LT33-36. 85 good questions, nicely answered!

Q: What is the function of monocytes?

0.1-1.0 x 10 9 Phagocytosis (bacteira, cell debris) secretion of cytokines (TNF, IL-1) Antigen processing and presentation to T-lymphocytes Stimulated by GM-CSF (gran-mono colony stimulating factor

Q: What is the life span of erythrocytes?

120 d. 2.4 x 10 6 per second, 207 x 10 9 per day

Q: What is the life span of granulcoytes?

few days 3-150 x 10 6 per second 0,3-12 x 10 12per day

What are the main functions of blood?

Body supply: oxygen, nutrients
Waste collection: carbon monoxide, acid, metabolites
Homestasis: temperature, pH
Defense: innate anda adaptive

What is the function of monocytes?

0.1-1.0 x 10⁹Phagocytosis (bacteira, cell debris)
secretion of cytokines (TNF, IL-1)
Antigen processing and presentation to T-lymphocytes
Stimulated by GM-CSF (gran-mono colony stimulating factor

What is the life span of erythrocytes?

120 d.

2.4 x 10⁶ per second, 207 x 10^{9 per day}

What is the life span of granulcoytes?

few days

3-150 x 10^{6 per second}0,3-12 x 10^{12per day}

What is the life span of thrombocytes?

10-11 d

1x 10^6 per second
86 x 10^9 per day

What is an omni-potential stem cell, pluri-potential stem cell and committed stem cell?

Omni-potential: differentiate into any type of cell
Pluri-potential: differentiatie into more than one type of cell
Committed: only differentiate into one type of cell

What are hematopoietic stem cells?

HSC
Have the capacity for self renwal
Have the capacity to differentiate into all the lympho-myeloid lineages
Repopulate the marrow of a secondary lethally irradiated animal/person

What is necessary for maintence of balanced number of HSC?

Proliferation of the cells must be accurately regulated
Inhibition of apoptosis
INhibition of differentiation

Which factors regulate the HSC stemness?

Intrinsic factors:
- transcription factors
- chromatin modifiers
- cell cycle regulators: cyclin E, c-myc, notch 1, mTor, which are reportedly important for G0 state maintenance in HSCs

Extrinsic factors
- cytokines
- growth factors

Tell something about the development of hematopoietic stem cells?

3rd week: first HSC in extra-embryonal mesenchyma, chorion and cord. in intra-embyronal: arota-gonad, mesonephros

5-6 weeks: liver
8-9 weeks: migration to lymfoid organs, thymus, spleen, lymph nodes
10-12 week: mesenchymal outrgrowht in marrow cavity; formation of stroma
Few weeks later: homing of stem cells to bone marrow
5th month: production of blood cells
birth: hematopoiesis in all bones
chilrden: decreased production in long bones
adults: no production in long bones, only in the proximal regions of the long bone

site of hemapoeis: yolk sac, liver, marrow

Erythropoieesis. What is the stem cell? What is the committed cell? What is the developmental pathway?

Stem cell: hemocytoblast
Committed cell: proerythroblast
Developmental pathway:
- phase 1: ribosome synthesis -> early erythroblast
- Phase 2: hemoglobin accumulation -> late erythroblast, normoblast
- phase 3: ejection of nucleus -> normoblast, reticulocyte

Erythrocyte

What is the influence of growth factors on hematopoietic?

Growth factors can directly influence hematopoiesis via inhibiton or stimulation of HSC and HPC

SCF, FLT3
GM-CSF
IL2,3,7, thrombopoietin

What is the influence of cytokines on hematopoietic?

Cytokines can indirectly influence hematopoiesis via inhibition/stimulation of production of hematopoietic growth factors

What ist he function of SCF?

Stem cell factor: effect on the early hemapoietic stem cells

What is the function of GM-CSF?

gran/monocolonly stimulating factor
late stage of the maturation

What is the function of thrombopoietin?

production of platelets from megakaryocytes, can stimulate the early stem cells

What is the role of erythropoietin? EPO

EPO promotes the proliferation of erythroid progenitor cells by reducing the level of cell-cycle inhibitors and augmentin transcription cyclins
and supports their survival by increasing the anti-apoptosis protein BCXL

It increased the number of erytrhoid progenitor cells which differentiate into normoblast, enucleate and leave the bonemarrow

main production source is the kidney

Which cells produce G-CSF?

endothelial cells, fibroblast, macrophages

Activated monocytes producte inflammatory cytokines, such as
- TNF alfa
- IL-1
- IL-6

This stimulates G-CSF production (explains the leukocytosis in patients with infection or inflammation)

How do you administer EPO, G-CSF and TPO in a patient with kidney failure?

EPO: subcutaneous, renal anemia, myelodysplasia
G-CSF: subcutaneous, stemcell mobilization and granulocytopenia, prevent bacterial infections
TPO: subcutaenous or orally, imune induced thrombocytopenia ITP. Stimulates a large production of new platelets

What are the effects of G-CSF?

Granulopoiesis

- functional stimulation
- maturation induction
- differentiation commitment
- proliferation
- survival

Tell something about homing and mobilition of HSC.

G-CSF induces increased neutrophil production and activation, release of elastases and athepsin-G to facilitate mobilization (cuts the binding between HSC and their micro-environment)

SDF-1 is a homing facot (cytokines) produced by the mbone marrow micro-environment, inducing HSC's to transendothelial migration in to the bone marrow niche by E- and P-selectins expressed on vessel walls

HSC has receptor for SDF-1 and CXCR4

For what is this a marker: CD45, CD34, SSC

CD45 = marker for leukocytes (hematopoietic origin of cells)
CD34 = marker for human HPC, not all CD34 + cells are HSC!
SSC: side scatter: granularity of the cells

How is the stem cell mobilization and transplantation?

Donor is first treated with G-CSF.
Harvest of hematopoietic stem cells
For autologous Tx, freeze in liquid nitrogen.
You give chemotherapy to ablate the immune system
Stem cell reinfusion to rescue from heatopoeitic apasia.
These stem cells will move from the circulation into the bone marrow

How is the granulocte count during transplantation

First you give chemotherapy , so that the patient becomes aplastic. Than you give the stem cell infusion and you generate new blood cells.
You need at least 4 x 10^{9 per liter to be healthy}

How is proto-oncogene activatoin by VDJ recombination?

When VDJ recombination goes wrong,
the gene that is being joined to the immunoglobulin genes, becomes irreversibly activated, because the primary purpose for lympohcytes is to express antigenreceptor.

What translocation in mantle cell lymphoma?

t(11;14)
IgH CCND1

What is the germinal center reaction?

Increase affinity for a certain antigen.
Centroblast morphology: gives somatic hypermutation (SHM) for th eantibody genes. ONly the ones with the highest affinity will survive.

Than you get class switch recombination.
Plasmacells can produce antibodies.
Mmeory Bcells go to the periphery or the marginal zone.

What are the clinical presentation patterns of lymphoma?

Nodal clinical presentation of a disease.
Patients have prominent lymphadenopathy (enlarged, hard, indolent), you find bone marrow infiltration when the disease spreads further.

Gernal symptoms: B symptoms
- local pain, because some other organs are being involed.

Leukemic pattern: frequently involve the spleen and other lymph nodes.
those lymphomas have a chronic immuno deficiency.

What diseases are often nodal?

lymphadenopathy +++
bone marrow infiltration ++
Splenomegaly ++

LL
MCL
LBL
nML.
HL

What diseases are leukemic?

immunodysfunction +++
bone marrow infiltration +++
splenomegaly +++

CLL
MCL
ALL
sMZL
HCL

What are the four stages of malignant lymphoma with nodal presentation?

Stage 1: lymph node region, 1 extranodal focus
Stage 2: > 1 lymph node regions w or w/o extranodal focus, supradiaphragmal or infradiaphragmal
Stage 3: lymph node regions w or/wor extranodal focus supradiaphragmal and infradiaphragmal
Stage 4: diffuse extranodal infiltration

B cell lymphoma in mantle zone?

burkitt lymphoma
diffuse large b-cell lymphoma
mntle cell lympoma

B cell lymphoma in marginal zone?

marginal zone lymphoma, immunocytoma

How can you make a discription and discrimination of really existin lymphoma entities?

morphology (histopathology/cytophatology)
immunophenotype
genetic aberrations
clinic (history, physical examination, laboratory, staging)

What are immature lymphomas?

lymphoblastic lmphoma LL/ALL
very rare

Why are B cells lymphomas more frequently than T cell lymphomas?

Only B cells use somatic hypermutation machinery in the germinal center and class switch recombination.
they have more processes to develop than T cells

DLBCL often --> 21.2%
MM 15.3%
CLL/SLL 17.5%
FL 8.8%

Explain why follicular lymphomas are incurable?

Follicular lymphomas have an anti-apototic gene (BCL-2)
Chemotherapy works by inducing apoptosis in malignant cells.
So this diseaes is incurable. We can treat it effectively, lymphomas regresss, but we can't rule it out.

What is the prognosis of follicular lymphoma?

Age > 60
Stage III + IV
LDH > normal
Hb < 6 mmol/L
> LN regions

Stage: if you have a 3-5 stage, the overall survival is only 50%
We treat with anti CD20 in combination with chemotherapy.
If follicular lymphoma is only 1/2 lymphod nodes, than we treat with radiation therapy

Stage I/II: radition therapy, 40% chance of cure
Stage III/IV
- no symptoms: watch and wait
- symptomatic: anti-CD20 antibody rituximab iv
cheotherapy
>cyclofosfamide, vincristin, prednison
> bendamustine

What is the pathophysiology of diffuse large B-cell lymphoma?

Uniform standard treatment for this with clear results.
Tumor develops in germinal center.
In the cause of somatic hypermutation mechanisms, you get activated point mutations.
In the end you get a quite agressively growing tumor .

How is the overall survival of a diffuse large B-cell lympohma?

50% survival
CHemotherapy cocktail of three apoptosis inducing agents: anti-CD20, rituximab, cortison
Suppress auto-immunity and contributes to killing lymphoma cells
The curves become flat, so it is curative.
5 year curative is between 95% and 59%

What is chronic lymphocytic leukemia?

Some lymphocytes have been damaged and destroyed by the smear preparation. So the cells are not healthy lypohcytes
Cells expres a normal B cell receptor.
Receptor is able to auto-activate itself
This gives an activated signal , and chronic lymphocytic leukemia, which is not dependent on antigens.

Rituximab improves outcome along the chemotherapy.

For what do they use classical chemotherapy in lympohas?

Cocktails of certain drugs
For proliferating cells.

They're effective against:
- Hodgkin Lymphoma ABVD
- T-lymphomas: CHOEP
- B-lympohmas: CHOp

For what do we use passive imunotherapy?

Lymhocyt subset -> rituximab

For what do we use targeted therapy?

Activated oncogene
- BCL-
- 2 MYC

Signaling pathway

In what stadia is the VDJ recombination?

Pre- B cell

What is philadelphia chromosome?

Lower part of chromosome 9 (ABL) breaks off and is exchanged with the lower part of chromosome 22 (BCR)

You get a changed chromosome 22 = Philadephia chromosome
BCR-ABL, it is a fusion gene.
This fusion gene give rise to constituitive tyrosine kinase acitivity
->
phosphorylation of multiple substrates
->
mitogenic signalling and genomic instability increaed apoptois and stroma regulation decreased
->
chronic myelogenous leukemia

What is chronic myeloid leukemia?

BCR-ABL fusion.
This activates a pathway.
- increased proliferation via RAS, JAK-STAT, CBL-PIK3
- inhibition of apoptosis
- changes in adhesion to stroma

Call the epidemiology of chronic myeloid leukemia?

+/- 14% of all adults leukemias
Member of myeloproliferative disease
Slowly progressive course
incidnece: 1-1,5 / 100.000
Male : female = 2 : 1
Mediage age 53 years, increase in younger patients
Cause: unknown

What are symptoms with chromische myeloide leukemie?

Fatigue, weight loss
- 40% is asymptomatic
- 50% splenomegaly = enlarged spleen
- 20% heaptomegaly = enlarged liver
- 50% anemia
- 20% thrombocytosis , too high platelet count

What is the course of the disease of BCr-ABL?

chronic phase
accelerated phase:
- >15% blasts
- blasten + promyelo's > 30%
- baso's > 20%
- platelets < 100
- additional cytogenetic abnormalities
blast phase: blood or bone marrow: > 20% blasts

Philadelphia positive cells have a proliferative advantage, they increase in number whereas normal cells decrease

What are subsequent mutations involved in progression to blast crisis CML?

29% regulation of trancription
9% chromatin modification
24% intracellular signaling cascade
5% cell proliferaton/differentiationn
16% cell metabolism and protein/iontransport
6% intracelluar organization
11% miscellaneous

What therapy can be given when you have BCR-ABL fusion gene?

imatinib = TKI

second generation TKI = dasatinib, nilotinib

allogenic stem cell transplantation when there is no response to TK inhibitor activity: mutation in binding pocket of BCR/ABL intolerance for TI, you can have a HLA identical sibling or a MUD = matched unrelated donor

conventional chemotherapy: hydroxyurea, busulphan

What are differentiation factors in normal hematopoiesis?

cell division is influenced by: signals of stromal cells in stem cellniche (cell-cell contact + soluble factors)

proliferation + differentiation of committed progenitors is influenced by: hematpoietic growth factors

What is a cytochemical stain for granules in myeloid cells?

myeloperoxidase

What classes of mutation in AML?

mutation giving a proliferation advantage (class I)
mutation giving a differentiation stop (class II)

What are characteristics of a leukemic stem cell?

- self renewal
- multi-potential
- highly proliferative
- CD34+, CD38-, CD123+, CD117-

How is the neoplastic progression over time?

Develoment of a first mutation in a cell resulting in a large number of abnormal cells.
Other mutations occur
In the subclone you get multiple new hits, so you get a polyclonal population of blasts.
If you treat the patients, you get rid of the subclones, but some persist and you can develop a relapse

What is secondary AML?

Derived from myelodysplastic syndrome.
Development of new mutations in time. First you have MDS, but than you develop AML, since there will be more mutations in time

What is a frequent route of clonal progression in MDS following treatment with Lenalidomide?

Lenalidomide has influence on a particular set of abnormalities within the whole population of different mutations of AML cells.
As long as you give treatment, particular clones get suppressed, but if you stop, other clones will grow again.

Call examples of class I mutations.

Is on the cell surface and within the cytoplasm.

Cell surface: growth factor receptors
Cytoplasm: signal tranuction molecules , apoptosis

Call examples of class II mutations

Nucleus:
- cell cycle
- gene transcription
- gene splicing

Acute Myeloid Leukemia, which groups?

Group1 : AML with recurrent cytogenetic abnormalities
- we will find new mutations in the recurrent situation

Group 2: AML with dysplasia in at least 2 lineages

Group 3: AML en MDS following chemotherapy

Group 4: AML not otherwise specified

Group 5: acute leukemia of ambigous origin

What drugs for induction course?

Doel: complete remission

cytarabine, antracycline

What rugs for consolidation course?

Stabilization of complete remission

high dose cytarabine, antracycine

What kind of therapies are there for AML?

induction course: cytarabine + antracycline
consolidation course: cytarabine + antracycline
autologous SCT
allogeneic SCT

What are complications of high dose chemotherapy?

mucositis
infections
pancytopenia -> bonemarrow starts slowly to recover and other damaged cells after three weeks

What is the conclusion of different researches to ttreament of AML?

Last 15 years no improvement in overall survival with conventional teratment

What is a forward scatter?

Size of the cell, by hydrodynamic focusing that makes sure that all cells are lined up by size . It passes a light source and when a cell passes it, this wil give an intrruption.

What is a side scatter?

Granularity of the imperium of a cell. Cell with a lot of granules, you have a light beam, when it hist a granule, the light beam will go side wards.

What are reasons for high blood cell counts?

acute leukemia
infections

Hoe maak je onderscheid tussen acute leukemia chronic leukemia, acute infections, als iemanda nemie heeft (laag Hb)?

acute infections: geen severe anemia
chronic leukemia: vaak is Hb niet zo laag en zijn de platelets hoger.

How can you do diagnostic pathology in cancer in tissues, cells, molecules?

Tissues - resections/biopsy
Cel - cytology
Molecules - molecular pathology

What are indications for a bone marrow examination?

Provide additional information for diagnostic clues seen in the peripheral blood

Increse or decreased leukocytes, Hb or platelets, presene of abnormal or immature cells

How do we get a bone marrow biopsy?

Needle in the posterior iliaca crest.
Biopsy is a piece of tissue in which the cells can be looked with their internal adherence.
You can also take single cells.

What diagnostic tests can you do on a bone marrow aspirate?

Hematomorphology/cytology
IMmunophenotyping
cytogenetics
molecular diagnostics

What diagnostics tests can be performed on biopsies?

standard histology (HE)
special stains (histochemical stianings, pas, alcian blue)
immunohistochemistry (proteins)
molecular assays

What is immune phenotyping?

Clinical diagnosis and scientific research

- FACs machine, stained cells: proteins cell emmbrane, cytoplasm and nucleus
- distinguish cell types based on presence/absence of proteins/antigens: blasts, mature and immature ells, lymphocytes, pasmacells, macrophages, mast cells

Count: 100.000-1.000.000 cells
Sensitivity: 0,01-0,001%
expertise, experience pattern recognition

CD = cluster of differentiation (cluster of designation or classification determinant)

What is cluster of differentation in immunophenotyping?

For example: CD1a, CD1b/c/d/e, CD2, CD3, CD4 --> CD100

- what kind of cells express these particular markers
- sometimes a lot about the function is known
- alternative names provided in history
- to which type of cell surface protein family do they belong

How does a flow cytometer work?

Cellsuspension + mAB
Cells pass the laser beam, hits the fluorchrome
Detection of emitted light and data processing
Data analysis gives a dotplot

What are markers for B celsl?

CD10 and CD19

What are markers for myeloid cells?

CD13.33
CD117

if theyre double positive you probably have AML

What is translocation of chromosome 15 and 17?

PML alfa, acute promyelocytic leukemia

What are molecular diagnostics?

-Look at specific parts of whole DNA/RNA content of cells
-No distinction between different cell types
-Starting with >- 5 million cells
-Distinction due to DNA/RNA probes: biased approach
-In advance determine what you want to look at
-PCR machine
oPrimers which bind to a chromosome/gene
oDuplication process starts
oFirst cycle, second cycle, 36 cycle.
oBeads have been amplified to a large extent
-New development: NGS >> unbiased approach

What are the markers CD45 and CD34?

Expression of CD34, those are the cells that contain the hemapoietic stem cells. All the hemapoietic stemcells are positive for CD45. These are the cells you need to perform stem cell transplantation.

CD45: marker for leukocytes (hematopoitic origins of cells)
CD34: marker for human HSC

What cells can you see in vitro assays of hematopoietic stem cells?

LTC-IC = long term culture initating cells
CAFC = cobble stone area forming cells
CFU = colony forming units

LT33-36

85 important questions on LT33-36