Inherited BMFS: Recent Advances esp, in Fanconi Anemia

Hoon Kook, M.D.

Chonnam National University Children’s Hospital Gwangju, Korea

Scientific Session 2018. 3. 30.

Introduction

• IBMFSs: genetic heterogeneous group of disorders

– Bone marrow failure

– One or more somatic abnormality

– Inherited or early embryonic development

• The precise incidence/prevalence: unclear

• Usually presents in childhood

but the age at presentation variable

• Malignant transformation

– MDS, AML, various epithelial cancers

IBMFS

• IBMFS associated with pancytopenia – Fanconi anemia

– Dyskeratosis congenita

– Shwachman-Diamond Syndrome

– Cartilage-Hair Hypoplasia

– Pearson's syndrome

– Down syndrome

– Familial marrow dysfunction

• IBMFS associated with isolated cytopenia – Diamond-Blackfan Anemia

– Congenital dyserythropoietic anemia

– Severe congenital neutropenia

– Inherited thrombocytopenia

– Amegakaryocytic thromobocytopenia

– Thrombocytopenia with absent radii

Fanconi Anemia (FA)

Introduction

Inherited bone marrow failure syndrome characterized by - Pancytopenia - Predisposition to malignancy - Physical abnormalities

In 1927, Guido Fanconi first reported 3 brothers with macrocytosis, pancytopenia, and physical abnormalities.

Clinical features of FA

1/200,000; estimated carrier frequency of 1/181 in North Am.

(1) Somatic abnormalities

- No overt physical/somatic abnormalities: 1/4~1/3

(2) Bone marrow failure

- Cytopenia: at 5-10 yrs of age

- Macrocytosis, thrombocytopenia: often

- Risk of full blown marrow failure: 50% at age of 40.

(3) Malignancies

- RR for AML & MDS: 700-6,000 x higher (Alter, 2014)

cf. CI of AML at age 40: 15-20%; CI of MDS at age 50: 40% (Alter, 2010)

- RR for H&N SCC (600 x); Esophagus (2,000 x); Vulva (3,000 x)

cf. Median age of SCCs: 33 yr for FA vs. 60-70 yr for general (Velleur, 2014)

CI of gynecological & H&N cancer: 30% at age 40 (Alter, 2014)

BRCA2 biallelic mutation: CI of cancers at age 7: 90%; CI of AML at age 10: 80% (Alter, 2014)

Alter, Cancer in Fanconi anemia, 1927-2001, Cancer, 2003

37% 50%

46%

76%

Incidence of Functional & Somatic Abn in FA

Dufour , Br J Haematol, 2017 No or subtle abnormalities in 25-30% of FA

Facial features, short stature, typical radial ray abnormalities and café au lait

spots and hypopigmentation in patients with FA.

PBS

Pancytopenia, and hypocellular BM

Development of AML in FA patient

BM Section

Diagnosis of FA

(1) Suggestive features

- Presence of BMF & somatic malformations - Family history or personal history of BMFS

- Macrocytosis or thrombocytopenia - Spontaneous chromatid breakage

- Unbalanced 1q, 3q or 7q translocations on BM karyotyping during MDS or AML Dx work-up

- Unexpected occurrence of classical FA malignancies in young patients

- Excessive toxicity after standard chemotherapy for acute leukemia

(2) Chromosomal breakage test w/ DNA cross-linking agents - Gold standard for the Dx of FA

- Mitomycin C (MMC) or Diepoxybutane (DEB) exposure on PB lymphocyte culture

Hematopoietic somatic mosaicism: seen in upto 25% of FA cases

- Correction of mutated allele in a HSC or in a lymphocyte progenitor

- eg., back mutation, intragenic crossover, gene conversion or compensating

deletion/insertion

Test on skin fibroblasts to confirm the Dx in mosaic FA patients

cf. Exact correlation btw somatic mosaicism and hematopoietic function: not fully established

(3) Flow cytometry for cell cycle: auxiliary diagnostic test

- S2 delay or block

(4) Complementation analysis

(5) Next generation sequencing

Diagnosis of FA

- ↑ Chr. breakage or aberration (breaks, gaps, rearrangements, radials, exchanges,

endoreduplications) in PB cells after culture w/ diepoxybutane (DEB) or

mitomycin C (MMC)

Blood lymphocytes (~90%) by somatic mosaicism or Skin fibroblasts (100%)

DEB 0.1 μg/mL

Chromosome breakage test (clastogenic assay)

FA genes

Nalepa & Clapp, Nat Rev, 2018

21 genetic subtypes identified - 5 new entities added recently - Autosomal recessive disorder except for FANCB (XR), FANCR (also known asRAD51): dominant-negative mutation - FANCA (60%), FANCC (12%), FANCG (10%)

Genotype-Phenotype correlation

FA: Guidelines for diagnosis and management, 4th edition (2014)

• FANCA – null alleles 1) earlier onset of hematologic abnormalities 2) higher risk of developing MDS &AML 3) shorter survival after diagnosis than with at least one hypomorphic mutation

• FANCC 1) less severe hematologic course 2) lower incidences of congenital microcephaly and radial ray abnormalities

• FANCG 1) more severe cytopenias 2) higher rates of MDS and AML

FANCD2-FANCI ubiquitylation regulates the DNA

damage response

FANCT: E2 ubiquitin-conjugating Enz FANCL: E3 ubiquitin ligase

The FA pathway orchestrates the interphase DNA

damage response (interstrand crosslink repair pathway)

Chromatin localization of FANCD2-FANCI heterodimer

Downstream FA Effector pathway

Function of FA proteins

Multifunctional

(1) DNA repair

- Interstrand crosslink (ICL) repair pathway through mono-ubiquitylation

and chromatin localization of the FANCD2-FANCI heterodimer.

(2) Reactive oxygen species (ROS) detoxification (Zhang, 2005; Du, 2012)

(3) Energy metabolism (Cappelli, 2013)

(4) Cytokine signaling (Li, 2007; Anur, 2012)

Management of FA at Diagnosis

Initial Work-up of FA

patients

Dufour, BJH 2017

Management based on cytopenia severity (at least one of the followings)

- Mild: Hgb (>10, but < lower limit for age);

ANC (1000-1,499), Plt (50k-149k) - Moderate: Hgb (8.0-9.9); ANC (500-999), Plt (20k-49k) - Severe: Hgb (< 8.0); ANC (<500), Plt (<20k) Lowest value of the 3 lineages defines the

level of severity

Dufour, BJH 2017

Growth factors: G-CSF, EPO

- Not recommended for long-term use d/t potential clonal evolution

- Anti-TNF-α: safe, but not effective against advanced BMF (Mehta, 2012)

Anabolic steroids:

(1) Danazole: 68% response in 37 FA (Paustian, 2016)

but hold d/t cytogen abn, lack of response, liver adenoma, MDS/AML

(2) Oxandrolone: 7/9 early response, but virilization in 3, ↑ LFT in 2 (Rose, 2014)

(3) Oxymetholone (0.5-1 mg/kg/d) in 49 Brazil FA

- Transfusion independence in 83% (median 3 mo), multilineage response, 55%

- 12% maintain hematologic response off Tx; Virilization in 100%

- 29/49 subsequent HSCT & 25/29 (86%), alive (Ribeiro, 2014)

Supportive care

(1) Iron chelation to start if RBC transfused >200 ml/kg or liver iron >7 mg/g dry wt

Or s-ferritin: >1,000 μg/L (Barone, 2015)

TPO mimetics, eltromopag or romiplostim:

Gene therapy using lentiviral vectors w/o conditioning

Treatment options

HSCT

- Only curative option to correct marrow failure

- Better outcomes in last 2 decades

- Fludarabine containing conditioning

- BM preferred to PB due to less GvHD

- Post-transplant HNSCC: 4.4 x ↑ & 16 yr acceleration than non-TPL

- Malignancies: found in 40% 15-20 yr after SCT (Rosenberg, 2005)

34% at 20 yr after SCT (Peffault de Latour; EBMT; 2013)

Treatment options

Peffault de Latour, Blood 2013

Allogeneic HSCT in FA: EBMT N = 795 (1972-2010)

OS = 49% at 20 yr

cGvHD, 2nd mal: time-dependent Poor Px factor

83%

64%

Peffault de Latour, Blood 2013 EBMT

HSCT from an HLA-matched sibling donor

Irradiation vs. Nonirradiation Conditioning

for MST in FA 148 FA from CIBMTR data btw 1990 & 2001

Supporting the use of nonirradiation conditioning Pasquini, BBMT 2008

Patients - All BM graft without T-cell deletion

Results Irradiation Non-irrad P value

Number 77 71

Median ANC recovery day 13 17 NS

ANC recovery 94% 89% NS

Platelet recovery at D+100 92% 92% NS

aGVHD, gr II-IV 23% 21% NS

cGvHD 18% 24% NS

5-yr OS 78% 81% NS

Factors ass w/ high mortality: 1) Age >10 (RR, 4,24; P<0.001) 2) Androgen therapy (RR, 2.62; P= .008) 3) CMV (+) either in donor or recipient (RR, 4.67; P= .39)

Factors for Outcome of Related HSCT for FA

94 FA children: related HSCT at FSPRC, Saudi Arabia

MDS a/o abn cytogenetics / AML: 12 cases

Fluda + CY 20 mg/kg: best survival for

Matched familial donor HSCT for FA

OS: 92.5%, 89%, 86% at 1, 5, 10 yrs

Ayas, BBMT 2014

10 yr OS: CY 60 mg/kg vs. CY 20 mg/kg = 91% vs. 82% (P<.05) Rad conditioning vs. non-rad = 76% vs. 91% (P<.01) Fludarabine + CY 20 mg/kg: highest OS (95.2%; P<.05) Higher CY: ↑ hemorrhagic cystitis (20%) Squamous cell Ca in 3

MSD in 43 FA conditioned w/ Cy 60 mg/kg

Bonfim Brazil, BBMT 2007

43 FA, marrow transplant from HLA-matched related donor

(Sib, 27; others, 6)

Conditioning: Cy alone 60 mg/kg (15 mg/kg x 4 d) GvHD prophylaxis: MTX + CyA Cytogenetic abn in 12 including 3 with rejection OS: 93% (40/43) median f-up 3.7 yr Graft failure: Primary (n=1) - Retransplant : Alive Late (n=4) - Retransplant in 3 : Alive in 2 TRM: 7% aGvHD (III-IV): 2% cGvHD (extensive): 24.5% Mucositis (high grade): 60%

HSCT from an HLA-matched sibling donor

Dufour, BJH 2017

SCT from healthy MSD using non-irradiation regimens with Flu + Cy, and BMT: Tx of Choice w/ excellent OS

UBMT for FA

Wagner, Blood, 2007

CIBMTR: 98 FA UBMT btw 1990-2003

Flu (+) Flu (-) P value

3-yr adjusted OS 52% 13% <.001

ANC recovery 89% 69% .02

Plt recovery 73% 23% <.001

TRM at D+100 24% 65% <.001

Flu-conditioning w/ TCD BM graft Early referral for TPL w/o excessive Tf

Peffault de Latour, Blood 2013

EBMT

77%

57%

HSCT from an HLA-matched unrelated donor

Dufour, BJH 2017

SCT from MUD: Best results w/ Flu-conditioning w/ ex vivo or in vivo TCD before excessive transfusions and clonal abnormalities Inferior OS outcome for MUD (64%) vs. MSD (83%) (P =.011)

Alternative Donor HSCT for FA

130 FA patients at U. Minnesota btw 1995 & 2012

Alternative donor HSCT: excellent for FA w/o prior opport inf. or transfusions

MacMillan, Blood 2015

Patients - Age: 9.0 (1-48) - Clonal abn (28%), late MDS/AML (8%) - Median f-up: 7.7 yr - Conditioning: CY + single TBI + ATG +/- Fluda

Results - 5-yr OS: 58% - aGvHD 2-4, 20%; cGvHD, 10% - Better survival - Flu-containing, esp w/ TBI 3Gy: RR, 0.1 - Higher mortality - Older age (10-17, RR = 2,2; >18, RR = 2.7) - Prior opportunistic infection (11%, RR = 3.5) - CMV + (RR = 2.3) - Prior RBC or Plt transfusions (74%, RR = 2.3)

N= 17

Gluckman, BBMT 2007

HSCT from Cord blood

93 FA patients Eurocord Registry & EBMT

Patients : 93 FA, median age at transplant: 8.6 yr - HLA matching: 6/6, 13%; 5/6, 37%; 4/6, 43%; 3/6, 5% - Median NC: 4.9 x 107/kg of recipient CD34+: 1.9 x 105/kg of recipient - Conditioning: various, but Flu-based in 61%

Flu+Cy±TBI: m.c. (< 5Gy in 13; w/o TBI in 28) - GvHD prophylaxis: CsA + pred ± ATG in 58% Results : - Neutrophil recovery: 60% at D+60 - aGvHD 2-4, 32%; cGvHD, 16% - OS: 40% ± 5%

OS OS OS

Donor selection & Flu-conditioning can improve survival

Zecca, BBMT 2014

Haplo- TCD HSCT in FA

12 FA patients from Pavia & Roma, Italy

Patients : 12 FA, 14 SCT - RI conditioning: Flu (120 mg/m2) + Cy (1200 mg/m2) + fATG (40 mg/kg) ± single TBI (2Gy) (11/14) - TCD (CD34+ selected) PBSC - Donor: Father, 8; Mother, 6 Results : - Graft rejection in 2 (17%) : retransplanted from other parent - aGvHD 2-4, 17%; cGvHD, 35% - No regimen-related mortality - TRM: 17% - 5-yr OS, 83%; EFS, 67%; DFS, 83%

Fluda conditioning + high-dose TCD SC: engraftment w/ good OS & DFS

Haplo SCT in FA: feasible

Haplo-BMT w/ Post-Transplant CY for FA

30 FA patients at Federal Univ. of Parana, Brazil btw 2008 & 2015

Haplo-BMT w/ PT-CY: suitable option for FA w/o MRD or MUD Bonfim, BBMT 2017

Patients - 26 FA w/o MRD or MUD 4 FA w/ graft failure after HSCT - Median age: 10 yrs (4 – 16 yr) - Conditioning 1) CY 10 mg/kg + Flu 150 mg/m2 + TBI 2-3 Gy (n=5) 2) Flu 150 mg/m2 + TBI 2 Gy (n=9) 3) Flu 150 mg/m2 + TBI 2 Gy + rATG 4-5 mg/kg (n=13) 4) Flu 150 mg/m2 + TBI 1 Gy + rATG 4-5 mg/kg (n=3) - Un-manipulated BM - GvHD prophylaxis: PT-CY 25 mg/kg/d on D+3 & D+4 CyA 3 mg/kg from D+5; MMF at 15 mg/kg/d 3 x/d - Donor: mother (n=17), sibling (n=5), father (n=4) - Donor specific Ab (DSA, n=3): Tx w/ rituximab & pheresis

Results 1-yr OS: 72.6% w/ median follow-up of 30 months - CMV reactivation: 75% at D+32 - No PTLD, No VOD - Hemorrhagic cystitis (50%) 14 FA w/o rATG vs. 16 FA w/ rATG - OS, P=NS; severe GvHD ↓ w/ rATG

SCT for FA transformed to MDS/AML

(1) Low-dose chemotherapy followed by SCT

Cincinnati group - 3 AML patients + 1 CMML patient - “mini-FLAG induction regimen Low-dose Flu (30 mg/m2 x 3 d) + Cy (300 mg/m2 x 3 d) + G-CSF (5 µg/kg x 5 d) - Conditioning: Flu + Cy + TBI 450 cGy + ATG Results : - No major toxicity - 2 CR & 1 PR at SCT

- Survival in 1 with a f-up of 8 mo (Mehta, PBC 2007)

Hannover, Germany - 3 AML patients - Minimal antileukemic treatment 6-TG (40 mg/m2 x 8 d) + ara-C (40 mg/m2 x 7 d) + single VCR None 6-TG (40 mg/m2 x 24 d, then half x 30 d) + ara-C (40 mg/m2 x 4 d) - Conditioning: Flu (150-180 mg/m2) + Bu (1.6 mg/kg) + Cy (20-40 mg/kg) ± ATG 40-80 mg/kg or Campath 35 mg/m2 Results : - 2 chemo patients showing marrow aplasia at the time of transplant - 3/3 alive at f-up of 14-174 mo

- All with cGvHD (Beier, BMT 2015)

SCT for FA transformed to MDS/AML

(2) Full-dose chemotherapy followed by Reduced intensity SCT

Saint Louis - 8 patients (AML, 7; MDS, 1) - “Full dose FLAG regimen Flu (30 mg/m2 x 5 d) + ara-C (2.0 g/m2 x 5 d) + G-CSF (5 µg/kg x 5 d) - Reduced intensity conditioning within 3 weeks:

Flu (120 mg/m2) + Cy (40 mg/kg) + TBI 200 cGy + rATG only in MUD Results : - 5/8 alive - Cause of deaths: relapse, 1; cGvHD, 1; infection,1)

(Talbot, Hematologica, 2014; Peffault de Latour, 2016) Saitama, Japan: Addition of Ara-C in conditioning 4 patients (AML, 3; MDS with 12% blasts, 1) - Antileukemic treatment No chemotherapy (MDS, blast 12%) Reduced AML chemo: CR MDS progression: (blast 27%) No chemo AML after ALL: (blast 30%) No chemo - Conditioning: Flu (120-150 mg/m2) + Cy (40 mg/kg) + Ara-C (6-20 g/m2) + TBI (4.5-6 Gy) ± rATG (5 mg/kg in 2) Results : - 4/4 alive (8 mo- 14yr) - SE: Gr IV in 1 (IPS); aGvHD (Gr >2): none; extensive cGvHD; none

(Aoki, BBMT 2016)

SCT for FA transformed to MDS/AML

(3) Full-dose chemotherapy followed by Reduced intensity SCT

Minnesota - 21 patients (AML, 12; ALL, 1; MDS, 8), BRCA2 biallelic mutation in 6 - Chemotherapy prior SCT: 8

Remission in 3 Toxicities in 6, severe in 3 Survival: 2

- 5-yr OS after HSCT: 33% (Mitchell, BJH, 2014)

Current Policy for FA with MDS/AML from Minnesota

- Without chemotherapy prior to SCT - TCD SCT - Conditioning: Flu + Cy+ ATG + TBI 300 cGy

(MacMillan, Blood 2015)

Some subsets might benefit from cytoreduction before HSCT - eg., MDS with excess blast, overt AML or BRCA2/FANCD1 patients (Peffault de Latour 2016)

HSCT for FA w/ Cytogen abn, MDS, or A Leukemia

IBMTR data from 1985 to 2007

113 FA (Cytogen abn, n =54; MDS, n =45; AL, n=14)

Younger patients & HLA-related SCT w/ cytogen abn only:

best survival Ayas, JCO 2013

64%, 55% at 1, 5-yr OS

Patients - Complex cytogen abn (n =18/54, 33.3%) - Acute leukemia (n=14; AML, 12; ALL, 2) - Stem cell: BM, 75%; PB, 12%; CB, 13% - Conditioning: Rad, 60% (TBI, 42%; TLI/TAI, 19%) Fluda, 27%; ATG, 50% Results - Neutrophil engraftment: 78% at D+28, 85% at D+100 - 2ndary GF: 9% - aGvHD ≥ 2 in 26%, ≥ 3 in 12% at D+100 - cGvHD: 20%, 23% at 1, 5 yrs; extensive in 12 Px factors for 5-yr OS - Age < 14 yr: 69% vs. 39%; P<.05 - In related SCT (n=82) Cyt abn only vs. MDS/AL = 67% vs. 43%; P<.05

Related SCT (n=82)

Dufour, BJH 2017

Thank you for your attention!