*Head of Stem Cell Transplant Program Clinica di Ematologia, Dipartimento Scienze Cliniche e

Molecolari, Università Politecnica delle Marche Azienda Ospedali Riuniti di Ancona

leukemias are characterized by the alteration of two sets of genes

•  those associated with a block of differentiation (AML1-ETO, CBFB, PML-RARA, MLL gene rearrangements…)

•  those that give the malignancy a proliferative advantage

(activating mutations in FLT3 or RAS…) •  recent detection of the NPM1 mutations revealed a third

category of mutations affecting genes implicated in cell-cycle regulation or apoptosis

…the recent detection of the NPM1 mutations revealed a third category of mutations affecting genes

implicated in cell-cycle regulation or apoptosis

Molecular heterogeneity of cytogenetically normal AML

GENE REARRANGEMENTS AND MUTATIONS IN AML

• Fusions (CBF, RAR-alpha, MLL protein )

• Activating mutations (FLT3, RAS, CEBPa)

• Loss of functions (GATA-1 etc, deletions))

1-Fusions

core binding factor (CBF), retinoic acid receptor-α (RARα),

HOX family members, the MLL protein,

transcriptional co-activators: CBP, MOZ, and TIF2

2-POINT MUTATIONS IN ACUTE MYELOID LEUKEMIA 1-Activating Mutations in RAS Family

Members

2-Mutations That Constitutively Activate Receptor Tyrosine Kinases (FLT3)

3-Other Mutations: CEBPA,WT1*, NPM

*WT1 mutation very rare; most common the overexpression of the Wild gene transcript

3-LOSS OF GENETIC MATERIAL

Certain genetic alterations that result in loss of genetic material are specific to AML:

loss of the long arms of chromosomes 5, 7, and 20, occurs commonly in therapy-related AML and AML

associated with previous MDS

and is associated with a poor prognosis

CBF •  Multiple translocations target the core binding

factor (CBF) in acute leukemias

•  CBF is a heterodimeric transcription factor comprised of the RUNX1 (also known as AML1)

and CBFß subunits

the gene rearrangements or mutations that result in CBF loss of function impair hematopoietic

differentiation. ….

AML1/ETO e INV16

•  MLL gene is rearranged in both common and rare

11q23 translocations in acute leukemia.

•  These rearrangements have

been associated with 5–10% of adult and pediatric cases of primary acute leukemias.

•  They are frequently found in

most patients with secondary leukemias after prior treatment with DNA

topoisomerase II inhibitors (e.g., etoposide)

Pathway activated by FLT3

NPM is a nucleolar protein that shuttles between the nucleus

and cytoplasm

In cytoplasm, NPM binds to centrosome

and regulates its duplication during cell

division.

Furthermore, NPM interacts with p53 and

its regulatory molecules

NPM mutations can occur with other

mutations

The NPM1 gene

MRD Assessment by Quantitative PCR in AML

Targets: •  fusion-gene transcripts •  (PML-RARA, AML1-ETO, and CBFB-MYH11) •  gene mutations •  (FLT3-LM/ITD, MLLPTD, CEBPA,NPM1) •  aberrantly over-expressed genes •  (WT1)

Molecular heterogeneity of cytogenetically normal AML

NPM1-mutated subsets

CEBPA-mutated subsets

•  Lack of specific molecular markers for several AML groups

•  Interlaboratory differences in: •  pre-PCR variables (eg, sample type, sample stabilization during shipment,

use of lysis or Ficoll, RNA preparation method, or method of cDNA synthesis)

•  during PCR (selection of primers, probes, buffers, enzymes, nucleotides, PCR

machines, labeling with hydrolysis probes, hybridization probes or SYBR

green, standard-curve etc)

•  What is the cut-off for the diagnosis of molecular relapse? (different

targets!)

•  There is an optimal source (BM or PB) for MRD detection?

•  What is the timing for MRD detection? After induction? Post.Cons? F-U?

•  Can we define a standard of Complete Molecular Remission? Like CML?

•  What is the impact of MRD for long-term prognosis?

Chritical issues and unanswered questions in MRD assessment by PCR in AML patients

Stability of markers at relapse

•  Fusion genes are extremely stable between diagnosis and relapse with only rare cases reported with a ‘‘relapse’’ as a different type of AML.

•  Also MLL- PTD, NPM1, and overexpressed genes like WT1 and EVI165 in general do not change at relapse.

•  In contrast, some studies have reported instability of FLT3-LM, whereas others have found a high stability of 95.9%.

•  A combined evaluation of all studies indicates that 88% of cases maintained the same FLT3-LM positivity at relapse.

Standardized MRD markers based on genetic changes account for no more than 65% of patients; overexpressed

genes (WT1) may be usefull in >80% of AML cases

HOKLAND and OMMEN BLOOD, 3 MARCH 2011 VOLUME 117, NUMBER 9

Overexpressed genes   >30% of all AMLs lack the genetic targets   For such cases, genes highly overexpressed in AML sometimes are

feasible as MRD targets   These genes have a basic expression level also in normal cells, therefore

they never can be ‘‘undetectable’’ like fusion genes or gene mutations.   The sensitivity is 1:1000 to 1:10,000 in very rare cases with extreme overexpression at diagnoses.

Wilms’ Tumor Gene (WT1) •  Overexpression in many types of hematological

malignancies •  Marker of leukemic haematopoiesis •  Low level of transcript in normal hematopoietic cells

CTRL BM

CTRL PB

AML BM

AML PB

ALL BM

ALL PB

CML BM

CML BP

CMML BM

CMML PB

IMF BM

IMF PB

HES BM

HES PB

RA BMRA PB

RAEB BM

RAEB PB

RAEB-T BM

RAEB-T PB0

5000

10000

15000

20000

25000

30000

WT1

cop

ies/

1000

0 AB

L co

pies

WT1 expression in haematological disorders (mean value)

CMPD MDS

Bone marrow Periph Blood

Courtesy of D. Cilloni

WT1 in reactive conditions

•  Reactive thrombocytosys •  Reactive leukocytosys •  Secondary erytrocytosis •  Reactive hypereosynophilia •  Polyclonal anaemias •  Secondary thrombocytopenias

CMPD

MDS

Negative (i.e. very low)

Courtesy of D. Cilloni

  WT1 is overexpressed in the majority of AML patients   Kinetics of WT1 response following induction therapy predicts risk of

subsequent relapse   The failure to achieve normal levels after induction chemotherapy is

highly predictive of relapse   Increasing of WT1 during follow-up predicts subsequent relapse

SUMMARY

normal BM normal PB normal PBSC0.01

0.1

1

10

100

1000W

T1

copi

es/1

0.00

0 A

BL

copi

es

WT1 expression in 204 normal samples (61 BM, 118 PB, 25 PBSC)

ELN WT1 assay

Median 19,8 0,01 6,1

Range 0-200 0,01-47 0-39

WT1

cop

ies/

104

ABL

copi

es

Upper normal limit 200 copies

Courtesy of D. Cilloni

BM PB0.01

0.1

1

10

100

1000

10000

100000

1000000

1.0×1007

WT1 expression in 729 samples from AML at diagnosis (collected by the European Leukemia Net) ELN WT1 assay

(588 BM 141 PB)

11% 12% WT1

cop

ies/

104 A

BL c

opie

s

Courtesy of D. Cilloni

•  No significant difference in WT1 expression at diagnosis by stratifying the patients according

to: –  cytogenetic risk groups (except for APL patients who

show significantly higher WT1 values) –  mutations of NPM1 or FLT3

favorable intermediate adverse10

100

1000

10000

100000

1000000

WT1

cop

ies/

1000

0 A

BL

copi

es

Courtesy of D. Cilloni

AML patients during follow-up (ELN study)

•  114 patients evaluated at diagnosis and during follow-up

 All the patients have been previously

characterized by cytogenetic and molecular analysis   Clinical data available  All the patients were treated with intensive

anthracycline and ARA-C   91/114 (80%) showed WT1 copies 2 logs higher

than normal controls)

Assessment of MRD using the ELN WT1 assay after induction CHT*

predicts relapse in AML *anthracycline and cytarabine-based

chemotherapy

Cilloni D. et al JCO 2009

•  Quantification of WT1 in PB on days 1 and 5 of treatment

•  WT1 ratio defined as the

ratio of copy number measured on day 1 and on day 5.

•  The median WT1 ratio was greater in

patients attaining CR as compared to non-responders

(11.68 vs. 2.14; P=0.0006).

•  DFS and OS significantly longer in patients with WT1 ratio >5.82

CCR

RELAPSED •  WT1 expression in normal PB is

1 log lower than in normal BM •  WT1 expression levels at

diagnosis and during F-U in 82 AML patients treated with standard CHT; 71 achieved CR

44/71 relapsed after a median of 12 months from diagnosis

(range 6-44). 27/71 pts persisted in CCR

after a median of 28 months of F-U (range 12-60).

48 pts achieved normal WT1 in

PB; no difference between WT1 levels at CR after

induction in the 27 in CCR, compared with the 21 who

relapsed during F-U (p=0.33).

The persistence of abnormal WT1 values after consolidation chemotherapy is predictive of relapse

Candidated for in vivo Purging and/or AlloTX

Use MPCF +/- other specific markers

If available

Courtesy of D. Cilloni

Approximately half of the patients

who reach normal WT1 values

after induction/consolidation will relapse,

although the reappearance of disease

occurs later compared with those

who do not reach the normalization of WT1.

Therefore, only the

abnormal WT1 values after induction treatment

are unfailingly predictive of relapse!

Other molecular markers useful for MRD detection in some AML subtypes

•  (CBF)+ leukemias comprise AML with t(8;21) (q22;q22) and inv(16) (p13q22)/t(16,16) (p13;q22), characterized by the presence of RUNX1-RUNX1T1 (AML 1-ETO) and CBFB-MYH11 fusion transcripts.

•  Heterozygous mutations of NPM1 can be detected in approximately one

third of all AML patients and up to 60% of CN-AML patients. Thus, NPM1 gene mutations represent the most common molecular alteration found in adult AML

•  Internal tandem duplication of FLT3 (FLT3-ITD) as MRD marker has

been questioned for its instability based on semi-quantitative methods

•  Several studies have shown that using nested RT-PCR in patients with t(8;21), RUNX1-RUNX1T1 transcripts could be detected after CHT, ASCT or allo-SCT in many patients in long-term remission.

•  The phenomenon has been ascribed to quiescent populations of SC,

monocytes and B cells harboring the fusion gene. •  Similarly, in AML with inv(16), MRD studies have reported persistence

of MRD in some long-term remitters, although most patients in prolonged remission remain PCR-negative.

Is it worthwhile to monitor MRD in CBF leukemias? NO!

YES!

RI: 15 vs 28% OS

TRM: 24% vs 6%

325 patients transplanted in CR1 (159 patients with inv16

and 166 patients with t(8;21);

145 alloTX and 180 ASCT

Howmany patients

received AlloTx with MRD- and howmany

patients received ASCT with MRD+?

(A) Relapse-free survival at checkpoint I (B) RFS at checkpoint II

OS in patients with inv(16) according to their PCR status at checkpoints I (PCR- in BM during consolidation) and II

(at least two PCR- samples in BM and/or PB during consolidation and F-U)

GORBACIOGLU et al

Serial MRD monitoring in CBF AML by quantitative RT-PCR prospectively assessed in 278 patients [163 with t(8;21) and 115 with inv(16)] entered in the MRC AML 15 trial

After induction CHT a < 3 log reduction in RUNX1-RUNX1T1 transcripts in BM in t(8;21) patients and a > 10 CBFBMYH11 copy number in PB in inv(16) patients were the most useful prognostic variables for relapse risk on multivariate analysis

During F-U, cut-off MRD thresholds in BM and PB associated with a 100% relapse rate were identified: for t(8;21) patients BM > 500 copies, PB > 100 copies; for inv(16) patients, BM > 50 copies and PB > 10 copies. During F-U, PB sampling was equally informative as BM

Outcomes of log reduction in BM at remission in t(8;21) patients

•  In AML t(8;21), RUNX1-RUNX1T1 transcripts could be detected after CHT, ASCT or allo SCT in many patients in long-term remission.

<1 log red 1-2 log red 2-3 log red >3 log red

<1 log red 2-3 log red >3 log red

<1 log red

>3 log red

1-2 log red

2-3 log red

>3 log red

<1 log red

Outcomes by CBFB-MYH11 copy numbers in PB at remission in

inv(16) patients

<10 copies 10-500 copies >500 copies

<10 copies 10-500 copies >500 copies

>500

<10

10-500

>500

10-500

<10

OS from CR by copy numbers

Sequential MRD monitoring during F-U in t(8;21) patients

CIR and OS in patients with >/<500 RUNX1-RUNXITI copies in BM

CIR and OS in patients with >/<100 RUNX1-RUNXITI copies in BM

Sequential MRD monitoring during F-U in inv(16) patients

CIR and OS in patients with >/< 50 CBFB-MYH11 copies in BM

CIR and OS in patients with >/< 10 CBFB-MYH11 copies in BM

Quantitative RT-PCR analysis in paired BM and PB samples after induction, during consolidation, and at F-U

•  Optimal schedules for MRD monitoring should include assessment after induction, during consolidation, and at 3 monthly intervals in BM and/or PB, at least during the first 18 months of follow-up.

•  During F-U, MRD monitoring

should ideally be carried out in paired BM and PB samples, to avoid missing a positive PCR result in BM with a negative PCR in the PB, in 10%-15% of patients.

•  However, in most patients, PB

sampling was equally informative and can therefore be used as a suitable alternative to BM.

t(8;21)

inv(16)

Clinical outcome confirmed the good prognosis of CBF-AML, but intensified induction was not associated with a better OS

MRD response remained the only significant factor in

multivariate analysis.

<3 log >3 log

  Various independent studies have shown a favorable impact of NPM1 gene mutations on clinical outcome in the CN-AML setting.

  Nevertheless, a substantial proportion of patients with

NPM1 mutations still show disease recurrence.   In the relapse situation, several studies have shown

stability of NPM1 gene alterations, implicating them as a potentially useful marker for minimal residual disease (MRD) detection.

  >50 different mutations in exon 12 of the NPM1 gene have

been identified, however, mutation types A, B, and D can be detected in about 95% of patients.

NPM1 MUTATIONS BY RQ-PCR

Irrespective of the mutation type, all mutations cause a distinct sequence change at the C-terminus of NPM1, elongating the protein and substituting one or two tryptophan residues which, in turn, lead to the abnormal accumulation of the protein in the cytoplasm of NPM1-mutant cells.13 As a consequence of this altered localization, the NPM1 protein loses its important function in the regulation of several key proteins

Optimized qPCR procedure to detect the three most common mutations (types A, B, and D3) to analyze the impact of MRD (residual

mutant NPM1) in 174 patients treated in two prospective trials

An increase of more than 1% NPM1mut/ABL1 was most prognostic for relapse after chemotherapy, whereas an increase of more

than 10% NPM1mut/ABL1 was most prognostic for relapse after allogeneic transplantation.

Median time to relapse was 121 days for the MRD >1% NPM1and 66 days for patients exceeding the 10% NPM1mut/ABL1cutoff after therapy

Time points of MRD sample collection

138 samples were paired BM and PB samples collected from individual patients at the same time point. By using data from this data set, we performed 4 separate subsample analyses: 1-receiver operating characteristic (ROC) analysis,2-prognostic value of MRD after completion of treatment (excluding allo-SCT), 3- prognostic value of MRD after allo-SCT, 4-prognostic value of MRD level at the time of the first complete response (CR1)

Sensitivity and specificity of RT-qPCR

Results were adjusted to ABL1 as the reference gene and expressed as percent NPM1mut/ABL1. A minimum ABL1 copy number of 1000 copies was required for inclusion of a sample

Using the optimized RT-PCR conditions, one NPM1mut OCI-AML3 cell was detected in a background of 100 000 MV4-11 cells.

MRD in PB was generally associated with higher levels of MRD in the corresponding BM sample; in general, the median NPM1mut levels in PB samples were almost 1-log10 lower.

CIR for patients carrying the FLT3-ITD mutation and MRD 1% was increased in comparison with that of the other subgroups

The median time to relapse was 85 days.

Median time to relapse: 121 days (range, 70 to 172 days) for the MRD >1% NPM1mut/ABL1

66 days (range, 34 to 98 days) for MRD >10% NPM1mut/ABL1

OS and DFS by 1% NPM1mut/ABL1 cutoff

DFS and OS by MRD level at CR1

Outcome according to MRD levels

after Allo- SCT

FLT3-ITD disappeared at relapse in 17% of patients and none in those harboring

mutant NPM1 compared with 29% in those with wild-type NPM1

Induct CHT Consolidation CHT

course 1 2 3

CR F-U CCR

Induction CHT Consolidation CHT

course 1 2 3

CR F-U CCR

MRD evaluation by WT1, Flow Cytometry and NPM1

Induct ion CHT

Consolidation CHT

ALLO-SCT

Induct CHT

2nd CHT course

MRD evaluated by MPFC LAIP: CD34+/DR+/CD7+/CD117+

triple neg genotype AML

Can we state today that close q-PCR monitoring in AML patients in CR will detect MRD in time for

interventions to prevent overt relapse? •  Despite an impressive body of data showing that RQ-PCR is

excellently suited for early detection of AML relapses, few investigators have taken clinical action on these findings, although it is by now evident that the risk of a false positive result of a RQ-PCR is minimal, especially when molecular conversion is confirmed in a subsequent sample.

•  To date, benefit from early salvage after conversion has been shown in PML-RARA APL, and in a preliminary report on the use of donor-lymphocyte infusion upon recurrent WT1 positivity after allogeneic transplantation.

•  Today it could be argued that a patient with CBF-AML and a ,3-log reduction of MRD level associated with a relapse risk of around 50% should be offered an allogenic HSCT if the estimated TRM is 10- 15%.

Figure 3. Conversion to PCR positivity. x-axis, Time, HR occurred at t ! 0. y-axis, Fraction-positive samples in each prerelapse interval. Median number of evaluable MRD profiles in each interval is shown for each molecular marker (panels A-D; range in parentheses). Solid lines, BM; broken lines, PB.A: NPM1c: blue, FLT3-ITD!; red, FLT3-ITD!. B: PML-RARA. C: CBFB-MYH11. D: RUNX1-RUNX1T1.

Doubling time of the CBFB-MYH11 clone was significantly longer (36 days) than that of clones harboring other markers (RUNX1-RUNX1T1,

14 days; PML-RARA, 12 days; and NPM1c, 11 days)

Cost-benefit estimations •  PB sampling every 6 months in patients with CBFB-MYH11 Leukemia will result

in a Relapse detection of 90%. •  With this sampling cadenza, only 10% of Mol Relapses will be missed before

hematologic relapse. •  Moreover, if the first sample is obtained 3 months after discontinuation of

therapy and each patient is followed for 3 years (given the literature: AML CBFB-MYH11 relapses in the MRC study cohort occurred during the first 3 years after diagnosis), only 14 MRD samples would have to be taken to detect 1 Mol Rel.

•  Given such considerations, molecular monitoring might prove to be cost

effective with its promise of early, possibly less intensive, intervention. •  In conclusion, the specific markers are generally superior to non-leukemia-

specific marker WT1 and could be more effective in early detection of Mol Rel.

Can we extrapolate to AML groups other than the CBF-AML? The German Austrian AML study group showed that NPM1(mut) transcript levels were significantly associated with prognosis after each treatment cycle.5 Mutations in FLT3, WT1, and CEBPa offer other molecular markers potentially useful for MRD detection. However, robust data in prospective studies are currently lacking.

Studies showing the prognostic value of flow-cytometric MRD were mostly performed in a single-institute setting, resulting in well-known potential pitfalls such as bias in patient groups and subjective judgment.

CONCLUSIONS •  There is still, except for APL a paucity of large prospective trials demonstrating the

clinical utility of MRD in AML. •  Potentially useful applications of MRD monitoring include: -early assessment of response to therapy to improve risk stratification -guide post-remission therapy (SCT?); -post-treatment monitoring to detect impending relapse and guide preemptive

therapy (GO?). •  The kinetics of RUNX1-RUNX1T1 and CBFBMYH11 decline has been found to

correlate with risk of relapse, or to represent a prognostic factor independent of other pretreatment variables.

•  NPM1 mutations likely provide one of the most promising new targets and studies

are ongoing to evaluate the clinical utility of MRD monitoring in AML with NPM1 mutation.

•  RQ–PCR assays have been developed for other fusion gene targets such as

MLLT3-MLL and DEK-NUP214, but data are very scarce due to the low frequencies of these leukemias.

Clinical options based on MRD+ results

 Intensified Consolidation  In vivo purging before harvesting SC  Allogeneic SCT  Graft purging before ASCT?  Maintenance after ASCT?  Immunotherapy after ASCT?

++ +-/-- ++ ?? ?? ??

We need prospective

studies

in all these settings