Hematopathology

Monika Klimkowska MD PhD

Klinisk patologi/cytologi

monika.klimkowska@karolinska.se

1. Normal hematopoiesis

- WHO Classification of Tumours of Haematopoietic and

Lymphoid Tissues

2. Introduction to hematopathological

diagnostics

Hematopoesis

• From Greek: hem (αἷμα) = blood, poes

(ποιεῖν) = make, create

• Generation of all types of blood cells

• All blood cells – derived from embryonic

connective tissue (mesenchyme)

• Hematopoiesis appears around 14th day

of gestation

Embryonal hematopoiesis

• First blood islands – present in yolk sac 3-4 wks after conception

– contain hemangioblasts (precursors to blood cells & endothelial cells)

• First hematopoietic line in embryo: red cell series – primitive megaloblastic erythropoiesis

– definitive normoblastic e-poiesis

• Progenitors & pluripotent stem cells migrate via vessels to liver (from 5th-6th wk), then to bone marrow (from 4th-5th mo)

• Fetal hematopoiesis – higher turnover, shorter cell lifespan, no or few growth factors required

Erythropoietic cells, 55 days of

gestation

Hematopoiesis during lifetime

Distribution of red BM during lifetime

Stem cell concept

• Stem cells

– reside in specific locations (niches)

– are not fully differentiated (as opposed to mature cells in

same tissue)

– have controlled but robust proliferative potential for the

lifetime of host tissue

– each SC can regenerate both stem & differentiated cells

– have capacity to divide into two daughter cells

• one that retains all properties of parental cell (self-renewal)

• the other that undergoes differentiation specific for the tissue

Stem cell hierarchy

• Totipotent SC – gives rise to both embryo & placenta

(fertilized oocyte, zygote or first blastomere)

• Pluripotent SC – gives rise to all three germ layers of the

embryo (ICM of blastocyts, embryonic SC, embryonic germ

cells EG, epiblast derived stem cells ESC)

• Multipotent SC – gives rise to one germ cell layer only

(ecto-, endo- or mesoderm)

• Monopotent SC – tissue-committed SC, gives rise to cells

of one lineage, eg hematopoietic SC (HSC), intestinal

epithelium SC, neural SC, liver SC, skeletal muscle SC

Stem cell hierarchy

Nobel Prize 2012

• AN important problem in embryology is whether the differentiation of cells depends upon a stable restriction of the

genetic information contained in their nuclei. The technique of nuclear transplantation has shown to what extent

the nuclei of differentiating cells can promote the formation of different cell types (e.g. King & Briggs, 1956;

Gurdon, 1960c). Yet no experiments have so far been published on the transplantation of nuclei from fully

differentiated normal cells. This is partly because it is difficult to obtain meaningful results from such experiments.

The small amount of cytoplasm in differentiated cells renders their nuclei susceptible to damage through exposure

to the saline medium, and this makes it difficult to assess the significance of the abnormalities resulting from their

transplantation. It is, however, very desirable to know the developmental capacity of such nuclei, since any nuclear

changes which are necessarily involved in cellular differentiation must have already taken place in cells of this

kind.

• The experiments described below are some attempts to transplant nuclei from fully differentiated cells. Many of

these nuclei gave abnormal results after transplantation, and several different kinds of experiments have been

carried out to determine the cause and significance of these abnormalities.

• The donor cells used for these experiments were intestinal epithelium cells of feeding tadpoles. This is the final

stage of differentiation of many of the endoderm cells whose nuclei have already been studied by means of

nuclear transplantation experiments in Xenopus. The results to be described here may therefore be regarded as

an extension of those previously obtained from differentiating endoderm cells (Gurdon, 1960c).

• GURDON JB. J Embryol Exp Morphol. 1962 Dec;10:622-40.

Nobel Prize 2012

• Differentiated cells can be reprogrammed to an embryonic-like state by transfer of

nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is

known about factors that induce this reprogramming.

• Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or

adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES

cell culture conditions. Unexpectedly, Nanog was dispensable.

• These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the

morphology and growth properties of ES cells and express ES cell marker genes.

• Subcutaneous transplantation of iPS cells into nude mice resulted in tumors

containing a variety of tissues from all three germ layers. Following injection into

blastocysts, iPS cells contributed to mouse embryonic development. These data

demonstrate that pluripotent stem cells can be directly generated from fibroblast

cultures by the addition of only a few defined factors.

Takahashi and Yamanaka, Cell 126(4):663-676, 2006

revoseek.com

Stem cell niches

Bone marrow HSC

CMP – common

myeloid

progenitor

CLP – common

lymphoid

progenitor

HSC niche in bone marrow

BM blood supply

Blood cell release from BM

Mobilization of HSCs

Stem cell

Bone marrow aspirate

Bone marrow aspirate - smear

megakaryocytes

Hematopoiesis overview

LT-HSC = long term

hematopoietic stem cell

ST-HSC = short term

HSC

TFs involved in hematopoiesis

Hematopoiesis - cytokines

2008 4th Edition

2016 Update (not published

yet, 3 papers in Blood)

New edition planned 2018?

WHO 2008

Myeloid diseases

• Myeloproliferative neoplasms

• Myeloid/lymphoid neoplasms with

eosinophilia & abnormalities of

PDGFRA, PDGFRB or FGFR1

• Myelodysplastic/myeloproliferative

neoplasms

• Myelodysplastic syndromes

• Acute myeloid leukemia and related

precursor neoplasms

• Acute leukemias of ambiguous lineage

Lymphoid diseases

• Precursor lymphoid neoplasms (B-, T-

cells)

• Mature B-cell neoplasms (B-NHL),

including plasma cell diseases

• Mature T- cell (T-NHL) and NK-cell

neoplasms

• Hodgkin lymphoma

•Histiocytic and dendritic cell

neoplasms

•Post-transplant lymphoproliferative

disordes

Erythropoiesis

Erythroid island

Chasis Blood 2008

Erythron

Proerythroblast Basophilic

erythroblast

Polychromatophilic

erythroblast

Orthochromatic

erythroblast

Reticulocyte

Erythropoiesis

Granulocytopoiesis

Granulocytopoiesis - cytokines

Granulocytopoiesis - localisation

Monocytopoiesis/dendritic cells

Granulocytes

• Have phagocytic properties

• Act ”on demand”, answering to tissue

injury/inflammation/infection

• Move to inflammation site within several hours)

• Relatively short-lived (up to 6 days, die at the site)

Granulocytes

• Neutrophils:

– azurophilic granules – myeloperoxidase, defensins, proteases

(elastase, cathepsin), BPI

– specific (seondary) granules – lysozyme, collagenase, alkaline

phosphatase, NADPH oxidase, lactoferrin

– tertiary granules – gelatinase, cathepsin

• Eosinophils: MBP, peroxidase, lipase, Rnase,

plasminogen, histamine

• Basophils: histamine, elastase, phospholipase,

proteoglycans (heparin, chondroitin)

• Mast cells: also granulated but not granulocytes(!),

contain histamine, heparin

Monocytes/macrophages

• Move actively

• Undergo terminal differentiation at the site

• Major phagocytes (direct phagocytosis or followed by

opsonisation)

• Major antigen presenters

• Dendritic cells – multiple subpopulations for different

purposes/at different locations (skin, lymph nodes,

respiratory tract, GI tract)

• Lymphoid vs myeloid vs plasmacytoid DCs

Megakaryocytopoiesis

Megakaryoblast Promegakaryocyte Megakaryocyte

(with emperipolesis)

How a MGK produces platelets?

Thrombocytes

• Granules:

– dense (delta) – Ca, serotonin, ADP, ATP

– lambda – hydrolytic enzymes

– alpha – P-selectin, PF4, vWF, fibrinogen,

PDGF, TGF-beta, coagulation factors V and

XIII

Produce TXA2 (arachidonic acid pathway)

Glycoprotein receptors

B-cell development

B-cell distribution

Primary vs secondary lymphatic organs

MATURE B-CELL NEOPLASMS

• Chronic lymphocytic leukemia /small lymphocytic lymphoma

• Monoclonal B-cell lymphocytosis*

• B-cell prolymphocytic leukemia

• Splenic marginal zone lymphoma

• Hairy cell leukemia

• Splenic B-cell lymphoma/leukemia, unclassifiable

• Splenic diffuse red pulp small B-cell lymphoma

• Hairy cell leukemia-variant

• Lymphoplasmacytic lymphoma

• Waldenstrom macroglobulinemia

• Monoclonal gammopathy of undetermined significance

(MGUS), IgM*

• Mu heavy chain disease

• Gamma heavy chain disease

• Alpha heavy chain disease

• Monoclonal gammopathy of undetermined significance

(MGUS), IgG/A*

• Plasma cell myeloma

• Solitary plasmacytoma of bone

• Extraosseous plasmacytoma

• Monoclonal immunoglobulin deposition diseases*

• Extranodal marginal zone lymphoma of mucosa-associated

lymphoid tissue (MALT lymphoma)

• Nodal marginal zone lymphoma

• Pediatric nodal marginal zone lymphoma

• Follicular lymphoma

• In situ follicular neoplasia*

• Duodenal-type follicular lymphoma*

• Pediatric-type follicular lymphoma*

• Large B-cell lymphoma with IRF4 rearrangement*

• Primary cutaneous follicle center lymphoma

• Mantle cell lymphoma

• In situ mantle cell neoplasia*

• Diffuse large B-cell lymphoma (DLBCL), NOS

• Germinal center B-cell type*

• Activated B-cell type*

• T cell/histiocyte-rich large B-cell lymphoma

• Primary DLBCL of the CNS

• Primary cutaneous DLBCL, leg type

• EBV positive DLBCL, NOS*

• EBV+ Mucocutaneous ulcer*

• DLBCL associated with chronic inflammation

• Lymphomatoid granulomatosis

• Primary mediastinal (thymic) large B-cell lymphoma

• Intravascular large B-cell lymphoma

• ALK positive large B-cell lymphoma

• Plasmablastic lymphoma

• Primary effusion lymphoma

• HHV8 positive DLBCL, NOS*

• Burkitt lymphoma

• Burkitt-like lymphoma with 11q aberration*

• High grade B-cell lymphoma, with MYC and BCL2 and/or

BCL6 rearrangements*

• High grade B-cell lymphoma, NOS*

• B-cell lymphoma, unclassifiable, with features intermediate

between DLBCL and classical

• Hodgkin lymphoma

T-cell development in thymus

T-cell subsets

NK-cells

MATURE T-AND NK-NEOPLASMS

• T-cell prolymphocytic leukemia

• T-cell large granular lymphocytic leukemia

• Chronic lymphoproliferative disorder of NK cells

• Aggressive NK cell leukemia

• Systemic EBV+ T-cell Lymphoma of childhood*

• Hydroa vacciniforme-like lymphoproliferative disorder*

• Adult T-cell leukemia/lymphoma

• Extranodal NK/T-cell lymphoma, nasal type

• Enteropathy-associated T-cell lymphoma

• Monomorphic epitheliotropic intestinal T-cell lymphoma*

• Indolent T-cell lymphoproliferative disorder of the GI tract *

• Hepatosplenic T-cell lymphoma

• Subcutaneous panniculitis- like T-cell lymphoma

• Mycosis fungoides

• Sezary syndrome

• Primary cutaneous CD30 positive T-cell lymphoproliferative

disorders

• Lymphomatoid papulosis

• Primary cutaneous anaplastic large cell lymphoma

• Primary cutaneous gamma-delta T-cell lymphoma

• Primary cutaneous CD8 positive aggressive epidermotropic

cytotoxic T-cell lymphoma

• Primary cutaneous acral CD8+ T-cell lymphoma*

• Primary cutaneous CD4 positive small/medium T-cell

lymphoproliferative disorder*

• Peripheral T-cell lymphoma, NOS

• Angioimmunoblastic T-cell lymphoma

• Follicular T-cell lymphoma*

• Nodal peripheral T-cell lymphoma with TFH phenotype*

• Anaplastic large cell lymphoma, ALK positive

• Anaplastic large cell lymphoma, ALK negative *

• Breast implant-associated anaplastic large cell lymphoma*

HODGKIN LYMPHOMA

• Nodular lymphocyte predominant Hodgkin lymphoma

• Classical Hodgkin lymphoma

• Nodular sclerosis classical Hodgkin lymphoma

• Lymphocyte-rich classical Hodgkin lymphoma

• Mixed cellularity classical Hodgkin lymphoma

• Lymphocyte-depleted classical Hodgkin lymphoma

POST-TRANSPLANT LYMPHOPROLIFERATIVE

DISORDERS (PTLD)

• Plasmacytic hyperplasia PTLD

• Infectious mononucleosis PTLD

• Florid follicular hyperplasia PTLD*

• Polymorphic PTLD

• Monomorphic PTLD (B- and T/NK-cell types)

• Classical Hodgkin lymphoma PTLD

HISTIOCYTIC AND DENDRITIC CELL NEOPLASMS

• Histiocytic sarcoma

• Langerhans cell histiocytosis

• Langerhans cell sarcoma

• Indeterminate dendritic cell tumour

• Interdigitating dendritic cell sarcoma

• Follicular dendritic cell sarcoma

• Fibroblastic reticular cell tumour

• Disseminated juvenile xanthogranuloma

• Erdheim/Chester disease*

Hematopathology - diagnostics Types of samples Possible analyses What cannot be done?

Bone marrow aspirate morphology (histology + cytology),

immunophenotyping, molecular analyses (FISH,

PCR), cytogenetics

Bone marrow biopsy morphology (histology + cytology),

immunophenotyping, molecular analyses (FISH)

Cytogenetics, PCR

Peripheral blood morphology (cytology), immunophenotyping,

molecular analyses

Cerebrospinal fluid morphology (cytology), immunophenotyping Molecular analyses?

Fine needle aspirate (LN, focal lesions) morphology (cytology), immunophenotyping,

molecular analyses

Tissue biopsies morphology (histology + cytology),

immunophenotyping, molecular analyses (FISH,

PCR)

Tissue biopsies = core biopsies, small biopsies or resectates - LN, parenchymatous organs,

endoscopic material

Other body fluids (ascitic fluid, fluid from pleural cavity/pericardial sac etc) – same as FNAB

Cytogenetics, flow cytometry – only on fresh material

BM provtagning – när och

varför? • Utredning av cytopenier/abnormt förhöjda blodvärden

• Efter lymfomdiagnos – staging (bedömnign av

sjukdomens utbredning)

• M-komponent i blod/urin

• Kontroll efter behandling av hematologiska maligniteter

• Kontroll efter SCT för t ex aplastisk anemi

BM provtagning – biopsi eller

aspirat? • Aspirat

+ tunnare nål (mindre smärta?)

+ snabbare bearbetning

- endast cellsuspension (=cytologisk analys)

- svårare att ta ut celler vid t ex fibros (dry tap)

• Biopsi + ger vävnadsmaterial (=histologisk analys, med information om bentrabeklar, stroma, topografi, typ av patologisk infiltration)

+ enda möjlighet om inget aspirat fås

+ materialet kan arkiveras

- grövre nål

- tar längre tid att bearbetas, kräver urkalkning

Rutiner kring materialhantering

• Om ”hematologisk” (Lymfom? Cancer?)

frågeställning på remissen

– fallet hanteras av hematopatologigruppen

= färskhantering & utskärning, med biobanking

• → FACS svar samma/nästa dag

• → svar på små biopsier inom max två

dagar

Routine analysis - morphology

Peripheral blood - smear

Bone marrow – AML

Bone marrow – aplastic anemia

Peripheral blood - abnormalities

anemi leukocytos

PB – abnormalities (2)

Akut lymfoblastisk leukemi Akut myeloisk leukemi

PB – abnormalities (3)

Kronisk lymfocytisk leukemi Kronisk myeloisk leukemi

PB – abnormalities (4)

Hemolysis Iron-deficiency anemia

Special techniques

Molecular analyses

• Detect certain DNA/RNA fragments

• Detect RNA translation products – abnormal proteins

resulting from e.g. translocations between two genes on

different chromosomes

• Qualitative analysis (yes/no) → diagnosis

• Quantitative analysis (how many copies/aberrant cells)

→ follow-up after treatment

• MRD = minimal residual disease

Phenotyping

• Flow cytometry (suspension of living cells)

– markers on cell membranes

– markers in the cytoplasm

– markers in the nucleus

– phenotype, cell size & granulation

• Immunohistochemistry (fresh frozen or FFPE material)

– markers on the surface/inside cells

– topographical information (phenotype+location)

– material can be stored for many years

• FFPE = formalin-fixed paraffin.embedded

Immunologic background

Major histocompatibility complex (MHC)

• Family of molecules on cell surface

• Present in all vertebrates

• Aim: to help cells recognize own/foreign cells (self/non-self)

• In humans: human leukocyte antigen (HLA) system.

• Classes of MHC protein molecules

– class I MHC – on almost every cell in the organism

– class II – only on leukocytes

– class III – complement cascade, interleukins

• Flow cytometry/IHC detect MHC antigens using monoclonal antibodies

Flow

cytometry

BMWS0289

FACS of lytic bone lesion Much fewer cells available for analysis with intracellular markers

but clonality pattern consistent with mIg

CD138

CD20 CD79a

BMWS0289

MC2317-13

CD138 CD79a

CD20 IgM

BMWS0289

T12608-13