changed with the implementation of TKIs as a

targeted therapy: CR rates improved and also evidence of a survival

benefit emerges. Now BCR-ABL is not only a prognostic marker but

also predicts response to TKI. In addition, this new therapeutic approach

changes MRD kinetics in Ph-positive ALL. Whereas in the pre-TKI era

studies suggested a good correlation between MRD and outcome, MRD

data in the TKI setting are more conflicting, and optimal time-points and

thresholds are still a matter of debate. A potential reason for the

discrepancy between initial MRDresponse and outcomeis the existence

of low-levelBCR-ABL kinase domain mutations before treatment.54,72,73

Whereas the leukemic bulk may well respond to treatment the small-

sized resistant subclone may be the origin of relapse potentially

necessitating MRD analysis of subclones in the future.

Even though many of new potential predictive markers are not

ready to be used as treatment targets in clinical routine because of

issues related to reproducibility, statistical significance, and practi-

cal applications, the possibilities of targeted therapy are intriguing.

However, when implementing new elements into treatment proto-

cols, the sensitivity of leukemic cells to a particular drug is not

known in advance. Traditional risk groups may well respond

differently to targeted therapies than they do to classic chemo-

therapy, potentially leading to unpredictable treatment responses in

different subgroups of ALL. For instance, CD20 expression in

B-lineage ALL, which is under debate to be an adverse prognostic

factor,74,75 is increasingly targeted by protocols including ritux-

imab. Data from the MD Anderson Cancer Center indicate that

addition of rituximab to polychemotherapy alters MRD kinetics

and significantly improves MRD response.76 However, the effect of

the same rituximab dose seems to be different in SR compared with

HR patients.77 Therefore, it is particularly important to evaluate

treatment success separately within the different subgroups to

identify sensitive subpopulations. Ignoring baseline risk factors

means obscuring a possibly relevant treatment effect in distinct

biologic subsets by diluting in the whole population. Therefore,

Figure 2. Prospective therapeutic shifts according to conventional pretreatment stratification criteria and MRD. (A) Potential treatment decisions based on

pretreatment factors. (B) Potential treatment decisions based on MRD. Cons indicates consolidation; DLI, donor lymphocyte infusion; and TD, treatment decision.




10/13

assessment of pretherapeutic markers cannot be skipped in diagnos-

tic workup of ALL, even if MRD is closely monitored as

pretherapeutic and MRD markers increasingly become complemen-

tary tools to tailor therapy.

MRD as new primary end point

In times of targeted and sequential therapies, definition of an

adequate and discriminatory primary variable for judging treatment

success and planning subsequent treatment steps becomes more

and more important. OS, the classic gold standard for a primary end

point, as reliable, objective, and easily determined parameter gets

problematic in times of multimodal and sequential therapies,

including SCT. The other endpoint, the CR rate, is less objective in

particular in B-cell precursor ALL where the distinction between

leukemic cells and hematogones is exceedingly difficult in bone

marrow during recovery after chemotherapy or SCT. The value of

CR assessment is also limited by the fact that current treatment

protocols lead to CR rate of 90% in adult ALL. In contrast, MRD

offers itself as an endpoint in this setting as it is a clinical parameter

that integrates different leukemia, host, and treatment aspects into

one highly sensitive parameter. As a prerequisite for a usage as

primary endpoint, there has to be (1) a plausible biologic relation-

ship between reduction of MRD and response of the disease to

therapy, (2) the prognostic value of the surrogate for the clinical

outcome has to be validated, and (3) the evidence from clinical

trials has to exist that treatment effects on the surrogate correspond

to effects on the clinical outcome.78 A necessary technical precondi-

tion is the availability of a standardized technology for MRD

measurement regarding both standardized analysis and interpreta-

tion. This is currently primarily fulfilled for MRD assessment using

DNA-based quantitative RQ-PCR analysis according to EuroMRD

standards36 and related consensus definitions obtained at the second

international symposium on MRD assessment.16

Ten years ofinternational quality controls with 40 participating laboratories

demonstrated the robustness of the system with intra- and inter-

assay variability of less than half a log. In addition, FCM-based

MRD quantification and quantitative RT-PCR of BCR-ABL tran-

scripts are increasingly standardized within different international

collaborations. These analyses have to be performed in accredited

specialized laboratories as tests used in decision-making in clinical

trials generally have to conform to the Organisation for Economic

Co-Operation and Development (OECD) Guidelines on Good

Laboratory Practice79 or the Clinical Laboratory Improvement

Amendments.80 As shown in large-scale pediatric trials, this does

not hamper a broad application to all eligible ALL patients.

Although not yet generally accepted as a primary endpoint of

clinical trials by the European Medicines Agency and the Food and

Drug Administration, MRD measurement in combination with

adaptive trial designs may overcome part of the current difficultiesin evaluating the efficiency of new agents in ALL.

In conclusion, published data on MRD assessment in adult ALL

have shown a strong correlation between MRD response and

outcome as well as the prognostic value of MRD reappearance for

hematologic relapse. In times of individualized targeted therapies,

MRD will not substitute baseline risk factors but evaluate their

impact within different treatment regimens and will help to

optimize treatment sequence. In this context, MRD has also to be

considered as quantitative and objective extension of established

endpoints of hematologic remission and relapse more than a

substitute of pretherapeutic risk factors.

Acknowledgments

The authors thank Nicola Gokbuget, Dieter Hoelzer, and the

participants of the German Multicenter Study Group for adult ALL

for their close collaboration in the MRD studies.

This work was supported in part by the Wilhelm Sander Stiftung

(2001-074.1 and 2001-074.2) and the Deutsche Krebshilfe

(702657Ho2).

Authorship

Contribution: M.B. wrote the first draft of the manuscript; T.R.contributed to the writing and prepared figures; and M.B., T.R., and

M.K. finalized the manuscript.

Conflict-of-interest disclosure: The authors declare no compet-

ing financial interests.

Correspondence: Monik a Bruggemann, De partment of Hema-

tology, University Hospital Schleswig-Holstein, Campus Kiel,

C hemnitzs tr as se 33, D-24116 Kiel, Ger many; e-mail:

[email protected].

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13/13

online October 2, 2012originally publisheddoi:10.1182/blood-2012-06-379040

2012 120: 4470-4481

Monika Brggemann, Thorsten Raff and Michael KnebaHas MRD monitoring superseded other prognostic factors in adult ALL?

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