John E. Wagner, M.D. · 2.7-8.1 x 10. 8 . NC/kg • 8.1-24 x 10. 8 . NC/kg. CD34- Cryopreserved CD34+-15. DUCBT Platform . Uncultured UCB 1 UCB 2. NC >1.5 x 10. 7 /kg (smaller

Blood and Marrow Transplant for FA John E. Wagner, M.D.

175

150

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75

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25

0 1976 1979 1982 1985 1988 1991 1994 1997 2000 2003 2006 2009 2012 2015

Cum ulative

175

150

125

100

75

50

25

0

Cum ulative

U R D R D

Num

ber o

f Tra

nspl

ants

1st Umbilical cord blood transplant

1st T cell depletion for URD BMT

Dose reduced therapy

High dose therapy

TBI dose de- escalation study

1st use of fludarabine

1st successful PGD/IVF

Next steps

Elimination of MP (Siro/MMF)

Accomplishments (n=226)

Tissue Repair

Quality of Life

Cancer Prevention

Immune system Other tissues

Curing Fanconi Anemia

Grand Challenges

• Universal donor availability

• Reliable engraftment

• Absence of GVHD

• Prompt immune recovery

• Minimal late effects

• Earlier diagnosis / referral

- Done (BM/UCB)

- Done (FLU)

- Better (TCD)

- Needs work

- Better

- Improving

University of Minnesota Referral

• Confirmatory DEB / MMC

• HLA typing

• Evaluation of siblings (DEB / HLA)

• Q3M CBC / Q1Y BMA-BX (cytogenetics)

• Organ / endocrine evaluation

• Reproductive / genetic counseling

University of Minnesota Treatment Options by Disease Status and

Donor Type

Aplastic Anemia MDS/Leukemia

FLU-CY CY-FLU-TBI 300 cGy

HLA matched sibling

Alternative donor type

Aplastic Anemia MDS/Leukemia

CY-FLU-TBI 300 cGy [BU-FLU-CY if BRCA2]

If no HLA matched sibling Best Available Donor

Diagnosis

Search

HLA 8/8

HLA 7/8

Bone Marrow

Peripheral Blood

Cord Blood

HLA 6/6

HLA 5/6

>3.0

>4.0

2 weeks 3-4 mos

University of Minnesota

Cleave the bead from the cells

Prevention of GVHD

T cell depletion

CD34+ cells University of Minnesota

Matched Sibling Donor HSCT For FA/SAA

-5 -4 -3 -2 -1 0 1 2 3

CY 5 HSCT

FLU 35

ATG ATG ATG ATG ATG

G-CSF

(ANC 2500)

CSA (Day 100)

(Day -5 to 15) MP

University of Minnesota

TCD BM

UCB Eliminated Radiation TCD to reduce risk of GVHD

Matched Sibling Donor HSCT For FA/SAA

-5 -4 -3 -2 -1 0 1 2 3

CY 5 HSCT

FLU 35

ATG ATG ATG ATG ATG

G-CSF

(ANC 2500)

CSA (Day 100)

(Day -1 to 30) MMF

University of Minnesota

TCD BM

UCB

Eliminated Radiation TCD to reduce risk of GVHD Eliminated MP

11/2011

Cum

ulat

ive

prop

ortio

n

• 100% engraftment • 0% severe RRT • 3% acute GVHD (1 patient) • 0% chronic GVHD

0 2 4 6 8 10 12

Months after HSCT

0.0

0.2

0.4

0.6

n=28 0.8

1.0

14 16 18 20 22 24

97%

Survival Patients <18 years with a genotypic identical donor

One with grade 3 GVHD

COD infection due to GVHD

University of Minnesota

-6 -5 -4 -3 -2 -1 0 1 2 3

CY10 CY10 CY10 CY10 HCT

UCB

TCD BM

FLU 35 FLU 35 FLU 35 FLU 35

ATG ATG ATG ATG ATG

TBI 300

Unrelated Donor HCT For FA Since 2006

CsA / MP Rapa / MMF

Fanconi Anemia Thymic Shielding

Since 2003

Thymus block Prior to HCT, CT is performed to locate the thymus

Thymus blocks attached to TBI stand brackets securing the lung compensators

Thymus Shielding

University of Minnesota

Similar Survival after MSD HSCT and Alternative Donor HSCT for Standard Risk Patients with FA

92% Matched sibling, n=24

67% Alternative donor: high risk n=6 (MDS/Acute leukemia or renal failure)

87% Alternative donor: std risk, n=45

4 6 Months After HSCT

Cum

ulat

ive

Inci

denc

e

0.4

0.2

0.0

0.6

0.8

1.0

0 2 8 10 12 University of Minnesota

y Matched Sibling Donors

Standard Risk FLU-CY

Alternative Donors

Standard Risk TBI 300+TS

Alternative Donors

High Risk TBI 300+TS

N 24 45 6

Engraftment 100% 93% 100%

Acute GVHD 4% 11% 17%

Chronic GvHD 8% 4% 0%

1 Year Survival 92% 87% 67%

Secondary Malignancies

2/45 = 4% (2 SCC)

Neutrophil Recovery Cu

mul

ativ

e In

cide

nce,

%

Days

100

10

0

30

20

50

40

70

60

90

80

0

100

90

80

UCB mismatched, 79%

0 20 50

Eapen / Wagner et al. 10 30 40

PBPC matched, 96%

PBPC mismatched, 96% BM matched, 92%

BM mismatched, 94% 13 18

25

Slow recovery in adults and children Increased risk of graft failure

Adults

14%

Lancet Oncol 2010; 11: 653-60

Cell dose is critical Correlation between recovery and cell dose

0 7 14 21 28 35 42 49

Days after UCBT

56 63 70 77

Neutrophil Engraftment by Cell Dose (x107/kg)

Target: >20 x 107/kg

Median cell dose transplanted: 3.6 x 107/kg in children

-7 -6 -5 -4 -3 -2 -1 0 7 14

Speed of PMN Recovery

100

Advantages • Infusion timing

• Tracking progeny Limitations • Immunological

graft-graft interactions

BMBx BMBx

360

Days

Trial Design HSC835 Dose Escalation

• 0.3-0.9 x 108 NC/kg

• 0.9-2.7 x 108 NC/kg

• 2.7-8.1 x 108 NC/kg

• 8.1-24 x 108 NC/kg

CD34-

Cryopreserved

CD34+

-15

DUCBT Platform

Uncultured UCB 1

UCB 2

NC >1.5 x 107/kg (smaller unit)

HLA < 2 ag mm NC >1.5 x 107/kg (larger

unit)

TBI FLU FLU FLU

CY CY

Ex vivo culture begins

HSC835 engraftment accelerates neutrophil and platelet recovery

Significant correlation between HSC835 CD34 dose and time to engraftment

HSC835 Engrafted (12/17 patients)

Unmanipulated Engrafted (5/17 patients)

Wagner et al. Submitted

Patient Age

CY FLU TBI 300 cGy Engraft GVHD Survival COD

Outcomes in Adults

1 19.4 11 none Dead (2.2 y) SCC (1 y)

2 21.2 14 none Alive (5.3 y) -

3 24.1 16 none Alive (2.6 y) -

4 (MDS) 33.1 15 GI (stg 2) Alive (2.5 y) -

5 34.3 13 none Alive (2.0 y) -

6 (MDS) 26.1 38 none Dead (59 d) hem

7 (MDS) 28.6 22 GI, Liv (stg 4) Dead (57 d) GvH

8 (MDS) 25.2 30 none Dead (34 d) tox

Fanconi Anemia Advanced MDS/Leukemia

HCT TCD BM

UCB

G-CSF

(Day 30)

(Day 180)

(ANC 2500)

CSA

MMF

FLU 35 FLU 35 FLU 35 FLU 35

-6 -8 -7 0 -5 -4 -3 -1

ATG ATG ATG ATG ATG

-2

BU 0.8 BU 0.8

CY 10 CY 10 CY 10 CY 10

Overall Survival and Relapse FA MDS / Acute Leukemia

Cum

ulat

ive

Prop

ortio

n

2 Years

1.0 0.8 0.6 0.4 0.2 0.0

0 1 3 4 5

Survival: 50% (95% CI, 21-74%)

Relapse: 21% (95% CI, 1-44%)

Impact of BRCA2 / Leukemia

2 Years

Cum

ulat

ive

Prop

ortio

n

BRCA2

0.2 0.0

0.6

0.8

1.0

0 1 3 4 5

II I

I I

No BRCA2

Two additional pts

BU/CY/FLU; single UCB

MDS, 6.5 months out, well BU/CY/FLU; single UCB

Timing of Transplant When to consider alternatives

to transplant

Very poor organ function Kidney Cardiac

Pre existing infections

Lung Blood

Older Patient Age

>40 (sibling donor) >35 (unrelated donor)

Alternatives Androgens

Long term use of blood growth factor

Experimental therapy (gene therapy or novel transplant protocol)

No donor

Cancer

Historical Observations

Conclusions • Transplant is associated with

high rate of solid tumors, particularly SCCs

•Association between GVHD [or its treatment] and SCCs

- 33-fold higher risk AGVHD - infinity higher risk CGVHD

Rosenberg, Blood 2005; 105: 67-73

Relative to non transplanted FA patients

Earlier onset and higher frequency of SCC after BMT

Additional Findings

Median age 19 years (BMT) Association with grade 3-4 acute and extensive chronic GVHD

96% pts with aGVHD died

All SCC patients had h/o cGVHD

Median age 31 years (non BMT)

Higher rate of SCC after BMT is due to

Hypothesis

• GVHD or its treatment Bonfim (Curitiba) • Conditioning regimen?

11 patients with cancer - Radiation

- Cyclophosphamide

- Busulfan

MTecdeialnl daegep1le8tyioeanrs

MediaTnBinI tdeorvsael (dBeM-T toeSsCcCa)la7tyioeanrs

All patients transplanted at the U Minnesota from 1990 to 2012

• All donor sources

• 174 transplants

• 100 alive

• 95% contacted to verify current survival and cancer status

Patient Population

Post-BMT Cancers

Pt/Gender Age Age Cancer Interval 2994, M 41.2 42.1 SCC larynx 0.9 yrs 7303, F 28.6 41.0 SCC sal gl 12.4 yrs 8443, F 19.4 20.5 SCC tongue 1.1 yrs 1680, M 16.8 32.3 SCC lung 15.5 yrs

9/25/2012 31

Shaded areas show 95% confidence limits. Hatch marks show censor times in the Minnesota Cohort. Not shown is one SCC in Pre-Transplant FA cohorts at age 49.9 years.

NO significant difference in the rate of SCC, overall (P=0.96), or up to age 20 (P=0.94), or after age 20 (P=1.0); rate ratios are 0.8, 0.0, 0.9 with VERY broad limits.

9/25/2012 32

Conclusions: • BMT using more modern treatment packages may

not be associated with an increased risk of cancer

• Cancer remains an important issue for long term FA survivors [with or without BMT] requiring heightened surveillance and improved treatments

FA Roadmap Sh

ort

Inte

rmed

iate

Long

Clinical Trials in FA

T regulatory cells

FA HSC expansion AML targeted therapy

Thymic progenitor cells iPS cell derived FA HSC

Survivorship Clinic

Genetic Testing Center

Developmental Studies

Assessment of GVHD and Dual Chimerism

Mycophenolate - 3 to + 30

G-CSF until ANC >2500/uL

Fludarabine 200 mg/m2

-3 -2 -1 0 7 14 21 28 -4 -8 -7 -6 -5 -9

sTBI 200 cGy

Day +28 BMBx

Cytoxan 50 mg/kg

University of Minnesota

Treg Unit 3

CSA - 3 to + 100 RAPA - 3 to + 100

HLA mismatched double UCBT

Doses on day 1 1 x 105/kg 3 x 105/kg 1 x 106/kg 3 x 106/kg

10 x 106/kg 30 x 106/kg 100 x

106/kg

Treg Dose Escalation

Treg Expansion Culture Alternatives

KT64/86

Purified UCB CD4+25+

cell

10,000 RADS

CD28

CD3

TCR

Treg cell

Anti-CD3

Anti-CD28

Anti-CD3/28 beads

Re-stimulat ion increases clin ical UCB nTreg yield w ithout decreasi ng Foxp3 or suppressive fu nction (n=3).

I Expansion I Day o

I IL 2

3

(300 /nll) Stimulate lvith

KT64/86

12

l ± Re-stimulation

\ \ ith KT64/86 1

19

1 Assay for phenotype

and suppression

10000 100 100 2843

N + 80 .. 80

c 60

c 1000 or-t m c. © ·- v-, ) ( c 0 60 nJ LL ·-0

v-, v-,

100 I c. N"""' ) (

w or-t 40 40 "'C c c. - u

'*' c. ::::s

V ' )

'*' 0 10 LL 20 20

1 0 0 RO Rl RO Rl RO Rl

Higher Treg Dose Results in Greater Proportions of Treg in the PB

Prevention of Acute GVHD Potency of Treg >3 x 106/kg vs Controls

Inci

denc

e

Days

CsA/MMF 62%

Treg/Siro/MMF 12%

80 100 0.0

0.4

0.6

0.8

1.0

0 20 40 60

Siro/MMF 48%

Immune Reconstitution

Protect the thymus

Increase T progenitors

Eliminate post transplant immune suppression

Thymic Protection and Increasing Thymic Progenitors

Culture conditions OP9-DL1 Flt3 IL7

thymus

Gene Therapy

• Isolate and expand autologous FA HSC for gene correction

• iPS cell-derived HSC

• Outcomes after Alternative Donor HSCT same as for MSD HSCT – engraftment, GVHD and survival similar

• High risk patients = advanced MDS (>5% blasts), acute leukemia, or renal failure have lower survival

• Lymphoid recovery is the biggest hurdle

Summary

When to Transplant Adults with FA

• before advanced MDS or AML • before transfusions • before major infections

• Next steps – Eliminate use of steroids (AVN) – Shield ovaries

Acknowledgements Fanconi Anemia Comprehensive Clinic

- Margy MacMillan, MD - Pat Fidler, RN -Heather Zierhut, MS - Lucie Turcot, MD – late effects - Anna Petryk, MD - endocrinology - Lynda Polgreen, MD - endocrinology - Frank Ondrey, MD - ENT - Rahel Ghebre, MD - gynecology - Sarah Jane Schwarzenberg, MD - gastroenterology - Jo-Anne Young, MD - infectious diseases - Ann Van Heest, MD – hand surgeon -neuropsychologists, ophthalmology, audiology, cardiology, pulmonology, nephrology, dentistry