Bone Marrow Pathology 2.pdf

Bone marrow pathology Educational course, Belgian Society of Hematology 18 Nov 2017 Dr. J. Somja, CHU Sart Tilman, Liège, [email protected]

Learning objectives When ?

Why ?

Bone marrow examination

How ?

What ?

I. When ? Indications for BM examination

Indications for BM examination • Haematological abnormalities that cannot be explained by available clinical and laboratory data. • A good indication is essential for an accurate diagnosis • 3 main indications : – Diagnostic purposes – Staging for malignant diseases – Monitoring Riley et al., JCLA, 2004

II. What? What specimens to collect ?

(Bone marrow Particle clot)

BONE MARROW

Bone marrow aspirate

Smear Preparation

+ Heparin

Bacteriology if necessary

+EDTA

Bone marrow trephine biopsy

Heparin

FFPE 2-10 days

MGG cytology (hours)

Cytochemistr y (hours)

FISH If necessary

(days)

Flow cytometr y (hours)

Molecular biology (clonal expansion, NGS, …) Days-Weeks

Morpho, IHC, Cytogenetics

days-weeks Karyotyping FISH

FISH, (clonal expansion, NGS)

BM biopsy touch imprint (hours) Usefull if dry tap

BM aspirate or trephine biopsy ?

• • • •

•

BM ASPIRATE Quick results Fine cytological detail Enumeration of marrow cellular elements Wider cytochemical stains can be used Ideal for flow cytometry, cytogenetics/molecular studies

• • • • •

BM BIOPSY Complete assessement of cellularity and architecture More sensitive for focal lesions Allows grading of fibrosis Use of IHC Useful for assessment of AA, metastasis, some infections

BM aspirate or trephine biopsy ? A thorough bone marrow examination includes both BM aspiration and trephine biopsy. – – – – – – – –

MDS: MPN: MPN/MDS AML: NHL: HL: MM: Carcinoma:

BM aspirate >>> BM biopsy BM aspirate = BM biopsy BM aspirate > BM biopsy BM aspirate >>> BM biopsy BM aspirate << BM biopsy BM aspirate <<< BM biopsy BM aspirate < BM biopsy BM aspirate << BM biopsy

III. How? How to collect these specimens ? 1. Anatomic sites 2. Collection procedure 3. Adequacy

Anatomic sites • Crest of the posterior superior iliac spine – Preferred site

• Sternum – Experienced operator – Only marrow aspiration ! – Not in MM

• Anterior superior iliac spine – Rarely performed

• Anterior tibial plateau – Very young children Riley et al., JCLA, 2004

Collection procedure BM aspirate adequacy • Adequate aspirate – – – – – – – –

6-12 slides Bone marrow particles ! Not crushed Not too thick Not clotted Allow to dry quickly Dry tapes represent 2-7% of the cases If an adequate aspirate has not been possible, considerations should be given to preparing touch imprints of the core biopsy prior to placing it in fixative

BM Aspirate smears

Riley et al., JCLA, 2004

Touch imprints of the core biopsy

Riley et al., JCLA, 2004

Touch imprints of the core biopsy Be gentle, be delicate ! Do NOT CRUSH !

Riley et al., JCLA, 2004

Collection procedure BM trephine biopsy adequacy

• 11-gauge needle AT LEAST • If osteopenic, a 8-gauge needle allows the collection of an intact core biopsy with minimal crush artifact • 13-gauge biopsy needle for paediatric patients • Adequate core biopsy, – At least 1.6 cm to 2 cm long • Prior to fixation • Exclusive of cortical bone, cartilage, or periosteum • Free of crush artifact or interstitial hemorrhage or fragmentation

Riley et al., JCLA, 2004

I. Why? Understanding of the BM examination reports

Shemes like this should not have any secrets …

BM trephine : Generalities

Jaffe, Hematopathology, second edition, 2017

Erythropoïesis

Courtesy of Pr. Tassin and Dr. Keutgens

Granulopoïesis

Jaffe, Hematopathology, second edition, 2017

Mégacaryocytes

Courtesy of Pr. Tassin and Dr. Keutgens

Other? • Monocytes • Macrophages • Plasma cells • Lymphoid cells • Mast cells • Oestoclasts • Bone • Iron • …

Jaffe, Hematopathology, second edition, 2017

Immunohistochemistry • • • •

Erythroid: GlycophorinA, LMO2, CD71 Myeloid: MPO Megacaryocytes: CD61, Factor VIII Blasts: CD34, CD117, CD33 – CD34+ cells are rare in normal marrow – CD34 does not equal blast • not all blasts are CD34+ • not all CD34+ cells are blasts

– Not all AML’s are CD34+ – CD34 is not lineage specific

• • • •

Mastocytes: Tryptase, CD117, CD25, CD2 Plasma cells: CD138, IgKappa, IgLambda Lymphocytes: CD20, CD3, CD30, … …

Cellularity 100-age = expected cellularity for age

Severe AA is characterized by a markedly hypocellular bone marrow (<25% of normal for age or 25 to 50% of normal with <30% hematopoietic cells) accompanied by two of the following: granulocytes <0.5 × 109/L; platelets <20 × 109/L; or corrected reticulocyte count <1% Riley et al., JCLA, 2009

Jaffe, Hematopathology, second edition, 2017

Bone marrow (neoplastic) pathology

WHO 2016 Myeloid Neoplasms

WHO 2016 Myeloid Neoplasms

1. Myeloproliferative neoplasms • Clonal hematopoietic disorders • Characterized by proliferation of cells of one or more of the myeloid lineages; erythroid, granulocytic, or megakaryocytic • Initially, the proliferation in the bone marrow is effective and associated with maturation of the neoplastic cells • Leads to increased numbers of mature granulocytes, red blood cells (RBCs), and platelets in the peripheral blood • Splenomegaly and hepatomegaly are common and caused by the sequestration of excess blood cells, extramedullary hematopoiesis or both in these organs.

Chronic Myeloid Leukaemia, BCR-ABL1+ • Blood findings – – – – – – –

Leukocytosis Plts N or ↗ Often anaemia Spectrum of maturing granulocytes with a « myelocyte bulge » Blasts usually <2% if WBC Absolute basophilia No significant dysplasia

• BM findings – – – – – –

Hypercellularity Increased M:E ratio Blasts usually <5%, always <10% Widening of maturing granulocytes with myelocyte bulge but no dysplasia Megs N or ↗ in number, with « dwarf » morphology Reticulin fibers N to moderately increased

• Genetics – 100% have Phi chromosome or BCR-ABL1 fusion gene

Courtesy of Pr. Tassin and Dr. Keutgens

Jaffe, 2017

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Jaffe, 2017

Essential Thrombocytaemia

From major mixed to the 4th to minor

Jaffe, 2017

Jaffe, 2017

Possible cause of Thrombocytosis • Secondary – – – – – – – – – –

Infection Inflammation and AI diseases Blood loss, hemorrhage Chronic iron deficiency Post-splenectomy Hyposplenism Trauma (brain injury) Post-surgical procedures Neoplasms (Non-hemato and non-myeloid hemato) BM regeneration, rebound, following chemotherapy

• Myeloid neoplasm related – – – – – – –

MPN CML, BCR-ABL1+ PV ET AML with t(3;3)(q21.3;q26.2) or inv(3)(q21.3q26.2) MDS with isolated del(5q) abnormaly MDS/MPN-RS-T

Polycythaemia Vera Was >18.5 g/dL

Was >16.5 g/dL

From minor to major criteria

Endogenous erythroid colony formation in vitro

Jaffe, 2017

Jaffe, 2017

Jaffe, 2017

Primary Myelofibrosis

Jaffe, 2017

Diagnostic criteria of distinctive value regarding WHO-defined ET (left) versus earlyprefibrotic stage of PMF (right), including standardized morphologic features (Table 1 contains more details), allowing the generation of characteristic histologic BM patterns

Jürgen Thiele et al. Blood 2011;117:5710-5718

©2011 by American Society of Hematology

Jaffe, 2017

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Bone marrow fibrosis European consensus on grading bone marrow fibrosis and assessment of cellularity. J.Thiele et al. Haematologica 2005; 90:1128-1132

Standard grading Bauermeister 1971; Manoharan et al. 1979

Jaffe, 2017

2. Myelodysplastic syndromes Definition • Sustained unexplained anemia, neutropenia or thrombocyopenia • And at least one of the following (Hb<10g/dL; Abs. Neutrophil count <1.8 x109 or platelets <100 x109/L)

– Dysplastic morphology in erythroid cells, granulocytes or megacaryocytes, affecting at least 10% of the cells of at least one of these lineages – Acquired conal MDS-associated cytogenetic abnormality in hematopoïetic cells and absence of de novo AML-defining cytogenetic abnormalities t(15;17), inv (16)/t(16;16) or t(8;21) – Increased blasts (at least 5% of marrow cells) not attributable to exogenous GF administration or transient marrow recovery

Acquired conal MDS-associated cytogenetic abnormality

Del(20q), +8 and –Y abnormalities, although common findings in MDS, are not considered MDS defining and cannot in isolation be used to make a diagnosis of MDS

Dysplastic Erythropoeisis • Nuclear – – – – – –

Nuclear budding Internuclear bridgking Karyorrhexis Multinuclearity Nuclear hyperlobation Megaloblastic changes

• Cytoplasmic – Ring sideroblasts – Vacuolisation – PAS positivity

Jaffe, Hematopathology, 2017 Cantù Rajnoldi et al., Ann Hematol, 2005

Ring Sideroblasts •

3 types of sideroblasts – Type 1 : <5 siderotic granules – Type 2 :>5 siderotic granules but no perinuclear distribution – Type 3 : Ring sideroblasts : 5 or more perinuclear granules in a perinuclear distribution, surrounding the nucleus or at least 1/3 of the nuclear circonference

• Reactive ring sideroblasts – – – –

Alcohol (plasma cell iron) Zinc Pyridoxine deficiency Drugs (isoniazid, cycloserine)

• MPN and MDS/MPN – PMF, ET – MDS/MPN-RS-T

• Hereditary – Hereditary sideroblastic anaemia Mufti et al., Haematologica, 2008

Dysplastic Myelopoiesis • Small or unusually large size • Nuclear hypolobation (pseudo-Pelger-Huët; pelgeroid) • Irregular hypersegmentation • Decreased granules; agranularity • Pseudo-Chédiak-Higashi granules • Auer rods Jaffe, Hematopathology, 2017 Cantù Rajnoldi et al., Ann Hematol, 2005

Dysplastic Megacaryotes • Micromegacaryocytes • Nuclear hypolobulation • Multinucleation – Normal megacaryocytes are uninucleate with lobulated nuclei

Jaffe, Hematopathology, 2017 Cantù Rajnoldi et al., Ann Hematol, 2005

Hasserjian, XIII EBMWG , 2017

MDS : Pitfalls Non-MDS conditions associated with cytopaenia and >10% dysplasia • Drugs/toxins – Recent (<6 m) chemotherapy – Heavy alcohol intake

• Metabolic deficiencies: B12, folate, copper • « Stress erythropoiesis » due to haemoglobinopathy or acquired/congenital haemolytic aenemia • Infections (HIV, HepC, …) • Autoimmune diseases • Concurrent neoplasm – Infiltrating marrow (especially MM and HCL) – Rare paraneoplastic dysplasia for remote tumour Castello A et al., Haematologica, 1992

MDS : WHO update 2016 Cytogenetivs by conventional karyotype analysis

Cytopaenias

RS as % of marrow erythroid elements

BM and PB blasts

1

1 or 2

<5%

BM

2 or 3

1-3

<5%

BM

Any, unless fulfills all criteria for del(5q) Any, unless fulfills all criteria for del(5q)

MDS-RS with single lineage dysplasia (MDS-RS-SLD)

1

1 or 2

≥15%/≥5% and SF3B1 mutation

BM

Any, unless fulfills all criteria for del(5q)

MDS-RS with multilineage dysplasia (MDS-RS-MLD)

2 or 3

1-3

BM

Any, unless fulfills all criteria for del(5q)

MDS with single lineage dysplasia (MDS-SLD) MDS with multilineage dysplasia (MDS-MLD)

Dysplastic lineage

≥15%/≥5%and SF3B1 mutation

BM

Del(5q) alone or with one additional abnormality except -7 or del(7q)

MDS with isolated del(5q)

1-3

1-2

None or any

MDS-EB-1

0-3

1-3

None or any

MDS-EB-2

0-3

1-3

None or any

1-3

1-3

None or any

BM, no Auer rods

Any

1

3

None or any

BM

Any

0

1-3

<15%

BM

MDS-defining abnormality

BM 5-9% or PB 2-4% No auer rods BM 10-19% or PB 5-19% or Auer rods

Any

Any

MDS, unclassifiable (MDS-U) -with 1% blood blasts -with single lineage dysplasia and pancytopenia -based on defining cytogenetic abnormality

MDS : Recurrent somatic genetic mutations -Not sufficient to diagnose MDS in a cytopenic patient (CHIP)! -May support an MDS diagnosis suspected by other observations

Genomic architecture of MDS. (A) Frequency of driver mutations identified in the sequencing screen or by cytogenetics in the cohort of 738 patients, broken down by MDS subtype. Elli Papaemmanuil et al. Blood 2013;122:3616-3627 ©2013 by American Society of Hematology

Indolent Myeloid Haematopoietic Disorders ICUS

IDUS

CHIP

CCUS

MDS

-

-

+/-

+/-

+/-

Clonal karyotypic abnomality

-

-

+/-

+/-

+/-

Marrow dysplasia

-

+

-

-

+

Cytopaenia

+

-

-

+

+

Somatic mutation

ICUS : idiopathic cytopaenia of unknown significance IDUS: idiopathic dysplasia of unknow significance CHIP: clonal hematopoeisis of indeterminate potential CCUS : Clonal cytopaenia of unknown significance

•Heterogeneous group •Can evolve into MDS or AML •Frequent monitoring of blood count recommended

Valent et al., Am J Cancer Res, 2011

5. Acute Myeloid Leukaemia • Heterogeneous group of diseases • Clonal proliferations of immature, non-lymphoid, bone marrow– derived cells • Most often involve the bone marrow and peripheral blood • May present in extramedullary tissues • Aggressive clinical course. • Diagnostic on the basis of a minimum blast cell count in bone marrow (>20%) or peripheral blood • Several specific AML types are now defined without regard to blast cell count – Acute myeloid leukemia with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 – Acute myeloid leukemia with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11 – Acute promyelocytic leukemia with PML-RARA

Still a role for morphology in the diagnosis of AML? • Blasts (in all cases) should be counted the old fashion way, not based on flow cytometry !

• May allow establishment of a quick diagnosis – Especially important in the diagnosis of acute promyelocytic leukemia so therapy can be started

• Leads to the ability to review all of hematopoiesis – AML with MDS related changes » » »

Previous MDS Dyspoiesis > 50% of the cells in 2 cell lines Cytogenetic MDS abnormalities

• Exclude relevant differential diagnoses • Clues to the diagnoses of AML with recurrent genetic anomalies can be obtained by evaluating morphology

Jaffe, 2017

AML with MDS-related changes

Jaffe, 2017

Survival data.

Olga K. Weinberg et al. Blood 2009;113:1906-1908

©2009 by American Society of Hematology

Jaffe, 2017

CONCLUSION • Integration of clinical, morphologic, immunophenotypic, genetic, and other biologic features is mandatory to define specific disease entities • The relative contribution of each feature varies, depending on the case • Make your cytologists/pathologists/geneticians good ! by providing them relevant clinical informations and optimal samples.

BIBLIOGRAPHY • Elaine S. Jaffe et al., Hematopathology, second edition, 2017, • Bain B., Bone Marrow Pathology 4th ed. 2009 • The WHO classification of tumors of the hematopoietic and lymphoid tissues. 2016 revision. Arber et al. Blood 2016; 127(20):2391-2405 • Swerdlow SH, Campo E, Harris NL, et al, eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th ed. Lyon, France: IARC Press; 2017. • XIII EBMWG International Course and Workshop on Bone marrow Pathology, Utrecht, 2017