haematological malignancies and CML

Question 1

what’s the reaction when someone is suspected of having a haematological malignancy (HM)?

Answer 1

Once a patient is suspected of having a haematological malignancy – it’s a race against time to classify the disease, as some classes of this cancer are capable of progressing extremely rapidly and in extreme cases can result in death in a matter of days or even hours

Question 2

what are HMs and give some figures on their rate of occurrence

ages for AML vs Lymphomas?

Answer 2

What - cancers of the blood/blood forming tissues (bone marrow and lymph nodes)

Occurrence -
HMs are most common in adults (50-70 ish), however, in children, some types of HM are the most common cancer seen, e.g. acute lymphoblastic leukaemia more common in children, peak around toddler age, and a median of roughly 15

Account for roughly 3% of people living with cancer (though % of cases may be higher as it is quite lethal), ~60 cases/100,000 individuals/year (tho often underdiagnosed, especially chronic lymphocytic leukemia)

AGES -
Acute myeloid leukaemia = more common in elderly, peaks around 75
Lymphomas - roughly affect all age groups equally

Question 3

what are the three categories of HMs (and the fourth-ish one)?

Answer 3

Leukaemia
Lymphoma
Multiple myeloma

Myelodysplastic syndromes (MDS) = precancerous disorder originating in BM, if left untreated can progress to AML. they’re basically leukaemias

Question 4

aside from genetics, what types/methods of diagnostics are used to classify HMs? (briefly for the first two)

Answer 4

Histology (morphology of whole tissues)

cytology and haematology - morphology of SCs in BM and lymph nodes (LNs), and on the differentiated derivatives in the peripheral blood (PB)

immunology/biochemistry -
monoclonal antibodies can be used to detect cell specific surface markers, as different blast cells have different CS markers, quantification can help determine which cell type and which blast cells are most affected

Question 5

go into more detail about how 1. histology is used in classifying HMs. What specifically do they look into?

Answer 5

in this case a BM sample -

Cellularity - often abnormal in HMs (density/number of cells)

density of chromatin staining and morphology of nuclei also expected to be abnormal, so the characterisation of these cellular features = important to understand the nature of abnormal growth, as there are cases of hypercellularity which have not been caused by cancer

Question 6

go into more detail about how 2. cytology and haematology are used to classify HMs - what looks different when there is a HM?

Answer 6

They report on the morphology of SCs in BM and lymph nodes (LNs), and on the differentiated derivatives in the peripheral blood (PB)

HMs often result in changed proportions of these cells (important to see which lineage is affected), typically affecting the SCs (blast cells), restricting differentiation and increasing proliferation.

Blast cells should remain in BM and divide in response to highly regulated stimuli, but in HMs they are seen in the PB (poor prognostic marker)

When the number, differentiation potential and location of blood stem cells is altered – the function of blood is altered – and as you can imagine this leads to serious life threatening illnesses in patients

Question 7

right from the beginning (HSC) explain haematopoiesis.

Answer 7

Haematopoietic Stem Cell (HSC)
The origin of all blood cells.
Differentiates into two major progenitors:
Common Myeloid Progenitor (CMP)
Common Lymphoid Progenitor (CLP)

Myeloid Lineage (from CMP)
Gives rise to cells involved in innate immunity, oxygen transport, and clotting:

Erythrocytes (Red Blood Cells)
Megakaryocytes → Platelets
Granulocytes -
Neutrophils: Phagocytose bacteria (first responders).
Eosinophils: Combat parasitic infections and allergies.
Basophils: Release histamine in allergic responses.
Monocytes → Macrophages/Dendritic Cells: Phagocytose pathogens and present antigens

Lymphoid Lineage (from CLP)
Gives rise to cells involved in adaptive immunity and some innate immunity:

B Lymphocytes: Produce antibodies (humoral immunity, plasma cells).
T Lymphocytes: Cell-mediated immunity; includes:
Helper T Cells: Coordinate immune responses
Cytotoxic T Cells: Kill infected cells.
Natural Killer (NK) Cells: Kill virus-infected and tumour cells (innate immunity)

Question 8

how does the structure of haematopoiesis effect what HMs look like?

Answer 8

HMs are clonal = a genetic change/mutation acquired in a SC is passed on to all cells in its lineage
Cancer progresses and the genetic changes become increasingly complex
Cytosis is observed, as expected, too many cells, in HMs it’s often of one/certain cell types
Cytopenia - too few cells - is observed in other cell types derived during hematopoiesis - because the process requires SPACE; organization of the bone marrow destroyed, crucial spaces filled up with abnormal SCs, less space available to normal tissue. Non malignant SCs in BM don’t have enough physical space to divide – so the descendants of these cells are produced in lower numbers (cytopenia of that lineage) – adding to the malfunction of the blood

Cytopenia of RBCs - often seen in HMs, = low oxygen levels in blood = chronic fatigue and weakness

Question 9

lymphoma - give me more information on what cells are affected and the kinds

Answer 9

Main cell effected - B-lymphocytes

Hodgkins - arises in lymph nodes, not BM (worse prognosis?). Characterised by reed-sternberg cells (They are large and binucleate, with a prominent eosinophilic nucleus that often has a clear halo around the nucleolus, giving it an “owl eye” appearance)

Non-hodgkin’s - originates in BM, looks more like a leukaemia

Question 10

multiple myeloma - what is it/what’s going wrong in it?

Answer 10

overproliferation/cytosis/clonal expansion of plasma cells

These plasma cells produce this one abnormal AB, a paraprotein, called M-protein

This protein can cause damage to healthy tissues

Also, the cytosis can disrupt haematopoiesis , cause cytopenia of other lineages (not enough space to divide)

Question 11

what is leukaemia? there are two things that characterise a leukaemia, what are they/what are they based on?

Answer 11

Overproliferation of malignant cells, originating in the BM

Either myeloid or lymphoid (depends on which lineage is effected)

Then either chronic or acute (chronic = the earlier, can progress into acute). Refers to proportion of blast cells in PB; If BCs < 20% of all nucleated cells in the blood, the leukaemia is classified as a chronic disease. If >20%, acute

Blast cells - just a clarification, there are different kinds, megakaryoblasts, erythroblasts and myeloblasts from CMP, and lymphoblasts from CLP

Question 12

give a bit of info on the four classes of leukaemia?

Answer 12

1. Chronic PHASE myeloid leukaemia:
   These are myeloproliferative neoplasms (MPNs)
   Must include ‘phase’ as ‘chronic myeloid leukaemia’ is a specific kind of leukaemia in itself
   8 types, different types of myeloid cells effected
2. Acute phase myeloid leukaemia:
   AMLs AND related precursor neoplasms (MDS). Some have defined genetic abnormalities, there is even a subclass for when the lineage effected is unclear
   Often chronic phase MLs develop into acute when BCs go over 20%, but doesn’t have to be, some acute MLs are so proliferative/fast they’re never chronic)
3. Chronic phase lymphoid leukaemia:
   Thought to be most common HM, with lots of elderly suspectedly undiagnosed
   Also - can progress to acute, but also can just originate as acute

4, Acute phase lymphoid leukaemia:
Very rare heterogenous group of diseases effecting lymphoid lineage with >20% BCs in PB

Question 13

explain what is meant when HMs are described as 1. clonal and 2. progressive diseases.

Answer 13

Clonal -
Mutations, which can act as biomarkers, are inherited from mother to daughter cells
This is useful because - the disease is then amenable to genetic analysis

Progressive -
When untreated the affected tissue expands as these malignant cells proliferate, resulting in a group of cells with same genotype occupying greater proportions of tissue (clonal expansion)
And the abnormal clones may change, gain more mutations and genetic abnormalities (due to a mutator phenotype e.g. in cell cycle checkpoint proteins, DNA repair proteins etc…) this is clonal evolution - poor prognosis, indicates cancer has progressed, likely that new and more complex clone will grow and proliferate more rapidly than OG clone

Question 14

does remission just mean the cancer is gone?

Answer 14

does NOT mean that the cancer has gone. various stages of remission – and each has been carefully defined – and is specific to a particular genomic technology. There are cytogenetic remissions and molecular genetic remissions. The latter of these being a deeper remission, as the molecular genetic techniques used to monitor disease load are more sensitive than cytogenetic methods

Question 15

are genetic diagnostics useful in HMs?

Answer 15

yes - it can provide, in some cases, sufficient evidence to indicate the best course of treatment to use

However, majority of abnormalities, while informative, aren’t enough to say which treatment is truly best, or to determine the specific cancer type (alone, other things used as mentioned)

Some genetic abnormalities don’t help with classification at all

Question 16

what are three obvious red flags in genetic diagnostics of HM?

Answer 16

the inappropriate and/or overexpression of genes that result in growth and proliferation (oncogenes)

The downregulation or absent expression or inactivation/deletion of genes that normally suppress cell growth and division (tumour suppressors)

Aneuploidy (or partial aneuploidy)

Question 17

give an example of aneuploidy that is useful in diagnostics but doesn’t provide a final answer on its own

Answer 17

trisomy 8 -
often seen in myeloid leukaemia, thought to be how proto-oncogene cMYC is upregulated

+8 is not diagnostic of any one particular type of cancer, but in BM it is strongly indicative of myeloid disease (mainly because it is very rarely observed in lymphoid related neoplasms).

So it is useful to narrow down a possible diagnosis.
Also associated with an intermediate/poor prognosis, informative regardless of the specific leukaemia which would eventually be characterised

Question 18

aside - what is cMYC?

Answer 18

cMYC is a member of the MYC family of proto-oncogenes, and many of these are observed at abnormal levels in cancer, such as n-MYC – which can be massively amplified in neuroblastomas and is involved in cell growth proliferation, and as covered briefly in the previous lecture - has a role in promotion of angiogenesis

Question 19

give an example of a loss of tumour suppressor gene

give an example of a gene translocation often seen in follicular cancer + why it contributes to cancer

Answer 19

isodicentric chromosome 17 that results in the loss of one copy of 17p – and deletion of the tumour suppressor gene TP53, which is located on this chromosome arm. This means you’ve lost one copy - so need to check expression and sequence of other copy as a single point mutation could render it ineffective

IGH-BCL2 in follicular lymphoma - translocation of enhancer elements - can increase expression of proto-oncogenes

Question 20

what type of genomic rearrangement can be really useful in genetic diagnostics of HMs?

Answer 20

Fusion genes can be rather specific to a disease and are therefore excellent diagnostic markers and therapeutic targets.

Some fusion genes are even thought to be SUFFICIENT for disease - meaning that the normal requirement for cells to accumulate abnormalities (and hallmarks) of long periods of time - can be done instantly

Question 21

what is chromothripsis and why is this a poor prognosis marker?

Answer 21

The mechanism by which massive levels of DNA damage/ genome fragmentation, which should normally result in apoptosis but cancer cells often protected from this, are instead pieced back together, making loads of new abnormalities in the process (not super common, associated with more processed malignancies, produces lots of very competitive clones)

Question 22

what’s another (more subtle) thing that genetic diagnostics look out for when looking at cancer?

Answer 22

changes to methylation status of proto-oncogenes and tumour suppressors

Question 23

hallmarks of cancer - what are the 6 main ones, and the added ones?

Answer 23

• Sustaining proliferative signalling
• Evading growth suppressors
• Resisting cell death
• Enabling replicative immortality
• Inducing angiogenesis
• Activating invasion and metastasis
• Deregulating cellular energetics
• Avoiding immune destruction
• Tumour-promoting inflammation
• Genome instability and mutation

Question 24

sustaining proliferative signalling -
1. normally?
2. 4 ways cancer gets around regulation?

Answer 24

Decision to divide is tightly regulated normally, requiring mitogenic signals, detection of these by cell-surface receptors, cascades leading to response causing growth and division

Ways cancer gets around this regulation:
- Upregulate and release mitotic signals
- Stimulate normal cells in surrounding tissues to do the same
- Become more sensitive to growth factors to divide uncontrollably under normal mitogenic signalling
- Activation mutations somewhere in signalling cascade uncouple cell from outside regulation, the pathway can be perpetually on

Question 25

give an example of cancer cells becoming more sensitive to growth factors to divide uncontrollably under normal mitogenic signalling

in terms of treatment, how is this relevant?

Answer 25

Overexpression of HER2 on cell surface in metastatic breast cancer as a result of amplification of HER2 gene on Chr 17
Amplification above a certain

threshold = pharmacokinetically significant and qualify for herceptin (prevents dimerisation of HER2 so can’t respond to growth factor, note - stops growth, does not kill, coupled with e.g. chemo)

Question 26

in breast cancer, explain how HER2 staining determines whether or not someone is treated with Herceptin

Answer 26

Staining of HER2 shows how much is amplified, split into four categories (no and low both not given herceptin, class 3+ = needs more testing, and 4+ is given herceptin)

FISH for borderline (3+) category -
HER2 gene in red
Centromeric marker for Chr 17 in green
Blue = Dapii counterstain to see DNA

Normal cell should have 2 red and 2 green signals.

In order to be given Herceptin a sample must exceed ratio of 2, so twice amount of HER2 as Chr 17

Question 27

name five ways cancer cells can evade growth suppressors

Answer 27

1. Mutation inactivation (of a tumour suppressor or gene in its cascade)
2. Transcription suppression via methylation
3. Deleted via chromosome structural abnormalities/aneuploidy (loss of chromosome)
4. Altered expression of tumour suppressor regulators
5. Evasion of cell-cell contact inhibition

Question 28

what are two common tumour suppressors lost in cancer and what is needed for this loss to drive cancer?

Answer 28

TP53 (Chr 17) and RB (Chr 13)
Two tumour suppressors very commonly deactivated in most cancers
Both copies (alleles) need to be lost to drive cancer, and typically one copy is deleted and the other inactivated (mutant)

Question 29

when RB (chr 13) is lost/deactivated, what happens to result in ‘evasion of growth suppressors’?

what’s another gene related to RB that is commonly inactivated in cancers and how does it have its effects?

Answer 29

when lost, no longer inhibits E2F, a TF that goes and activates a load of genes involved in cell cycle stage transition (G1-S and G2-M)

P16 is another one often inactivated in cancers, it inhibits this molecule CDK that inhibits RB (so without it CDK is always inhibiting RB, so E2F always active)

Question 30

Resisting cell death - what does this really mean/specifically, and what three classes of genes are involved?

Answer 30

this is the evasion of apoptosis

1. Apoptotic regulators - sense the environment to decide if apoptosis should be triggered. Some respond to extrinsic cues, and other intrinsic
2. Apoptotic effectors - cause the cell death, the caspases
3. Apoptotic triggers - any of the molecules in between those^

Question 31

what are three key examples of genes (or gene combos) that may be affected to cause an evasion of cell death?

Answer 31

BAX-BAK - normally pro-apoptotic:
form the pore on the mitochondria to release Cyt-C, activates caspases and APAF (LOF in cancer)

Bcl2 family - anti-apoptotic:
This family of proteins inhibits the BAX/BAK pore formation, so the Bcl2 family is often affected somehow in cancer to prevent apoptosis (upregulated)

TP53 - seen mutated in around 50% of all cancers

Question 32

give an example of TP53 being involved in cancer

Answer 32

located on Chr 17, so an isodicentric chromosome (where the short arm is lost, got two long arms instead) results in loss of TP53

often occurs in MDS (myelodysplastic syndrome, a precancerous disorder that can become a myeloid leukaemia)

Question 33

give an example of how BCL2 can be affected in cancer

Answer 33

gene rearrangement, fusion of BCL2 (Chr 18) and IGH (Chr 14, the immunoglobulin heavy chain, which is transcribed A LOT)

So high expression of the anti-apoptotic BCL2 = drives cancer

Question 34

enabling replicative immortality - why aren’t normal cells immortal/what do they do?
What happens differently in cancer cells?

Answer 34

Normal cells telomeres get shorter, and the cell enters senescence, an irreversible non-proliferative sate
Telomeres get so short that you get chromosome-chromosome fusions, unstable structures that trigger apoptosis

in cancer cells - they overproliferate, so telomeres should shorten, and they should enter senescence, become unstable and undergo apoptosis (but of course must avoid this)
How?
Turning on telomerase. Enzyme that lengthens telomeres

Question 35

why is telomerase a potential, but not ideal target for treating cancer?

Answer 35

telomerase is thought to be a good target to kill cancer cells without harming healthy ones, as normal cells don’t typically express it much
BUT then you’re waiting for overproliferation to occur (before telomerase is activated) and there’s a suspected long delay before senescence/crisis initiation,(so impact would take a while) and we don’t yet have a 3D structure to assist in drug design?

Question 36

inducing angiogenesis - why is this useful to cancer cells?

what key genes are involved?

Answer 36

Rapidly proliferating cancer cells need to sequester a load of nutrients in order to grow into a tumour (and once in a tumour the inner cells are often in hypoxic and nutrient-deprived environments)
So the cancer cells stimulate growth of blood vessels

Loads of genes involved, but…

Key genes = RAS and MYC -
These two genes also feature as reasons for many other hallmarks as well so are key for diagnostics. RAS is also involved in cell growth and proliferation

Question 37

activating invasion and metastasis - this trait is acquired by…?

Genes commonly Upregulated?

Genes commonly deleted/inhibited (give 2 examples, one for upregulation and one for deletion. Hint - cad___)?

Answer 37

This trait is acquired by late stage cancers, after a load of these other hallmarks ^ have already been gained

Genes upregulated = ones involved in cell-migration

Genes typically inhibited/deleted = cell-cell interactions or cell-ECM interactions

E cadherin - normally acts to help assemble epithelial cell sheets, and is commonly depleted in metastatic cancers
N cadherin - normally expressed in migrating neurons and mesenchymal cells during embryonic development - is often upregulated in high grade carcinomas

Question 38

what are the six stages of metastasis? And…

Answer 38

1. Local invasion (to close by tissues)
2. Entry to blood/lymphatic system
3. Transition through blood/lymphatic system
4. Escape into distant tissues
5. Formation of small cancerous nodes
6. Growth of malignant lesions into more mature tumours

Once the cell reaches the point of metastasis, establishing a secondary tumour is often fast, as the cells have acquired a load of these hallmarks

Question 39

deregulating cellular energetics - why is this needed by cancer cells?
What common changes are seen?

therapeutic implications?

Answer 39

Cells growing and proliferating at extremely high rates = very high energy demand and metabolic demand (for lipids/proteins/nucleic acids etc…)
Also in tumours some cancer cells are surviving in hypoxic environments

Switch to glycolysis - the Warburg effect.

Therapeutic Implications:
Deregulated energetics can be targeted therapeutically.
Strategies include:
Inhibiting glycolytic enzymes.
Targeting pathways that regulate metabolic flexibility (e.g., HIF-1, mTOR)

*** FURTHER READING - PDAC CELLS, USING PROLINE AS AN ENERGY SOURCE FEEDING INTO THE TCA CYCLE

Question 40

genome instability and mutation - explain why this hallmark is needed for all the others in a way, covering two key terms, C_Ex and C_Ev

Answer 40

In order to acquire the hallmarks of cancer listed, cells must gain a ‘critical mass’ of mutations/genomic alterations, whether that be point mutations, chromosomal rearrangements, loss of heterozygosity etc…

Some mutations will be advantageous e.g. increases rate of proliferation somehow - the cell with this mutation will divide faster/outcompete its neighbours and give rise to a population/clone of cells that is very competitive and can become a major clone - this is clonal expansion

These cells are already genomically unstable, so will acquire additional mutations, and so possible additional advantages, leading to the evolution of that clone - clonal evolution

So - cells with an unstable genome can potentially develop mutations/mutator phenotype that give rise to these hallmarks needed for a mature tumour to form and metastasise
Result - the tumour is a heterogeneous population of major and minor clones competing amongst themselves and with normal tissue

Question 41

using BRCA 1 and 2 as an example, explain how the hallmark ‘genome instability and mutation’ can be obtained

include some stats on risk of cancer with BRCA 1 or 2 issues

Answer 41

These genes are involved in homologous recombination - a high fidelity (very accurate) mechanism used to repair DSBs mostly during DNA replication (as a duplicate is required to act as a template)
If these genes become inactive/mutated - and DNA breaks are not repaired properly - you get an unstable genome/mutator phenotype and acquire more mutations, increasing the chances of getting one that gives you a hallmark of cancer - and becoming cancerous

Inheritance of either inactivated gene, leads to a high risk of early onset BC - and a lifetime risk of 50-80% BC, and 30-50% OC. Other cancers are also predisposed such as prostate, stomach and pancreatic