How to diagnose myeloproliferative neoplasms and PNH in vascular diseases of the liver Valerio De Stefano

Transcription

1 How to diagnose myeloproliferative neoplasms and PNH in vascular diseases of the liver Valerio De Stefano Institute of Hematology, Catholic University School of Medicine, Academic Hospital A. Gemelli, Rome, Italy

2 Outlines Incidence of SVT in the general population and in the MPN and PNH patients Diagnosis of MPN in the SVT patients Diagnosis of PNH in the SVT patients

3 Splanchnic vein thrombosis Hepatic vein thrombosis Portal-mesenteric vein thrombosis

4 Definition Budd Chiari Syndrome (BCS) Occlusion of hepatic veins, from the small hepatic veins to the entrance of the right atrium Extra Hepatic Portal Vein Obstruction (EHPVO) Obstruction of the extrahepatic portal vein: With or without thrombosis of the intrahepatic portal veins With or without thrombosis of the splenic or superior mesenteric veins

5 Clinical features Budd Chiari Syndrome (BCS) Heterogeneus clinical presentation (from asymptomatic to fulminant liver failure) Main complications related to portal hypertension: ascites, variceal bleeding, portosystemic encephalopathy High morbidity/mortality Extra Hepatic Portal Vein Obstruction (EHPVO) Acute Abdominal pain, intestinal infarction, ascites or fever in the absence of portal cavernoma and varices Chronic Variceal bleed, splenomegaly, hypersplenism, jaundice, ascites, portal biliopathy (rare) De Stefano & Martinelli, Intern Emerg Med 2010; 5:487

6 Epidemiology Budd Chiari Syndrome (BCS) Annual incidence per million individuals in Western countries Extra Hepatic Portal Vein Obstruction (EHPVO) 1 % autopsies (one-third non-malignant and non-cirrhotic EHPVO) Annual incidence 0.7 per 100,000 individuals Superior Mesenteric Vein Thrombosis Annual incidence 2.7 per 100,000 individuals Reviewed in Martinelli & De Stefano et al, Thromb Haemost 2010; 103:1136

7 Martinelli & De Stefano, Thromb Haemost 2010;103:1136

8 Martinelli & De Stefano, Thromb Haemost 2010

9 MPN are frequent in SVT De Stefano et al, Thromb Haemost 2016;115:240

10 Arber et al, Blood 2016

11 Arber et al, Blood 2016

12 Arber et al, Blood 2016

13

14 Tefferi, A. & Pardanani, A. Nat. Rev. Clin. Oncol. 11, (2014);

15 Kampfl et al, NEJM 2013

16 Diagnosis of MPN in SVT Difficulties to meet WHO criteria: hemodilution occult bleeding hypersplenism related to portal hypertension possible elevation of Epo in BCS (liver ischemia)

17 JAK2 V617F is frequent in SVT De Stefano et al, Thromb Haemost 2016;115:240

18 JAK2 V617F is frequent in SVT De Stefano et al, Thromb Haemost 2016;115:240

19 CALR mutations are not frequent in SVT De Stefano et al, Thromb Haemost 2016;115:240

20

21 2011

22

23 93 patients with SVT (73 with EHPVO and 20 with BCS). The JAK2 V617F mutation was present in 34 of them (36%); additional 3 had Humara test positive for clonality. Primignani et al, Hepatology 2006

24 Kiladjian et al, Blood 2008

25 Role of Bone Marrow Biopsy (BMB) In a cohort of 74 SVT patients, BMB alone allowed a diagnosis of MPN in 29% (12/41) of the JAK2 V617F mutation-negative SVT patients (Primignani et al, 2006). In a larger cohort of 241 SVT patients, the rate of ET and PMF diagnosed by BMB alone in the absence of JAK2 V617F, JAK2 exon 12, and MPL 515 mutations was 7% (10/144) (Kiladjian et al, 2008). On the other hand, the JAK2 V617F mutation has been found to be positive in 6-18% of the SVT patients with negative BMB (Primignani et al, 2006; Kiladjian et al, 2008). Such high discrepancy in the reported diagnostic yield obtained by BMB could mirror a substantial interobserver variability. Primignani et al, Hepatology 2006;44:1528 Kiladjian et al, Blood 2008;111:4922

26 Role of measurement of Red Cell Mass (RCM) In a cohort of 241 patients with SVT, the measurement of RCM allowed for a definite diagnosis of PV in the JAK2V617F-positive cases with masked erythrocytosis. RCM demonstrated absolute erythrocytosis in 11 JAK2 V617Fnegative EHPVO patients and no evidence for MPN in BMB. In 10 of them, serum erythropoietin was elevated, suggesting reactive erythrocytosis. Therefore, RCM seems of limited value in the diagnostic work-up of JAK2 V617F-negative patients. In conclusion, RCM could be useful in refining diagnosis of the patients JAK2 V617F-positive and maybe could be applied in the patients JAK2 V617F-negative who have bone marrow histology for MPN. Kiladjian et al, Blood 2008;111:4922

27 Non-cirrhotic and non-malignant splanchnic vein thrombosis Laboratory investigation for inherited and acquired thrombophilia JAK2 V617F + 25% - 41% JAK2 V617F BMB + 67% - 93% BMB BMB + 7% - 28% BMB - [RCM] [RCM] CALR MPL JAK2 Exon12 [RCM] De Stefano et al, Thromb Haemost 2016;115:240

28 SVT predicts MPN during the follow-up 280 of 831 patients with SVT had the JAK2V617F mutation, for a mean prevalence of 33.7% Five studies provided data on development of MPN during follow-up in patients with JAK2 mutation and without an overt MPN at the time of SVT diagnosis (21 of 41 patients, 51.2%, developed overt MPN) Dentali et al, Blood 2009;113:5617

29 SVT predicts MPN during the follow-up Colaizzo D et al, Thromb Res 2013;132:e99

30 SVT predicts MPN during the follow-up Danish National Health Service [Sogaard K et al, Blood 2015;126:957)

31 SVT IN MPN AND PROGNOSIS MPN patients with previous SVT have an increased risk of: rethrombosis, bleeding, transformation to myelofibrosis Gangat et al, Eur J Haematol 2006;77:327 Hoekstra et al, J Thromb Haemost 2011; 9:2208

32 Antithrombotic prophylaxis after SVT A minimum of 3 to 6 months for EHPVO and lifelong for BCS are suggested; patients with EHPVO should receive life-long anticoagulation in the presence of permanent risk factors (e.g. MPN) for thrombosis (DeLeve LD et al, Hepatology 2009;49:1729)

33 SVT IN MPN AND CYTOREDUCTION In MPN patients with previous thrombosis, cytoreduction is warranted (Barbui T et al, J Clin Oncol 2011;29:761)

34 Chemotherapy and SVT Whether is justified to give cytoreduction to patients with SVT and JAK2 V617F not meeting the WHO criteria is unexplored. Therefore caution is due in prescribing hydroxyurea, as for MPN high-risk patients, in the absence of hypercythemia.

35 Non-cirrhotic and non-malignant splanchnic vein thrombosis Diagnostic work-up of thrombophilia Thrombophilia JAK2 V617F and/or MPN No systemic risk factors Overt MPN MPN criteria unmet BCS EHPVO Long-term VKA Long-term VKA +/- aspirin + cytoreduction Long-term VKA +/- aspirin Long-term VKA 6 months VKA De Stefano et al, Thromb Haemost 2016;115:240

36 What is PNH Mutation? PNH is due to a mutation in a gene in a blood stem cell. The gene is called the PIG-A gene (phosphatidylinositol glycan complementation group A) and is located on the X chromosome. >100 mutations in PIG - A gene known in PNH The mutations (mostly deletions or insertions) generally result in stop codons - yielding truncated proteins which may be non or partially functional - explains heterogeneity seen in PNH

37 Pathogenesis - The Defect GPI Anchor PIG - A gene codes for 60 kda protein glycosyltransferase which effects the first step in the synthesis of the glycolipid GPI anchor (glycosylphosphatidylinositol). Results in clones lacking GPI anchor - in turn, attached proteins PIG - A protein

38 What is PNH? As a result of the PIG-A mutation, there is little of no GPI anchor produced. PNH II cells- mild reduction PNH III cells- severely reduced. When the anchor is reduced, certain proteins can t attach to the cells. The most important proteins for PNH are CD 59, CD55.

39 Proteins Deficient from PNH Blood Cells CD55 CD58 CD59 PrPC AChE JMH Ag Dombroch HG Ag CD55 CD58 CD59 CD109 PrPC GP500 Gova/b CD55 CD58* CD59 CD14 CD16 CD24 CD48 CD66b CD66c CD87 CD109 CD157 LAPNB1 PrPC p50-80 GPI-80 ADP-RT NA1/NA2 RBC Platelets PMN Haematopoietic Stem Cell CD59, CD90, CD109 B cells Monocytes CD14 CD55 CD58* CD59 CD48 CD52 CD87 CD109 CD157 Group 8 PrPC GPI-80 CD16 T cells CD24 CD58 CD48 CD73 NK cells CD55 CD58 CD59 CD48 CD52 PrPc CD16 CD55 CD59 PrPC CD108 CD55 CD58* CD59 CD48 CD52 CD87 CD108 PrPc ADP-RT CD73 CD90 CD109 CD16* QuickTime and a GIF decompressor are needed to see this picture. (Courtesy of Lucio Luzzatto)

40 Laboratory Evaluation of PNH PNH Diagnosis by Flow Cytometry (1986) Considered method of choice for diagnosis of PNH (1996) Detects actual PNH clones lacking GPI anchored proteins More sensitive and specific than Ham and sucrose hemolysis test

41 PNH Diagnosis by Flow Cytometry Of the long list of GPI anchored protein, monoclonal antibodies to the following antigens have been used in the diagnosis of PNH The most useful Abs are to CD14, 16, 55, 59, and 66. Are all required? Probably not - more studies needed Antigen Cell Lineage Function CD14 monocytes LPS receptor, MDF CD16 neutrophils Fc III receptor CD24 neutrophils B-cell differentiation marker CD55 all lineages DAF CD58 all lineages possible adhesion CD59 all lineages MIRL, HRF, protectin CD66b neutrophils CEA-related glycoprotein

42 PNH Diagnosis by Flow Cytometry Antigen expression is generally categorized into three antigen density groups type I Normal Ag expression type II Intermediate Ag expression type III No Ag expression Patient samples that demonstrate cell populations with diminished or absent GPI-linked proteins (Type II or III cells) with multiple antibodies are considered to be consistent with PNH. Should examine multiple lineages (ie granulocytes & monocytes)

43 ICCS PNH Testing Guidelines Borowitz M, Craig F, DiGiuseppe J, Illingworth A, Rosse W, Sutherland R, Wittwer, C and Richards S Cytometry Part B (Clinical Cytometry). 2010:78B:

44 Leucocyte Analysis: Reagents CD55 and CD59 were used historically but these are not optimal CD16, CD66b, CD24 are most commonly used GPI-linked markers for granulocytes CD14 is often used for monocytes but some normal dendritic cells are CD14-negative and gate like monocytes FLAER is the most versatile reagent for detecting PNH white cells

45 WHAT IS FLAER? FLuorescent AERolysin Aerolysin is a pore-forming toxin secreted by Aeromonas hydrophila GPI-anchor serves as receptor FLAER A488-conjugated mutant aerolysin binds to GPI -anchor rather than surrogate protein and is inactive so doesn t form channels a-cd59 FLAER FLAER

46 Incidence and Prevalence of PNH in Britain Yorkshire population 3,742,835 (2001 census) Incidence 1.3/ million/ year Estimated prevalence 15.9/ million Great Britain population 57,105,375 (2001 census) estimated 75 new cases of PNH per year predicted prevalence of 908 patients 25% had PNH neutrophil clone size of > 50% Hill et al., Blood, November 2006, 294a

47 Pathogenesis CD 55 Also known as decay accelerating factor Accelerates decay of enzyme that destroys red cell membranes CD55 inhibits the formation or destabilizes complement C3 convertase (C4bC2a) When GPI anchor absent, CD55 not available and RBC membranes hemolyze

48 Pathogenesis CD59 Also known as membrane inhibitor of reactive lysis, protectin, homologous restriction factor, and membrane attack complex inhibitory factor CD59 Protects the membrane from attack by the C5- C9 complex Absence or reduction of CD59 leads to increased hemolysis and perhaps thrombosis

49 Thrombosis in PNH Pathophysiology COMPLEMENT INJURY NO PS COMPLEMENT C5b-9 HEMOLYSIS C5a THROMBIN GENERATION MONOCYTES PLATELET Endothelial cells TF TF = Tissue factor. CYTOKINES IL-6 INFLAMMATION

50 Incidence of thrombosis, % PNH Clone Size and Thrombosis (excluding warfarin prophylaxis patients) Incidence of Thrombosis is Highest in Patients With a Large PNH Clone thromboses/100 patient years P= Granulocyte clone size >50% (n=67) Granulocyte clone size <50% (n=55) Follow-up (years) Hall C et al. Blood 2003;102(10):

51 Malato et al, Blood transfusion 2012 Risk of VTE in PNH

52 Ziakas et al, J Thromb Haemostas 2007

53 Ziakas et al, J Thromb Haemostas 2007

54

55 Two (1.4%) cases had large PNH clones, both with PVT

56 From the total study cohort of 163 patients, 10 patients were already known to have PNH before diagnosis of BCS (6.1%). Additionally, 67 patients were tested for the presence of PNH by either flow cytometry (84%), Ham s test (7%) or both (9%). Five of these tests (7.5%) were positive for PNH.

57 Hoekstra et al, J Hepatol 2009

58 Conclusions - 1 SVT and MPN are strongly associated All SVT events have to be investigated for an underlying MPN either at the time of thrombosis and during the follow-up JAK2 V617F is the diagnostic cornerstone in this setting, whereas other MPN-associated mutations are of scarce relevance However BMB can capture additional cases of MPN in the absence of the JAK2 V617F

59 Conclusions - 2 Systematic investigation for PNH in patients with SVT produced variable results. PNH clones were detected from 1% up to 7.5% of patients without a pre-existent diagnosis of PNH Selection of patients according to the presence of hemolysis as demonstrated by increased LDH levels and/or cytopenia can be the clue to improve the diagnostic yield