KW seminar: Hematology clinic: fast implementation of science Flashcards
What is an advantage of the hematology department, in comparison to solid tumors?
It is easy accessible, you can take a blood sample which can be used for diagnosis, treatment results and follow-up.
Story of a patient: a woman of 68 years old was more fatigued when walking/on a bicycle. She had hematoma without trauma. When the general practitioner draws blood, he finds anemia, thrombocytopenia and leucocytosis. The lab also calls because there are blasts seen in the peripheral blood. What are these blasts?
These are immature blood cells (such as neutrophils, monocytes, lymphocytes and erythrocytes). In normal situations, they are in low number in the blood marrow, and are not found in the blood.
Thus, this woman has acute leukemia, but the type is still unknown
In order to examine what type of leukemia the patient has, a sample of the bone marrow needs to be taken. Explain how this works.
The patient will get local anesthesia at the site of the bone marrow. A sample is taken at the iliac crest because the bone is very thick there (these are the dimples on your low back). The bone marrow that is taken looks like blood, and is put on a slide for further examination for the pathologist
What is shown in this figure?
This is what normal hematopoiesis looks like, a variety of many different cells. A trained eye would recognize a blast, promyelocyt, myelocyt, metamyelocyt, eosinofiel, basofiel, monocyt, lymfocyt, plasmacel and erytroblast
What are the different techniques that are used in the clinic for diagnosis and testing in the hematology department?
- Morphology
- Cytogenetics
- Immunophenotyping
- Cytochemistry
- FISH
- Molecular biology
Why is it necesary to do further testing, besides morphology, after acute leukemia has been diagnosed?
Because you need to determine wether it’s a lymphoblast or myeloblas (ALL or AML). It is very difficult to determine what type of leukemia based on the morphology. This is why immunephenotyping is used
Explain how immunephenotyping (in context of hematology) works
With use of antibodies, the cell surface proteins are determined. We know for each cell type which surface proteins they express and can therefore determine wether it’s an immature/mature cell, and then if it’s a lymphoid or myeloid cell. An overview of the different markers is shown in the figure
Explain which cytogenetics is used in the hematology department. (2 techniques)
This can be done on a broad scale: karyotyping (right figure). This gives a picture of all the chromosomes, and large chromosomal translocation/duplications can be studied.
On a smaller scale: FISH is used (left figure). Fluorescence in situ hybridization (FISH) is a laboratory technique for detecting and locating a specific DNA sequence on a chromosome. The technique relies on exposing chromosomes to a small DNA sequence called a probe that has a fluorescent molecule attached to it (Leukemia can have many mutations, but these are all known and thus a probe can be used for diagnosis)
Molecular diagnostics also makes use of a probe, and by PCR, a DNA sample can be identified. However, nowadays we use this technique through another approach. How is this technique called?
Next generation sequencing
True/false: AML is a heterogeneous disease
True! It is thus important to realize that genetics play a huge role since there are so many mutations possible. The type of AML has an effect on the treatment, potential resistance and prognosis of the disease
What is Clonal hematopoiesis of indeterminate potential (CHIP)?
Clonal hematopoiesis of indeterminate potential, or CHIP, is a common aging-related phenomenon in which hematopoietic stem cells (HSCs) or other early blood cell progenitors contribute to the formation of a genetically distinct subpopulation of blood cells.
As the name suggests, this subpopulation in the blood is characterized by a shared unique mutation in the cells’ DNA; it is thought that this subpopulation is “clonally” derived from a single founding cell and is therefore made of genetic “clones” of the founder
It is thought that the development of leukemia is a multi-step process. Why do they think that?
Because certain mutations that patients with leukemia have, are also seen in the healthy population. This means that an accumulation of mutations is needed for leukemia to occur
How many targeted therapies are there for adult AML?
3, shown by the red circle in this figure: FLT3m, IDH2m and IDH1m
Through genetic analysis, classification has been made of certain mutations. Explain how this is used in the clinic
- The ‘Favorable’ group is sensitive to chemotherapy, and are thus ‘good-risk’. These patients are treated with chemotherapy.
- The ‘Adverse’ group has mutations making them insensitive to chemotherapy. These patients receive bone marrow transplantation
- The ‘Intermediate’ group has uncertainty wether they are sensitive or not. Therefore, they are first treated with 2 rounds of chemotherapy and then are intensively investigated to determine if the leukemia is still present. If not, they are placed in the ‘adverse’ group
Back to the story of our patient: after investigation it was shown she has Acute Myeloid Leukemia (AML), with a FTL3-ITD high ratio and IDH2 positive. Chemotherapy has many different action sites. Which are often used for AML?
Often a combination of chemotherapy is used, and almost always alkylating agents
What are common side effects of chemotherapy?
Mostly the mucosa is harmed in the process. You can imagine that since the blood flows through the whole body, it can create side effects everywhere (see image)
What is FLT3? What does an FLT3 mutation mean?
- The FLT3 is a class III receptor tyrosine kinase that regulates hematopoiesis. This receptor is activated by binding of the fms-related tyrosine kinase 3 ligand to the extracellular domain, which induces homodimer formation in the plasma membrane leading to autophosphorylation of the receptor. The activated receptor kinase subsequently activates pathways involved in apoptosis, proliferation, and differentiation of hematopoietic cells in bone marrow.
- Mutations that result in the constitutive activation of this receptor result in acute myeloid leukemia and acute lymphoblastic leukemia.
- There is factor-independend growh
- There is a block in myeloid differentiation
- This mutation is associated with adverse prognosis
A drug, midostaurin, was invented for the treatment of patients with FLT3 mutation. Explain what this drug does.
- Midostaurin is a multikinase inhibitor, that is a potent FLT3 inhibitor for both the wild-type and ITD and TDK mutation. However, it also inhibits other receptors (e.g. PKC, VEGFR2, KIT and PDGFR
- It specifially inhibits growth of leukemic cell lines made factor independent by transfection of activating FLT3 mutation (ITD/D825Y)
- Increased survival in a murine bone marrow transplant model of FLT3-ITD myeloproliferative disorder
What were the complete remission (CR) result of a phase III study where Midostaurin (multi-kinase inhibitor) together with chemotherapy were studied?
Complete remission betekent dat iemand schoon van kanker is verklaard
Although a difference of 5-7% looks small, this is a huge win. Also, the overall survival was 10% higher than in the placebo group. An even better survival was shown with chemotherapy, transplantation and midostaurin
What is the down-side of midostaurin?
There are many side-effects. Although not represented in this figure, the lecturer told us that she saw lots of nausea in the clinic.
To illustrate: Midostaurin is a big pink pill, and the patients got so nauseous every time, that after some time they developed nausea when only looking at the colour pink. They could even vomit, which is a huge problem
Since 2017, drugs have been made that are more specific to the kinase. Currently, the effects of this drugs are studied and compared
This is an international study, but the results are not yet available
Explain the role of IDH in Cancer
- IDH is a critical metabolic enzyme in the cirtric acid cycle
- IDH1 is present in the cytoplasm, IDH2 is present in the mitochondria
- Cancer-associated (mutated) IDHm produces 2-hydroxyglutarate (2-HG)
- 2-HG blocks DNA and histone demethylases
- This causes hypermethylation and thus modulation of gene expression
- Normal cellular differentiation is blocked
- Blasts (immature cells) develop, and so leukemia is developed
Explain how/why a mutant IDH inhibitor can be used in the clinic
An IDHm inibitor blocks the formation of 2-HG. This means that DNA and hystone demethylases are no longer blocked, causing active demethylation. This releases the blocked differentiation, and differentiated myeloid cells can develop
True/false: IDHm-positive mutations are seen in multiple cancer types
True.
The mutation is also seen in other cancer types, such as myelodysplastic syndromes, myeloproliferative neoplasms, angio-immunoblastic T-cell lymphoma, low-grade glioma and secondary GBM, chrondrosarcoma and intrahepatic cholangiocarcoma
To determine how intensive a treatment needs to be, a Measurable Residual Disease (MRD) is determined. How does this MRD work?
MRD is a postdiagnosis marker/indicator that states the number of leukeamic cells during/after treatment when the patient is in remission (=no symptoms/signs of disease). With very sensitive molecular biology tests are available, based on DNA, RNA or proteins. These can measure minute levels of cancer cells in tissue samples, sometimes as low as one cancer cell in a million normal cells.
MRD negativity is associated with improved long-term survival
Please take a good look at the detection thresholds of various MRD modalities compared to traditional clinical complete remission
How to look at this figure: The horizontal black line just below 10^10 is the threshold for clical complete remission, meaning that <5% of leukemic cells are present. With microscopy, FISH and chimerism, the detection rate was not low enough to fully determine if all cells are gone (orange lines/box), and often relapse occurs. With the advanced technology, this is much more specific, and with greater certainty can be said if there is complete remission, or more chemotherapy is needed
Explain what happens in autologous stem cell transplantation, aka this figure
An autologous stem cell transplant uses healthy blood stem cells from your own body to replace your diseased or damaged bone marrow. These cells are stored until after intensive chemotherapy. The aim of this total-body irradiation is to get rid of all the cells, but healthy cells might also be damaged, so that is why healthy cells are kept apart. This only works if the patient is sensitive to chemotherapy
Explain what happens in allogeneic stem cell transplantation (aka this figure)
This treatment is done in patients that are insensitive to chemotherapy. After two dosis of intense chemotherapy, the donor’s immune system is used make a new immune system to attack the cancer cells. This is called the “graft-versus-tumor effect.” This is done because chemotherapy alone will not do the trick
Although chemotherapy is very effective, it cannot always be done. This is because sometimes the patient is too old and will simply not survive such a heavy treatment. Another type of therapy that can then be given is by using epigenics. How does this work?
Often tumor suppressor genes are silenced by DNA methylation. This silencing of tumor suppressor genes promotes cancer development and progression. By using a DNA-methyltransferase (DNMT) inhibitor, these silenced genes can become activated again
A drug that inhibits DNA methylation is AZA (5-Azacytidine). Can you name the characteristics of this drug?
- AZA incorporation into DNA requires actively dividing cells
- Serial cycles of DNA replication needed
- Hypomethylation occurs gradually
- Extensive demethylation requires multiple exposures to the drug
- This drug is much less intensive than chemotherapy, and only gives little side effects like nausea
How did the overall survival look of AZA (DNA methylation inhibitor)?
There was an increased amount of surival months (10.4 vs 6.5) and the 1-year survival increased from 45.5 to 34.2%. It was also seen that some patients respond during a long time after treatment, and some don’t respond at all
A drug that could be used together with Azacitidine (DNA methylation inhibitor) is Venetoclax. This is a selective BCL-2 inhibitor that is used in CCL. How does this BCL-2 (inhibitor) work?
Cancer cells often (overexpress) BCL-2. Some family members of BCL-2 bind and inhibit pro-apoptotic proteins. This mean that apoptosis is inhibited. By using Venetoclax, the active site of BCL-2 is occupied/inhibited. Pro-apoptotic proteins are free to bind to Bax and induce apoptosis. The cancer cells which have evaded apoptosis, now are forced to go into apoptosis.
What increase in response rate was seen when using the combination Venetoclax (BCL-2 inhibitor) and AZA (DNA methylation inhibitor)?
That 80% of the patients had complete remission after this treatment. Also the survival rate was prolonged very significantly
