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Haematology > Module 2 Summary > Flashcards

Module 2 Summary Flashcards

1
Q

Acute Leukaemia Cytochemistry

A

Positive in AML:
Naphthol AS-D Chloroacetate esterase
Myeloperoxidase stain (MPO)
Sudan Black B stain (peroxidase dependent)
Acid phosphatase
Demonstration of esterases with Naphthyl acetate &
Naphthyl Butyrate
Acid alpha Naphthyl esterase
Positive in ALL:
Periodic Acid Schiff reaction (PAS) (Bind to the glycogen in leukaemia cells)

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2
Q

What were the principals of FAB classification?

A
  1. blast count > 30% (blast morphology was correlated to the myeloid/lymphoid
    maturation sequence)
  2. Morphology
  3. Cytochemistry: > 3% of blasts have to be positive either for MPO or SBB to qualify as myeloid blasts
  4. cases which appeared non myleoid, were classified as LL
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3
Q

What are the subtypes in AML FAB classification?

A 
AML
M0 - AML, MPO negative 
M1 - AML without maturation
M2 - AML with maturation
M3- APML 
M4- - Acute myelomonocytic leukaemia 
M5a - Acute monocblastic l
M5b -Acute monocytic l 
M6 -Acute erythryoleukaemia  
M7 - Acute megakaryblastic l
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4
Q

What are the subtypes of ALL in FAB classification?

A

ALL
L1 - small monomorphic cells
L2 - large heterogeneous cells
L3 - burkitt’s

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5
Q

What advances led to WHO classification?

A
  1. Metaphase cytogenetics in 1960ies- interphase fluorescence in situ hybridisation (FISH) and polymerase chain reaction (PCR) came later
  2. Flowcytometrical phenotyping since mid 1970ies; examines DNA content and cell membrane expression of lineage associated membrane / cytoplasmic proteins
  3. the assignment to subtypes of acute leukaemias is important
  4. Improvement in standardisation
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6
Q

WHO AML classification?

A

AML with recurrent genetic abnormalities
AML with myelodysplasia related changes
Therapy-related myeloid neoplasm
AML, not otherwise specified

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7
Q

WHO ALL classification?

A
  1. Precursor lymphoblastic leukaemia
    B – lymphoblastic leukaemia, not otherwise specified
    B – lymphoblastic leukaemia, with recurrent genetic abnormalities
    T – lymphoblastic leukaemia
  2. Mature lymphoblastic leukaemia
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8
Q

Major concepts of WHO classification?

A

Stratification of acute leukaemias by major lineage (lymphoid, myeloid,
biphenotypic)
Cell of origin is suggested for each leukaemia
Lowers the blast count from 30% to 20% in blood or bone marrow

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9
Q

What does flow measure?

A

relative size
relative granularity
relative fluorescence intensity

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10
Q

What is flow made up of?

A

Fluidics system transporting particles to the laser beam
Optics system consisting of lasers to illuminate particles in the sample stream
Electronics system which converts the detected light signal into electric signals,
which are processed by a computer

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11
Q

What are fluidics?

A

Blood or bone marrow, or other cells in liquid suspension, are injected into a stream of sheath
fluid within the flow chamber:
- One cell at the time moves through the laser beam at any time
- The laser beam will then interact with the cell

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12
Q

How are fluorescent dyes used in flocyto?

A

Fluorescent dyes used in flow cytometry are conjugated to monoclonal antibodies, so that a particular antigen on a cell can be identified. (they are incubated with the sample from beforehand)
In a mixed population of cells, different fluorochromes can be used to distinguish separate subpopulations

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13
Q

What is the purpose of flo cyto? What can it be used for?

A

Blast identification
Lineage assignment
Classification

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14
Q

Myeloblast CD numbers

A

CD117 (myeloid/stem cell), CD34 (early precursor),

Myeloid: CD13, CD33, HLA-DR, -ve CD19

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15
Q

B lymphoid precursors CD

A

CD34 and TdT, CD19

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16
Q

T Lymphoid precursors CD

A

CD34, TdT, cyCD3

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17
Q

CLL lab findings

A

• Lymphocytosis between 5 and 300 x 109/l
• Smear cells
• Normocytic normochromic anaemia
• Thrombocytopenia
• Bone marrow: lymphocytic replacement of normal
marrow elements

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18
Q

CLL immunophenotyping + CLL score

A

•The main thing with mature CLL B cells is that they express CD5, which is usually exclusively expressed on T cells - normal mature B cells do not express it
• CLL is +ve for CD5, CD19, CD20, CD22, CD23, CD79b.
•The CLL score gives 1 point to CD5+. CD23+, FMC7 negative and weak expression of SmIg and negative/weak expression of CD79b.
A score of 5/5 = CLL likely

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19
Q

What cancers have Mature B cells CD5 +ve?

A

Mantle cell
Marginal zone NHL
B NHL unclassified
CLL

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20
Q

Other tests for CLL

A

•Other tests include antigolubulin test (coombs test), reticulocyte count, serum Ig, bone marrow aspirate/ LN biopsy
oSerum Ig - reduced concentrations/ immuneparesis (Hypogammaglobulinaemia)
oBM aspirate - lymphocytic replacement of normal cells

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21
Q

CLL Cytogenetics

A

Karyotpyping is impossible in CLL because cells are too slow diving - you need FISH
•Good prognosis - normal karyotype, del(13q), trisomy 12
•Worse prognosis - del 11q, del 17p

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22
Q

Role of NGS in CLL

A

Can identify other mutations such as:
NOTCH1- encodes tf that regulates MYC,
and SF3B1, which encodes a important part of the splicesome

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23
Q

CLL Poor prognostic mutations/factors

A 
17p (TF53) deletion 
11q (ATM) deletion in pnts under <55
ZAP70 and CD38 infer poor prognosis
Unmutated IgH status 
High B2-microglobulin (Keating, 1995)
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24
Q

Why is unmutated IgH status a poor prog factor?

A

CLL with Mutated Ig heavy chain genes has a better survival than non-mutated. The principle behind this is that Ig somatic hyper-mutation is a normal process by which Ig’s transform to bind antigens better. Lack of mutation = worsenes B cell function

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25
Q

Good Prognostic factors for CLL

A

•Good prognosis - normal karyotype, del(13q), trisomy 12

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26
Q

Staging systems for CLL

A

Binet (A, B, C) A: <3 lymphoid areas are enlarged; B: >3; C: anaemia or thromboyctopenia are present
Rai (Stage 0-IV)
0 is lymphocytosis only
1 Lympho + enlarged LN
2 Lympho + enlarged spleen ± enlarged LN
3 Lympho + anaemia
4 Lympho + low plts + 1 enlarged (spleen/LN/liver)

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27
Q

CLL prognosis

A

HIGHLY variable!!
> Indolent disease 10-20 year course, never require treatment, death from unrelated cause (30% patients)
> Initially 5-10 years good health until progression to a 2-3 year terminal phase
> Rapid progression to death within 2-3 years

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28
Q

When do we begin treatment for CLL?

A

Watch and wait for all patients until they show an indication for intervention (5):
o Progressive BM failure
o Massive/progressive lymphadenopathy
o Progressive lymphocytosis defined as 50% increase of 2months and/or a doubling time less than 6 months
o >10% weight loss in 6months, fever >38 for >2weeks, fatigue, night sweats
o Autoimmune cytopenias

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29
Q

MBL?

A

Monoclonal B lymphocytosis (MBL) is defined as the presence of a clonal B-cell population in the peripheral blood with fewer than 5 × 10^9/L B-cells (>5 is the cut off for CLL) and no other signs of a lymphoproliferative disorder. The majority of cases of MBL have the immunophenotype and resemble CLL. MBL does not require treatment. 1% of cases become CLL per year.

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30
Q

What is the Mx in CLL?

A

o First line treatments - steroids, alkylating agent, purine analogues, anti CD20
o Second line - purine analogues, anthracyclines, allogeneic BMT
o Treatment of patients who are refractory - high dose steroids, anti-CD52
• Young patients may be cured by allogeneic SCT
• Supportive management for people who would not benefit from intensive chemo e.g. Prophylaxis/treatment of infections (infections account for 50% CLL deaths)

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31
Q

Why do 50% of pnts get recurrent infections in CLL?

A

Remember CLL is clonal proliferation of nonfunctional B cells!!!
Multifactorial:
Hypogammaglobulinaemia (due to b cells not working)
Impaired innate immunity
Neutropenia
Leukaemic infiltration of bone marrow
Therapy related

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32
Q

What increases in the incidence of infections in CLL?

A

Advanced age
Disease progression
Repeated therapies (Steroids, Cytotoxic chemotherapy reduces T and B cell number and function, B cell specific therapy worsens B cell function as affects CLL and residual normal B cells)

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33
Q

What complications in terms of infections can CLL pnts experience?

A

Rec sinopulmonary
CMV reactivation
Rec herpes zoster
Pneumocystis Jirovecii

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34
Q

CML pathogenesis

A

BCR-ABL fusion over-activates tyrosine kinase this activates signalling pathways: JAK STAT, Ras Mek, mtor, src kinases and PI3K/AKT. This leads to proliferation, survival and clonal expansion.

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35
Q

CML PC

A 
Incidental finding 
Fatigue, night sweats, weight loss
LUQ pain
Splenomegaly 
Less frequent: bone pain, priapism, retinal haemorrhage, thrombosis, hepatomegaly
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36
Q

Accelerated phase definition

A 
blasts in BM or PB 10-19%
basophils >= 20%
Persistent thrombocytopenia
Ph+ on treatment
Thrombocytosis unrelated to treatment
Increasing spleen size and wcc unrelated to therapy
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37
Q

Blast phase definition

A

> 20% in PB or BM
Extramedullary blast proliferation, apart from spleen
Large foci or clusters of blast seen in the BM biopsy

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38
Q

CML diagnosis

A
  1. FBC + Blood film (mature leucocytosis with blasts <2% of nucleated cells in BM)
  2. FISH for BCR-ABL (24hr)
  3. RT-PCR for BCR - ABL (For monitoring)
  4. BM asp (morphology, flow, cytogen analysis) count blasts, ph+, rule out other cytogenetic abnormalities)
  5. BM trephine (histopathological confirmation + blast count)
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39
Q

Different scores for CML

A

bap bap bap bap to the top, CML and slide that rhythm
SOKAL (1984) - BAPS (blast % on pb, age, platelet count, spleen size)
HASFORD (1998) - BAPS + eo & basophil % on pb
EUTOS (2011) - Spleen size + basophil % on pb

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40
Q

Describe BM aspirate in CML

A

HYPERCELLULAR
o Megakaryocytes can increase in number and appear hypo-lobular
o The percentage of erythroid cells falls (increased myeloid:erythroid ratio)
o Increased deposition of reticulin fibres
o Pesudo Gaucher cells (macrophaged that have phagocytosed other cells) can be present

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41
Q

CML Mx

A

c - monitor white cell count, cytogenetics and molecular genetics.
m - imatinib is a useful treatment (interferons used to be used).
S - BM transplant is the only cure but it can not be used in all patients

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42
Q

What is the definition of MM?

A

Multiple myeloma is a cancer of transformed plasma cells, terminally differentiated B cells that secrete Ig and are the effector cells of the specific humoral immune response

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43
Q

MM pathogenesis

A

Transformation results from a range of numeric and structural genetic aberrations that accumulate from a pre-malignant condition (“MGUS”) to terminal progression

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44
Q

MM incidence + epidemiology

A 
>4000 people every year in the UK
incidence 5/100.000 per year
 15% of blood cancers and 1% of cancers
median age at diagnosis 65-70y
2x more frequent in blacks than in whites, less common in Asians
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45
Q

Prognosis of MM

A

Poor. Median survival 4-7years (age)

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46
Q

What is MGUS?

A

Monoclonal Gammopathy of Undetermined Significance
Monoclonal serum protein < 30g/L
BM plasma cells < 10%
annual risk of progression to MM 1-2%
Rare in young, increasing incidence with age (5% >70y)

47
Q

What is the next stage after MGUS?

A

Smouldering Myeloma
Monoclonal serum protein ≥ 30g/L
BM plasma cells ≥ 10%
annual risk of progression to MM 10%

48
Q

MM aetiology

A

Crap Performance, try harder if you can okay
Chronic inflammation, radiologys, asbestos, petroleum, pesticides, takes of laxative, high-dose radiation, inflamattion chronic, obesity

49
Q

Normal plasma cell development

A

> Encountering Ag drives a virgin B cell to generate a low-affinity plasma cell or stimulates its migration to a LN GC
In GC, affinity maturation via: somatic hypermutation, VDJ recombination and Ag selection (all incl ds-breaks i.e. has a propensity to mutate)
Class switch recombination occurs, leading to the development of Ig isotypes. (Centrocytes with poor match are apoptosed)
After, the plasmablast either enters circulation to release Ab or migrates to the BM where it becomes a long-lived plasma cell that produces Ab.
Each plasma cell produces one type of specific antibody IgM> IgG eventually

50
Q

Myeloma plasma cell development

A

Key challenges for a plasma cell include switching off cellular characteristics that are no longer required, e.g. cell cycling, activating programmes that are essential for antibody production, and undergoing apoptosis if they do not find a receptive niche in the bone marrow. Genomic instability (e.g. t14q32 or del 13, can cause failure to complete these programmes correctly & could potentially leave active cellular processes, which may result in the features of myeloma.

51
Q

How does the myeloma cell grow in the bone marrow?How does this lead to end stage myeloma?

A

As malignant plasma cells accumulate in the BM, +ve CK- and cell adhesion-mediated feedback loops are established between the plasma cell and stromal cells that facilitate the growth of the myeloma clone. These feedback loops support the survival of the myeloma clone and mediate drug resistance and constitute therapeutic targets.
Plasma cells produce lots of IG and, therefore, are heavily reliant on protein handling pathways e.g. proteasom pathway, the unfolded protein response (UPR) etc.
Numerous signalling pathways are constitutively activated and/or deregulated in myeloma.
The end results are the hallmarks of myeloma, which include abnormal plasma cell differentiation, deregulation of the cell cycle, decreased apoptosis and increased myeloma cell growth and survival

52
Q

Which signalling pathways are constitutively activated/deregulated in MM?

A

PI3K, (NF-κB), RAS–RAF–MAPK and (JAK)– (STAT).

In addition, over-expression of MAF and MYC are important.

53
Q

Where are plasma cells usually found? found in MM?

A

•Plasma cells are dependent on interactions with Bm stroma for survival and proliferation hence it is not normally found in the peripheral blood
oThey have a low labelling index and make Ig inefficiently
oPlasma cell leukaemia is a progression where plasma cells become independent of stroma and hence begin to appear in peripheral blood

54
Q

What do MM cells look like?

A

In MM cells, the nucleus is pushed to one side as the golgi and ER are so big. You can see a pale area around then nucleus (Golgi) and then lots of ER on the outside. Thus, have low n:c. Plasma cells are MATURE and have clumped chromatin with rare nucleoli. Sometimes, plasmablasts can be seen -> immature, diffuse chromatin, prominent nuceoli etc.

55
Q

Immunophenotype of MM

A

Positive: CD38, CD138, CD56/58, monotypic cytoplasmic Ig, LC restricted
Negative: CD19 and CD20, surface Ig
CD56; this is absent in normal plasma cells and in plasma-cell leukaemia

56
Q

MM Mx

A

Ultimately, the only treatment for active myeloma patients is autologous BM transplant (If patients are not candidates for autografts, use melphalan for autologous SCT)
Chemotherapy regime for autologous SCT:
•Standard therapy - Melphalan +/- prednisolone
oLow CR [complete response] rate but oral therapy
and produces response in 50-60% Px
•High dose therapy - Cyclophosphamide, thalidomide and dexamethasone has now replaced VAD as high dose regimen in the UK

57
Q

What was VAD?

A

oVAD - vincristine, Adriamycin, dexamethasone + autologous SCT
•High response rate 65%, CR achieved in 10%
•Bm suppression, high dose steroids, hickman line and unsustainability of response are weaknesses (median survival is about the same)

58
Q

Why don’t we use allogenic SCT in MM pnts?

A

Allogeneic SCT only applicable to a minority of patients [because most people get myeloma when they are old]. Does offer a cure but has a high relapse rate and high treatment-related-mortality. Should be considered for anyone

59
Q

Treatment options in relapsed myeloma/ refractory myeloma?

A

Thalidomide, Lenalidomide and Bortezomib

60
Q

Thalidomide moa and adv and disadv

A
  1. INHIBIT PRODUCTION OF IL-6 (which is a GF for myeloma cells and they @ apoptotic pathways through caspase 8-mediated cell death. By @ T cells to produce IL-2, thalidomide and IMiDs alter (NK) cell numbers and function, thus augmenting the activity of NK-dependent cytotoxicity (Anderson, 2005)
  2. Stimulates IL10, T-cells, which blocks NF-KB, inhibits pro-inflammatory CKs and inhibits TNFa and thus angiogensis (Riley, 1999). This is particularly important as Morphology of advanced disease shows microvessels - hence anti-angiogenesis may be its most important mechanism.
    oLow renal excretion; particularly useful in MM because renal failure is a problem
    oTeratogenicity, constipation, somnolence, peripheral neuropathy, and thromboembolism are side effects
61
Q

What is lenalidomide? How is it different?

A

Lenalidomide is more potent than thalidomide in enhancing Nk cell activity, has less incidence of side effects but may produce cytopenias. It is also very expensive.

62
Q

Wtf bortezomib

A

oProteasomes degrades proteins that are ‘polubiquinated’ [tagged in many sites by ubiquitin]
oTumour cells are highly dependent on proteasome activity because they are high turnover cells
oProteasome inhibiton leads to growth retardation and apoptosis of tumour cells
•Inhibition of NfKB = decreased proliferation, adhesion molecule and IL6 production, and increased apoptosis
oBortezomib is potent, reversible and selective; it binds to proteasomes with a high affinity and a low turnover rate

63
Q

Supportive treatment for MM

A

Give Bisphosponates because they reverse bone disease and block mevalonic acid pathway which may trigger an anti-myeloma response

64
Q

When do we start MM treatment?

A

There is no treatment benefit by starting early in MGUS/ indolent patients - watch and wait unless there is signs of active /progression of disease

65
Q

What are the cues for MM progression when we should start treatment?

A

oPresence of M component in serum (as kappa or lamda light chains) and/or urine (as bence jones proteins)
•NB, M proteins don’t correlate disease progression
oIntact Ig, BJP
oPresence of plasma cells in BM
oRaised serum light chains in absence of >10% plasma cells in BM
oCa>2.65, bone disease
oCreatinine >177
oHb <10

66
Q

MM Clinical manifestation?

A

•Proliferation of plasma cells within BM
oSymptoms of BM overcrowding/failure; anaemia,
neutropenia, thrombocytopenia
•Cytokine production by plasma cells
oBone resorption = pain. Fractures, hypercalcaemia
•Paraprotein production by plasma cells can lead to Hyperviscous blood
•Excess light chain production may lead to amyloidosis
•RENAL FAILURE caused by rec infections (neutropenia), hyperCa, paraprotein and light chain deposits

67
Q

MDS Definition

A

MDS is a group of stem cell disorders, RIDIC:
Recurrent genetic abnormalities
Increased risk of transforming to acute myeloid malignancies
Dysplasia in one or more lineages
Ineffective haematopoiesis
Cytopenias

68
Q

Explain the ineffective haematopoiesis

A

Increased apoptosis > cells die inside the BM space, before they enter the blood stream, resulting in stream, resulting in cytopenias
(in contrast to cytopenias, the underlying bone marrow is hyperactive hypercellular)

69
Q

Epidemiology & Mortality of MDS

A

3-4/100,000 in UK
Incidence rises with age (approx 30 per 100K in age group 70+)
median = 75
M>F
median survival is extremely variable, ranging from over 5 years to less than 6 months after diagnosis
50% die because of cytopenia, 30% progress to AML (esp those who present with blasts at diagnosis)

70
Q

Diagnostic criteria

A

 HB < 10 g/dl
 Neutrophil count < 1.8 x 10^9/l
 Platelets < 100 x 10^9/l
But if definite cytogenetic findings and dysplasia are present, MDS can be diagnosed without significant diagnosed without significant cytopenias

71
Q

Cytogenetics of MDS

A

 loss of chromosome 7, or del(7q)
 del (5q) or t(5q) del (5q) or t(5q) – typically typically thrombocytosis thrombocytosis > 450 x 10^9/l > 450 x 10^9/l
 isochromosome 17q, or t(7q)

72
Q

Threshold for dysplasia in MDS

A

the required percentage of erythroid, and/or granulocytic cells and /or megakaryocytes. Manifesting dysplasia must be > 10% of all nucleated cells (> 10% of all megakaryocytes resp.) Dysplasia is often accompanied by an increase in blast percentage

73
Q

Etiology of MDS?

A

age related accumulation of genetic damage
Environmental exposures to
 benzene
 chemicals, solvents, insecticides, pesticides in agriculture
 cigarette smoking

74
Q

What haematological disorders is MDS associated with?

A

Fanconi’s anaemia
Dyskeratosis congenita
Down’s syndrome

75
Q

Clinical presentation of MDS

A

ANT Anaemia neutropenia and thrombocytopenia

76
Q

BM biopsy for MDS

A
  1. Cellularity - hypercellular particles + trails
  2. Dysplastic granulopoiesis - agranular hypolobated forms, clumped chromatin, nuclear lobation
  3. Megakaryopoiesis reduced in numbers
  4. Abnormal cells: infiltration with polymorphic blasts of medium to large size, faintly granular cytplasm, multiple visible nucleoli, 15% of all nucleated cells
77
Q

MDS Diagnosis summary

A

Presence of persistent (> 6 months) and significant cytopenias:
- Haemoglobin < 10 g/dl
- Neutrophil count < 1.8 x 10^97l
- Platelet count < 100 x 10^9/l
Bone marrow findings:
- Dysplasia affecting > 10% of any one lineage
- blast excess, but not > 20%, otherwise qualified as AML
Typical cytogenetic abnormalities
Exclusion of differential diagnoses

78
Q

Detail dyserythropoeisis in MDS

A 
Cytoplasm alteration
- Ringed siderblast
- PAS positive
- Vacuolisation
Nuclear alteration:
- budding
- karyorrhexis 
- megaloblastoid changes (asynchronous nuclear cytoplasic alteration)
79
Q

Neutrophil maturation stages

A 
 Myeloblast
 Promyelocyte
 Myelocyte
 Metamyelocyte
 Band cell
 Segmented neutrophil 
 Neutrophil
80
Q

Detail Dysgranulopoiesis in MDS

A

Nuclear alterations
 Nuclear hyposegmentation (Pseudo Pelger – Huet anomaly) (hyposegmented neutrophils v common)
 Nuclear hypersegmentation
 Presence of Auer rods (automatically classed as high risk MDS)
Cytoplasmic alterations
 Cytoplasmic plasmic hypogranularity
 Pseudo Chediak-Higashi granules (giant autophagosomes)
 Small size

81
Q

Detail megakaryocytic dysplasia in MDS

A
  • Widely separated nuclei

- Hypolobated and monolobated, small, megakaryocytes

82
Q

State the factors known to cause dysplastic changes in MDS?

A 
NIP Dadeedoodad NIP Dadeeday
Nutrtional (Severe b12, folate or copper def)
Infectious (HIV, parovirus)
Poisoning (arsenic, lead, zinc)
Drugs (cotrimoxazole, isoniazid)
83
Q

How are the risk factors of MDS related to differential diagnoses?

A
  1. Methotrexate, Hydroxycarbamide can cause cytopenias, macrocytosis and neutrophil neutrophil dysplasia
  2. Cotrimoxazole can cause Pseudo Pelger abnormalities
  3. ring sideroblasts sideroblasts can be seen in healthy individuals with benign conditions
    such as:
     vitamin B12 deficiency
     copper deficiency
     alcohol excess
  4. Paroxysmal Nocturnal Haemoglobinuria can present with cytopenias and dysplasia
84
Q

Cytogenetics MDS

A 
In roughly 50% one of these occur:
 del 5q
 monosomy 7
 isochromosome 17
 loss of chromosome 13
 del 11q
 del 12p, t(12p)
85
Q

what is 5q syndrome

A

Characterised by hypolobated/monolobated megakaryocytes, refractory macrocytic anaemia, and normal to elevated platelet count. FAVOURABLE prognosis.

86
Q

WHO Classification of MDS

A

 MDS with single lineage dysplasia (SLD)
 MDS with multilineage dysplasia (MLD)
 MDS with ring sideroblasts (RS)
 MDS with RS and SLD
 MDS with RS and MLD
 MDS with isolated del(5q) MDS with isolated del(5q)
 MDS with excess blasts class 1 or 2 MDS with excess blasts class 1 or 2 (5-9%, 10-19% blasts in the BM)

87
Q

Lymphoma definition

A

A lymphoma is a neoplastic condition arising in lymphoid cells (lymphocytes or their precursors)
It can arise in a cell of T lineage, B lineage or natural killer (NK) lineage

88
Q

B lineage lymphomas can arrise from which cells?

A 
o	Naïve/pre germinal - CLL/SLL
o	Pre-germinal/manle zone - MCL
o	Germinal - Burkitt, Follicular
o	Post-germinal - MZL/ extranodal MALT lymphomas
o	Plasma cell - myeloma
89
Q

Follicular lymphoma (defn and epidemiology)

A
  • Mature b cell (non Hodgkin) lymphoma - accounts for 22% of NHL, low grade germinal centre
  • Affects elderly as an indolent disease that can transform into an aggressive lymphoma
  • F>M
90
Q

Morphology of FL

A
  • Nuclear cleft presence is indicative of germinal cell lymphomas
  • Chromatin is evenly condensed - this helps differentiate it from CLL, where chromatin is patchy
  • Trephine biopsy shows paratrabecular infiltration
91
Q

Immunophenotype of FL (flow and immunohisto)

A

Flow:
o CD10+ (germinal cell), CD19, 20,22 + (B), CD 45+ (WCC)
o CD5 - (identifies it from CLL), CD3-, CD15,30- (these are
HL markers), CD34, Tdt- (lymphoma cells are mature)
•NB CLL will also display CD23
o One of kappa or lamda light chain

Immunohisto:
o BCL2+ - this is key to diagnosis

92
Q

FL Cytogenetics

A 
Cytogenetics shows the rearrangement that brings BCL2 (cr18)  under the influence of the enhance of the IG gene
Mutations: t(2;18), t(14;18) ,t(18;22) Crisnan (1993) 
The t(14;18) translocation usually arises from an error in VDJ recombination so that BCL2 is juxtaposed to one of the JH regions
93
Q

What is the molecular pathophysiology of FL?

A

T14;18: BCL2 encodes an anti-apoptotic protein. BCL2 stabilizes the mitochondrial membrane and prevents efflux of cytochrome C and subsequent activation of caspases in response to DNA damage, triggers the intrinsic pathway of induction of apoptosis.

94
Q

FL diagnosis

A

You can detect this with FISH & PCR
oUse a breakapart probe for 14q32 and this will detect any of the 3 translocations
RQ-PCR is useful for MRD monitoring

95
Q

FL staging

A

Determined by examination, bloods, biopsies, CXR, CT and PET (radioactively labelled glucose - picks up high metabolism tissues inc. heart, liver, bowel)
•1 - single LN/ structure affected
•2 - >2 structures on same side of diaphragm
•3 - involvement of both sides of diaphragm
•4 - invasion of non lymphoid tissues
o All - A/B depending on whether ‘B’ symptoms are
present or not

96
Q

FL Grading

A
  • Grade 1 & 2 are low grade. 3 a can also behave indolently
  • Grade 3b behaves more like a high grade lymphoma
97
Q

FL Rx

A

• Most options are aimed at palliation. AlloSCT + local radio offers the only hope for cure but very few patients can survive the treatment
• Involved field radiotherapy - stages 1 & 2
• Chemo-immunotherapy is now considered the optimum treatment for symptomatic stages 3/4
o CVP + rituximab
• We still don’t know what to do for advanced stage, asymptomatic patients - the rationale for delaying treatment until the patient experiences symptoms is to avoid drug resistance

98
Q

MDS Subtypes

A 
Subtypes are determined by presence of:
Ringed sideroblast (lower risk) (Patnaik, 2017)
99
Q

Cytogenetics of leukaemia brief history

A

Pre 1960s - use giemsa stain to show unbanded chromosomes in metaphase. (the cr would be recognised by their size and centromere position)
Trypsin exposure followed by giemsa stain -> allowed band metaphase and digitally arranged karyograms
This allowed recognition of translations and inversions -> which led to molecular genetic analysis

100
Q

Uses of FISH

A

Can use single probe to identify trisomy
Can use one probe for gene and one probe for centromere to distinguish deleted gene vs whole chromosome lost
Can use double colour double fusion probe for 2 fusion genes or one
Double colour breakapart probes can identify a gene that has split into 2

101
Q

Types of probes

A 
Oncogene probe (to show split or amplification)
TSG probe (is it lost)
Centroemric probe (Is there trisomy/monosomy)
Whole chromosome paint
102
Q

Role of cytogenetics in acute leukaemia

A

Cytogenetic abnormalities sometimes indicate the cause of the leukaemia
Often they indicate the prognosis
Cytogenetic abnormalities sometimes indicate the cause of the leukaemia
Often they indicate the prognosis
Cytogenetic analysis can indicate that a leukaemia will respond to a specific form of treatment, e.g. to all-trans-retinoic acid

103
Q

Detail Alkylating agent-related AML

A

These two broad groups have genetic/ cytogenetic and prognostic differences
Alkylating agent-related AML is characterized by abnormalities of chromosomes 5 and 7, inv(3), t(3;3), t(6;9), t(8;16) and complex rearrangements

104
Q

Topoisomerase II inhibitor T-AML

A

Topoisomerase II-inhibitor-related AML is characterized by balanced translocations with 21q22.12 (RUNX1) and 11q23.3 (KMT2A)* breakpoints including t(3;21), t(8;21), t(4;11), t(9;11) and t(11;19)(q23.3;p13.1) —but not t(11;19)(q23.3;p13.3). Topo tml is also associated with other translocations usually found in de novo AML, e.g. t(15;17) and inv(16)

105
Q

Why is it important to distinguish t-aml?

A

Therapy-related AML needs to be distinguished from de novo AML, even with the same cytogenetic abnormality as prognosis tends to be worse e.g. with t8;21 its a lot worse if tAML
It is also important, for the benefit of future patients, to recognising that t-AML/MDS are occurring

106
Q

How to apply the WHO classification?

A

First risk factors are considered, then genetic features and thirdly phenotype is used to categorise the remaining pts (initially separate the MDS changes and categorised rest using FAB classification)

107
Q

WHO AML flow chart for classification?

A

Prev chemo/radio? Y- t-AML
N- Rec. genetic abnormality? Y- AML with re.c. genetic abnormally
N- Previous MDS or MDS related cytogenetic abnormalities or multilineage dysplasia?
Y- MDS related AML
N- AML, not otherwise categorised.

108
Q

What is the next step in who classification?

A

Classifying the rec gen abnormalities seen in AML.
Good prog:
1. t(8;21) (q22;q22)[RUNX/RUNX1T1]
2. inv(16) or t(16;116)
Assoc with eosinophilia and increase basophil with granulation
3. APML - t15;17 (PML RARA) has poor survival rate initially because of DIC
4. t(9;11) bad prognoasis

109
Q

What is the PML RARA variant in the FAB classifcation?

A

o APML results from 515;17 forming 2 fusion genes PML RARA and RARA PML
o variant M3 is very (but not 100%) similar to true M3 on a genetic and clinical basis. it is ‘variant’ because of MORPHOLOGICAL differences – granules cannot be seen on LM but they can on EM
o the diagnosis between classic and variant M3 lies with using a fluorescent Ab against PML, which appears microparticulate in variant M3
o RARA is involved in a number of leukaemias

110
Q

Prognostic role of Cytogenetics

A

a. Demonstrates monoclonality of leukaemia
b. Shows which lineages are involved in the neoplasm
c. Provides evidence of mechanisms in leukaemia (over-expressed oncogene/ suppressed tumour suppressor gene)

111
Q

Diagnostic role of cytogenetics

A

a. Provides evidence of aetiology of leukaemia (because certain aetiological agents cause distinctive genetic changes; finding these changes can reveal the cause)
b. Very specific at identifying subtypes of AML (eg M3/APML)
c. Distinguishes therapy-related and recurrent AML
d. Can provide evidence for underlying/predisposing disease to a leukaemia

112
Q

Monitoring role of cytogenetics

A

a. Can provide evidence of regression in response to treatment
b. Can provide evidence for engraftment in response to BMT

113
Q

What is the significance of KMT2A rearrangement

A

Acute leukaemia with 11q23.3/KMT2A involvement is many diseases not one
This does not yet have much therapeutic significance but maybe it will have in the future

114
Q

What are the AML not otherwise categorised?

A 
Acute myeloblastic, minimally differentiated
Acute myeloblastic without maturation
Acute myeloblastic with maturation
Acute myelomonocytic leukaemia
Acute monocytic/monoblastic leukaemia
and many more...

Haematology (11 decks)

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