Remission, treatment failure, and relapse in pediatric ALL: An international consensus of the Ponte-di-Legno Consortium

Swantje Buchmann1*, Martin Schrappe1*§, Andre Baruchel2, Andrea Biondi3, Michael Borowitz4, Myriam Campbell5, Gunnar Cario1, Gianni
Cazzaniga3, Gabriele Escherich6, Christine J. Harrison7, Mats Heyman8, Stephen P. Hunger9, Csongor Kiss10, Hsi-Che Liu11, Franco Locatelli12,
Mignon L. Loh13, Atsushi Manabe14, Georg Mann15, Rob Pieters16, Ching-Hon Pui17, Susana Rives18, Kjeld Schmiegelow19, Lewis B. Silverman20, Jan
Stary21, Ajay Vora22 and Patrick Brown23 on behalf of the Ponte-di-Legno Consortium

Supplementary material

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Supplementary data

Examples for confirmatory tests for diagnosing relapse:

1. Recommendations of COG:

- FCM showing leukemia ≥1%

- PCR or NGS-based demonstration of Ig or TCR rearrangement that matches diagnosis and is quantifiable as 1.0E-2
- PCR- or NGS-based demonstration of validated leukemogenic lesion (e.g., fusion, mutation) that matches diagnosis and is quantifiable as
1.0E-2
- karyotypic abnormality (must display at least 1 metaphase similar/identical to diagnosis)
- FISH abnormality identical to one present at diagnosis (must be above the level of sensitivity of that specific FISH probe)

2. Recommendations of AIEOP-BFM:

- ≥ 5 % leukemic cells by FCM

- > detection limit for FISH
- at least 2 aberrant metaphases for cytogenetics
- MRD increase by at least one log (true value) to ≥ 1 % (≥ 1 x 10-2) by ASO RQ-PCR

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Supplementary tables

Supplement table 1: Remission and treatment failure definitions in current ALL studies

Study group Current/Recent Trial Remission (CR) Treatment Failure (TF) Additions Events Reference
definition definition timepoint(s) technique definition technique
AIEOP-BFM AIEOP-BFM ALL 2017 <5% blasts in BM ≥5% blasts in BM d33, end of induction cytomorphology Late Response Death NCT03643276
CR after d33 up to end of 3rd HR/2nd
CNS1 blasts in CSF cytomorpholgy Relapse
Blina
no localized leukemic infiltrates/
reduction of initial mass to at least 1/3 of persistent leucemic mass clinical examination, imaging, biopsy Morphological Non Response Second malignancy
initial mass
nCR after 3rd HR/2nd Blina cytomorphology Morphological Non Response

Molecular Non Response Molecular Non Response

MRD ≥ 5x10-3 after 3rd HR/2nd Blina PCR-MRD

MRD ≥ 5x10-4 after Myocet/FLA PCR-MRD

ALL-IC-BFM ALL-IC-BFM 2009 <5% blasts in BM ≥5% blasts in BM d33, end of induction cytomorphology Late Responders Death EudraCT 210-019722-13
no blasts in CSF blasts in CSF CR after d33 up to end of 2rd HR cytomorphology Relapse

no localized leukemic infiltrates/masses persistent leukemic mass clinical examination, imaging, biopsy Non Responders Second malignancy

nCR after 2rd HR cytomorophology Morphological non remission

CR regardless of timing
FCM/PCR-MRD, cytomorphology if no Resistant disease/Protocol therapy
ALLTogether ALLTogether1 <1% blasts in BM ≥1% blasts in BM end of induction (at the earliest), TF after Induction Death NCT04307576
markers (5% cut-off) failure
Cons (Down) or HR-blocks (all others)

CSF: no blasts on Cytospin 2 consecutive cytomorphology, confirmation by FCM- FCM/PCR-MRD, cytomorphology if Protocol Therapy Failure (RD)
still Cytospin pos EOC (d71) EOC (d71) ≥1% blasts in BM after 3 HR blocks
samples MRD allowed if cells are seen no markers (5% cut-off) (not "refractory")
still no reduction (viable tumour by PET-
mediastinal mass: reduction to <1/3 EOC (d71) radiology +/- PET-CT, biopsy Refractory disease (T-ALL) Relapse
CT/biopsy)
≥5% d29 and ≥0.5% day 50 or
US not normal, biopsy proven remaining FCM/PCR-MRD, cytomorphology if
testis: bilateral US normalize EOC (d71) US +/- biopsy <5% d29 and ≥0.05% d71 or Death in CR1
infiltration no markers (5% cut-off)
≥5% d29, and ≥0.01% day 71
unchanged/increasing MR-
CNS clinical/MR: MR-findings EOC (d71) clinical assessment, MR Second malignancy
findings/symptoms
eye: Ophtalmoscopy normalised remaining infiltrates EOC (d71) clinical assessment
CoALL CoALL 08-09 <5% blasts in BM ≥5% blasts in BM d 29, end of induction cytomorphology Late Response Death EudraCT 2009-012758-18
CNS1 blasts in CSF CR after d29 up to end of 2rd HR1 cytomorphology Relapse
no localized leukemic infiltrates/
reduction of initial mass to at least 1/3 of persistent leukemic mass clinical examination, imaging, biopsy Non Response Second malignancy
initial mass
nCR after 2nd HR Morphological Non Response
COG AALL1731, AALL1732 <1% blasts in BM ≥1% blasts in BM d29, end of induction (for early CR) FCM-MRD Early CR Death NCT03914625
FCM-MRD, cytomorphology and
resolution of CNS leukemia (CNS1) CNS2 or CNS3 End of consolidation (for late CR) cytomorphology achievement of CR by d29 evaluation Relapse NCT03959085
clin. examination
NOTE: end of consolidation evaluation is
resolution of other extramedullary disease non-resolution of other EM disease clinical examination/imaging Late CR Second malignancy
done after day 56 once APC>500/uL
If not early CR, achievement of CR by FCM-MRD, cytomorphology and
Consolidation failure
end-of-consolidation evaluation clin. examination

Induction Failure
d29 M3 marrow, or d29 MRD ≥5%, or
residual EM disease
Consolidation Failure
d92 (d57) MRD ≥1% (CF-MRD), or
residual EM disease
FCM-MRD, cytomorphology and
clin. examination
FCM-MRD, cytomorphology and
clin. examination

DCOG DCOG ALL-11 <5% blasts in BM ≥5% blasts in BM d33, end of induction cytomorphology Non Responders Death in induction/death in CR EudraCT 2012-00006725

without extramedullary leukemia extramedullary leukemia clinical examination,imaging M2/M3 or cytomorphology, imaging, biopsy Relapse

testicular enlargement is excluded, documented extramedullary

Second malignany
retesting after Prot. M leukemia after HR-1
Induction failure
cytomorphology conf. by FCM-MRD,
DFCI DFCI 16-001 <5% blasts in BM with PB recovery > 25% blasts in BM d32 Induction failure Death NCT03020030
FISH when unclear
> 25% blasts in BM at Day 32, or
cytomorphology conf. by FCM-MRD, cytomorphology conf. by FCM-
no blasts in peripheral blood blasts in peripheral blood 5-24% blasts in BM at Day 32, and > 5% Relapse
FISH when unclear MRD, FISH
blasts in BM at Week 10
CNS-3 at Day 32, or persistent CNS-2
no blasts in the CSF blasts in CSF cytomorophology cytomorophology Second malignancy
at week 10
>70% reduction in size of initial leukemic
< 70% reduction of leukemic mass imaging, biopsy <70% reduction in leukemic mass imaging, biopsy Induction failure
mass
Assessed at end-IB and again at end- Consol
EsPhALL/COG EsPhALL2017/COGAALL163 <5% blasts in BM CAVE: nCR after end of Cons.block 3 PCR-/FCM-MRD Resistant disease Death NCT03007147
Block 3 (if no CR by end-IB)
MRD ≥ 10-2 by PCR-MRD (or ≥ 1% by FCM- MRD ≥ 10-2 PCR-MRD (or ≥ 1% by FCM-
no extramedullary leukemia PCR-MRD, FCM-MRD PCR-MRD, FCM-MRD Secondary malignancy
MRD) MRD) after Cons. 3
cytomorophology conf. by FCM-MRD,
M2 morphology M2/M3 marrow after Cons. 3 cytomorophology Relapse
FISH
M3 marrow cytomorphology Progressive disease
MRD ≥ 10-2 PCR-MRD at 2 timepoints
PCR-MRD, FCM-MRD
after HSCT
www.ncbi.nlm.nih.gov/pubmed/2986
JCCG JPLSG-ALL-B12 <5% blasts in BM ≥5% blasts in BM day 33, end of induction cytomorphology Induction failure Death
0341
no clinical symptoms due to leukemia clinical examination ≥5% blast in BM cytomorphology Relapse
no organ infiltration of leucemia imaging, biopsy Second malignancy
Induction failure
cytomorphology, confirmed by FCM- Death in induction/death in
NOPHO NOPHO-ALL 2008 <5% blasts in BM ≥5% blasts in BM d29, end of induction Resistant disease NCT00819351
MRD remission
(CNS status has different influence on risk cytomorphology, FCM-MRD,
blasts in CSF after 3rd i.t. injection (d29) (or after HR block A) cytomorphology no CR after Block C Relapse
stratification) cytogenetics

extramedullary disease (testis and CNS: blasts in CSF after one Depocyte
imaging and biopsy cytomorphology Second malignancy
mediastinal mass) will be tested at d 85 d15 due to remaining blasts in CSF

testis: test. disease at d 85/after

ultrasound, clinically, biopsy Resistant disease
1st block B
other sides: persistent, vitale
imaging, biopsy
leucemic mass
cytomorphology, confirmed by FCM- www.sehh.es › documentos › guias ›
SEHOP-PETHEMA LAL/SEHOP-PETHEMA 2013 <5% blasts in BM ≥5% blasts in BM end of induction Induction failure Death
MRD LAL_SEHOP_PETHEMA_2013
no blast in the CSF cytomorphology confi. by FCM-
presence of extramedullary mass cytomorphology, imaging ≥5% blast in BM at d33 Relapse
no clinical CNS involvement MRD and/or genetics

no extramedullary disease extrammedullary disease

imaging, biopsy Second malignancy
> 75 % reduction of mediastinal mass less than 75% reduction of mediastinal mass

Induction failure
<5% blasts in BM reinforced by MRD < 5x10- Treatment failure/
SFCE CAALL-F01 ≥5% blasts in BM reinforced by MRD ≥5x10-2 d35-d42, end of induction cytomorphology AND PCR-MRD Death NCT02716233
2 Induction failure at d 35
cytomorphology, confirmed by
no blasts in the CSF blasts in CSF cytomorphology ≥5% blasts in BM AND Relapse
PCR-MRD
absence of leukemic mass/reduction of MRD ≥5x10-2 (if not possible,
persistence of leukemic mass ≥30% imaging, biopsy Secondary malignancy
mass to at least 30% cytomorphology only)
Treatment failure
SJCRH TOT-XVI <5% blasts in BM ≥5% blasts in BM d38-42, end of remission induction FCM-MRD Induction failure at day 42 Death NCT00549848
cytomorphology, confirmed by
≥5% blasts in BM Relapse
FCM-MRD
Second malignancy
Induction failure
cytomorophology, confirmed by FCM-
TPOG TPOG-ALL-2013 <5% blasts in BM ≥5% blasts in BM d35-d42, end of remission induction Induction failure at day 42 Death no NCT or EudraCT
/PCR-MRD
cytomorphology, confirmed by
no blasts in CSF blasts in CSF ≥5 % blasts in BM Second malignancy
FCM-/PCR-MRD
Relapse
Induction failure
UKALL UKALL 2011 Trial <5% blasts in BM ≥5% blasts in BM d29, end of induction PCR-MRD True induction failure at day 29 Death www.isrctn.com/ISRCTN64515327
no blasts in the CSF blasts in CSF cytomorphology ≥25% blasts in BM PCR-MRD Relapse
initial testsis involvement is controlled at
week eight, without impact on risk Partial response at d 29 Secondary tumour
stratification

< 35 % reduction of mediastinal mass > 35 % reduction of mediastinal mass 5- < 25% blasts in BM PCR-MRD True induction failure

Blina: Blinatumomab, BM: bone marrow, CNS: central nervous system, CSF: cerebral spinal fluid, Cons.: Consolidation, CR: complete remission, CT: clinical trial, d: day, EM: extramedullary
sites, EOC: end of consolidation, EOI: end of induction, FCM-MRD: flow cytometric minimal residual disease, FISH: fluorescence in situ hybridization, HR: high risk, i.t.: intra thecal, MRD:
minimal residual disease, NCT: number of ‘Clinical Trial’, PB: peripheral blood, PCR-MRD: polymerase chain reaction minimal residual disease, TF: treatment failure, US: ultra sound

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Supplement table 2: Relapse definitions in current ALL studies

Study group Current/Recent Trial Relapse localization Definition Technique Additions

≥ 25% blasts without extramedullary
AIEOP-BFM AIEOP-BFM ALL 2017 Isolated BM cytomorphology
involvement
cytomorphology and FCM-/PCR-MRD, FISH,
≥ 5% blasts and two confirmatory tests
genetics
≥ 5% blasts in BM and ≥ 1
Combined cytomorphology, biopsy, imaging
extramedullary site localization
>5/μl nucl. cells in CSF and evidence of
Isolated CNS cytomorphology
blasts
intracranial mass imaging, biopsy
uni-/bilateral painless hard testicular
Isolated testicular clinical examination, imaging, biopsy
tumor
Other sides imaging, biopsy
ALL-IC-BFM ALL IC-BFM 2009 Isolated BM ≥ 25% blasts cytomorphology
≥ 2 compartments, BM > 5% cytomorphology, biopsy, imaging (FCM-MRD
Combined
lymphoblasts is not accepted)
>5/μl nucl. cells in CSF and evidence of if blasts ≤ 5/μL, repunction
Isolated CNS cytomorphology
blasts after 2-4 weeks
Intracerebral mass w/o blasts in CSF CT/MR, biopsy
Uni-/bilateral painless hard swelling of
Isolated testicular clinical examination, imaging, biopsy
testicle(s)
Other sides biopsy is necessary
ALLTogether ALLTogether-1 Isolated BM >5% blasts confirmed by one method cytomorphology, FCM-MRD, genetics
>1% blasts confirmed by two
methods/one method repeated
imaging (CT/MR), cell count +
Isolated CNS >5/μl blasts in CSF cytospin+cytomorphology, confirmed by
FCM/genetic markers
Combined BM and cytomorphology + FCM/genetic markers,
BM (definition above) and EM
CNS/other EM biopsy, imaging
Isolated testicular clinally suspiciuos clinical finding confirmed by biopsy
Isolated other biopsy
CoALL CoALL 08-09 Isolated BM ≥ 25% blasts cytomorphology
≥ 5% blasts and a confirmatory tests
cytomorphology, FCM/genetic markers
with rising blast count
≥ 5% blasts in BM and ≥ 1
Combined cytomorphology, biopsy, imaging
extramedullary site localization
>5/μl nucl. cells in CSF and evidence of
Isolated CNS cytomorphology, confirmed by Flow
blasts
intracranial mass imaging, biopsy
uni-/bilateral painless hard testicular
Isolated testicular clinical examination, imaging, biopsy
tumor
Other sides imaging, biopsy
COG AALL1731, AALL1732 Isolated BM ≥ 25% blasts cytomorphology
5-24% blasts and one confirmatory FCM-/PCR-MRD, cytogenetics, proof of
molecular test diagnostic leucemic lesion
<5% blasts and two confirmatory
molecular tests
<5% blasts and one confirmatory test
Equivocal bone marrow cytomorphology
plus
marrow repeated 1-4 weeks later, to
convert to relapse, either:
FCM-MRD, FISH testing in
- meet criteria for relapse or cytomorphology, FCM-MRD, FISH
COG-lab
- demonstrate ≥ 1% blasts by one
method
Combined BM and at least one other side
Islated CNS relapse ≥ 5/μL blasts in CSF
clinical signs of CNS leukemia
Equivocal CNS <5/μL blasts in CSF plus cytomorphology
CSF repeated 1-4 weeks later, to
convert to relapse, either:
FCM-MRD, FISH testing in
- meet criteria for relapse or cytomorphology, FCM-MRD, FISH
COG-lab
- CNS2 with blasts confirmed by one
method
Testicular relapse biopsy proven only
Other sides biopsy proven only
cytomorphology conf. by immunoph. or
DCOG DCOG ALL11 Isolated BM > 5% (1%) blasts in BM/PB
genotyp.
if blasts ≤ 5/μL, repunction
cytomorphology conf. by immunoph. or after 4 weeks or confirmation
Isolated CNS ≥ 5/μL blasts in CSF
genotyp. to be leukemic by second
method
Other sides biopsy
cytomorphology, FCM-MRD, genetics, FISH,
DFCI DFCI 16-001 Isolated BM ≥ 5% blasts with one confirmatory test
immunhistochemistry or other tests
Isolated CNS ≥ 5/μL with blasts in CSF or
2 consecutive CSF with WBC <5/μL but
without confirmatory test
blasts present (at least 3 weeks apart)
biopsy-proven extramedullary
Other sides biopsy
leukemia

EsPhALL/COG EsPhALL2017/COGAALL163 Isolated BM ≥ 25% blasts cytomorphology

≥ 5% blasts with one confirmatory test cytomorphology, FCM-/PCR-MRD, FISH
Isolated CNS CSF WBC ≥ 5/µL cytomorphology
clinical signs of CNS leukemia
MRD ≥ 10-2 or ≥ 1% at two post-HSCT
Molecular relapse (post-
time points separated by at least 2 PCR-MRD, FCM-MRD
HSCT)
weeks
JCCG JPLSG-ALL-B12 Isolated BM ≥ 50% blasts cytomorphology

reexamination in intervals of
≥ 5-50% blasts cytomorphology
one week, if increase or >25%
≥ 25% blasts in BM AND >2% in PB cytomorphology
cytomorphology AND confirmation by
≥ 25% blasts in BM
genetic tests
>5/μl nucl. cells in CSF and evidence of
Isolated CNS cytomorphology
blasts
intracranially mass excluding
imaging AND biopsy
hemorrhage
≥ 5% blasts in BM and ≥ 1 if BM relapse is diagnosed, no
Combined
extramedullary site localization biopsy needed
Other sides biopsy/imaging
cytomorphology, conf. by histochemistry,
NOPHO NOPHO-ALL 2008 Isolated BM ≥ 5% blasts
FCM-/PCR-MRD or genetics
cytomorphology, conf. by histochemistry,
Isolated CNS ≥ 5/μL blasts in CSF
FCM-/PCR-MRD or genetics
bulky CNS leukemia imaging, biopsy
Isolated testicular imaging, biopsy
Other sides clinically suspiious imaging, biopsy

≥ 25% blasts without extramedullary

SEHOP-PETHEMA LAL/SEHOP-PETHEMA 2013 Isolated BM cytomorphology
involvement
>5/μl nucl. cells in CSF and evidence of
Isolated CNS cytomorphology
blasts
imaging or clinical evidence of neurol.
imaging
Involvement
testicular enlargement or increased
Isolated testicular imaging/biopsy
consistence
≥ 5% blasts in BM and ≥ 1
Combined
extramedullary site localization
Other sides biopsy

≥ 5% blasts in BM and two confirmatory cytomorphology, conf. by

SFCE CAALL-F01 Isolated BM tests immunophenotyping AND PCR-MRD
without leukemic mass/blasts in PB (mandatory if blasts >5-20%)
imaging, biopsy, conf. by
Isolated extramedularry presence of leucemic infiltration
immunophenotyping and/or genotyping
cytomorphology, conf. by
<5% blasts in BM
immunophenotyping and/or genotyping
cytomorphology, conf. by
Combined relapse ≥ 5% blasts in BM
immunophenotyping AND PCR-MRD
imaging, biopsy, preferely confirmed by
presence of leucemic infiltration
imm. and genotyping
SJCRH TOTXVI Isolated BM ≥ 25% blasts cytomorphology
cytomorphology and FCM-/PCR-MRD, FISH,
≥ 5% blasts
genetics
Isolated CNS ≥ 5/μL blasts in CSF cytomorphology
< 5/µL x 2 within 2-4 weeks
Isolated testicular clinically suspicious biopsy
no biopsy of testis is needed, if testis is
Combined ≥ 5% blasts
enlarged

TPOG TPOG-ALL-2013 Isolated BM ≥ 25% blasts in BM cytomorphology

Isolated CNS ≥ 5/μL blasts in CSF cytomorphology
Isolated testicular clinically suspicious imaging, biopsy
≥ 5% blasts in BM and ≥ 1
Combined
extramedullary site localization
Other sites biopsy
cytomorphology conf. by
UKALL UKALL 2011 Trial Isolated BM ≥ 5% blasts in BM immunoph./genotyp./FISH/FLOW or PCR-
MRD
Isolated CNS ≥ 5/μL with blasts in CSF or cytomorphology, FCM-MRD, FISH
2 consecutive CSF with WBC <5/μL but
blasts present

BM: bone marrow, CNS: central nervous system, CSF: cerebral spinal fluid, CR: complete remission, EM: extramedullary sites, FCM-MRD: flow cytometric minimal residual disease, FISH:
fluorescence in situ hybridization, i.t.: intra thecal, MRD: minimal residual disease, PB: peripheral blood, PCR-MRD: polymerase chain reaction minimal residual disease, US: ultra sound

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Supplement table 3: Common/currently used relapse criteria

Localization Definition/Threshold Method

Isolated BM ≥ 25% blasts without extramedullary involvement) cytomorphology

Combined ≥ 5 % blasts in BM and ≥ 1 EM site cytomorphology and imaging, biopsy

Isolated CNS > 5/μl nucleated cells in CSF and evidence of blasts cytomorphology
OR
intracranial mass imaging, biopsy

Others clinical examination, imaging, biopsy

BM: bone marrow, CNS: central nervous system, CSF: central spinal fluid, EM: extramedullary sites