Deficit di piruvato kinasi

Disclosures

Research support ALEXION, NOVARTIS

Advisory Board AGIOS, GENZYME, ALEXION, TNT, BIOVERATIV

Invited speaker NOVARTIS, GSK, ALEXION, BIOVERATIV

Wilma BarcelliniFondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milano

Università di Milano

Hemolytic anemias: congenital vs acquired

Barcellini W, Fattizzo B. Dis Markers. 2015

If the diagnostic flow chart turns negative for congenital hemolytic anemias, reconsider acquired causes and vice-versa

RBC: red blood cells; AIHA: autoimmune haemolytic anemias; DHTR: delayed haemolytic transfusion

reactions; CDAs: congenital dyserythropoietic anemias; PNH: paroxysmal nocturnal hemoglobinuria.

• RBC need to continuously catabolize glucose to have energy during their life via: • the glycolysis (Embden–Meyerhof pathway), accounts for 90% energy• the pentose phosphate pathway (10%)

Congenital hemolytic anemias due to RBC enzyme defects

Pyruvate Kinase Deficiency (PKD)

• Hereditary autosomal recessive disorder• Prevalence 1:20.000 in the general white population, world-wide distribution• Heterogeneous clinical presentation, from compensated to very severe transfusion-dependent

anemia (hydrops fetalis in rare cases, miscarriages)• Usual signs of chronic hemolysis (splenomegaly, jaundice, cholelitiasis)• Frequent iron overload (independently from transfusion)• Rarely, aplastic crisis, splenic abscess, acute pancreatitis, spinal cord compression by extramedullary

hematopoiesis, infections and thrombosis in splenectomised• Exacerbations (acute infection/pregnancy)• Anemia tends to improve with ageing in infants, and is in general well tolerated (2,3 DPG)• Treatment: transfusions, splenectomy, iron chelation

• Median Hb• 9.8 g/dL in unsplenctomised• 7.3 in candidates for splenectomy

• Median Hb increase after splenectomy• 1.8 g/dL (0.4-3.4)

• Median Retics• 166x109/L in unsplenctomised• 796x109/L in splenectomised

Zanella & Bianchi, 2000, Zanella et al, 2005

Blood 2018 131:2183-2192

www.clinicaltrials.gov: #NCT02053480.

Transfusion status at enrollment

• 254 patients enrolled at 31 sites in USA and Europe from 2013, retrospective-prospective study (annual controls)

• Median age at diagnosis 0.4 yrs (0-60), 88% diagnosed age<18

• splenectomy performed in 59%, at 3.2 yrs (0.4-37), with a median rise in hemoglobin of 1.6 g/dl, reduction in the transfusion burden in 90% of cases, but transfusion dependence persisted in 10%

• Predictors of response to splenectomy: higher presplenectomy hemoglobin (P = .007), lower indirect bilirubin (P = .005), and missense PKLRmutations (P = .0017).

• Simultaneous splenectomy and cholecystectomy performed in 87/254 (34%) patients. In those who had a splenectomywithout simultaneous cholecystectomy, 48% later required a cholecystectomy.

• Peculiar characteristics of the Amish cohort, 51/55 (93%) of patients had a splenectomy, at a median age of 1.6 years (range: 0.6-28.2)

Complications:• iron overload (48%); correlate with disease

severity but also occur in milder cases

• gallstones (45%)

• thrombosis (11%, all splenectomized). No occurrence in not splenectomized (p=0.0001)deep vein (n=9), pulmonary embolism (n=6), stroke (n=3), and portal vein (n=2) (5 pts had >1 event).

• aplastic crisis (14%)

• osteopenia/bone fragility (17% bone fractures)

• sepsis (7%)

• extramedullary hematopoiesis (9%)

• Other more rare: endocrine disfunction (5%), pulmonary hypertension (3%), leg ulcers (2%)

6

Type of PK-LR mutations (n=123, 50 not previously

described) found in 192 cases enrolled in the PKD NH

Missense/missense

58%

Missense/non-missense

27%

Non-missense/non-missense

(del/ins/non-sense/splicing)15%

79 missense, 36 non-missense mutations (12 splicing, 13 frameshift, 7 stop codons, and 4 large deletions), 5 inframe indel, and 3 promoter variants.

NM/NM, N=29 M/NM, N=52 M/M, N=111p-

value

Hb, median (range) 7.9 (6.5-8.9) 8.4 (6.4-12.8) 9.2 (4.3-12.3) 0.003

Total N transfusions 65 (3-991) 25 (1-721) 16 (1-1915) 0.0013

ferritin (ng/ml) 1787 (423-13,409) 604 (22-8,220) 573 (31-9,679) 0.0001

Rate of splenectomy 21/29 (72%) 26/52 (50%) 49/111 (44%) 0.024

Blood 2018 131:2183-2192

www.clinicaltrials.gov: #NCT02053480.

• Patients with NM/NM genotype:• were more anemic, • had a higher transfusion need and iron overload (defined as

ferritin >1000 ng/mL or having received chelation in the year before enrollment), and

• had a higher frequency of previous splenectomy• no evidence of an association of PK enzyme activity with genotype

7

• iron overload as defined by MRI or chelation was 82%(67/82), and 45% as defined by ferritin or chelation; cardiac iron overload was present in 7%

• 34% had been prescribed chelation therapy during their lifetime (initiation at median age 10.4 years)

• Ferritin levels below 1000 ng/ml, hemoglobin levels above 9 g/dL, young age, and/or an absence of regular transfusions do not exclude the possibility of iron overload

• iron screening, monitoring, and treatment is important starting in childhood.

Ferritin >1000 ng/ml or chelation LIC >3 mg/g DW or chelation

Absent

(n=85)

Present

(n=53)

p Absent

(n=15)

Present

(n=67)

p

CHARACTERISTICS (median, range; or rate %)

Hemoglobin

(g/dl)

9.3

(6.2-14.1)

8.7

(6.5-12)0.003

9.9

(7.6-10.9)

9

(6.2-12)

Absolute

reticulocyte

count (106/μL)

0.2

(0.1-5.3)

0.5

(0.1-1.2)

0.9

(0.9-1)

0.6

(0.1,-0.9)

Bilirubin (mg/dl) 3.6

(0.9-9)

4.3

(1.3-17.6)0.01

3.5

(1-7.2)

3.6

(1.1-17.6)

LIC (mg/g DW)4.0

(1-8.4)

8.0

(2.2-33.4)0.0003

2

(1-3)

6.4

(2.2-33.4)0.0001

Transferrin

saturation (%)

43.3

(8.8-100)

62

(18.4-100)0.02

42.4

(23.2-96.6)

50.1

(12.1-100)

Splenectomized 51/85 (60%) 47/53 (89%) 0.0002 14/15 (93%) 63/67 (94%)

sensitivity/specificity to predict LIC >3 mg/g DW: 53% / 100% with a ferritin cut off of 1000 ng/ml90% / 67% with a ferritin cut off of 500 ng/ml

Search for signsof chronic hemolytic anemia

Identify the hemolyticnature of anemia

Perform PK enzymatic activity

•Family and personal hystory• Infections•Transfusions•Drugs

•Reticulocytosis•Hyperbilirubinemia•Reduced haptoglobin•Increased LDH•hyperferritinemia

PKD diagnosisreduced

Reconsider other more rare causes of hemolytic anemia•Congenital memebrane defects: HE, HSt, CDA, HPP (perform membrane protein analysis, ectacytometry, molecular testing),•Congenital enzyme defects: chronic forms of G6PD, PFK, TPI, PGK, HK, GPI, P5N (specific enzymatic activity and molecular testing)•DAT-negative AIHA (use more sensitive DAT methods)•Mechanical, infectious,toxic causes , drugs (anamnesis, specyfic tests)•Perform familiar studies

Exclude acquired hemolyticanemias, hemoglobinopathies, and

most common congenital RBC membrane defects

•AIHA (DAT or Coombs test)•PNH (CD55/CD59 on granulocytes)•Hemoglobinopathies (HPLC )•RBC membrane defects (blood smear morphology,

osmotic fragility tests/EMA binding)

Perform PK moleculartesting

+

Also present in hemorrhage, pregnancy, acclimationAbsent/inadequate in CDAs, associated BM disease, AIHA with

autoimmunity against BM

Poor/absent in recessive forms, may be absent in dominant hemolytic disorders

Increased also in diseases with cellular necrosis or tissue turnover Highly increased in intravascular hemolysis (PNH, thrombotic

microangiopathies, prosthetic valves) sis

Increased also in metabolic and inflammatory diseases, hemochromatosis carrier, transfusions

Perform a carefullanamnesis

•Signs of anemia• Jaudice•Splenomegaly•Gallstones

Increased also in Gilbert syndrome

Reduced also in congenital hypohaptoglobinemia

normal

Consider coexistance of: iron and vitamin deficiency, blood lossLiver and renal disese

Testing algorithm and differential diagnosis of PKD

Hereditary elliptocytosis(HE)

Pyruvate kinase deficiency (PKD)

Pyrimidine 5’nucleotidase(P5’-N)

Hereditary pyropoikilocytosis(HPP)

Heinz bodies in G6PD

Hereditary Stomatocytosis (HSt)Hereditary spherocytosis (HS)

Pros Cons

Enzyme testing

Test availability in different Centres

Fast (time-frame about 2 hr; results available in 2-5 working days)

Cheap (cost of analysis 15-80€)

Able to detect functional abnormality of PK activity

Interference of recent transfusions (at least 40 days from the

last transfusion is advisable)

Reticulocytosis may give falsely normal/elevated values

Leucocyte/platelet interference (need to remove buffy coat)

Amount of blood required (1 ml minimum, problematic in

neonates)

Inability to discriminate between heterozygous carriers and

affected patients (homozygous/compound heterozygous)

Shipping and storage issues (PK activity is considered stable

up to 14 days at 4°C)

Variability of reference ranges among Centres, need to

include normal controls in each test, need to refer to other

enzymatic activities (hexokinase)

Inability to demonstrate a reduced activity in some very rare

variants of dysfunctional thermolabile enzyme

Molecular analysis

Requirement of small sample volume (prenatal diagnosis)

Easy handling and shipping of samples

No interference of transfused red blood cells

Time consuming and relatively expensive

Need to confirm the pathogenic role of new mutations by in silico analysis or other functional tests

Pros/Cons of enzyme versus molecular testing in pyruvate kinase deficiency

Current treatment options for Pyruvate Kinase Deficiency

• Transfusion • Decision guided by symptoms or growth delay

• Splenectomy• Some benefit on Hb, risk of infections and thrombosis

• Management of secondary complications • cholelithiasis, iron oveload, osteopenia/osteoporosis

• AG-348, oral pyruvate kinase activator

• Stem cell transplantation • 16 patients with PK deficiency who underwent transplant in Europe and Asia; 74% cumulative

survival but also a high rate of graft versus host (van Straaten, et al 2018).

• Gene therapy• Safe and effective in mouse model (Garcia-Gomez, et al 2016, Kanno, et al 2007, Meza, et al

2009, Tani, et al 1994).

• future trials planned

Effects of AG-348, a pyruvate kinase activator, in patients with pyruvate kinase deficiency: Updated results from the DRIVE PK study

Rachael F Grace*1, D Mark Layton2, Frédéric Galactéros3, Christian Rose4,

Wilma Barcellini5, D Holmes Morton6, Eduard van Beers7, HassanYaish8,

Yaddanapudi Ravindranath9, Kevin HM Kuo10, Sujit Sheth11, Janet L

Kwiatkowski12, Bruce Silver13, Charles Kung14, Marvin Cohen15, Hua Yang14,

Penelope A Kosinski14, Lei Hua14, Ann Barbier14, Bertil Glader 16

1Dana-Farber Boston Children's Cancer and Blood Disorders Center, Boston, MA USA; 2Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, UK; 3Unité

des Maladies Génétiques du Globule Rouge, CHU Henri Mondor, Créteil, France; 4Hôpital Saint Vincent de Paul, Lille, France; 5Fondazione IRCCS Ca' Granda

Ospedale Maggiore Policlinico, Milan, Italy, 6Central Pennsylvania Clinic, Belleville, PA,

USA, 7Universitair Medisch Centrum Utrecht, Utrecht, Netherlands, 8University of Utah,

Salt Lake City, UT, USA 9Wayne State University School of Medicine, Children's

Hospital of Michigan, Detroit, MI, USA; 10University of Toronto, Toronto, Canada; 11Weill Cornell Medical College, New York, NY, USA; 12Children's Hospital of

Philadelphia and Perelman School of Medicine of the University of Pennsylvania,

Philadelphia, PA, USA;13Bruce A Silver Clinical Science and Development, Dunkirk,

MD, USA; 14Agios Pharmaceuticals, Inc., Cambridge, MA, USA; 15 MBC Pharma

Solutions, Newtown, PA, USA; 16Stanford University School of Medicine, Palo Alto, CA,

USA

Presented at the 22nd Congress of the European Hematology Association, 22-25 June 2017, Madrid, Spain

S451

Active PK-R is a tetramer.

Mutations (green) impair the

catalytic activity

AG-348is an orally active

allosteric activator of pyruvate

kinase R

AG-348 (yellow) binds at the

PK-R dimer-dimer interface,

away from the active site and

the most common mutations

14

Study design

Arm 1

300 mg BID

Arm 2

50 mg BID

Transfusion-independent PK-deficient adults (ClinicalTrials.gov NCT02476916) n=25 in each arm

Ran

do

miz

ati

on

Str

atified

by P

K-R

ge

no

typ

e (

no

ne

exclu

de

d)

6 month core dosing period

1 2 3 6 9 12 16 20 24

Assessment points

(weeks)

Extension arm

Transfusion independence = no more than 3 units of red blood cells transfused

in 12 months prior to the first day of study dosing and no transfusions within 4

months of first day of study dosing

Primary endpoints:

• Safety and tolerability

Secondary endpoints:

• Pharmacokinetics of AG-348

• PD response: ATP, 2,3-DPG

• Indicators of clinical activity:

hemoglobin, reticulocyte count,

and other hematologic parameters

Fully enrolled as of November, 2016This phase 2 study evaluated the safety and efficacy of AG-348 in non-regularly transfused adults with PK deficiency (≤3 units of red blood cells in the prior 12 months). A total of 52 patients were randomized (1:1) to open-label 50 or 300 mg AG-348 twice daily over a core treatment period of 24 weeks.

15

• Twenty-six (50%) patients achieved a maximum hemoglobin increase of 1.0 g/dL with a mean maximum hemoglobin increase of 3.4 g/dL (range 1.1–5.8 g/dL)

• the median time to hemoglobin response >1.0 g/dl was 10 days (range 8–24 days) and the response was sustained in the majority of patients.

Maximum Hb increase observed during the Core period

The baseline value is the average of all central assessments within the screening period (42 days prior to Day 1)

- 1

0

1

2

3

4

5

6

M a x i m u m H b c h a n g e f r o m b a s e l i n e

P a t i e n t

Ma

x H

b c

ha

ng

e (

g/d

L)

< 2 5 m g B I D

2 5 m g B I D

5 0 m g B I D

1 0 0 m g B I D

1 5 0 m g B I D

2 0 0 m g B I D

3 0 0 m g B I D

16

- 1

0

1

2

3

4

5

6

M a x i m u m H b c h a n g e f r o m b a s e l i n e

P a t i e n t

Ma

x H

b c

ha

ng

e (

g/d

L)

M is s e n s e / m i s s e n s e

M is s e n s e / n o n - m i s s e n s e

N o n - m is s e n s e / n o n - m is s e n s e

• Hb responses were observed only in patients with at least one missense mutation and correlated with baseline PKLR protein level.

• Improvement in markers of hemolysis were observed in patients with Hb increase.

Maximum Hb increase observed by genotype

The baseline value is the average of all central assessments within the screening period (42 days prior to Day 1)

17

• Range of doses (5 mg QD–300 mg BID) used

– Decreased for AEs or Hb exceeding midpoint of normal range (M: Hb >15; F>13.5 g/dL)

– Increased for lack of Hb response

• Adverse events grade 3 occurring in at least 2 patients were hypertriglyceridemia (6%), insomnia (4%), hemolysis (4%), and pharyngitis (4%).

Safety

•minimum of 6 transfusion episodes in the 52-week period prior to date of informed consent •Excluded subjects homozygous for the R479H mutation or having 2 non-missense mutations in the PKLR gene

• documented presence of at least 2 mutant alleles in the PKLR gene, of which at least 1 is a missense mutation, 5. • no more than 4 transfusions in the 12-month and no transfusions in the 3 months prior to the first day of study treatment

20

• Pyruvate kinase deficiency is a greatly heterogeneous disease, frequently presenting in childhood with severe hemolytic anemia requiring transfusions

• Splenectomy ameliorates anemia and transfusion need but is associated with thrombotic and infectious risk

• Iron overload is a frequent and underestimated complication

• Diagnosis needs genotype testing that may be useful for prognosis and therapy

• AG-348, a new oral PK activator, induces rapid and sustained hemoglobin responses in about half adults with PK deficiency, with a good safety profile

Take home messages

Blood 2018 131:2183-2192

Paola BianchiFrancesca BindaFrancesca CavallaroBruno FattizzoValeria FerlaElisa FermoValeria FerriJuri GiannottaAnna GregoriniAnna MarcelloGiulia MilesiTommaso RadiceVeronica SciumbataGiulia SoveriniViviana ValliCristina VercellatiAnna Zaninoni

Agostino CortelezziAlberto Zanella

Thank you for your attention!