hematopathology -...
TRANSCRIPT
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