on behalf of the sub-committee

Disease-Specific Methods and Strategies for Monitoring Relapse Following Allogeneic

Stem Cell Transplantation

Pediatric Acute Lymphoblastic Leukemia

on behalf of the Sub-Committee

Peter Bader, Wendy Stock,Andre Willasch, Alan Wayne

Surveillance of Remission

Two principle approaches: Chimerism

Characterization of post transplant hematopoiesis MRD

Direct detection of the underlying malignancy

Hematopoietic Chimerismin Children with ALL

Bader et al., J Clin Oncol 33: 1696 (2004)

Studies on Chimerismand Intervention

AuthorNumber

of patients

Diagnosis Interval of investigations

Methods Relapses

FormakovaHaematologica

200354

AL, CML and MDS

children

weekly to +100;

monthlySTR

MC associated with rejection and relapseImmunotherapy was

possible

GorczynskaBMT 2004

14ALL, AMLchildren

weekly to +100;

monthlySTR

In-MC could be converted by immunotherapy to CC

BaderJCO 2004

163ALL

children

weekly to +100;

monthlySTR

MC associated with rejection and relapseImmunotherapy was

possible

HornBMT 2008

20AL

children

1,3,6,12 months;In MC bi-weekly

STRMC associated with

relapseIT was not possible

Conclusions I

Immunotherapy (WD of immunosuppression, DLI) is principally effective as pre-emptive treatment

Chimerism can be used as surrogate marker for identifying patients at risk for impending relapse However:

Not in all patients! Additional role for MRD?

Retrospective Studies - MRD prior to SCTLiterature

AuthorNumber of patients

Diagnosis Time of investigation

MethodsSurvival

according to MRD status

KnechtliBlood 1998

64 ALLprior to

conditioningIg / TCR

PCR

high level pos. – 0%low level pos. – 36%

negative – 73%

BaderLeukemia 2002

41 ALLprior to


PCR


negative – 78%

UzunelBlood 2001

30 ALLprior to


PCR


negative – 100%

SramkovaPed Blood

Cancer 200725 ALL

prior to conditioning

Ig / TCRPCR

positive – 0%negative – 94%

Prospective Study: MRD Prior SCT ALL REZ BFM Group: CR2

EFS CI

Years after SCT

MRD < 10-4

MRD ≥ 10-4

0 1 2 3 4 5 6

0.0

0.2

0.4

0.6

0.8

1.0

Eve

nt-f

ree

Su

rviv

alP

rob

ab

ility

Years after SCT

MRD < 10-4

MRD ≥ 10-4

0 1 2 3 4 5 6

0.0

0.2

0.4

0.6

0.8

1.0

Eve

nt-f

ree

Su

rviv

alP

rob

ab

ility

Years after SCT

MRD < 10-4

MRD ≥ 10-4

0 1 2 3 4 5 6

0.0

0.2

0.4

0.6

0.8

1.0

Cum

ula

tive

Inci

de

nce

Years after SCT

MRD < 10-4

MRD ≥ 10-4

0 1 2 3 4 5 6

0.0

0.2

0.4

0.6

0.8

1.0

Cum

ula

tive

Inci

de

nce

EFS CIR

MRD < 10-4: n = 46; cens.= 29; pEFS = .60 .08 CI (relapse) = .13 .06≥ 10-4: n = 45; cens.= 14; pEFS = .27 .07 CI (relapse) = .57 .08

p = .0004 p < .001

Bader et al.: JCO 2009

Conclusions II

MRD prior to stem cell transplantation has a profound impact on post transplant outcome!

What adds MRD post transplant?

Retrospective Studies -MRD Post SCTLiterature

AuthorNumber

of patients

DiagnosisTime of

investigationMethods

Survival and MRD status

KnechtliBJH 1998

68 ALLup to 24 months

post SCTIg / TCR

PCRrelapse – 88% pos.

remission – 22% pos.

UzuelBJH 2003

23 ALL 24 monthsIg / TCR

PCRMRD pos. associated with

relapse

SanchezBJH 2002

40 ALLd30, 60, 90,every 2-3 months

Flow cytometry

positive – 33%negative – 74%

Prospective StudyBFM Group

N 92

Diagnosis ALL

Remission ≥ CR2

Transplant Period Jan 1999 May 2006

Evaluation January 15th 2009

Follow up Median Min Max

[Years] 5.13 3.44 6.48

MRD - Highest Level post SCTAll Patients

pEFS pRFS

< 10-6: n = 46; cens.= 26; pEFS = .55 .08 n = 46; cens.= 37; pRFS = .77 .07

≥ 10-6- <10-4 n = 25; cens.= 12; pEFS = .48 .10 n = 25; cens.= 17; pRFS = .62 .11≥ 10-4: n = 21; cens.= 03; pEFS = .09 .06 n = 21; cens.= 03; pRFS = .11 .07

P=0.002 P=0.000

Event free survival [years]

1086420

Cu

m E

FS

1,0

0,8

0,6

0,4

0,2

0,0

Relapse free survival [years]

1086420C

um

RF

S

1,0

0,8

0,6

0,4

0,2

0,0

MRD ≥ 10E-4MRD ≥ 10E-4

MRD < 10E-6

MRD < 10E-6

MRD <10E-4 - 10E-6

MRD <10E-4 - 10E-6

Conclusions III and Summary

MRD assessment in BM post transplant is predictive for relapse Serial BM investigations are warranted. Current working recommendations of the BFM: days

30, 60, 100, 200, 300, 365, at 18 months and 24 months.

Summary: Patients with mixed chimerism have a high risk for

relapse Patients, who become/remain MRD positive >10-4,

have a very high risk to develop relapse Additional treatment in these patients is warranted

PB-04/06tk05.06

MRD in adults with ALL Shown to be useful predictor of DFS in

many studies (non-transplant) Independent prognostic feature Mostly using PCR techniques – IgH/TCR, fusion

genes “Informative” assay available in 60-90% of patients

Early CR time-points predictive of outcome: from 4-22 weeks following initiation of treatment

Fewer studies evaluating role of MRD in setting of alloSCT

PB-04/06tk05.06

AlloSCT improves outcome of MRDpos in CR1 but much room for improvement

0.0

00

.25

0.5

00

.75

1.0

0C

um

ula

tive S

urv

iva

l

0 12 24 36 48 60 72 84Months

Kaplan-Meier survival estimates, by allo_iper2

SCT or H/C (n = 36)

rest )n = 18(

SCT or H/C-auto SCT or H/C-auto vv chemo chemo

Bassan, R. et al. Blood 2009;113:4153-4162

PB-04/06tk05.06

MRD following alloSCT in Adults with ALL

AuthorNumber

of patients

Diagnosis Time of investigation

Methods DFS and MRD status

MortuzaJCO 2002

19ALL

(B-lineage)From 1-20

mos.

Ig / TCRPCR

Semi-quant.

positive – 0%negative – 100% CCR

SpinelliHaematologica

200737 ALL Day +100

Ig/TCR or fusion gene

PCRQuantitativ

e

positive >10-4: 20%negative: 93%

Bassan*Blood 2009

18

ALL*All were

PCR+ prior to

transplant

Not definedIg / TCR

PCRpositive >10-4: 0negative: 50%

PB-04/06tk05.06

Dombret et al: Blood 100:2002

MRD status prior to transplant predicts DFS

Achievement of Molecular Remission Prior to AlloSCT is Important in Ph+ ALL

PB-04/06tk05.06

Combination of ChemoRx + Imatinib

Produces Molecular Remissions Group N Age Time-point % PCR

negative

MDACC 20 <75 After consolidatio

n

60

Korea 20 <67 After 1st consolidatio

n

70

JALSG 77 <63 Day 63 50

GMALL 92 <65 After 2 inductions

52

GRALL 45 <60 After 2 inductions

29 “neg”

64 “low”

PB-04/06tk05.06

Yanada, M. et al. J Clin Oncol; 24:460-466 2006

Is Transplant in CR1 Still Treatment of Choice for Ph+ ALL?

Transplanted patients

No transplant

PB-04/06tk05.06

Wassmann, B. et al. Blood 2005;106:458-463

Imatinib Treatment of Molecular Relapse with Following Allo-SCT for Ph+ ALL

PB-04/06tk05.06

Summary MRD detection both prior to and following alloSCT for

adults with ALL is associated with poor DFS

Clinical interventions based on MRD measurements suggest utility but data are very limited:

Allocation to alloSCT in CR1 Post-transplant intervention to prevent relapse

Targeted therapy (e.g. imatinib) following transplant

Challenge: implementation of standardized MRD assays that can be done in “real-time”

IgH/TCR qPCR assays are laborious Data on flow cytometric measurements of MRD in adults with

ALL are lacking

Disease-Specific Methods and Strategies for Monitoring Relapse Following Allogeneic

Stem Cell Transplantation

Chronic Lymphocytic Leukemia

Sebastian Böttcher, Issa Khouri, Peter Dreger

Overview

• Techniques

• MRD kinetics

• Clinical significance of MRD

Techniques

ASO IGH qPCR and MRD flow in CLL- Comparative analysis in 530 samples -

10-5 10-4 10-3 10-2 10-1 100 101

10-5

10-4

10-3

10-2

10-1

100

+ < qr0

0

344

140 67

278

7

r = 0.95

ASO IGH qPCR

MR

D f

low

Böttcher, Leukemia, 2009

IgH-consensus PCR - Sensitivity -

Böttcher, Leukemia, 2004

polyclonal monoclonal10-4

10-3

10-2

10-1

100

Consensus primer IGH-PCR

MR

D f

low

le

ve

ln = 43 n = 106

Techniques for MRD in CLL

ASO IGHqPCR

MRD flow Consensus IgH PCR

Sensitivity 10-5 10-4 10-2 – 5 x10-4

Quantitative range 10-4 10-4 n.a.

Quantitative reproducibility

high high poor

Standardization

van der Velden,

Leukemia, 2007

Rawstron, Leukemia,

2007poor

iwCLL guidelines accepted accepted Not mentioned

Turn-around time weeks hours days

MRD kinetics

MRD patterns after allogeneic SCT I

SCT↓

CSA red.↓

Ritgen, Leukemia, 2008

1 2 3 4 5 6 7

Landmark

1E-6

1E-5

1E-4

1E-3

1E-2

1E-1

1E+0

1E+1

MR

D l

ev

el

A

MRD patterns after allogeneic SCT II


1 2 3 4 5 6 7

Landmark

1E-6

1E-5

1E-4

1E-3

1E-2

1E-1

1E+0

1E+1

MR

D l

ev

el

B

SCT↓

CSA red.↓

MRD patterns after allogeneic SCT III


1 2 3 4 5 6 7

Landmark

1E-6

1E-5

1E-4

1E-3

1E-2

1E-1

1E+0

1E+1

MR

D l

ev

el

C

SCT↓

CSA red.↓

DLI↓

MRD patterns after allogeneic SCT

1 2 3 4 5 6 7

Landmark

1E-6

1E-5

1E-4

1E-3

1E-2

1E-1

1E+0

1E+1

MR

D l

ev

el

C

1 2 3 4 5 6 7

Landmark

1E-6

1E-5

1E-4

1E-3

1E-2

1E-1

1E+0

1E+1

MR

D l

ev

el

B

1 2 3 4 5 6 7

Landmark

1E-6

1E-5

1E-4

1E-3

1E-2

1E-1

1E+0

1E+1

MR

D l

ev

el

A

Prognostic significance

Prognostic significance MRD +12 months after alloSCT

Dreger, ms. in prep.

12 24 36 48 60 72 84 960

50

100

+12 MRD- (27)+12 MRD+ (11)

HR 0.047 (0.007-0.3); p 0.0011

Months from SCT

Perc

en

t re

lap

sed

MRD –ve (1/27)

MRD +ve (5/11)

Prognostic significance MRD +6 months after alloSCT

Farina, Haematologica, 2009

MRD –ve (1/16)

MRD +ve (8/13)

MRD kinetics after RIC alloSCT

Ritgen et al., 2008

Farina et al., 2009

Moreno

et al., 2006*

Caballero et al., 2005

n 28 29 20 21

MRD kinetics– neg / mixed

– pos79 %

21 %

55 %

45 %

70 %

30 %

94 %

6 %

Relapse by MRD– neg / mixed

– pos4 %

83 %

6 %

62 % n.d. n.d.

Evidence for delayed clearance

YES YES YES YES

* and Moreno personal communication 2009

Summary: MRD after alloSCT• Techniques: have to be quantitative & sensitive ( 10-4)

• MRD flow • ASO IgH qPCR

• Retrospective analyses show that:• delayed, likely GVL-mediated MRD clearance occurs• MRD clearance:

• predicts of very low relapse risk• is durable• might serve as surrogate marker for cure

• MRD persistence after CsA tapering can be used as trigger for preemptive immun-therapy (DLI)

Treatment aim to be tested prospectively : MRD negativity (< 10-4) 12 months after alloSCT

Perspective: MRD after alloSCT

• Test MRD negativity (< 10-4) 12 months after alloSCT prospectively

• Treat MRD after alloSCT using • DLI• alternative treatment options (e.g. Rituximab)

• Delineate mechanisms of MRD clearance

Relapse Monitoring after Allogeneic Stem Cell Transplantation for Lymphomas

Issa Khouri, Julie Vose

Response Definitions in LymphomaResponse Definitions in Lymphoma

ResponseResponse DefinitionDefinition Nodal MassesNodal Masses Spleen, LiverSpleen, Liver Bone MarrowBone Marrow

SDSD Failure to attain CR/PR Failure to attain CR/PR

or PDor PD(a) FDG-avid or PET positive prior to (a) FDG-avid or PET positive prior to

therapy; PET positive at prior sites of therapy; PET positive at prior sites of

disease and no new sites on CT or disease and no new sites on CT or

PETPET

(b) Variably FDG-avid or PET (b) Variably FDG-avid or PET

negative; no change in size of negative; no change in size of

previous lesions on CTprevious lesions on CT

Relapsed Relapsed

disease or PDdisease or PDAny new lesion or Any new lesion or

increase by 50% of increase by 50% of

previously involved previously involved

sites from nadirsites from nadir

Appearance of a new lesion (s) > 1.5 Appearance of a new lesion (s) > 1.5

cm in any axis, 50% increase in SPD cm in any axis, 50% increase in SPD

of more than one node, or 50% of more than one node, or 50%

increase in longest diameter of a increase in longest diameter of a

previously identified node > 1 cm in previously identified node > 1 cm in

short axisshort axis

Lesions PET positive if FDG-avid Lesions PET positive if FDG-avid

lymphoma or PET positive prior to lymphoma or PET positive prior to

therapytherapy

> 50% increase > 50% increase

from nadir in the from nadir in the

SPD of any SPD of any

previous lesionsprevious lesions

New or recurrent New or recurrent

involvementinvolvement

Abbreviations: CR, complete remission; FDG, [Abbreviations: CR, complete remission; FDG, [1818F]fluorodeoxyglucose; PET, positron emission tomography; CT, computed tomography; PR, partial F]fluorodeoxyglucose; PET, positron emission tomography; CT, computed tomography; PR, partial

remission; SPD, sum of the product of the diameters; SD, stable disease; PD, progressive disease.remission; SPD, sum of the product of the diameters; SD, stable disease; PD, progressive disease.

Cheson, JCO 2007

““False Positive” PET Scans in Therapy False Positive” PET Scans in Therapy of Lymphomasof Lymphomas

996 PET scans in 706 patients with lymphoma996 PET scans in 706 patients with lymphoma PET to evaluate recurrence after treatmentPET to evaluate recurrence after treatment 31/134 scans (23.1%) were False Positive31/134 scans (23.1%) were False Positive

7 brown fat7 brown fat 5 thymic hyperplasia5 thymic hyperplasia 4 muscle contraction4 muscle contraction 4 non-specific inflammation of the colon4 non-specific inflammation of the colon 4 pulmonary/mediastinal inflammation4 pulmonary/mediastinal inflammation 4 intestinal: gastritis (2), colitis (2)4 intestinal: gastritis (2), colitis (2) abscess, lactating breast, abscess, lactating breast, H. zosterH. zoster (1 ea) (1 ea)

Castellucci et al. Nuc Med Commun 26: 689-794, 2005.Castellucci et al. Nuc Med Commun 26: 689-794, 2005.

BM Involvement Present No BM Involvement, GCSF(+)BM Involvement Present No BM Involvement, GCSF(+)

Examples of Bone Marrow Findings on Examples of Bone Marrow Findings on PET in Two Patients with NHLPET in Two Patients with NHL

Message: The films look the same!Message: The films look the same!

0 20 40 60 80 100 120

Months Since Disease Progression

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Cu

mula

tive

Pro

po

rtio

n S

urv

ivin

g

Follicular, 54% (28-74)

T-Cell, 42% (15-66)

MCL, 31% (13-52)

DLCL/Other, 6% (12-17)

Survival in NHL Relapsing post AlloUsing CT Criteria

Khouri et al. unpublished data

Detection Early relapse in Lymphoma

Quantitative PCR

-IgH in b-cell disease

-t(11,14) in MCL and t(14,18) in FL

-t-cell receptor in t-cell lymphoma Chimerism

Chimerism at day 90 and Outcomepost NST

(6)1(6)1No. of relapse, (%)

(69)11(59)10Chronic GVHD, no. (%)

(100)16(100)17Achieved CR, no. (%)

38637129CR: PR at NST,%

1617No. of patients

P valueFull DonorMixed

Chimerism

0.06

0.5

Khouri, Blood 2008

Yes

1. Failure of disease response

2. Responding, but failing to achieve CR at 6 months

No

1. If stable mixed chimera (SMC) in the absence of measurable disease or disease progression

2. SMC definition:

- > 50% donor cells

- No significant decrease of >20% on two consecutive analysis

Donor Lymphocyte Infusion with Rituxan (for b-cell) after Allo

Nicolaus KrögerNicolaus Kröger

Disease specific Monitoring of Relapse after Disease specific Monitoring of Relapse after Allogeneic Hematopoietic Cell TransplantationAllogeneic Hematopoietic Cell Transplantation

Multiple MyelomaMultiple MyelomaNCI Workshop 1./2.-11.2009NCI Workshop 1./2.-11.2009

Conventional techniques for MonitoringConventional techniques for Monitoring

• Bone marrow aspiration: infiltration often Bone marrow aspiration: infiltration often underestimatedunderestimated

• Serum/24h urine electrophoresis (agarose gel or Serum/24h urine electrophoresis (agarose gel or capillary zone): lowest detectable level of M-capillary zone): lowest detectable level of M-component: 0.2 - 0.6 g/Lcomponent: 0.2 - 0.6 g/L

• Immunofixation (serum/urine): lowest detectable level Immunofixation (serum/urine): lowest detectable level of M-component: 0.12 - 0.25 g/Lof M-component: 0.12 - 0.25 g/L

• Free light chain assay (Free light chain assay (κκ//λλ ratio) : useful in light chain ratio) : useful in light chain disease and non-secretory, necessary to determine disease and non-secretory, necessary to determine sCR, early response assessment due to short half time sCR, early response assessment due to short half time (6h)(6h)

Imaging monitoring Imaging monitoring

• More than 80% of the pts develop osteolytic bone lesionsMore than 80% of the pts develop osteolytic bone lesions

• The hallmark of myeloma bone disease is an increased The hallmark of myeloma bone disease is an increased osteoclastic bone resorption and an exhausted osteoblast osteoclastic bone resorption and an exhausted osteoblast function resulting in a reduced bone formation even in patients in function resulting in a reduced bone formation even in patients in complete remissioncomplete remission

• Standard: conventional radiology as skeletal survey involving Standard: conventional radiology as skeletal survey involving cervical, thoracic and lumbar spine, skull, chest, pelvis, humeri cervical, thoracic and lumbar spine, skull, chest, pelvis, humeri and femoraand femora

• Disadvantage: low sensitivity, no exact response assessmentDisadvantage: low sensitivity, no exact response assessment

• CT: high sensitivity, but higher radiation doseCT: high sensitivity, but higher radiation dose

• MRI: high sensitivity, no radiation dose, detect extramedul-lary MRI: high sensitivity, no radiation dose, detect extramedul-lary diseasedisease

• PET-CT: highest sensitivity for extramedullary diseasePET-CT: highest sensitivity for extramedullary disease

Flow-cytometryFlow-cytometry

• Flow cytometry has become an easy applicable method Flow cytometry has become an easy applicable method to detect residual myeloma cells The European Myeloma to detect residual myeloma cells The European Myeloma Network recommends a minimal panel including Network recommends a minimal panel including

• CD19, CD56, CD20, CD117, CD28 and CD27.CD19, CD56, CD20, CD117, CD28 and CD27.

• Plasma cell gating should be based on CD38 vs. CD138 Plasma cell gating should be based on CD38 vs. CD138 expressionexpression

• This method is less sensitive (10This method is less sensitive (10-4-4) than allele-specific ) than allele-specific oligonucleotides PCR (ASO-PCR) oligonucleotides PCR (ASO-PCR)

Rawstron 2008Rawstron 2008

Allele-specific oligonucleotides PCRAllele-specific oligonucleotides PCR(ASO-PCR)(ASO-PCR)

• Patient-specific primers (IgH rearrangement)Patient-specific primers (IgH rearrangement)

• High sensitivity of (10High sensitivity of (10-5 -5 - 10- 10-6-6) and highly specific (100%) ) and highly specific (100%)

• Time-consuming (for each patients), does not detect Time-consuming (for each patients), does not detect extramedullary diseaseextramedullary disease

Rate of molecular remission basedRate of molecular remission basedon rearranged immunoglobulin heavyon rearranged immunoglobulin heavychain geneschain genes

In CR:In CR: after allograft:after allograft: 50% molecular CR50% molecular CRafter autograft:after autograft: 7% molecular CR7% molecular CR

In CR:In CR: after allograft:after allograft: 50% molecular CR50% molecular CRafter autograft:after autograft: 16% molecular CR16% molecular CR

Martinelli et al., JCO 2000Martinelli et al., JCO 2000

Corradini et al., JCO 1999Corradini et al., JCO 1999

No. of ptsNo. of pts 1616 1919 1313

5 year cumulativ5 year cumulativ

risk of relapserisk of relapse 0% 0% 33% 33% 100% 100%

Minimal residual disease after allogeneic stem cell Minimal residual disease after allogeneic stem cell transplantationtransplantation

Multiple Myeloma (EBMT-Studie): Pat with CRMultiple Myeloma (EBMT-Studie): Pat with CR

PCR negPCR neg PCR mixedPCR mixed PCR posPCR pos

Corradini et al., Blood 2003Corradini et al., Blood 2003

ChimerismsChimerisms

• Not specific for relapse, in majority of relapse donor Not specific for relapse, in majority of relapse donor cell chimerism persistedcell chimerism persisted

• Lineage specific chimerism (plasmacell-chimerism: Lineage specific chimerism (plasmacell-chimerism: CD138+ BM cells)CD138+ BM cells)

• By using real-time PCR the sensitivity of the method is By using real-time PCR the sensitivity of the method is 1010-4-4 to 10 to 10-5-5. The disadvantage of the methods is the . The disadvantage of the methods is the lack of specificity.lack of specificity.

Quantitative donor plasma-cell chimerism in Quantitative donor plasma-cell chimerism in patients with negative immunofixationpatients with negative immunofixation

Predictive value of donor-plasma-cell chimerism for Predictive value of donor-plasma-cell chimerism for relapse relapse

• 93% with stable or increasing donor-plasma-cell 93% with stable or increasing donor-plasma-cell chimerism remained immunofixation-negative.chimerism remained immunofixation-negative.

• 83% with a decrease of donor-plasma-cell chimerism 83% with a decrease of donor-plasma-cell chimerism was associated with relapse in the sense of becoming was associated with relapse in the sense of becoming immunofixation-positivity (in 2: 3 and 6 months prior immunofixation-positivity (in 2: 3 and 6 months prior than immunofixation becomes positive)than immunofixation becomes positive)

Kröger et al., 2006

CRCR

non CRnon CR

Depths of remission and survival post allograftingDepths of remission and survival post allografting

p=0.03

According EBMT criteriaAccording EBMT criteria

58%

According Flow cytometry According Flow cytometry

74%CR

Non-CR

p=0.001

According to molecular methodsAccording to molecular methods

81%

CR

p=0.001Kröger et al., 2009Kröger et al., 2009

Nicolaus Kröger Nicolaus Kröger

Dept. of Stem Cell Transplantation, University Hospital HamburgDept. of Stem Cell Transplantation, University Hospital Hamburg

Hamburg, GermanyHamburg, Germany

Relapse DefinitionRelapse DefinitionNCI Workshop 1./2.11.2009NCI Workshop 1./2.11.2009

CML Standard DefinitionCML Standard Definition

Molecular relapse Molecular relapse (The date of molecular relapse is the date of the first positive RT-PCR assay.)(The date of molecular relapse is the date of the first positive RT-PCR assay.)

Is said to be present in a CML patient lacking any other evidence of the disease (i.e. patient in hematological remission Is said to be present in a CML patient lacking any other evidence of the disease (i.e. patient in hematological remission

and cytogenetic remission) at least 4 months after SCT when any of the following apply:and cytogenetic remission) at least 4 months after SCT when any of the following apply:

Three samples over a minimum of 4 weeks show a BCR-ABL/ABL ratio higher than 0.02% as measured by Three samples over a minimum of 4 weeks show a BCR-ABL/ABL ratio higher than 0.02% as measured by

quantitative RT-PCR tests. quantitative RT-PCR tests. Three samples over a minimum of 4 weeks show clearly rising levels of BCR-ABL/ABL ratio with the last two Three samples over a minimum of 4 weeks show clearly rising levels of BCR-ABL/ABL ratio with the last two

higher than 0.02% as measured by quantitative RT-PCR tests.higher than 0.02% as measured by quantitative RT-PCR tests. Two samples over a minimum of 4 weeks show a BCR-ABL/ABL ratio higher than 0.05% as measured by Two samples over a minimum of 4 weeks show a BCR-ABL/ABL ratio higher than 0.05% as measured by

quantitative RT-PCR tests.quantitative RT-PCR tests.

Cytogenetic relapseCytogenetic relapse

Any of the following in a patient lacking any clinical or hematological evidence of the disease (i.e. patient in Any of the following in a patient lacking any clinical or hematological evidence of the disease (i.e. patient in

hematological remission):hematological remission):

Presence of one or more Ph-positive metaphases with standard cytogenetics or hypermetaphase FISH;Presence of one or more Ph-positive metaphases with standard cytogenetics or hypermetaphase FISH; >2% cells with the BCR-ABL fusion gene by interphase FISH>2% cells with the BCR-ABL fusion gene by interphase FISH

Hematological relapseHematological relapse

All of the following:All of the following:

Abnormal blood or marrow counts or morphology consistent with CML.Abnormal blood or marrow counts or morphology consistent with CML. Cytogenetic and/or molecular confirmation of the presence of the disease.Cytogenetic and/or molecular confirmation of the presence of the disease.

Hematological relapse is sub-classified into chronic phase, accelerated phase or blastic phase according to WHO Hematological relapse is sub-classified into chronic phase, accelerated phase or blastic phase according to WHO

criteriacriteria

CML CML ProposalProposal

DiseaseDisease Definition of Definition of

CRCRDefinition of Definition of

RelapseRelapseMolecular Molecular

markermarkerChromosomeChromosome ChimerismChimerism ImagingImaging Flow cytometryFlow cytometry

CMLCML

applicableapplicable

Comment:Comment:

HematologicHematologic

CytogeneticCytogenetic

MolecularMolecular

All patientsAll patients

HematologicHematologic


MolecularMolecular


BCR-ABL RT-PCRBCR-ABL RT-PCR


qPCR identifies qPCR identifies

relapse risk groupsrelapse risk groups


(incl FISH )(incl FISH )


Not as sensitive as Not as sensitive as

qPCR for MRD qPCR for MRD

detectiondetection

PCR or PCR or

VNTR/STRVNTR/STR

All patientsAll patients Not Not


4-6 color flow4-6 color flow

subgroupssubgroups

Only helpful in Only helpful in

identifying aberrant identifying aberrant

blasts in advanced blasts in advanced

phase diseasephase disease

Myelofibrosis Standard DefinitionMyelofibrosis Standard Definition

Progressive Disease: Progressive Disease: Requires one of the following:Requires one of the following:

•Progressive splenomegaly that is defined by the appearance of a Progressive splenomegaly that is defined by the appearance of a previous absent splenomegaly that is palpable at greater than 5 cm previous absent splenomegaly that is palpable at greater than 5 cm below the left costal margin or a minimum of 100% increase in below the left costal margin or a minimum of 100% increase in palpable distance for baseline splenomegaly of 5-10 cm or a palpable distance for baseline splenomegaly of 5-10 cm or a minimum of 50% increase in palpable distance for baseline minimum of 50% increase in palpable distance for baseline splenomegaly of greater than 10 cm.splenomegaly of greater than 10 cm.

•Leukemic transformation confirmed by bone marrow blast count of Leukemic transformation confirmed by bone marrow blast count of at least 20%at least 20%

•Increase in peripheral blood blast percentage of at least 20% that Increase in peripheral blood blast percentage of at least 20% that lasts for 8 weekslasts for 8 weeks

•RelapseRelapse: : Changes from CR to PR or CR/PR to Clinical improvement Changes from CR to PR or CR/PR to Clinical improvement



RelapseRelapseMolecular markerMolecular marker ChromosomeChromosome ChimerismChimerism ImagingImaging Flow cytometryFlow cytometry

MyelofibrosisMyelofibrosis


Comment:Comment:

IWG-MRTIWG-MRT

All ptsAll pts

Not fully Not fully

applicable applicable

IWG-MRTIWG-MRT

All ptsAll pts

Not fully Not fully


JAK2/MPLJAK2/MPL

SubgroupsSubgroups

High sensitivity and High sensitivity and

predictive for predictive for

relapserelapse


(incl FISH)(incl FISH)

SubgroupsSubgroups

Not investigatedNot investigated

PCR/VNTRPCR/VNTR

All ptsAll pts

Correlates with Correlates with

molecular marker, molecular marker,

but less specificbut less specific

MRTMRT

All ptsAll pts

Correlates with Correlates with

fibrosis fibrosis

regressionregression

Flow-cytometryFlow-cytometry

All ptsAll pts

Circulating CD34+ Circulating CD34+

cells may be usefulcells may be useful

Myelofibrosis Myelofibrosis ProposalProposal

AML Standard Definition AML Standard Definition (Cheson et al., 2003)(Cheson et al., 2003)

ParametersParameters Complete remissionComplete remission RelapseRelapse

Morphological/Morphological/

hematological criteriahematological criteria

BM blasts < 5%;BM blasts < 5%;

thrombocytes ≥ 100 x 10thrombocytes ≥ 100 x 1099/L; /L; neutrophils ≥ 1.0 x 10neutrophils ≥ 1.0 x 1099/L/L

Reappearance of blasts post CR Reappearance of blasts post CR (BM: > 5%; PB) (BM: > 5%; PB)

Cytogenetic criteriaCytogenetic criteria Major cytogenetic remission: Major cytogenetic remission: Disappearance of cytogenetic Disappearance of cytogenetic alterationalteration

Minor cytogenetic remission: > 50% Minor cytogenetic remission: > 50% reduction of abnormal metaphasesreduction of abnormal metaphases

Reappearance of cytogenetic Reappearance of cytogenetic alteration alteration

Molecular remissionMolecular remission Disappearance of molecular mutationDisappearance of molecular mutation Reappearance of molecular Reappearance of molecular mutationmutation

Flow cytometryFlow cytometry Disappearance of cells with previously Disappearance of cells with previously determined LAIPdetermined LAIP

Reappearance of cells with LAIPReappearance of cells with LAIP

Criteria of remissionCriteria of remission ParametersParameters

Morphologic and Morphologic and

hematological responsehematological responseComplete remission (CR): bone marrow blasts <5% Complete remission (CR): bone marrow blasts <5%

without dysplasia, hemoglobin ≥11 g/dL, platelets without dysplasia, hemoglobin ≥11 g/dL, platelets

≥ 100 x 10≥ 100 x 1099/L, neutrophils ≥ 1.5 x 10/L, neutrophils ≥ 1.5 x 1099/L/L

Partial remission (PR): reduction of blasts by at least 50% Partial remission (PR): reduction of blasts by at least 50%

or achievement of lower risk category than prior to or achievement of lower risk category than prior to

treatmenttreatment

Cytogenetic responseCytogenetic response Major cytogenetic response: disappearance of a Major cytogenetic response: disappearance of a

cytogenetic abnomalitycytogenetic abnomality

Minor cytogenetic response: ≥50% reduction of abnormal Minor cytogenetic response: ≥50% reduction of abnormal

metaphasesmetaphases

MDS Standard Definition MDS Standard Definition (Cheson et al., 2006)(Cheson et al., 2006)

AML / MDS ProposalAML / MDS Proposal



RelapseRelapseMolecular Molecular

markermarkerChromo-Chromo-

somesomeChimerismChimerism ImagingImaging Flow cytometryFlow cytometry

AML/MDSAML/MDS


Comment:Comment:

IWGIWG

All ptsAll pts

Well Well

establishedestablished

IWGIWG

All ptsAll pts

Well Well

established, established,

but less but less

sensitivesensitive

Mol. MarkerMol. Marker

SubgroupsSubgroups

Expansion of Expansion of

MRD marker MRD marker

panel for post-panel for post-

transplant transplant

monitoring in monitoring in

AML (e.g. AML (e.g. NPM1NPM1

–mutations) or –mutations) or

MDS (e.g. MDS (e.g.

RUNX1RUNX1//AML1AML1

mutations) mutations)


(incl FISH )(incl FISH )

SubgroupsSubgroups

No No

standardization standardization

for MRD for MRD

monitoring, monitoring,

useful for useful for

specific specific

aberrationsaberrations

PCR or VNTR/STRPCR or VNTR/STR

All ptsAll pts

Well established, lack Well established, lack of specificity: of specificity: investigation of lineage investigation of lineage specific chimerism specific chimerism (e.g. CD34(e.g. CD34+ + cells); and cells); and standardization of standardization of techniques techniques

Not Not



All ptsAll pts

Few studiesFew studies

Progressive Disease: Progressive Disease:

An increase of at least 25% in the absolute number of circulating or bone marrow An increase of at least 25% in the absolute number of circulating or bone marrow

leukemic blasts or extramedullary disease burden; leukemic blasts or extramedullary disease burden; oror Development of new extramedullary disease.Development of new extramedullary disease.

Relapsed Disease: Relapsed Disease:

The reappearance of leukemia blast cells in the blood or the bone marrow (≥ 25%) The reappearance of leukemia blast cells in the blood or the bone marrow (≥ 25%)

or in any other extramedullary site after a CR with confirmation of lymphoid blasts or in any other extramedullary site after a CR with confirmation of lymphoid blasts

by morphology and flow cytometry, PCR for antigen receptor loci or fusion genes, by morphology and flow cytometry, PCR for antigen receptor loci or fusion genes,

or cytogenetics/FISH; or cytogenetics/FISH; oror Progression to > 25% leukemia blasts in the marrow after a PR. Progression to > 25% leukemia blasts in the marrow after a PR. Importantly, isolated extramedullary relapses (e.g., CNS) are considered relapse Importantly, isolated extramedullary relapses (e.g., CNS) are considered relapse

from a diagnostic standpoint, although these are commonly approached from a diagnostic standpoint, although these are commonly approached

differently in terms of therapy.differently in terms of therapy.

ALL Standard DefinitionALL Standard Definition

ALL ProposalALL Proposal

DiseaseDisease Definition Definition

of CRof CRDefinition of Definition of

RelapseRelapseMolecular markerMolecular marker ChromosomeChromosome ChimerismChimerism ImagingImaging Flow cytometryFlow cytometry

ALLALL


Comment:Comment:

Less than Less than

5% blasts 5% blasts

in BMin BM

All ptsAll pts

More thanMore than

5% blasts in 5% blasts in

BMBM

All ptsAll pts

TCR- and Ig- Gene TCR- and Ig- Gene

rearrangement rearrangement

90% of all patients 90% of all patients

- ASO primer- ASO primer

80-90% of patients80-90% of patients

- Ig VDJ for most - Ig VDJ for most

patients patients

- BCR-ABL for all - BCR-ABL for all

Ph+ ALLPh+ ALL


(incl .FISH)(incl .FISH)

subgroupssubgroups

clinical not clinical not

important for MRD important for MRD

assessmentassessment


All ptsAll pts

Gold standard: Singleplex Gold standard: Singleplex

PCR with fluorescent PCR with fluorescent

labelled STR primers. labelled STR primers.

importantly: product importantly: product

resolution using capillary resolution using capillary

electrophoresiselectrophoresis

Limited data on utilityLimited data on utility

Not Not



>95% of patients>95% of patients

Sensitivity in B-ALL Sensitivity in B-ALL

limited after SCT limited after SCT

because of large because of large

numbers of numbers of

hematogoneshematogones

• Relapse: Relapse: progression occurring 6 months or later after having achieved CR or PR progression occurring 6 months or later after having achieved CR or PR

• ProgressionProgression: : IW-CLL/NCI-WG criteria for CLL progression (at least one must apply)IW-CLL/NCI-WG criteria for CLL progression (at least one must apply)•• Appearance of any new lesion such as enlarged lymph nodes (> 1.5 cm), splenomegaly, Appearance of any new lesion such as enlarged lymph nodes (> 1.5 cm), splenomegaly, hepatomegaly or other organ infiltrates;hepatomegaly or other organ infiltrates;

•• increase of lymphadenopathy by 50% or more in greatest determined diameter of any increase of lymphadenopathy by 50% or more in greatest determined diameter of any previous site, or an increase of 50% or more in the sum of the product of diameters of previous site, or an increase of 50% or more in the sum of the product of diameters of multiple multiple nodes;nodes;

•• increase in the liver or spleen size by 50% or more or the de novo appearance of increase in the liver or spleen size by 50% or more or the de novo appearance of hepatomegaly or splenomegaly;hepatomegaly or splenomegaly;

•• increase in the number of blood lymphocytes by 50% or more with at least 5/nL B cells;increase in the number of blood lymphocytes by 50% or more with at least 5/nL B cells;

•• transformation to a more aggressive histology (e.g. Richter's syndrome).transformation to a more aggressive histology (e.g. Richter's syndrome).

•• occurrence of cytopenia (neutropenia, anemia or thrombocytopenia) attributable to CLL.occurrence of cytopenia (neutropenia, anemia or thrombocytopenia) attributable to CLL.

• Complete MRD response: Complete MRD response: clinical remission in the absence of one CLL cell per clinical remission in the absence of one CLL cell per 10,000 leukocytes in the peripheral blood or bone marrow 10,000 leukocytes in the peripheral blood or bone marrow

• MRD relapse: MRD relapse: Tumor cell recurrence or increases at the MRD level that does not Tumor cell recurrence or increases at the MRD level that does not exceed 5 B cells/nL in the peripheral blood.exceed 5 B cells/nL in the peripheral blood.

CLL Standard DefinitionCLL Standard Definition

CLL CLL ProposalProposal



RelapseRelapseMolecularMolecular

markermarkerChromo-Chromo-

somesomeChimerismChimerism ImagingImaging Flow cytometryFlow cytometry

CLLCLL


Comment:Comment:

iwCLL/NCIiwCLL/NCI

All ptsAll pts

iwCLL iwCLL

definition of definition of

MRD MRD

negativity: negativity:

MRD < 10MRD < 10-4 -4 by by

qPCR or MRD qPCR or MRD

FlowFlow

iwCLL/NCiwCLL/NC

All ptsAll pts

ASO-primer ASO-primer IGHIGH

qPCRqPCR

~90%~90%


sustained remission sustained remission

if < 10if < 10-4 -4 1 year post 1 year post

SCT.SCT.

More sensitive than More sensitive than

MRD flow belowMRD flow below

1010-4 -4



subgroupsubgroup

No role in No role in

relapse relapse

monitoringmonitoring

PCR/VNTRPCR/VNTR

All ptsAll pts

Complete donor Complete donor

chimerism usually chimerism usually

prerequisite for MRD prerequisite for MRD

negativity, but not negativity, but not

suitable as MRD suitable as MRD

markermarker

CTCT

All ptsAll pts

Only to be used if Only to be used if

CR by clinical CR by clinical

methods or in methods or in

clinical trialsclinical trials

MRD flowMRD flow

> 95% > 95%


sustained sustained

remission if < 10remission if < 10-4 -4 1 1

year post SCT.year post SCT.

Equally sensitive Equally sensitive

and specific as and specific as

qPCR up to10qPCR up to10-4-4

ResponseResponse DefinitionDefinition Nodal MassesNodal Masses Spleen, LiverSpleen, Liver Bone MarrowBone Marrow

CR Disappearance of all evidence of disease

(a) FDG-avid or PET positive prior to therapy; mass of any size permitted if PET negative

(b) Variably FDG-avid or PET negative; regression to normal size on CT

Not palpable, nodules disappeared

Infiltrate cleared on repeat biopsy; if indeterminate by morphology, immunohistochemistry should be negative

Relapsed Relapsed disease disease

or PDor PD

Any new lesion Any new lesion or increase by ≥ or increase by ≥ 50 % of 50 % of previously previously involved sites involved sites from nadirfrom nadir

Appearance of a new lesion(s) > 1.5 Appearance of a new lesion(s) > 1.5 cm in any axis, ≥ 50 % increase in SPD cm in any axis, ≥ 50 % increase in SPD of more than one node, or ≥ 50 % of more than one node, or ≥ 50 % increase in longest diameter of a increase in longest diameter of a previously identified node > 1 cm in previously identified node > 1 cm in short axisshort axis

Lesions PET positive if FDG-avid Lesions PET positive if FDG-avid lymphoma or PET positive prior to lymphoma or PET positive prior to therapytherapy

> 50 % increase > 50 % increase from nadir in the from nadir in the SPD of any previous SPD of any previous lesionslesions

New or recurrent New or recurrent involvementinvolvement

Lymphoma Standard Definition Lymphoma Standard Definition (Cheson et al., 2007)(Cheson et al., 2007)

Lymphoma ProposalLymphoma Proposal

DiseaseDisease Definition of CRDefinition of CR Definition of Definition of

RelapseRelapseMolecularMolecular

markermarkerChromosomeChromosome ChimerismChimerism ImagingImaging FlowFlow

cytometrycytometry

LymphomaLymphoma


Comment:Comment:

Cheson criteriaCheson criteria

All patientAll patient

Well established Well established

for all for all

lymphomaslymphomas

ChesonCheson criteriacriteria


Well Well

established for established for

all lymphomasall lymphomas

ASO-primer (IgH ) for ASO-primer (IgH ) for

B-cell NHLB-cell NHL

subgroupssubgroups

Bcl-2 for FLBcl-2 for FL

Bcl-1for about 30% of Bcl-1for about 30% of

MCLMCL

T cell receptor for T-T cell receptor for T-

NHLNHL



subgroupssubgroups

t(14;18) for FLt(14;18) for FL

t(11,14) for MCLt(11,14) for MCL



Monitoring T-cell Monitoring T-cell

by PCR useful in by PCR useful in

NHL. Role not NHL. Role not

established in HDestablished in HD

CT/PETCT/PET


Well Well

establishedestablished

in all in all

lymphomaslymphomas


SubgroupsSubgroups

Could be helpful Could be helpful

for FL and MCLfor FL and MCL

Multiple Myeloma Standard DefinitionMultiple Myeloma Standard Definition

RelapseRelapse: : EBMT criteria (Bladè et al) requires at least one of the following:EBMT criteria (Bladè et al) requires at least one of the following:

• Reappearance of serum or urinary paraprotein on immunofixation or routine Reappearance of serum or urinary paraprotein on immunofixation or routine electrophoresis, confirmed by at least on further investigation and excluding oligoclonal electrophoresis, confirmed by at least on further investigation and excluding oligoclonal immune reconstitution.immune reconstitution.

• ≥ ≥ 5 % plasma cells in a bone marrow aspirate or on trephine bone biopsy.5 % plasma cells in a bone marrow aspirate or on trephine bone biopsy.

• Development of new lytic bone lesions or soft tissue plasmacytomas or definite increase Development of new lytic bone lesions or soft tissue plasmacytomas or definite increase in the size of residual bone lesions (development of a compression fracture does not in the size of residual bone lesions (development of a compression fracture does not exclude continued response and may not indicate progression).\exclude continued response and may not indicate progression).\

• Development of hypercalcaemia (corrected serum calcium > 11.5 mg/dl or 2.8 mmol/l) not Development of hypercalcaemia (corrected serum calcium > 11.5 mg/dl or 2.8 mmol/l) not attributable to any other cause.attributable to any other cause.

IWG Criteria (Durie et al):IWG Criteria (Durie et al): Relapse from CR requires at least one of the following:Relapse from CR requires at least one of the following:

•• Reappearance of serum or urinary M-protein by immunofixation or electrophoresisReappearance of serum or urinary M-protein by immunofixation or electrophoresis

•• ≥ ≥ 5 % plasma cells in a bone marrow.5 % plasma cells in a bone marrow.

•• Appearance of any other sign of progression (i.e new lytic bone lesions or soft tissue Appearance of any other sign of progression (i.e new lytic bone lesions or soft tissue plasmacytomas or hypercalcemia).plasmacytomas or hypercalcemia).

Multiple Myeloma Multiple Myeloma ProposalProposal


CRCRDefinition Definition

of Relapseof RelapseMolecular Molecular

markermarkerChromosomeChromosome ChimerismChimerism ImagingImaging Flow Flow

cytometrycytometryOther Other

methodsmethods

MultipleMultiple

MyelomaMyeloma


Comment:Comment:

1) EBMT1) EBMT

2) IWG2) IWG

All ptsAll pts

Accepted Accepted

but less but less

sensitivesensitive

1) EBMT1) EBMT

2) IWG2) IWG

All ptsAll pts

Accepted Accepted

but less but less

sensitivesensitive

ASO-primerASO-primer

(IgH)(IgH)

40-80%40-80%

Important, but Important, but

not included in not included in

EBMT and IWG EBMT and IWG

definitiondefinition



subgroupssubgroups

May be useful* May be useful*


All ptsAll pts

MNC-donor MNC-donor

chimerism not chimerism not

useful, lineage useful, lineage

specific donor specific donor

chimerism (CD138+ chimerism (CD138+

plasma cells) plasma cells)

predicts relapsepredicts relapse

MRIMRI

PET-CTPET-CT

All ptsAll pts

Not Not

established, established,

but useful but useful

for for

extramedullextramedull

aryary

diseasedisease


All ptsAll pts

More sensitive More sensitive

than than

EBMT/IWG in EBMT/IWG in

predicting predicting

relapserelapse

Free lightFree light

chainchain

assayassay

subgroupssubgroups

Proposed by Proposed by

IWG: no valid IWG: no valid

datadata

Sub-Committee on Disease-Specific Methods And Sub-Committee on Disease-Specific Methods And Strategies For Monitoring Relapse Following Strategies For Monitoring Relapse Following

Allogeneic Stem Cell TransplantationAllogeneic Stem Cell Transplantation

Panel Discussion

Relapse and Response Definitions After SCTRelapse and Response Definitions After SCTStandard diagnostic criteria used to define response and relapse Standard diagnostic criteria used to define response and relapse

Well validated in upfront clinical trialsWell validated in upfront clinical trials

Utility after allogeneic SCT is limited for most hematologic malignanciesUtility after allogeneic SCT is limited for most hematologic malignancies

Sensitive disease-specific detection methodsSensitive disease-specific detection methods

Methodologic standardization and validationMethodologic standardization and validation

Highly sensitive monitoring possibleHighly sensitive monitoring possible

Prognostic value in predicting continuous remission Prognostic value in predicting continuous remission vs.vs. relapse relapse

Facilitate early intervention Facilitate early intervention

Utility “pre-emptive” initiation of therapy prior to overt relapseUtility “pre-emptive” initiation of therapy prior to overt relapse

Proposed incorporation of sensitive detection methods to augment Proposed incorporation of sensitive detection methods to augment standard response/relapse definitions for use in allogeneic SCT trialsstandard response/relapse definitions for use in allogeneic SCT trials

Response endpoints Response endpoints

Relapse predictionRelapse prediction

Relapse prevention Relapse prevention

Relapse treatmentRelapse treatment

Discussion PointsDiscussion Points1. Are the standard diagnostic criteria for relapse and response

adequate for use after allogeneic SCT?

2.2. Proposed incorporation of sensitive detection methods to Proposed incorporation of sensitive detection methods to augment disease-specific definitions after allogeneic SCTaugment disease-specific definitions after allogeneic SCT

A. Methods included for specific diseases

B. Value of chimerism

C. Discordant results

D. Frequency of monitoring

3. Should achievement of molecular remission be the goal of allogeneic SCT?

4. When does molecular relapse or residual disease justify therapeutic intervention?

Research PrioritiesResearch Priorities1. Harmonization and standardization of molecular

monitoring and flow cytometry

2. Define the kinetics of molecular remission and molecular relapse after allogeneic SCT

3. Determine the predictive value of MRD and chimerism (incl lineage-specific) for clinical relapse

4. Apply and assess proposed definitions in studies designed to change the natural history of relapse after SCT

5. Apply and assess proposed definitions in trials of new treatments for prevention and treatment of relapse after SCT

on behalf of the sub-committee

Documents

mrd post transplant

mrd statusknechtliblood

cr2efsciefscir mrd

mrd statusknechtlibjh

low level

retrospective studies

bm post transplant

post transplant outcome