Platelet Esoteric Testing: When and Why

12/3/2020

Dong Chen MD PhD Special Coagulation Laboratory Division Of Hematopathology

A Case-based Workshop: Clinical and Laboratory Aspects of Hemophilia and Thrombosis Dec 4th, 2020 [email protected]

DISCLOSURES

Relevant Financial Relationship(s) Off Label Usage • None • None

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1. Know the platelet laboratory testing algorithm LEARNING OBJECTIVE 2. Understand the platelet esoteric testing 3. Use 2 cases to illustrate our experiences

1 Introduction

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Diagnosis of Inherited Platelet Disorder

Clinical Assessment Standard Laboratory Testing Esoteric Laboratory Testing

• Patient’s bleeding and other • CBC and Morphology • Esoteric Testing history • Peripheral blood CBC • Flow cytometry • Bleeding assessment and platelet indices • Transmission electron • Other histories • Peripheral blood smear microscopy • Cardiovascular • Genetic testing • Bone marrow status • Platelet Functional Tests • Malignancy • Platelet aggregation • Neuromuscular • Platelet function analysis (PFA-100) • Vision and pigmentations • Granule release (serotonin or ATP release) • Family history • Bleeding history • Other histories

Diagnosis

Platelet Surface Receptor Assessment by Flow Cytometry

Collagen receptor deficiency Glanzmann thrombasthenia

Collagen receptor TXA2, ADP receptors deficiency Bernard-Soulier syndrome

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Quantification of Platelet Surface Glycoproteins by Flow Cytometry

Platelets

Red cells

n=112; age:18-76 years

ୗୟ୫୮୪ୣ େୈସଵ ୑୊୍ CD41 expression level (%) = ୑ୣୢ୧ୟ୬ ୭୤ ୒୭୰୫ୟ୪ ୢ୭୬୭୰ େୈସଵ ୑୊୍

(MFI: Mean fluorescent intensityሻ

Glanzmann Thrombasthenia

GPIIb GPIIIa

GPIX GPIB-α GPIa/IIa

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Platelet Transmission Electron Microscopy (PTEM)

• Whole Mount • Dense granules

• Thin Section • Alpha granules • Other platelet ultrastructure

• Buffy Coat • Inclusions in leukocytes

Platelet Transmission Electron Microscopy (Normal Donor) Whole mount PTEM Thin-section PTEM

DG Gly AG: Alpha granule AG DG: Dense granule DG MT Gly: Glycogen MT: Mitochondria

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Molecular Testing of Inherited Platelet Disorders

Claire Lentaigne et al. Blood 2016;127:2814-2823

Quan Li and Kai Wang. InterVar: Clinical interpretation of genetic variants by ACMG- www.isth.org/page/GinTh_GeneLists AMP 2015 guideline

• Proven disease-associated gene (DIAGNOSTIC-GRADE) • 3 unrelated pedigrees with co-segregation data TIER1 • Functional data (animal model and cell/protein studies) 69

• Evidence from 1 or 2 pedigrees with insufficient co- TIER2 segregation data & without functional studies

• Evidence for role in platelet disorders (e.g. Functional TIER3 studies or KO mice)

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2 Case illustration

Case 1 Bleeding and life-long thrombocytopenia

Case 2 Bleeding and life-long thrombocytopenia

Case 1

A 51-year-old woman with life-long thrombocytopenia

Patient History Age 6 • She had severe epistaxis and was found to have thrombocytopenia • Her platelet count has been 30,000-90,000/μL 20s • Her ISTH bleeding score is 9: menorrhagia, epistaxis requiring ENT intervention, significant bruising

30s • She had 2 vaginal deliveries, and both had excessive bleeding requiring platelet transfusions

49 • She had a bone marrow biopsy which was morphologically normal • She had hysterectomy for a uterine mass and endometriosis. Her postoperative course (2 51 months) was complicated by bleeding, which required two surgical re-explorations and more than 10 units of platelet transfusion

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Family History

No family or personal history of immune deficiencies, bone or skeletal abnormalities, skin disease or warts, deafness, renal failure, idiopathic pulmonary fibrosis, or cirrhosis.

Standard Laboratory Testing

CBC Coagulation Hemoglobin (g/dL) 12.5 INR 1.0 Hematocrit (%) 37.4 APTT (sec) 33 Platelet count (10^9/L) 20 ↓ Thrombin time (sec) 16 MPV (fL) 11.8 ↑ VWF antigen (IU/dL) 150 White blood cell (10^9/L) 4.6 Fibrinogen (mg/dL) 342 VWF activity (IU/dL) 138 Platelet Laboratory Testing PFA-100 Canceled* Platelet aggregation Canceled* * Due to low platelet count.

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Peripheral blood smear

Platelet Glycoprotein Assessment by Flow Cytometry

GPIIb GPIIIa

GPIX GPIB-α GPIa/IIa

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Platelet Transmission Electron Microscopy

Whole mount Thin section

Mayo Clinic Platelet NGS Panel 69 genes/full exons (ISTH Tier 1)

ABCG5 BLOC1S6 FERMT3 GP9 ITGB3 P2RY12 STIM1 ABCG8 CD36 FLI1 HOXA11 LYST PF4 TBXA2R ACTN1 CHST14 FLNA HPS1 MASTL PLA2G4A TBXAS1 ADAMTS13 COL3A1 FYB HPS3 MLPH PLAU THPO ADRA2A DTNBP1 GATA1 HPS4 MMRN1 PRKACG

ANKRD26 ETV6 GATA2 HPS5 MPL PTGS1 TUBB1 ANO6 F2R GFI1B HPS6 MYH9 RAB27A VIPAS39 ANXA5 F2RL1 GP1BA ITGA2 MYO5A RASGRP2 VPS33B AP3B1 F2RL2 GP1BB ITGA2B NBEAL2 RGS2 VWF BLOC1S3 F2RL3 GP6 ITGB1 P2RY1 RUNX1 WAS

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NGS Testing on Both Blood and Skin Fibroblasts

Exon 1 and 2 deletion

Germline RUNX1 mutations: Mayo Clinic Cohort

Transcripts RUNX1a RUNX1b RUNX1c 12 3 4 5 6 78

Cavalcante de Andrade Silva M, et al. Cancer Genet. 2018;222-223:32-7.

Modified from DiFilippo, E.C. et al. Leukemia. 2020; 34, 2519–24

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Final Diagnosis

RUNX1 mutation-related inherited platelet disorder

At age of 54, she developed anemia and repeated bone marrow biopsy: hypercellular bone marrow with subtle dysplasia.

Bone marrow HemeOnc NGS testing showed additional mutations:

1. SRSF2: c.284C>A; p.Pro95His (48%) 2. TET2: c.3955G>T; p.Glu319* (42%)

2016 WHO Classification of myeloid neoplasms with germ line predisposition Myeloid neoplasms with germ line predisposition without a preexisting disorder or organ dysfunction AML with germ line CEBPA mutation Myeloid neoplasms with germ line DDX41 mutation

Myeloid neoplasms with germ line predisposition and preexisting platelet disorders Myeloid neoplasms with germ line RUNX1 mutation Myeloid neoplasms with germ line ANKRD26 mutation Myeloid neoplasms with germ line ETV6 mutation

Myeloid neoplasms with germ line predisposition and other organ dysfunction Myeloid neoplasms with germ line GATA2 mutation Myeloid neoplasms associated with BM failure syndromes Myeloid neoplasms associated with telomere biology disorders JMML associated with neurofibromatosis, Noonan syndrome or Noonan syndrome-like disorders The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia – Arber et al., 2016, 27069254 Myeloid neoplasms associated with Down syndrome ©2020 MFMER | slide-24

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Summary of the Assessment of Case 1

Clinical Assessment Standard Laboratory Testing Esoteric Laboratory Testing

Diagnosis

Case 2 Clinical Assessment A 35-year-old female with thrombocytopenia and bleeding diathesis since childhood.

Age Patient History 2 • Ecchymosis and right knee swelling after injury. CBC showed thrombocytopenia μ 5 • Diagnosed with Evans syndrome (platelets 30 to 40,000/ L), and was treated with prednisone 15 • She was treated with Cytoxan with weekly vincristine for 9 months 16 • Splenectomy due to ongoing low platelets 22 • First pregnancy, delivery was without complication 30 • Underwent second full-term pregnancy with birth. During delivery she had significant vaginal tearing, and severe bleeding and receive massive transfusion 35 • Since her second pregnancy: she has had many episodes of small hematomas on her hands and large hematomas on her legs

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Family history

Standard Laboratory Testing CBC Coagulation Hemoglobin (g/dL) 9.8 ↓ INR 0.9 Hematocrit (%) 36.3 APTT (sec) 31 MCV (fL) 89.7 Thrombin time (sec) 17 Platelet count (10^9/L) 57 ↓ VWF antigen (IU/dL) 75 MPV (fL) NA Fibrinogen (mg/dL) 344 White blood cell (10^9/L) 7.9 VWF activity (IU/dL) 74 Platelet Laboratory Testing PFA-100 Epi-cartridge 236 ↑ Platelet aggregation (Sec) ADP-cartridge 195 ↑ Arachidonate Normal* ADP Normal* Platelet glycoproteins Normal GPIIb, IIIa, Ia, Ib- Collagen Normal* alpha, VI and IX Epinephrine Normal* Ristocetin Normal*

* Normalized to low platelet counts

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Peripheral blood smear

Platelet Transmission Electron Microscopy Whole mount Thin section Thin section

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Bone Marrow Biopsy

H&E Reticulin

• Normal chromosome or BCR/ABL1 (FISH) • Negative JAK2 V617F, CALR, MPL mutation analyses

Congenital Myelofibrosis

• MPL Thrombocytosis • JAK2 • TPO

• G6B-b

Thrombocytopenia • GFI1B • NBEACH2 • VPS33b/VIPAS39

Neutropenia • VPS45

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Genetic analysis by WES NGS G6B-b (aka MPIG6B or C6orf25) Homozygous c.469G>A p.Gly157Arg

Father: Heterozygous Sister: Homozygous

HGMD: Macrothrombocytopaenia with focal myelofibrosis (DM – CM1816000)

Gene 1 2 3 4 5 6

G6B-b

Protein

Ig: Immunoglobulin like TM: Transmembrane domain ITIM: Immunoreceptor tyrosine-based inhibition motif ITSM: Immunoreceptor tyrosine-based switch motif

G6B-b Platelet Biology

Extracellular matrix proteins

SFK Src family kinase

Shp1/2 Src homology 2 domain-containing protein tyrosine phosphatases 1/2

Modified from Vogtle T, et al. Elife 2019;8; 1-43.

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Previously Reported Cases

HGB PLT G6B-b ID PB smear morphology Bone marrow findings (g/dL) x10^9/L genotype

Large hypogranular c.469G>A 4-II-2 5.4 96 platelets Grade 2 myelofibrosis p.Gly157Arg Abnormal red cells

Large hypogranular c.469G>A 4-II-3 12.5 97 platelets Grade 2 myelofibrosis p.Gly157Arg Myelofibrosis Abnormal red cells Thrombocytopenia

Hofmann I, et al. Blood 2018;132:1399-412.

Final Diagnosis

G6B-b mutation-related thrombocytopenia

Treatment options:

Observation, supportive care with platelet transfusion Uncertain future progression of myelofibrosis or a myeloid neoplasm

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Congenital Myelofibrosis

• MPL Thrombocytosis • JAK2 • TPO

• G6B-b

Thrombocytopenia • GFI1B • NBEACH2 Platelet alpha • VPS33b/VIPAS39 granule deficiency

Neutropenia • VPS45

Summary of the Assessment of Case 2

Clinical Assessment Standard Laboratory Testing Esoteric Laboratory Testing

Diagnosis

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1. Discussed the platelet laboratory testing algorithm Summary 2. Reviewed the platelet esoteric testing 3. Used 2 cases to illustrate our experiences

[email protected]