Transcript cells, exhibit the morphology and growth properties of

Hematopathology
Monika Klimkowska MD PhD
Klinisk patologi/cytologi
[email protected]
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
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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
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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
Lymphoid diseases
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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
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Precursor lymphoid neoplasms (B-, Tcells)
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 Reticulocyte
erythroblast
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
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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*
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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
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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
HISTIOCYTIC AND DENDRITIC CELL NEOPLASMS
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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)
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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 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
Bone marrow aspirate
morphology (histology + cytology),
immunophenotyping, molecular analyses (FISH,
PCR), cytogenetics
Bone marrow biopsy
morphology (histology + cytology),
immunophenotyping, molecular analyses (FISH)
Peripheral blood
morphology (cytology), immunophenotyping,
molecular analyses
Cerebrospinal fluid
morphology (cytology), immunophenotyping
Fine needle aspirate (LN, focal lesions)
morphology (cytology), immunophenotyping,
molecular analyses
Tissue biopsies
morphology (histology + cytology),
immunophenotyping, molecular analyses (FISH,
PCR)
What cannot be done?
Cytogenetics, PCR
Molecular analyses?
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)
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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
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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
BMWS0289
MC2317-13
CD138
CD20
CD79a
BMWS0289
T12608-13
CD138
CD79a
CD20
IgM