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FRCPath MPAD Part 1 > Haematological Malignancies > Flashcards

Haematological Malignancies Flashcards

1
Q

What are myeloproliferative neoplasms and what are the types

A

Characterized by the pathological accumulation of mature myeloid cells

Polycythemia vera, essential thrombocythemia, primary myelofibrosis
CML
Chronic neutrophillic leukaemia
Chronic eosinophillic leukaemia
MPN, unclassified

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2
Q

What are the classical ph- MPNs?

A

PV
ET
PMF

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3
Q

What are the WHO diagnostic criteria of PV?

A

Major criteria
- increased erthryocytes (>16g/dL) or increased red cell mass
- Bone marrow showing hypocellularity with trilineage growth
- JAK2 variant

Minor criteria
- Low erythropoietin level

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4
Q

What are the WHO diagnostic criteria of essential thrombocythemia (ET)?

A

Major critiera
- Increased platelets (>450 x10^9/L)
- Bone marrow showing hypercellular of megakaryocytes with hyperlobulated nuclei
- Not meeting criteria for other MPNs
- JAK2, CALR or MPL mutation

Minor criteria
- Clonal marker
- Reactive thrombocytosis

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5
Q

What are the risks associated with ET?

A

thrombosis and, less frequently, haemorrhage.

In common with PV, long-term problems include a risk of transformation to MF and acute leukaemia (although these are less frequent in ET).

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6
Q

What is PMF?

A

Can be de novo or as late stage ET

Myelofibrosis is characterized by proliferation of abnormal megakaryocytes and granulocytes with deposition of fibrotic tissue in the bone marrow and extramedullary haematopoiesis. 

Fibrosis is thought to arise from an interaction between clonal megakaryocytes, releasing mitogens such as platelet-derived growth factor (PDGF) and transforming growth factor that directly increase fibroblast proliferation.

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7
Q

What are the WHO diagnostic criteria for PMF?

A

Major criteria
- Presence of megakaryocytic proliferation with collagen fibrosis
- Not meeting other WHO criteria for MPNs
- Presence of JAK2, CALR or MPL

Minor criteria
- leukocytosis >11 x10^9/L
- Splenomegaly
- Aneamia

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8
Q

What are the clinical features of PMF?

A

50-60 years 

Symptoms relate to bone marrow failure (anaemia, infection, bleeding) or progressive splenomegaly and a pro-inflammatory state (pain, weight loss, sweating)

25% of patients progress to AML

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9
Q

What is chronic eosinophilic leukemia (CEL) NOS?

A

CEL is a rare chronic myeloproliferative neoplasm of unknown aetiology in which a clonal proliferation of eosinophilic precursors results in persistently increased numbers of eosinophils in the blood, bone marrow, and peripheral tissues

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10
Q

What is chronic neutrophilic leukemia (CNL)?

A

CNL is a rare distinct chronic myeloproliferative neoplasm defined by persistent, predominantly mature neutrophil proliferation, marrow granulocyte hyperplasia, and frequent splenomegaly,

Characterized by:
- sustained peripheral blood neutrophilia (>25 × 109/L)
- hepatosplenomegaly (absence of BCR::ABL1)
- The bone marrow is hypercellular. No significant dysplasia is in any of the cell lineages, and bone marrow fibrosis is uncommon.

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11
Q

What abnormalities are associated with CNL?

A

Activating membrane proximal mutations in CSF3R at exon 14, especially T618I and T615A; present in 50-80% of CNL.

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12
Q

What is MPN, unclassified?

A

MPN-U is an uncommon subtype consisting largely of cases that fail to meet the diagnostic criteria for a specific MPN subtype, or present with features that overlap with two or more subtypes. As such, most cases test positive for JAK2 V617F, CALR or other myeloid driver mutations

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13
Q

What is juvenile myelomonocytic leukemia (JMML)?

A

Median age at diagnosis is 2 years old

Increase in granulocytes and monocytes

Lymphadenopathy and skin rash

Associated with presence of RAS activating genes – NRAS/KRAS

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14
Q

What are the risk factors associated with MPN?

A

Most cases appear to be sporadic, but families with an increased incidence of ET have been described

Familial cases are thought to be due to a genetic predisposition to acquire somatic mutations rather than to direct inheritance of germline mutations.

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15
Q

How do MPNs present?

A

Splenomegaly
Anaemia
Bone marrow fibrosis

Fever
Night swears
Weight loss

Most patients are asymptomatic and are diagnosed from a routine blood test

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16
Q

What molecular abnormalities are important in ph- MPN?

A

Diagnostic:
- JAK2 V617F and Exon 12
- CALR Exon 9 insertion
- MPL W515

Exclude
- BCR-ABL1

Other molecular
- IDH1/2- treatment
- If undiagnosed, large panel may be requested

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17
Q

What is the significance of JAK2 in MPN?

A

JAK2 is a non-receptor tyrosine kinase involved in JAK/STAT pathway

The most common driver of MPN and present in 95% of all PV, 50% of ET and 60% of PMF

Common variants are JAK2 V617F and exon 12 variants

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18
Q

What is the significance of CALR in ph- MPNs?

A

CALR is a chaperone protein required for protein folding/calcium homeostasis
Frameshift variants allow binding to TPO-R and ligand independent activation of JAK2

Found in 20% of ET and 35% of PMF

Type 1 variants: 52bp deletion in exon 9

Type 2: 5bp insertion in exon 9

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19
Q

What is the clinical significance of MPL?

A

Encodes thrombopoietin receptor (TPO-R) on megakaoryocytes which regulates platelets
Activating mutations activated JAK/STAT

Occurs in 3% ET and 5% PMF

Codon 515 and 505 in exon 10 are most common

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20
Q

Is cytogenetics required for MPN?

A

Cytogenetic studies are not essential and many laboratories do not offer chromosomal analysis as most abnormlaities are just general myeloid markers

There is DIPSS plus prognostic scoring system for PMF

An unfavourable karyotype would be defined as:
- complex karyotype
-abnormalities including +8, -7/7q-, i(17q), -5/5q-, 12p-, inv(3) or 11q23 rearrangement.

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21
Q

What testing strategy is often used for diagnostic MPN?

A

Frontline: ddPCR or fragment analysis for JAK2 V617F

If negative: JAK2, CALR, MPL NGS
Myeloid NGS panel

Exclude CML: BCR-ABL1 RT-PCR

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22
Q

How is MPN monitored?

A

Not routinely monitored

Could monitor JAK2 V617F by ddPCR

Should monitor to check for progression to either PMF or AML

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23
Q

How is PV treated?

A

Reduce complications
- Venesection (remove RBC)
- Aspirin (anti-platelets)

Cytoreductive chemotherapy
- Hydroxycarbamide

Targeted treatment
- Ruxolitinib: JAK2 inhibitor

Progression
- Azacitidine
- Stem cell transplant

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24
Q

How is ET treated?

A

Aims to try and prevent thrombotic complications
- anti-platelets and aspirin

Chemotherapy
- hydroxycarbamide
- interferon-a

Ruxolitinib is not NICE approved for ET but may have some utility

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25
Q

How PMF treated?

A

Aspirin

Hydroxycarbamide
Interferon-a

Ruxolitinib if JAK2 positive

Candidates for stem cell transplant

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26
Q

How is CEL treated?

A

Transplant

Pegylated interferon-α (IFN-α)

Corticosteroids.

Hydroxyurea

Cyclophosphamide,

Imatinib: for those with FIP1L1-PDGFR alpha fusion tyrosine kinase translocations

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27
Q

How is CNL treated?

A

Transplant

Pegylated interferon-α (IFN-α)

Hydroxyurea

Transplant

JAK2 inhibitors currently user clinical tial for treatment of CNL.

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28
Q

What is mastocytosis?

A

No longer considered an MPN

Accumulation of mast cells

Aggressive disease that requires rapid treatment

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29
Q

How is mastocytosis treated?

A

Mast cell cytoreductive therapy to ameliorate disease-related organ dysfunction.

Midostaurin: small-molecule inhibitors that target mutant-KIT

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30
Q

How is the prognosis of MPN determined?

A

The Dynamic International Prognostic Scoring System (DIPSS) Plus uses the following eight risk factors. (Such scoring dictates NICE funding)

Age older than 65
Constitutional symptoms 
Hemoglobin lower than 10 g/dL
White blood cell count greater than 25 x109 /L
Peripheral blood blasts greater than 1 percent
Platelet count less than 100 x109 /L
Transfusion need
Unfavorable karyotype

The risk groups based on these eight risk factors are:

Low-risk - Patients with no risk factors 
Intermediate 1 risk (INT-1) - Patients with one risk factor 
Intermediate 2 risk (INT-2) - Patients with two or three risk factors 
High-risk - Patients with four or more factors

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31
Q

What is the rate of MPN transformation to AML?

A

MF: 10-20%
PV: 7.9–17%
ET: 8.1% for ET

Progression to myelofibrosis (MF) over a period of 20 years occurs in 26% of PV and 19.9% of ET.  

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32
Q

What is the role of MDT in MPN management?

A

All new patients should be referred to the MDT for confirmation of diagnosis, prognosis and management plan, taking into account their performance status, needs and co-morbidities

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33
Q

What guidelines are used in MPN?

A

WHO myeloid neoplasms

BJH 2021

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34
Q

What is CML?

A

Sub classification of MPN
Characterisation of BCR-ABL1
Leads to overproliferation of mature white blood cells- granulocytes

These granulocytes are abnormal and build up in the blood and bone marrow so there is less room for healthy white blood cells, red blood cells, and platelets. When this happens, infection, anaemia, or easy bleeding may occur.

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35
Q

What is the incidence of CML?

A

Chronic myeloid leukaemia (CML) accounts for approximately 15% of adult leukemias.

Median age of 57

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36
Q

What is the prognosis of CML?

A

The advent of tyrosine kinase inhibitor (TKI) therapy has transformed CML from a fatal disease into a chronic disease for the majority of patients.

With the introduction of TKI therapy in 2001, the 8-year survival is now 87% and continues to improve.

The life expectancy of a newly diagnosed patient with Philadelphia chromosome-positive (Ph+), BCR-ABL1+ CML in chronic phase (CP) is now very close to that of age matched individuals in the general population

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37
Q

What are the risk factors for CML?

A

There is evidence regarding one of the causes of CML being acute radiation exposure as it has been observed at a high incidence in atomic bomb survivor.

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38
Q

How does CML present?

A

Newly diagnosed cases can be asymptomatic and discovered as part of a routine medical examination.

Common findings at presentation: splenomegaly, fatigue, malaise, weight loss, night sweats, and anemia.

Splenomegaly is the most common finding on physical exam and is present in over half of the patients.

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39
Q

What are the three phases of CML and their diagnostic criteria?

A

Chronic phase
- Increased WBC >10
- Leukocytosis and/or basophillia
- BCR-ABL1
- <10% blasts

Accelerated phase
- increased WBC >25
- Platelets <100
- <20% blasts
- BCR-ABL1 and other abnormalities

Blast crisis
- increased WBC >25
- >20% blasts
- BCR-ABL1 and other abnormalities

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40
Q

What is the characteristic fusion seen in CML?

A

BCR-ABL1
- 95% of cases due to t(9;21)(q34.1;q11.2)
- 5% of cases are cryptic or complex rearrangements

Results in the Philadelphia chromosome on der 22

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41
Q

What are the types of BCR-ABL1?

A

p210: In CML, the breakpoint cluster region is almost always in the major BCR (M-bcr), forming an abnormal fusion protein, e13a2/e14a2

p230: Rarely, the breakpoint occurs in the μ-BCR region, between exons 17-20. This encodes for a larger fusion protein

p190: Ph-positive acute B-ALL, and occasionally in AML, but rarely in CML - e1a2, poor repsonse4 to imatinib

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42
Q

How does BCR-ABL1 drive oncogenesis?

A

Promotes cell proliferation and blocks apoptosis. The hybrid protein has constitutive activated tyrosine kinase activity.

Activates downstream pathways including RAS/MAPK, PIK3/AKT, JAK/STAT

The 3BP1 (binding protein) binds normal ABL1 on the SH3 domain, which prevents SH1 activation. For BCR/ABL1, the N-terminal exon of BCR binds to SH2, hiding SH3, which therefore cannot be bound to 3BP1, and thus activating SH1

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43
Q

What Additional chromosomal abnormalities (ACAs) are seen in CML and what is the significance?

A

+Ph
+8
i(17q)
-7
-5
3q26 abnormalities (MECOM)

Increased risk of transformation to blast phase

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44
Q

How is BCR-ABL1 detected in CML?

A

FISH- rapid detection, detects cryptic rearrangements
G-banding on bone marrow: identify Ph and additional chromosomal abnormalities (ACAs)
RT-PCR on blood: determine transcript type
RQ-PCR:

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45
Q

What is the turnaround time for diagnostic CML?

A

FISH- 3 days
G banding and RT-PCR- 14 days

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46
Q

What guidelines are used in CML?

A

BSH
ELN

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47
Q

Why is CML monitored and how often?

A

Measurable residual disease (MRD) can show response to treatment, predict relapse and suggest when it is suitable for treatment change or treatment free remission

Happens 3 monthly until MMR achieved and then 6 monthly

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48
Q

How is MRD monitored in CML?

A

RQ-PCR- BSH and ELN guidelines recommend this technology

Extracted RNA is converted to cDNA using reverse transcriptase- 2 separate reactions for BCR-ABL1 and ABL1

Uses Taqman technology. Fluorescently labelled probe for BCR-ABL1 and ABL1 and probe contains quencher molecule which prevents the fluorescence

The ROI is amplified by taq polymerase which digests the probe releasing the quencher. This releases the fluoresence

Each round of amplification therefore increases the fluorescence which is measured. The ct value is the amplification round it takes for the fluorescence to cross a specific threshold. This is then compared to a standard curve to find an absolute number of copies of BCR-ABL1.

This is then normalised to ABL1 to produce a ratio

Standardised to international scale

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49
Q

What is the international scale?

A

A lab specific coversion factor given that is used on the final BCR-ABL1:ABL1 ratio during MRD monitoring with RQ-PCR

This improves comparability between labs and allows for specific thresholds for MRD e.g. MMR, MR4

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50
Q

What percentages of BCR-ABL1 are considered molecular responses according to ELN guidelines?

A

> 35%- continuing disease
<35%- residual diseae
<1% CCyR (complete cytogenetic response)
<0.1% MMR (major molecular repsonse)
<0.01% MR4
MR4.5
MR5

10,000 copies of ABL1 transcripts are also required to ensure sensitivity of analysis

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51
Q

What are the treatment response milestones according to the BSH?

A

Optimal
- 3 months <10%
- 6 months <1%
- 12 months <0.1%
- >12 months <0.01%

If milestones are not met then they should consider changing treatment to a second or third generation TKI

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52
Q

Why do some CML patients experience treatment failure?

A

Poor tolerance- had to discontinue

Primary resistance- never responded to imatinib

Secondary resistance- acquired resistance variants in ABL1 TKD or clonal progression

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53
Q

What is AKD testing?

A

BSH and ELN guidelines state that ABL1 kinase domain testing should be varied out to look for resistance variants

The specific variant may suggest a specific TKI should be used

Often tested with nested RT-PCR to enrich for ABL1 KD, followed by sanger

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54
Q

How does imatinib work in CML?

A

These TKIs compete with ATP for the binding site of BCR::ABL1, thus rendering its phosphorylation ability inactive.

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55
Q

What 2nd and 3rd line TKIs are available for CML?

A

Nilotinib
Bosutinib
Dasatinib
Ponatinib- sensitive to all common AKD variants including T315L

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56
Q

What is treatment free remission in CML?

A

If patients have a deep molecular response (MR4 or more) for over 12 months, patients can stop treatment

This requires an increased in MRD monitoring to monthly for the first 12 months

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57
Q

What is MDS?

A

Myelodysplatic syndrome/neoplasm

Heterogeneous group characterised by clonal expansion of myeloid cells with impaired differentiation

Results in dysplasia in one or more myeloid lineages- leading to anaemia, neutropenia and/or thrombocytopenia

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58
Q

What is the incidence of MDS?

A

The incidence of MDS in the UK is 3.72/100 000 population/year; predominantly a disease of the elderly (median age at diagnosis 75.7 years)

The annual incidence is 1-2 per million children, with 10-25% presenting with increased blasts

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59
Q

What are the risk factors of MDS?

A

Exposure to cytotoxic agents (alkylating), PARP inhibitors or radiotherapy for an unrelated neoplasm (formally categorised as MN-pCT). Therapy-related myeloid neoplasms account for 10-20% of all cases of MDS/AML/MDS/MPN with a strong association with TP53 mutations.

Exposure to benzene (smoking), agricultural chemicals and solvents (19)

Germline predisposition

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60
Q

How does MDS present?

A

Cytopenias

Anaemia: fatigue, dizziness
Neutropenia: increased infections
Thrombocytopenia: bleeding/bruising

Can overlap with MPN and risk of transformation to AML

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61
Q

What are the diagnostic criteria for MDS?

A

Persistent cytopenias: <10g/dL Hb, <100 x 109/L platelet count, <1.8 x 109/L absolute neutrophil count

BM Morphology: dysplasia in 10% of cells, 5-19% blasts, ringed sideroblasts

Flow: Increase in CD34+, CD15, CD16, CD14 and CD56

Genetics: detection of MDS related abnormality

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62
Q

What are the WHO classifications of MDS?

A

MDS with defining genetic abnormalities
- MDS with isolated 5q del
- MDS with SF3B1
- MDS with bi-allelic TP53 del
MDS morphologically defined
- MDS low blasts
- MDS hypoblastic
- MDS-IB1 (10% blasts)
- MDS-IB2 (20% blasts)

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63
Q

What is the IPSS-R in MDS?

A

International prognostic scoring system
- uses karyotype/ cytogenetically visible abnormalities to put patients into prognostic groups

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64
Q

What are the IPSS-R prognostic groups?

A

Very good: -Y, del11q
Good: normal, del5q, del21p, del20q
Intermediate: del7q, +8, i(17q)
Poor: -7, inv(3), complex (3 abnormalities)
Very poor: Complex >3

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65
Q

What are the problems with IPSS-R?

A

Can only be used at diagnosis

Can’t be used for secondary MDS

Does not take into account sequence variants

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66
Q

What is the IPSS-M?

A

Updated prognostic scoring system that uses haematologic, cytogenetics and molecular genetics

Six categories with different prognoses and an online tool

Can be used for primary and secondary MDS

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67
Q

What cytogenetic abnormalities are seen in MDS?

A

Found in 50% of patients

Del5q- favourable
Monosomy 7 or del7q- poor prognosis, associated with alkylating agents
Del20q and del12p- favourable
3q abnormalities- MECOM rearrangements
-Y - non specific to MDS

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68
Q

What molecular abnormalities are seen in MDS?

A

Found in a range of genes

TP53- adverse
ASXL1- adverse
SF3B1- favourable

TET2, RUNX1, DNMT3A, EZH2

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69
Q

What germline predispositions are associated with MDS?

A

MDS can be caused by germline mutation (autosomal recessive and X-linked recessive) found in Fanconi anaemia (FA genes), severe congenital neutropenia (ELANE), Shwachman-Diamond syndrome (SBDS), Diamond-Blackfan anaemia (RPS genes) and telomere biology disorders (TERT, TERC). Germline mutations can also occur in RUNX1, ETV6 and DDX41

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70
Q

What is the testing strategy for MDS?

A

Cytogenetics
- G banding and SNP arrays used
- At WMRGL, SNP arrays used for all confirmed MDS cases (higher resolution and can detect CN-LOH) and G banding used for high risk cases (detects balanced translocations)

Molecular
- Myeloid gene panel

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71
Q

What is CHIP?

A

Age-related Clonal Haematopoiesis of Indeterminate Potential (CHIP)

Patient with CHIP has a better survival rate compared to MDS, and lower risk of progression to AML, but still have a risk of progression to a haematological neoplasm compared to individuals without mutations

Non -defining abnormalities e.g. DNMT3A, TET2, ASXL1

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72
Q

What is CCUS?

A

Clonal Cytopenias of Undetermined Significance (CCUS), where dysplasia has not been found, therefore cannot be classified as MDS. One or more unexplained cytopenias persistent for 4 months or longer, presence of one or more somatic mutations with VAF ≥2%

Can be non-malignant causes such as B12, folate deficiency

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73
Q

Are MDS patients molecularly monitored?

A

Karyotype on follow-up BM biopsies can detect clonal evolution where there was a cytogenetic marker at diagnosis or can use specific FISH probes, however, limited sensitivity.

Can use NGS data to monitor variant allele frequencies (VAF’s) and monitor emerge of sub-clones.

High-risk mutations (TP53, FLT3) should be assessed with every BM biopsy.

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74
Q

How is MDS treated?

A

Only curative treatment- stem cell transplant for high risk patients

Supportive therapy: transfusions, iron-chelating agents, anti-biotics

Chemotherapy: Azacytidine considered to be non-intensive, is a hypomethylating agent
Cytarabine- intensive

Targeted therapies:
- Lenalidomide available specifically for del5q patients, immunomodulation therapy acting on cytokine signalling pathways
- IDH inhibitors: Ivosidenib

Immunotherapy
- durvalumab (anti-PD-L1) in combination with aza- low risk MDS

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75
Q

What is the prognosis of MDS?

A

Median overall survival for MDS patients is 5 years

Patients with a high-risk karyotype (involving 3 or more unrelated abnormalities or chromosome 7 abnormality) had a median survival of four months

Around 30% of MDS patients progress to AML (21).

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76
Q

What guidelines are used in MDS?

A

BSH

WHO

IPSS-R/IPSS-M

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77
Q

What is AML?

A

Heterogeneous neoplasms

The accumulation of immature myeloid precursor cells which have derived from the unregulated proliferation of a single abnormal progenitor

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78
Q

What are the clinical features of AML?

A

Bone marrow becomes overcrowded with blasts leading to bone marrow failure
- Anaemia (fatigue)
- Neutropenia (Infections)
- Thrombocytopenia (bruising/bleeding)

Can be fatal in weeks/months if untreated

Can lead to organ infiltration including spleen, liver, CNS, bones, chloroma

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79
Q

What is the incidence of AML?

A

AML is relatively rare but is the most common acute leukemia in adults. The incidence is approximately 4 per 100,000 people per year.

Incidence increases with age

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80
Q

What are the risk factors for AML?

A

Risk factors may include cigarette smoking, past chemotherapy or radiation treatment/exposure

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81
Q

How is AML diagnosed?

A

Bone marrow
- >20% blasts
- Complete blood count

Flow
- CD13, CD33, CD34, CD117, HLA-DR
- Identify leukemia associated immunophenotype (LAIP) for MRD monitoring by flow

Biochemistry
- Myelo-peroxidase (MPO): an enzyme which is present in the primary granules of myeloid cells is an unequivocal marker of myeloid lineage

Genetic abnormlaities
- t(8:21) (q22;q22);RUNX1::RUNX1T1
- Inv(16) (p13.1q22) or t(16;16) (p.13.1;q22);CBFB::MYH11
- t(15;17)(q24.1;q21.2) ; PML-RARA
- t(9;11)(p22;q23); MLLT3:: KMT2A
- FLT3
- NPM1

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82
Q

What different classification systems are used in AML?

A

WHO 2022
- Genetics and morphology

FAB
- Morphology based

ICC 2022
- Diagnosis and prognosis

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83
Q

How does the WHO definition of AML differ between WHO 2017 and WHO 2022?

A

WHO 2017= 20% blasts OR presence of AML defining genetic abnormalities (t15:17 and t8:21, inv16), irrespective of blast percentage.

WHO 2022= regards blast cut-offs as largely arbitrary and does not require any blast threshold for the diagnosis of AML with defining genetic abnormalities (with the exception of AML with BCR::ABL fusion and with CEBPA mutation, which requires 20% blasts).

Newly recognized entities included AML with: KMT2A-r/ MECOM-r / NUP98 rearrangement

Abandoned entity: AML with mutated RUNX1 was mainly reclassified as AML-MR

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84
Q

How is AML classified according to the WHO 2022?

A

Acute myeloid leukemias with defined genetic abnormalities

Acute myeloid leukemias defined by differentiation

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85
Q

What are the AML defining genetic abnormalities according to WHO 2022?

A

APL with t(15;17)(q24.1;q21.2)/PML::RARA
AML with t(8;21)(q22;q22.1)/RUNX1::RUNX1T1
AML with inv(16)(p13.1q22)/CBFB::MYH11
AML with t(9;11)(p21.3;q23.3)/MLLT3::KMT2A
AML with t(6;9)(p22.3;q34.1)/DEK::NUP214
AML with t(1;22)(p13.3;q13.1); RBM1::MRTFA
AML with t(9;22)(q34.1;q11.2)/BCR::ABL1
AML with mutated NPM1
AML with mutated CEBPA
AML with KM2TA rearrangements
AML with NUP98 rearrangements
AML with MECOM rearrangements
AML associated with MDS
AML with other defined genetic abnormalities

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86
Q

What is the FAB classification?

A

FAB (French-American-British) . The 1976 FAB classification relied essentially on blast morphology. Divided into 8 subgroups M0-M7

M0: Undifferentiated AML
M1: AML with minimal maturation
M2: AML with maturation
M3: Acute promyelocytic leukemia (APL)
M4: Acute myelomonocytic leukemia
M5: Acute monocytic leukemia
M6: Acute erythroid leukemia
M7: Acute megakaryocytic leukemia

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87
Q

What is the AML International Consensus Classification (ICC) 2022?

A

ICC provides a hierarchical classification of AML, based on genetic determinants with clinical and prognostic relevance.

The ICC stratifies AML into five molecular subgroups:
(i) AML with recurrent genetic abnormalities (both gene rearrangements and gene mutations)
(ii) AML with mutated TP53
(iii) AML with myelodysplasia (MDS)-related gene mutations (a new category with 10%–19% blasts in the bone marrow or peripheral blood, in recognition of the similarities in biology and prognosis between these patients and those with ≥20% blasts)
(iv) AML with MDS-related cytogenetic abnormalities
(v) AML, not otherwise specified (NOS)

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88
Q

What are the differences between ICC and WHO classifications of AML?

A

Recurrent genetic abnormalities
- ICC and WHO have similar recommendations
- t(9:11) MLLT3::KTM2A is separate from other KMT2A mutations in ICC
- Mutated CEPBA vs. Bzip CEBPA- WHO 2022 allows any biallelic mutation (not only in-frame bZIP derangements) as well as monoallelic bZIP mutations for the diagnosis of AML with CEBPA mutation)

Blast cut-off for AML
- Both acknowledge the blurring boundary between MDS and AML
- WHO-regards blasts cut-off as arbitrary; any blast % with recurrent genetic abnormalities
- ICC- at least 10% blast threshold for recurrent genetic abnormalities (to support a differential diagnosis with MDS)
- ICC recognise MDS with 10-19% blasts as being MDS/AML but WHO keep MRS with 10-19% blasts as MDS-IB2

TP53 mutation
- Both acknowledge MDS with bi-mutated TP53 as distinct poor prognosis entity
- Unlike the ICC, the WHO does not recognize TP53 mutations as genetic classifiers and does not consider AML with TP53 mutations as a separate diagnostic category

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89
Q

What is the role of genetics in AML?

A

Classification (WHO, FAB, ICC)
Prognosis (ELN)
Treatment stratification
MRD monitoring
Demonstrate clonality and disease progression

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90
Q

How is AML risk stratified?

A

ELN 2022
- Genetics at initial diagnosis
- Split into favourable, intermediate and adverse

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91
Q

What are the AML ELN 2022 risk groups?

A

Favourable:
- RUNX1:RUNX1T1
- CBFB:MYH11
- NPM1 without FLT3-ITD
- CEBPA

Intermediate:
- NPM1 with FLT3-ITD
- wt NPM1 with FLT3-ITD
- MLLT3::KMT2A

Adverse:
- BCR::ABL1
- DEK::NUP214
- MECOM
- TP53
- KAT6A::CREBBP
- ASXL1, BCOR, EZH2, SF3B1

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92
Q

What are the 2 classes of abnormalities seen in AML?

A

Class I
- Variants that give a proliferative e.g. BCR-ABL1, FLT3, KIT

Class II
- Variants that block haematopoeitic differentiation
- PML-RARA, CBFB-MYH11, RUNX1::RUNX1T1, KMT2A

Both required for AML

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93
Q

What is APL?

A

Acute promyelocytic leukaemia
5-10% of AML diagnoses

Associated with PML-RARA translocation

Morphologically see promyeloblasts and auer rods

APL is treated with combinations including all trans retinoic acid (ATRA) and arsenic trioxide (ATO), with or without additional chemotherapy and has excellent prognosis as compared to other non-APL AML subtypes

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94
Q

How does PML-RARA drive APL?

A

RARA regulates myeloid differentiation

PML forms nueclar bodies which inhibit apoptosis

PML-RARA inhibits apoptosis and differentiation

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95
Q

What is CBF AML and what is its pathogenesis?

A

Core binding factor
- hematopoietic transcription factors characterized by heterodimers of two units (CBFA consisting of RUNX1 etc and CBFB)

RUNX1:RUNX1T1
- RUNX1 regulates differentiation and RUNX1T1 is a transcriptional repressor

CBFB-MYH11
- Tethers the CBF complex including RUNX1 outside of the nucleus, repressing differentiation

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96
Q

How is AML is treated?

A

Induction
- Myeloblative treatment to obtain remission
- 3+7 regime (cytarabine and daunorubicin)
- targeted therapies

Consolidation
- Deepen remission to eliminate any AML cells to prevent relapse

Maintenance
- maintain remission

Cure: stem cell transplant

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97
Q

What targeted therapies are available for AML?

A

ATRA-ATO: PML::RARA- forces differentiation as it is a more potent analogue of RA which forced binding

Venetoclax: BCL2 inhibitor, increased response with NPM1

Midostaurin: FLT3 inhibitor

Ivosidenib/Enasidenib: IDH inhibitor

CBF AML: favourable risk with Gemtuzumab Ozogamicin

AML with MDS changes: CPX-351

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98
Q

How is AML monitored using flow cytometry??

A

Flow cytometry
-LAIP Leukaemia associated immunophenotype- clonal markers identified at diagnosis
- DfN (different-from-normal) approach- aberrant surface antigen expression patterns not present in normal bone marrow are identified at follow-up

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99
Q

How is AML monitored using genetics?

A

RQ-PCR
- if MRD marker identified: PML-RARA, CBFB-MYH11, RUNX1-RUNX1T1, NPM1
- Monitored at diagnosis, post 2 cycles, and then every three months for 24 months

ddPCR and NGS can also be considered according to ELN

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100
Q

What is the prognosis of AML?

A

50-80% achieve complete remission after treament. ~50% who achieve CR develop recurrent AML.

Patients who have not responded (are resistant) to treatment and patients who have relapsed generally have a poor prognosis.

Survival rates for AML are lowest for all cancers (15% survive after 5years). Younger age groups have better prognosis.

<40yrs 60% survive 5yrs+ / >70yrs 5% survive 5yrs+

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101
Q

How is AML treatment determined?

A

AML may be a curable disease in young and older fit patients. It becomes harder to treat with age; fewer patients are cured as age advances and therapeutic complications are increasingly common.

All patients should be treated with therapy adjusted to performance status and AML risk stratification.

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102
Q

What testing strategies are used for a diagnostic AML sample?

A

G banding: Detect large rearrangements to aid classification

FISH: PML-RARA, KMT2A, NUP98, CBF

Fragment analysis: Rapid FLT3-ITD and NPM1 insertion detection

NGS: Detection of prognostic and recurrent AML abnormalities e,g, FLT3-TKD, TP53, ASXL1, CEBPA

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103
Q

Whys is FLT3 and NPM1 rapid testing performed?

A

FLT3
- in frame ITD varying in size
- determines an intermediate prognosis
- can be used for MRD monitoring
- treatment with midostaurin

NPM1
- 4bp insertion- frameshift
- Good prognosis
- WHO classification
- MRD marker
- Good response to venetoclax

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104
Q

What guidelines are used in AML?

A

ELN
WHO
ICC
FAB
ESMO

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105
Q

What is CLL?

A

Chronic lymphocytic leukaemia

Pathological accumulation of small mature lymphocytes

Small lymphocytic lymphoma is the same disease but is found mainly in lymph nodes rather than blood and bone marrow

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106
Q

What is the incidence of CLL?

A

CLL/SLL is the most common leukaemia in the Western world populations with an age-adjusted annual incidence of 4.9 per 100,000

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107
Q

What are the risk factors for CLL?

A

Although the overwhelming majority of CLLs occur sporadically there is clear evidence of an inherited predisposition to this disease in 10-15% of the cases.

First-degree family members of patients with CLL have an increased risk of developing CLL as well as various types of B cell NHL.

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108
Q

How does CLL present?

A

Most CLL patients are asymptomatic (diagnosed incidentally) but some may present with fatigue, increased infections (T cell suppression) autoimmune haemolytic anaemia, infections, splenomegaly, lymphadenopathy or extra-nodal infiltrates.

Ranges from a near-normal life expectancy to rapidly progressing disease and early death

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109
Q

What are the WHO diagnostic criteria of CLL?

A

> 5000 per ul monoclonal B lymphocytes in the blood

Flow: Matutes score- CD5+, CD19+, CD20+ CD23+, CD43+, CD10-

Morphology: small mature lymphocytes with dense nuclei filling almost all the cytoplasm. Smudge cells- artefacts from fragile cells and characteristic

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110
Q

When is genetics needed in the CLL pathway and why?

A

Most CLL patients do not receive treatment, instead watch and wait

When disease progresses and treatment is required, genetics is used to look at prognosis and treatment markers

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111
Q

What abnormalities are used to determine prognosis in CLL?

A

IGHV
TP53
ATM
Trisomy 12
del13q

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112
Q

What is the significance of IGHV in CLL?

A

IGHV-mutated (IGHV-M) – associated with good prognosis as cells are more mature

IGHV-unmutated (IGHV-UM) – associated with poor prognosis as cells more immature when disease started

113
Q

How is IGHV mutation status assessed?

A

NGS Lymphotrack assay
- IGHV VDJ domain sequenced
- if <98% homology to germline then it is considered mutated
- if >98% to germline than unmutated

114
Q

What is the prognostic significance of CLL abnormalities?

A

IGHV unmutated- poor prognosis
Chromosome alterations
- Del(17p) - TP53 - poor, reduced response to chemo-immunotherapy
- Del(11q) - ATM- poor
- Del (13q)- favourable
- Trisomy 12- intermediate

SNVs:
TP53, BIRC3- poor
NOTCH1, SF3B1- intermediate

115
Q

What is the significance of BTK and PLCG2 in CLL?

A

Resistance to ibruitinib treatment

116
Q

What is the international prognostic index for CLL (CLL-IPL 2016)?

A

Prognostic scoring system that uses age, stage, IGHV status, TP53 status

Gives a risk for %5 year survival

117
Q

What is the significance of TP53 in CLL?

A

Includes deletions and SNVs
- detected by FISH and/or NGS

TP53 abnormalities are associated with worse disease outcomes due to resistance to chemoimmunotherapy

Often biallellic loss

118
Q

How is CLL monitored?

A

MRD in CLL is not routinely used in practice but is recommended for trials

RQ-PCR for IGHV can be done
- done at diagnosis, after 6 cycles, end of therapy and then every 3 months

119
Q

When is CLL treated?

A

Watch and wait
- not treated until considered active disease

ESMO define active disease as
- evidence of progressive marrow failure as manifested by the development of, or worsening of, anaemia and/or thrombocytopaenia
- progressive lymphadenopathy
- hepatosplenomegaly and/ or splenomegaly
- constitutional symptoms
- symptomatic or functional extra nodal involvement

120
Q

How is CLL treated?

A

ESMO guidelines (dependant upon IGHV and TP53 status)

Chemo-immunotherapy
- Ibrutiinib (BTK inhibitor)
- venetoclax (BCL2 inhibitor)

121
Q

How many CLL patients relapse?

A

Approximately 6% of patients will relapse within six to 12 months and another 14% will do so within two years.

122
Q

What is hairy cell leukaemia?

A

Hairy cell leukaemia (HCL) is a rare lymphoid leukaemia and gets its name from the chracteristic short, thin projections that look like hair on its cells.

classical (cHCL) and variant (vHCL). CHCL and vHCL differ in therapeutic response and prognosis.

123
Q

What variants are associated with hairy cell leukaemia?

A

BRAFV600E mutations in ∼100% of cHCLs,

∼30% of vHCLs harbour activating mutations in MAP2K

124
Q

How is hairy cell leukemia treated?

A

Treatment with small-molecule inhibitors of active BRAF – eg vemurafenib or dabrafenib

125
Q

What is Monoclonal B-cell lymphocytosis?

A

Monoclonal B lymphocytosis (MBL) is defined as the presence of a clonal B-cell population in the peripheral blood with fewer than 5 × 109/L clonal B-cells and no other signs of a lymphoproliferative disorder. The majority of cases of MBL have the immunophenotype of chronic lymphocytic leukemia (CLL).

126
Q

What is B-cell prolymphocytic leukaemia (B-PLL)?

A

Rare aggressive mature B cell neoplasm that represents less than ~1% of all leukaemia

Characterized by the presence of large lymphoid cells (prolymphocytes) accounting for at least 55% of total circulating cells in the peripheral blood

Prolymphocytes are defined as large cells with clumped chromatin, a large single prominent vesicular nucleolus and abundant cytoplasm

127
Q

What is the prognosis of B-cell prolymphocytic leukaemia (B-PLL)?

A

Aberrations in MYC and TP53 portend worst prognosis

Poor prognosis in general; the degree of treatment response is dictated by anaemia, degree of lymphocytosis and molecular prognostic markers

128
Q

How is B-cell prolymphocytic leukaemia (B-PLL) treated?

A

Chemotherapy
- in the absence of 17p deletion or TP53 mutation, rituximab in association to FC (fludarabine, cyclophosphamide) or bendamustine is the recommended first line therapy
- For TP53 mutated, treatment with alemtuzumab or more recently ibrutinib are used

129
Q

What guidelines are used in CLL?

A

ERIC
ESMO
WHO

130
Q

What is ALL?

A

Disease is caused by genetic lesions in B and T progenitor cells resulting in clonal expansion of immature progenitor B and T cells

131
Q

What is B-ALL?

A

Accumulation of immature blast cells at the expense of blood cells in the bone marrow and also in the peripheral blood as well as in extramedullary sites (lymph nodes, testes, central nervous system (CNS) and spleen).

132
Q

What is T-ALL?

A

Accumulation of progenitor T cells.
Always involves bone marrow and nearly always peripheral blood. T-LBL frequently shows bulky mediastinal (thymic) involvement, although any lymph node or extranodal site may be involved including the skin, tonsils, liver, spleen, CNS, and testes.

133
Q

What is the incidence of B-ALL?

A

The B-ALL estimated annual incidence worldwide is 1-4.75 cases per 100,000 population

85% of ALL in children
75% of ALL in adults

134
Q

What is the incidence of T-ALL?

A

T-ALL accounts for about 15% of childhood ALL cases; it is more common in adolescents than in younger children, and more common in males than in females.

T-ALL accounts for approximately 25% of cases of adult ALL and 15% of children

135
Q

What are the risk factors for ALL?

A

Previous treatments with chemotherapy​
Genetic predisposition​
- Down syndrome (20-fold increased risk)​
- Fanconi Anaemia​
- LFS
Weakened immunity/Infections: ​
- HTLV-1 mainly associated with T-cell ALL​
- HIV
- Treatments after an organ transplant​

136
Q

How does B-ALL present?

A

B-ALL commonly presents with bone marrow failure
- thrombocytopenia (easy bleeding or bruising)
- anaemia (fatigue, dyspnoea)
- neutropenia (infections). The leukocyte count can be variable.

Lymphadenopathy and hepatosplenomegaly, bone pain, fever, weight loss, night sweats

137
Q

How does T-ALL present?

A

T-ALL typically presents with a high leukocyte count, and often a concurrent large mediastinal or other tissue mass- can grow rapidly and present as a respiratory emergency

Pleural and/or pericardial effusions are also reported. 

138
Q

What is the diagnostic criteria of B-ALL?

A

Flow: >20% B-lymphoblasts. CD19, CD20, CD22, CD79a, CD10 (common ALL antigen), TdT

Genetic testing do determine classification

139
Q

What are the diagnostic criteria of T-ALL?

A

Flow: >20% T-lymphoblasts. CD3, CD7, CD2, CD5, CD1a, TdT.

Genetic testing do determine classification
- FISH
- SNP arrays

140
Q

How is B-ALL classified according to the WHO 2022?

A

B-ALL with Recurrent Genetic Abnormalities:
- B-ALL with t(9;22)(q34.1;q11.2); BCR-ABL1
- B-ALL with t(v;11q23.3); KMT2A rearranged
- B-ALL with t(12;21)(p13.2;q22.1); ETV6-RUNX1
- B-ALL with hyperdiploidy
- B-ALL with hypodiploidy
- B-ALL with t(5;14)(q31.1;q32.3); IGH-IL3
- B-ALL with t(1;19)(q23;p13.3); TCF3-PBX1
- B-ALL with iAMP21 (intrachromosomal amplification of chromosome 21)
- B-ALL with BCR-ABL1-like (Ph-like)
- B-ALL with other genetic abnormalities
- B-ALL, Not Otherwise Specified (NOS)

141
Q

How is T-ALL classified according to the WHO 2022?

A

T-ALL with Recurrent Genetic Abnormalities:
- T-ALL, Not Otherwise Specified (NOS)
- T-ALL with recurrent genetic abnormalities (e.g., rearrangements of TLX1, TLX3, and TAL1 genes).

142
Q

What is the significance of aneuploidy in B-ALL?

A

B-ALL with hyperploidy
- >50 chromosomes
- Present in up to 30% childhood B-ALL
- Favourable

B-ALL with hypoploidy
- 24-43 chromosomes
- 1% of childhood and 10% of adults
- Unfavourable
- Associated with TP53 and/or RB1

Can be detected by G-banding, FISH, arrays and flow cytometry

143
Q

What is the significance of BCR-ABL1 in B-ALL?

A

WHO classification

Associated with IKZF1 deletions

2-5% in children and >25% in adults

Unfavorable outcome but some may respond to imatinib. Usually e1a2 which responds worse than CML transcripts

Can be used for MRD monitoring

144
Q

What is the significance of KMT2A rearranged B-ALL?

A

More than 90 different partners including AF4, MLLT3

Accounts for 70-85% in adolescents ALL, 2% in childhood and adult

Unfavorable prognosis

Presents with high WCC and CNS involvement

145
Q

What is the significance of TCF3 rearrangements in B-ALL?

A

Most commonly TCF3::PBX1- intermediate prognosis

TCF3::HLF- poor prognosis

Rare in adult, 5% in children

146
Q

What is the significance of IGH::IL3 rearranged B-ALL?

A

Rare

Associated with IKZF1 deletion, marked eosinophillia

Intermediate prognosis

147
Q

What is the significance of ETV6::RUNX1 B-ALL?

A

Favorable prognosis

25% of childhood cases, <3% of adults

148
Q

What is the significance of iAMP21 B-ALL?

A

Defined by >5 copies of RUNX1 with >3 o more copies of a single abnormal chromosome 21

1-2%

Unfavourable

149
Q

What is the significance of BCR-ABL1-like B-ALL?

A

ABL class fusions e.g. ABL1, ABL2, PDGFRA, PDGFRB
or
JAK/STAT abnormalities

Frequency increases with age 10-15% children, 20-25% adults

Unfavourable

150
Q

What abnormalities are seen in T-ALL?

A

NOTCH1 pathway (60% of cases)

Rearrangements involving T cell receptor (TCR) genes

Kinase signalling pathway (JAK/STAT, RAS/MAPK, PI3K/AKT, ABL1)

Epigenetic deregulation

Ribosomal dysfunction

Altered expression of oncogenes miRNAs or long-noncoding RNA

151
Q

What is Early T-cell precursor (ETP) ALL?

A

Subgroup of T-cell ALL
- Children- intermediate prognosis
Adults- very poor prognosis

152
Q

What is on the test directory for ALL?

A

M91 (lots of entries)

Including
- Karyotype
- Structural variants by NGS, FISH, RT-PCR
- WGS

153
Q

What is the turn around times for ALL?

A

Very urgent diagnostic: BCR::ABL1 by FISH - 3 calendar days

Urgent upfront treatment determining: FISH and karyotype - 7 calendar days; SNP array 14 days

Urgent monitoring: ALL concerns re relapse – FISH and karyotype - 7 calendar days

Routine monitoring: FISH and ddPCR – 14 calendar days

Non urgent for prognostication/stratification where not required pre-treatment: karyotype or SNP array – 21 calendar days

154
Q

How are copy number changes assessed in ALL?

A

Most commonly by SNP arrays

Also use G banding and FISH

G banding can be difficult due to rapid apoptosis- often need dir dir cultures which do not have many metaphases

155
Q

What is the ALLTogether trial?

A

The ALLTogether trial is a significant international clinical study focused on treating ALL in patients below 30. It puts patients into genetic risk groups which determines which treatment patients get

The Primary Objective of ALLTogether is to:​
- improve survival and quality of survival with ALL by a number of interventions​
- de-intensification of therapy ​
- experimental intensification​

156
Q

What screening methods are recommended at diagnosis according to the ALLTogether?

A

Array, NGS
- Hyper and hypodiploidy-
- iAMP21
- CNVs

FISH, G-banding, NGS, RT-PCR
- Fusions

157
Q

What ALL risk groups are there according to the ALLTogether?

A

Good risk
- ETV6-RUNX1
- Hyperdiploidy

Intermediate
- TCF3-PBX1
- B-other

Poor risk
- KMT2A rearranged
- BCR-ABL1
- iAMP21
- Hypodiploidy
- TCF3-HLF

158
Q

What is the UK CNA A2G classifier?

A

Alltogether trial (for patients <30 years) to classify into risk groups
– Looks at deletons in 8 key genes: ETV6, PAX5, BTG1, CDKN2A/CDKN2B, PAR1, IKZF1,RB1 and EBF1
- CNA-Good risk with no evidence high risk genetics will be used along with MRD to classify patients into the IR-Low (intermediate risk low) group and will receive different treatment to high risk patients
- The classifer does not apply to older adults or T-ALL

159
Q

Why do genetic/CNA risk groups matter for MRD monitoring in ALL?

A

Patients with good risk genetics can tolerate higher MRD levels during treatment and still have good outcomes compared to patients with poor risk genetics and the same MRD level.

Treatment is stratified according to various risk categories including genetic risk and MRD levels.

160
Q

Why monitor ALL?

A

ALL treatment has significant toxicity, therefore it is desirable to administer the least intensive treatment that will still cure disease.

MRD monitoring is crucial for treatment stratification into different intensities (treatment arms).

The relationship between genetic risk groups and MRD levels means that patients can be stratified into the appropriate treatment arm

161
Q

What biomarkers are monitored in ALL according to UKALL guidelines?

A

Fusions
- BCR-ABL1
- ETV6-RUNX1
- KMT2A

Monitoring of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements.
- Ig/TCR MRD uses surrogate markers i.e. the rearrangements are not disease causative, they just happen to be present in ALL cells.

162
Q

What are Ig genes?

A

Immunoglobulins are produced by B-cells and may be membrane bound as part of the B-cell receptor complex, or secreted as antibodies by plasma cells.

Formed from 2 heavy chains (encoded by the IgH genes) and 2 light chains (encoded by IgK or IgL genes)

Immunoglobulins are a component of the acquired immune system and have a constant region (formed by C gene segments) joined to a hypervariable antigen binding domain formed by joining of the V (variable), D (diversity) and J (joining) gene segments (heavy chain) or V-J joining (light chain).

Somatic recombination of these regions to produce variable region. D-J joining in IgH always occurs before V segment joining.

In ALL cells are immature and do not express functional immunoglobulins, therefore partial (incomplete) D-J rearrangements frequently occur

163
Q

What are TCR genes?

A

T-cells can be either have an αβ TCR (~90-95% of blood T-cells) or a γδ TCR (5-10% of blood T-cells, more abundant in epithelia).

Generation of TCR diversity occurs in the thymus and T-cells are more abundant in blood (10-25% of nucleated cells) than B-cells (3-10% nucleated cells).

TCRα is not generally used in MRD as very complex locus

164
Q

How can Ig/TCR rearrangments be used to monitor MRD in ALL?

A

Healthy individuals will be polyclonal for Ig/TCR gene rearrangements- millions of different rearrangements with no single one predominating

Clonal expansions of cells carrying particular Ig/TCR rearrangements can occur in response to infection or as a consequence of ALL leukaemogenesis (as ALL is a clonal expansion of immature lymphocytes).

We can perform a screening PCR across the V(D)J region which will pick up dominant clones in Ig and TCR genes. This clone will therefore be associated with the leukaemia cells and can be used as a biomarker and monitored using RQ-PCR

As B-ALL and T-ALL cells are immature, can see cross lineage rearrangements (e.g. IGH D-J rearrangements in T-ALL, TCRG, TCRD and TCRB rearrangements in B-ALL)

165
Q

How are ALL patients treated?

A

Treatment typically takes place in 3 phases: Induction, Consolidation and Maintenance. and is often dependent upon risk group

Induction (remission induction) – 1 month – hyper-CVAD
- Cyclophosphamide
- Vincristine
- Anthracycline drug such as doxorubicin (Adriamycin) or daunorubicin
- Dexamethasone or prednisone
- If BCR-ABL1 +- Imatinib

Consolidation (intensification) – a few months
- If the leukaemia goes into remission, the next phase often consists of another fairly short course of chemo, using many of the same drugs that were used for induction therapy. Stem cell transplant can be suggested to patients with poor prognostic factors.

Maintenance – two years
- After consolidation, the patient is generally put on a maintenance chemotherapy program of low dose of methotrexate and 6-mercaptopurine (6-MP). In some cases, this may be combined with other drugs such as vincristine and prednisone.

166
Q

What is the significance of targeted therapies in ALL?

A

Targeted therapies for T-ALL
- Venetoclax (anti Bcl2)​
- Navitoclax (Bcl-xL)​

Targeted therapy for B-ALL
- Imatinib for BCR-ABL1 +
- Immunotherapies: Rituximab (anti CD20)​, Ofatumumab (anti-CD20)​, Blinatumomab (CAR-T)​

167
Q

When are stem cell transplants considered in ALL?

A

Stem cell transplantation (autologous or allogeneic) is usually reserved for ALL in first remission that has a higher risk of relapse (e.g. BCR-ABL1, KMT2A), and for patients who have already relapsed and achieved a second remission.

In preparation for stem cell transplantation, patients with ALL are usually given very high doses of chemotherapy or total body irradiation (TBI), called myeloablative conditioning.

Reduced-intensity conditioning and nonmyeloablative conditioning are less intensive treatments that can prepare the bone marrow for transplantation and are being studied in older patients and others who are not candidates for myeloablative treatment.

168
Q

Why do children have a better prognosis in ALL than adults?

A

There is increasing evidence that adult ALL cells are more resistant to chemotherapy and have a higher MRD after therapy compared to children.

Older ALL patients can have lower rates of complete remission and more treatment-related toxicities due to decreased drug tolerance (such as asparaginase), resistance to treatment agents (corticosteroids, L-asparaginase, cytarabine, daunorubicin, vincristine) and alterations in drug metabolism.

The Cancer and Leukaemia Group B (CALGB) study demonstrated that the administration of L-asparaginase is well tolerated by adult patients and that patients with T-cell ALL had a better prognosis compared to those with pre-B-cell AL

In adults, MRD evaluation has only recently been incorporated into treatment algorithm. Using the MRD response can accurately select patients for HSCT, thus sparing adult patients with negative MRD from transplant-related toxicities

169
Q

What guidelines are used in ALL?

A

WHO
NCCN
ESMO
ALLTogether trial

170
Q

What are lymphomas and how do they present?

A

Tumours of the lymphoid tissue which may spread and infiltrate the bone marrow

Can affect B and T cells

Presentation:
- Enlarged lymph nodes, masses, splenomegaly, anaemia, thrombocytopenia, lethargy
- B symptoms include fever, weight loss, night sweats

171
Q

What determines a B cell from lymphoid precursors?

A

Early B cell factor 1 (EBF1), E2A, and PAX5 are the three main transcription factors for early B cell development.

The production of a functional and unique BCR through V(D)J recombination is the central process for the generation of a mature B cell.

172
Q

How are lymphomas classified?

A

Classified using clincial features, morphology, immunophenotype and genetics

First split into
- Hodgkins lymphoma: characterised by the presence of reed-sternberg cells which are large abnormal lymphocytes in a background of reactive cells
- Non-hodgkins lymphoma: split into B cell, T cell and NK cell lymphomas

Classified using WHO 2022 (updated from 2016) and ICC 2022 guidelines which are based on the REAL 1994 guidelines

173
Q

What molecular abnormality is a hallmark of B cell lymphomas?

A

Chromosomal translocations involving one of the Ig loci and a proto-oncogene. These translocations happen as by-products during the processes of V(D) J recombination, SHM, and class switching.
The DNA strand breaks occurring in each of these processes bear an inherent risk of generating translocations.
The translocation of a proto-oncogene into an Ig locus in B cell lymphomas deregulates the oncogene expression through the associated Ig enhancers, which are highly active in the B cells.

B-cell lymphomas depend on the expression of a B-cell receptor (BCR) for survival- BCR signalling important

174
Q

How are B cell lymphomas classified according to WHO 2022?

A

Mature (small) B cell neoplasms
- CLL/SLL
- Hairy Cell Leukaemia
- Marginal Zone Lymphoma
- Follicular Lymphoma (including pediatric)
- Mantle Cell Lymphoma
- Lymphoplasmacytic Lymphoma

Large B cell neoplasms (morphologically appear DLBCL, BL or HGBL)
- Diffuse Large B cell Lymphoma NOS
- High Grade B cell Lymphoma NOS
- High grade B cell lymphoma-11q
- Diffuse large B-cell lymphoma/ high grade B-cell lymphoma
with MYC and BCL2 rearrangements
- Large B-cell lymphoma with IRF4 rearrangement
- Burkitt Lymphoma

175
Q

What is follicular lymphoma?

A

Low-grade, indolent disease (chronic) with good prognosis
2nd most common NHL (20-30% of cases)

Morphology: Predominantly small centrocytes with few centroblasts.
Immunophenotype: CD10+, BCL6+, BCL2+.
Molecular Abnormalities: t(14;18)(q32;q21) IGH::BCL2

176
Q

What is paediatric follicular lymphoma?

A

Median age 15-18 and more common in males (10:1). Excellent prognosis and a conservative watch‐and‐wait approach is recommended.

Morphology: Follicular growth, has a blastoid morphology with highly proliferative follicle centres

Does not have BCL2, BCL6, IRF4 or Ig rearrangements. Does have MAP2K variants

177
Q

What are the types of paediatric follicular lymphoma?

A

GI Follicular lymphoma
- Duodenal-type FL
- Multiple polyps
- Good prognosis

Testicular Follicular lymphoma
- Increased frequency in children
- Lack BCL2 translocation
- Good prognosis
- Can be treated with surgery alone

Diffuse-Appearing Follicular Lymphoma
- Often large localised inguinal masses
- Largely diffuse pattern
- Low grade morphology
- Lack BCL2 rearrangement
- Have 1p36 deletion (not specific, can be seen in conventional FL)

178
Q

What is mantle cell lymphoma?

A

Low grade but aggressive, often has bone marrow involvement
3-10% of NHL cases
Prognosis is poor (2-5 years)

Morphology: Small to medium-sized lymphoid cells with monomorphic appearance.
Immunophenotype: CD5+, Cyclin D1+, CD19+, CD20+.
IHC: overexpression of Cyclin D1
Molecular Abnormalities: t(11;14)(q13;q32) translocation involving IGH::CCND1.

179
Q

How is the prognosis of mantle cell lymphoma determined?

A

Mantle Cell Lymphoma International Prognostic Index
- morphology (blastoid/pleomorphic variant), immunophenotype, proliferative index (labeling of Ki67) and presence of TP53 deletions/mutations since the occurrence of one or more of these variables associated with a poor prognosis
- TP53 mutations associated with resistance to chemotherapy

180
Q

What targeted therapies are used in mantle cell lymphoma??

A

BTK inhibitors
Venetoclax
CD-19 directed CAR-T

181
Q

What is marginal zone lymphoma?

A

Subtypes: Extranodal (MALT)- most common, Nodal, and Splenic.
MALT is associated with chronic infections

Morphology: Small B cells with marginal zone growth pattern.
Immunophenotype: CD20+, CD79a+; usually CD5-, CD10-.
Molecular Abnormalities: t(11;18)(q21;q21) BITC3::MALT1 associated with extra nodal. No recurrent assisted with nodal or splenic

182
Q

What is Lymphoplasmacytic lymphoma (LPL)?

A

Small B cell lymphoma, also called Waldenström macroglobulinemia (WM) when monoclonal IgM is present
1-2% of NHL

Morphology: Bone marrow involvement by small lymphocytes, plasmacytoid lymphocytes, and plasma cells.
Infiltration can be interstitial, nodular, or diffuse.
Immunophenotype: CD19+, CD20+, CD38+, CD138+.
CD5-, CD10-, CD23-, and Cyclin D1-, which helps differentiate LPL from other small B-cell lymphomas.
Molecular: MYD88 L265P mutation is present in over 90% of cases. Also has some IgH rearrangements

183
Q

What is the incidence of NHL?

A

Non-Hodgkin lymphomas (NHL) are the seventh most common type of cancer in the US, accounting for 4.3% of all newly diagnosed cancers.

In 2021, the incidence rate of NHL in the US was estimated at 19.6 cases per 100,000 per year, 4.68 of which were DLBCL

Incidence of different subtypes vary

Usually affects those in 7th or 8th decade

184
Q

What risk factors are there for DLBCL?

A

Age
Transformation from indolent condition e.g. CLL or follicular lymphoma
Immundeficiency e.g, HIV or immunosuppressant treatment
Chemicals such as pesticides

185
Q

What is diffuse large B cell lymphoma

A

Highly agrressive and most common NHL (30-50%)

Usually occurs in adults

Can be further classified into WHO classifications e.g. DLBCL NOS, DLBCL/HGBL with MYC/BCL2

186
Q

What can diffuse large B cell develop from?

A

Can be de novo or develop from another entity e.g. CLL (richter transformation) or Follicular lymphoma

CLL poor prognosis, candidate for stem cell transplant

Follicular better prognosis

187
Q

How does diffuse large B cell lymphoma present?

A

Presents with a rapidly enlarging tumour mass at a single or multiple nodal/extranodal sites. Some common sites for extranodal presentation include gastro-intestinal tract, bones, testes, spleen.

CNS DLBCL: cognitive dysfunction and psychomotor slowing, headaches, and blurred vision/floaters

Primary mediastinal (thymic) DLBCL (PMBL): mediastinal mass that may affect respiratory or cardiac systems,

Primary cutaneous DLBCL – leg type (PCLBCL): red rapidly growing tumours on one or both of the lower legs

188
Q

What is the significance of the cell of origin (COO) in diffuse large B cell lymphoma?

A

DLBCL can be classified by the cell of origin (when in B cell development did the lymphoma arise):

Germinal Center B-cell-like (GCB) DLBCL
- Cells were in a stage of active proliferation and somatic hypermutation.
- Improved prognosis and respond better to R-CHOP regime
- Often involves BCL2 rearrangements
Activated B-cell-like (ABC) DLBCL
- Cells were in are in a stage of differentiation towards plasma cells.
- Poor prognosis and resistance to R-CHOP
- Assocviated with activation of the NG-kB pathway which restricts apoptosis
- Looking at targeted therapies

189
Q

How can GC and ABC DLBCL be distinguished between?

A

The Hans algorithm uses 3 markers to distinguish GC from non-GC subtypes: CD10, BCL6 & IRF4/MUM1.

EzH2 SNVs or CNVs also used (included in test directory)- GC type

190
Q

What genes are commonly rearranged in diffuse large B cell lymphomas?

A

BCL2
BCL6
MYC
(double or triple hit- not longer classified like this in WHO 2022)

191
Q

How do BCL2 rearrangements drive DLBCL?

A

Anti-apoptotic oncogene.

Translocation t(14;18)(q32;q21), which juxtaposes the BCL2 gene with the immunoglobulin heavy chain (IGHV) gene enhancer (14q32), resulting in deregulated expression of BCL2.

This translocation is more frequent in GC DLBCL (30 – 40%) than in ABC DLBCL, although it is more commonly duplicated or amplified in the latter.

192
Q

How do BCL6 rearrangements drive DLBCL?

A

BCL6 is a transcriptional repressor in cell cycle control, proliferation and differentiation, apoptosis, and DNA damage response.

IGH::BCL6 t(3;14)(q27;q32)

BCL6 translocation, leading to a loss of gene regulation, is found in up to 35% of DLBCL cases, and is believed to contribute to malignant transformation in germinal center-derived B cells (GCB).

193
Q

How do MYC rearrangements drive DLBCL?

A

C-MYC acts as a transcriptional regulator involved largely with cell cycle progression (from G1 to S phase) and the inhibition of terminal differentiation.

Rearrangement of the C-MYC with IgH gene, or lambda (λ) and kappa (κ) light chain genes subsequently caused upregulation of gene expression, leading to resistance to apopotosis triggers.

Such translocations are observed in 7-15% of de novo DLBCL and 8% of post-transformation DLBCL. Note that other translocations partners can be involved.

194
Q

How are DLBCL diagnosed?

A

Biopsy

Morphology: partial architecture effacement, with a diffuse proliferation of medium or large lymphoid cells.

Immunophenotype: high Ki-67 proliferation index and expression of pan-B cells markers (CD19, CD20, CD79a & PAX5)

Molecular: MYC, BCL2 and BCL6 FISH can also be used to aid diagnosis

195
Q

How is the prognosis of DLBCL determined?

A

Prognosis is determined using IPI. This usually includes 5 factors:
- Age: ≤ or > 60 years
- LDH: ≤ or > upper limit of normal range
- Performance Status (ECOG) 0/1 or > 1
- Ann Arbor Stage: I/II (one or more sites, but same side of diaphragm) or III/IV (sites on both sides of the diaphragm and/or involvement of extra-lymphatic sites such as liver or bone marrow)
- Number of extranodal sites 0/1 or > 1

This scoring system puts in risk groups and gives a 5 year survival

COO classification and presence of double/triple hit also influence prognosis

196
Q

How is DLBCL monitored?

A

Currently, monitoring is performed by imaging (CT/PET scan) after end of treatment. As risk of relapse is higher within the first 2 years, patients are monitored every 6 to 12 months during the first 5 years following end of treatment.

ctDNA being monitored as part of clinical trials

197
Q

How is DLBCL treated?

A

R-CHOP
- rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone.

This regimen is generally given every three weeks for six cycles. This may be complemented by radiotherapy.

198
Q

How common is relapse for patients with DLBCL?

A

Relapse is relatively common, and usually occurs between 1 and 3 years after end-of treatment (~30%).

199
Q

How are DLBCL patients treated at relapse?

A

Second line therapy include immunochemotherapy, usually in combination with rituximab (anti-CD20) and bendmustine (alkylating agent).

CAR-T (trials)

200
Q

What is burkitt lymphoma?

A

High grade, highly aggressive disease

Rare in developed countries; more common in endemic areas (e.g., Africa).

Associatedwith Epstein Barr virus (EBV) and HIV

Previously subtyped as endemic, immunodeficiency-related, and sporadic but now classified as EBV+ and EBV- by WHO 2022

201
Q

How do patients with Burkitt lymphoma present?

A

Patients often present with a rapidly growing mass, elevated lactate dehydrogenase (LDH), and increased uric acid levels because of the tumour’s rapid doubling time

The primary site of sporadicBL is typically the abdomen

202
Q

What is the diagnostic criteria of Burkitt lymphoma?

A

Morphology: increased apoptosis resulting in starry sky appearance due to macrophagescontainingcellular debris, monotomous cells with round nuclei
Immunophenotype: CD10+, BCL6+, MYC+.
Molecular: MYC translocations - most commonly t(8;14)(q24;q32) MYC::IGH. IGL and IGK are less common partners

203
Q

What prognosis of burkitt lymphoma?

A

The prognosis for adults is usually poorerthanchildren, especially if they have bone marrow involvement (adults approx50%, children as high as 90% survival).

204
Q

Why is molecular testing of high grade lymphomas required?

A

BL and DLBCL can be indistinguishable and have different prognoses and different treatment regimes

The WHO 2022 also now classifies these lymphomas based on molecular abnormalities

205
Q

How are double and triple hit lymphomas classified according to the WHO 2022?

A

BCL6 is very variable and is not required for classification in WHO 2022. New category is DLBCL/High grade B lymphoma

Triple hit
- MYC, BCL2, BCL6
- Can be just MYC and BCL2 (+/- BCL6)
- DLBCL/High grade B lymphoma

Double Hit
- MYC and BCL2 (DLBCL/High grade B lymphoma)
- MYC and BCL6 (High grade B cell lymphoma NOS)
- BCL2 and BCL6 (Diffuse large B cell lymphoma, NOS)

206
Q

What genetic testing is carried out for high grade lymphomas?

A

FISH
- MYC, BCL2, BCL6 rearrangements
- on FFPE and/or bone marrow or if involved

If specifically Burkitt’s then IGH::MYC

Can also use IHC- less specific and does not confirm fusion partner

207
Q

What is high-grade B-cell lymphoma with 11q aberration (HGBL-11q)?

A

Previously known as Burkitt-like lymphoma with 11q

Morphology is similar to burkitt lymphoma but without MYC rearrangement

Characteristic chromosome 11q gain or loss

Immunophenotype: CD10+, BCL6+, BCL2-

208
Q

What targets are on the test directory for DLBCL?

A

Clonality
- Ig rearrangements to help diagnose

IGH, MYC, BCL2 and BCL6 rearrangments
- FISH, help classify

EZH2
- NGS for SNVs and CNVs
- help determine COO

BTK, PLAG2
- NGS for SNVs
- Resistance to BTK treatment

MYD88
- Subtyping and poor prognosis

209
Q

How are T and NK cell lymphoma classified?

A

There are 36 recognised distinct T-cell and NK-cell subtypes. Within the WHO22 Edition, these subtypes are grouped together on the basis of site of presentation, the T-cell of origin or shared morphologic and immunophenotypical features.

Divided into peripheral T-cell lymphomas
- Peripheral T-Cell Lymphoma, Not Otherwise Specified (PTCL-NOS)
- Anaplastic Large Cell Lymphoma (ALCL)

Cutaneous T-cell lymphomas
- Mycosis fungoides and Sezary Syndrome

10-15% of NHLs

210
Q

How do cutaneous t cell lymphoma present?

A

Enlarged lymph nodes, weight loss, night sweats, fever, itchy skin

Patches of dry skin and red rash, mushroom-like tumours

211
Q

How are T cell lymphomas diagnosed?

A

Histology and IHC (pan T-cell marker CD3, CD5; and lineage specific – CD4, CD8, CD2, CD7)

Clonality
- TCR loci rearrangements using PCR or NGS to confirm clonal population
- clonal T cells can be reactive so is not definitive

212
Q

What is Anaplastic large cell lymphomas (ALCL)?

A

Most common T-cell lymphoma in children

Morphology
- Large cells with abundant cytoplasm and horseshoe shaped nuclei, CD30+

Consists of ALK positive
- 10-15%
- Most commonly ALK::NPM1 in 80% of cases
- treatment with ALK TKIs

And ALK negative
- Rarer and seen in adults
- DUSP22 rearrangements in 20-30%- better prognosis
- TP63 rearrangements- chemorefractory

213
Q

What abnormalities are associated with follicular T cell lymphoma and peripheral T cell lymphoma?

A

Epigenetic regulators (TET2, DNMT3A, IDH2) TCR signalling and activation and PIK3/protein kinase B pathways

214
Q

What abnormalities are seen in T-large granulocytic lymphocyte lymphoma (T-LGLL)?

A

STAT3 and STAT5B

215
Q

What abnormalities are seen in T-cell prolymphocytic leukaemia (T-PLL)?

A

Inversions and translocations involving the TCL1 family of genes

Abnormalities in chromosome 8 and ATM is frequently disrupted.

Up to 75% of patients harbour mutations in STAT5B, JAK1, JAK3. Complex karyotype (70%) poor prognosis.

216
Q

How are T cell lymphomas monitored?

A

Once clonality has been associated with the lymphoma, T-cell clonality – TCR gene rearrangement can be used to monitor and evaluate recurrence; detecting and assessing any residual disease.

217
Q

How are Peripheral T-cell lymphomas treated?

A

Combination chemotherapy as initial treatment, CHOP, CHOEP, EPOCH

Autologous stem cell transplants can also be considered as consolidation therapy.

218
Q

How are cutaneous T-cell lymphomas treated?

A

Topical chemotherapy for cutaneous lymphomas + systemic if high grade

  • Small molecule inhibitors – JAK/STAT, SYK and P13K inhibitors
  • TKIs – crizotinib
  • Immunomodulatory – Lenalidomide
  • CAR-T
  • Monoclonal antibodies - Alemtuzmab
219
Q

What is the prognosis of T cell lymphomas?

A

Most PTCL subtypes are associated with poor outcomes with conventional chemotherapy and consideration for clinical trial and HSCT should be given.

Good outcomes for paediatric cases with ALCL

220
Q

What is multiple myeloma?

A

Multiple myeloma (MM) is a malignancy of terminally differentiated (post GC B cell) plasma cells (PC) and is the second most common haematological malignancy after non-Hodgkin lymphoma

Characterised by the secretion of a monoclonal immunoglobulin protein (also known as M protein or monoclonal protein), which is produced by the abnormal PCs.

The malignant PCs are primarily resident in the bone marrow (BM), but they can also be seen in the peripheral blood and other extramedullary sites, such as soft tissue and organs

221
Q

How does MM present?

A

Monoclonal Ig protein in the serum or urine

CRAB
Hypercalcaemia
Renal insufficiency,
Anaemia, and/or
Bone disease with lytic lesions

222
Q

What is monoclonal gammopathy of undetermined significance (MGUS)?

A

Characterised by the infiltration of clonal PCs into the bone marrow (<10% clonal BM PCs) and the secretion of monoclonal protein (M-protein <30g/L).

MGUS is completely asymptomatic and precedes the development of multiple myeloma.

20% of MGUS will develop in MM

223
Q

What is smouldering myeloma?

A

Intervening stage between MGUS and MM

224
Q

What is plasma cell leukaemia?

A

Rare and aggressive form of leukemia and plasma cell dyscrasia- can develop from MM

PCL can be divided into primary PCL (PCL) and secondary PCL (sPCL) following previously diagnosed multiple myeloma (MM); the latter typically occurring at a late and advanced stage of MM.

PCL is defined by at least 20% circulating plasma cells and a total plasma cell count in peripheral blood of at least 2 × 109/L.

225
Q

How is MM diagnosed?

A

PET/CT

Blood and urine test
- M protein (diagnostic marker in 97%). With evolving disease and de-differentiation of myeloma cells, M protein may decrease
- Detect Ig heavy and light chains by serum protein electrophoresis

Bone marrow
- Morphology >10%
- Flow CD138+, CD38-, CD45-, light chain restirction

226
Q

Why is genetic testing in MM needed?

A

MM is highly heterogeneous with some patients progressing rapidly, while others surviving more than 10 years.

This clinical diversity is mainly driven by genetic changes. These alterations are important prognostic factors and can be divided into primary, disease-initiating abnormalities and secondary events, related to further progression of the disease.

227
Q

What are primary and secondary events in MM?

A

Primary abnormalities occur early when the normal plasma cell transitions to a clonal, premalignant stage

Most secondary abnormalities occur later in the disease course with malignant transformation or during progression of the malignancy.

228
Q

What primary abnormalities are seen in MM?

A

Trisomies of odd number chromosomes (not 1, 13, 21)/ hyperdiploidy- 42%

IgH translocation (30%)
- CCND1
- FGFR3
- MAF
- CCND3

IGH and trisomy

Monosomy 14

Subgroups do not overlap- all mutually exclusive

229
Q

What secondary abnormalities are seen in MM?

A

Monosomy 13/del(13q)
Del 17p/ Monosomy 17
del 1p (CDKN2C)
Gain of 1q

230
Q

What test directory targets are there for MM?

A

Multi-target NGS panel - small variant (KRAS, NRAS, BRAF, TP53, DIS3, FAM46C, IRF4)

IGH-FGFR3, IDH-CCND3, IGH-CCND1, IGH-MAF, IGH-MAFB, IGH rearrangement FISH FISH

Hyperdiploidy copy number FISH

del(1p), gain(1q), del(17p) TP53 copy number FISH

231
Q

What must be done with bone marrow before carrying out FISH for MM?

A

MACS for CD18+ positive cells
- sort cells to only have plasma cells as there can be as little as 2% in bone marrow due to patchy disease

232
Q

What is the R-ISS?

A

Prognostic scoring for MM

Integrates the presence of high-​risk cytogenetic abnormalities [t(4;14), t(14;16), and del17p determined by interphase FISH and serum lactate dehydrogenase (LDH) levels i

233
Q

What adverse cytogenetic prognostic factors are seen in MM?

A

Adverse IGH translocations: t(4;14) + t(14;16) + t(14;20) (~15% of patients)

1q gain (usually assessed by the number of copies of the CKS1B gene at 1q21)

TP53 loss (17p13 deletion). The presence of a biallelic inactivation (i.e. by an additional mutation) of TP53 may particularly shorten overall survival, but at present TP53 mutation rarely requested in routine testing

1p32.3 (CDKN2C) loss, but the association of CDKN2C loss and poor outcome is confined to patients in receipt of intensive treatment/bone marrow transplantation

234
Q

What technologies are used to detect abnormalities in MM?

A

FISH
- most common and recommended by R-ISS
- Limited to specific targets

SNP arrays
- Select LOH and multiple abnormalities
- requires 15-20% more plasma cells
- Can’t detect balanced translocations

RQ-PCR
- identify IGH translocations

NGS
- Being developed to detect structural, CNVs and SNVs

235
Q

How is MM monitored?

A

Absence of serum and urine M-protein, absence of any soft tissue plasmacytomas and <5% PC in the BM

MRD by flow cytometry to detect malignant PC and discriminate aberrantly expressed cell surface markers

Imaging with PET/CT

RQ-PCR of IGH rearrangements but is not widely available

236
Q

How is MM treated?

A

VRD regime
Patients initially receive a combination of a proteasome inhibitor (usually bortezomib) and an immunomodulatory agent (commonly lenalidomide) along with dexamethasone.

Autologous and allogenic stem cell transplant ASCT is still the most common standard of care

237
Q

How often to MM patients relapse and how are they managed?

A

Almost all patients who initially respond to treatment will eventually relapse.

Triplet therapy, Bi-specific and CAR-T cell are common types of regimes used for relapsed patients.

238
Q

What guidelines are used in MM?

A

NICE guidelines
International Myeloma Working group
Best practise BMJ

239
Q

What are bone marrow failure syndromes?

A

Bone marrow failure and related syndromes are rare disorders characterized by ineffective bone marrow hematopoiesis and peripheral cytopenias

Can be acquired or inherited

Can lead to progression to MDS/AML

Aplastic anaemia is the most common form

240
Q

What is aplastic anaemia?

A

Aplastic anaemia is a rare and heterogeneous disorder. It is defined as pancytopenia with a hypocellular bone marrow (BM) in the absence of an abnormal infiltrate or marrow fibrosis.

241
Q

What is the incidence of aplastic anaemia?

A

The incidence is 2–3 per million per year in Europe, but higher in East Asia.

242
Q

What are the risk factors for aplastic anaemia?

A

Radiation and chemotherapy
Exposure to toxic chemicals such as pesticides
Drugs to treat rheumatoid arthritis and some antibiotics
Autoimmune disorders
Viral: hepatitis, EBV and HIV.
Pregnancy (rarely)

243
Q

How does aplastic anaemia present?

A

Anaemia and thrombocytopenia. Serious infection occasionaly

Peaks at 10-25 years and again at 60

244
Q

How is acquired aplastic anaemia diagnosed?

A

Diagnosis of exclusion so requires testing for testing for other etiology

  1. Exclude other causes of pancytopenia and hypocellular bone marrow e.g. MDS, lymphoma, B12/folate deficiency
  2. Exclude inherited failure syndrome
  3. Screen for underlying cause
  4. Document coexisting abnormal cytogenetic cones

Take BM aspirate and blood
- full blood count (PB haemoglobin concentration (Hb) <100 g/L,
and/or
PB platelet count <50 × 109/L
and/or
PB neutrophil count <1.5 × 109/L.
- reticulocyte count (young ref blood cells)
- hypocellular bone marrow
- Rule out viral infections and autoimmune diseases

245
Q

How can the severity of aplastic anaemia be assessed?

A

Camitta criteria

Severe AA
- Marrow cellularity <25%
- PB neutrophil count <0.5 x109/L
- BP platelet count <20 x109/L

Very severe AA
- <0.2 x 109/L

Non-severe AA
- Not fulfilling above criteria

246
Q

What is the pathology of aqcuired aplastic anaemia?

A

Dysregulated immune system (viral infection is the major driver of aAA) leading to autoreactive T cell destruction of hematopoietic stem and progenitor cells (HSPC)

An antigen is presented to a cytotoxic T cell (CD8+-CTL) via a Class 1 HLA molecule, and autoreactive T cells are activated then begin to expand.

T cells release cytokines including TNF-α and INF-γ, which have direct apoptotic effects on the HPSC, ultimately resulting in bone marrow failure

247
Q

What cytogenetic abnormalities are associated with aplastic anaemia?

A

Cytogenetic aberrations have been described in 5-15% of adult patients with severe aplastic anemia (SAA), and these patients were generally younger than patients with normal karyotype:

LOH6p
Trisomy 8
7q deletion
Monosomy 7
Trisomy 6
13q deletion

Increases risk of leukaemic transformation

248
Q

What sequence variants are detected in aplastic anaemia?

A

BCOR
BCORL1
DNMT3A
PIGA
ASXL1

ASXL1, DNMT3A and RUNX1 are associated with cloncal evolution and increased risk of transformation to MDS/AML and show poorer response to immunosuppressive therapy

249
Q

How are patients with aplastic anaemia treated?

A

Blood transfusions

Many patients do not undergo treatment but if become dependent on transfusions or have other symptoms:
- Immunosuppressive therapy (ATG with CSA)- suppresses T cells
- Eltrombopag (TPO receptor agonist) - stimulates more platelets
- Haematopoietic stem cell transplantation (HSCT)

250
Q

What is the prognosis of AA?

A

Five-year survival is >75% for patients who undergo bone marrow transplant from a suitable donor

Approximately 10% of patients with AA will later develop MDS or AML, and as many as 25% will develop PNH

251
Q

What testing is carried out for aplastic anaemia?

A

BSH guidelines
SNP arrays for copy number testing

  • important to help distinguish between AA and MDS- doesn’t exclude
252
Q

What is PNH- Paroxysmal nocturnal hemoglobinuria?

A

Bone marrow failure syndrome clinically characterized by acquired hemolytic anemia and thrombosis.

Cells have mutation in PIGA gene.

PIGA-mutated cells have defective cell surface expression of GPI)- anchored proteins including CD55 and CD59, making them vulnerable to complement mediated hemolysis.

253
Q

What is Pure Red Cell Aplasia (PRCA)?

A

Pure red cell aplasia (PRCA) is a rare disorder that designates anaemia secondary to failure of erythropoiesis.

PRCA is characterized by an isolated normocytic anaemia with severe reticulocytopenia in the peripheral blood, and defined by absence or near absence of erythroid precursors in the bone marrow (erythroid hypoplasia).

254
Q

What is the presentation of Pure Red Cell Aplasia (PRCA)?

A

There are no specific signs or symptoms associated with PRCA

The most common presentation is the same as that of anaemia.

Generalized fatigue, decreased exercise tolerance, palpitations, and in extreme cases, presyncope or syncope

255
Q

What is the congenital form of Pure Red Cell Aplasia (PRCA)?

A

Diamond - Blackfan anaemia

256
Q

What is primary and secondary PRCA?

A

Primary PRCA may be an autoimmune disorder resulting from an IgG inhibitor of erythropoiesis or it may be a clonal disorder most closely resembling MDS. The frequency of leukemic transformation in primary PRCA is less than 1%.

Secondary PRCA describes cases of PRCA associated with a wide array of autoimmune/ inflammatory, infectious, or neoplastic disorders In many cases the underlying mechanism is immunologic but generally not mediated by an antibody.

257
Q

What cases transient PRCA?

A

Parvovirus B19 infection
- member of the erythroviruses
- Very common virus
- The virus invades and destroys red cell progenitors, and the aplasia is terminated when neutralizing antibodies develop
- If immunocompromised, leads to a persistent infection that is toxic to the erythroid progenitor cells and leads to chronic red cell aplasi

258
Q

What is drug induced PRCA?

A

Drug-induced PRCA represents an acute and generally reversible form of isolated erythroid aplasia.At least 50 drugs and chemicals have been associated with PRCA
- Diphenylhydantoin
- Rifampicin
- rhEPO
- Azathioprine
- Isoniazid

259
Q

How is PRCA diagnosed?

A

Blood count
- reticulocytes <1%

Bone marrow
- absence of erythroblasts

Cytogenetics
-abnormal may suggest MDS

Parvovirus
- B19 parovirus testing

260
Q

How is PRCA treated?

A

Treatment of B19 parvovirus-associated PRCA
Intravenous immunoglobulin (IVIG), which contains a large amount of anti–HPV-B19 IgG, is the treatment of choice for HPV-B19 PRCA.

Treatment of drugs induced PRCA
Recovery of erythropoiesis usually occurs shortly after discontinuation of the offending medication

261
Q

What are Hemophagocytic LymphoHistiocytosis (HLH)?

A

Rare, life-threatening conditions characterized by overstimulation of the immune system (lymphocytes and macrophages) leading to systemic inflammation, hypercytokinemia and multi-organ failure.

Caused by EBV infection

Can also be familial

262
Q

How does HLH present?

A

HLH is characterized by fever, hepatosplenomegaly, bicytopenia or pancytopenia, hemophagocytosis in the BM and lymphoid tissues, liver dysfunction and central nervous system (CNS) symptoms.

Clinical manifestations of HLH are mainly a result of tissue infiltration by T cells and macrophages and the accompanying excessive cytokine storm.

263
Q

What is primary HLH (FHL)?

A

Autosomal recessive disease caused by mutations in PRF, UNC12D, STX11, STXBP2

264
Q

What is secondary HLH?

A

Associated with viral, bacterial infections, malignancy and autoimmune diseases

265
Q

What is a stem cell transplant?

A

Receiving hematopoietic stem cells to replace ablated malignant cells

This is often the only cure for haem malignancies

Can be allogenic or autologous

266
Q

What is reduced intensity conditioning?

A

Less intensive chemotherapy regime where not all host bone marrow is ablated prior to transplant- good for patients who cannot withstand intense chemo

This reduces graft vs host disease (GvHD) and increases graft vs leukaemia (GvL)

GvL is mediated by T cell which attacks leukaemia cells so patients can have T cell top ups

267
Q

What is a peripheral blood stem cell graft and what are the advantages?

A

PBSC collected by Apheresis . Donor given G-CSF for 4-5 days prior to donation to mobilise stem cell

Advantages
- Easier and safer for the donor (Apheresis)
- More rapid and efficient engraftment achieved (due to increased mature T-cell component which facilitates engraftment)
- increased graft versus tumour affect

Disadvantages
- Chronic graft versus host disease is more of a problem

268
Q

When are stem cells taken directly from bone marrow for HSCT?

A

Preferred in children due to the reduced long term GvHD

Children usually have myeloablative BMT (less risk of relapse (T-cell GvL effect less important))

Children tolerate infectious complications where immune reconstitution is delayed

269
Q

What are class I HLA antigens?

A

HLA class I molecules display peptides that result from the degradation of cytosolic proteins to the cell surface where they can be recognized by the CD8+T cells

270
Q

What are class II HLA antigens?

A

HLA class II molecules display peptides that result from the degradation of endocytosed proteins to the cell surface where they can be recognized by the CD4+ T cells

271
Q

What is required of a bone marrow donor?

A

HLA matching
- Human leukocyte antigens (HLA) are genes in major histocompatibility complexes (MHC) that help code for proteins that differentiate between self and non-self

CMV status
- Preference is to transplant CMV positive graft to CMV positive recipient and CMV negative graft to CMV negative recipient

Blood type
- A, B, O
- Delayed erythrocyte engraftment and pure red cell aplasia

Gender
- increased risk of GvHD

Age
- T cell decrease quality

272
Q

What are the different sources of bone marrow graphs and what HLA match is required?

A

HLA Matched sibling/ unrelated matched
- 7/8 (ideally 8) HLA required for

Haplo donor
- 4/8 from a first degree relative who shares at least one full haplotype with the recipient (one of the two high density HLA antigens match)

Umbilical cord blood
- HLA matching less stringent (due to low immunogenicity of the CB T-cells)
- 4/6 of HLA A, B and DRB1 is sufficient

273
Q

How is it decided whether to do a HSCT?

A

Looks at

i) Risk of Non-BMT related mortality (dependent on the patients health and co-morbidities)
Vs
ii) Risk of disease relapse

The HCT-CI scores co-morbidities (0-3) to assist the decision of whether to transplant or not

Examples include:
1) Arrythmia (1)
2) Diabetes (1)
3) Moderate/severe renal (2)
4) Moderate pulmonary (2)
5) Prior malignancy (3)
6) Heart valve disease (3)

274
Q

What is chimerism and why is it measured?

A

Mix of host and donor cell types after a stem cell transplant, can be monitored by microsatellite analysis to determine graft effect

If the % chimerism decreases it may suggest the graft is failing

Also measure the % of T cells- mediate GvL effect- can be given a top up of T cells

275
Q

How is chimerism measured?

A

Amplification of 16 short tandem repeat (STR) using semi-quantitative multiplex PCR DNA to generate a genetic profile for the pre-transplant patient and donor

Preliminary assessment of STRs unique to the Donor and patient is carried out in an initial analysis

The informative alleles are subsequently used to quantify the cells present in the post-HSCT sample

Sensitivity around 1%

276
Q

How is graft vs host disease reduced in HSCT?

A

T-cell inhibition
- Cyclosporin and methotrexate primarily used as effective prophylaxis of acute GvHD (less effective in controling chronic GvHD)

T-cell depletion
- Positive selection of CD34+ cells is the most common method
Essential in 2nd donor transplants as GvHD

Post-transplantation cyclophosphamide (PTCy) in Haplo HSCT
- PTCyis often used to prevent GVHD, especially in patients who have undergone haplo-identical transplantation
- PTCy effectively eradicates allo-reactive T cells after haplo-identical stem cell infusion, but hematopoietic stem cells are spared

277
Q

How does the graft vs leukaemia affect work?

A

Activated T cells have been associated with reduced capacity for GVHD after adoptive transfer.

Several different approaches using either anti-CD3/CD28 or anti-CD3 and IL-2 with interferon-γ termed cytokine induced killer (CIK) cells have resulted in expansion of T cells with antitumor properties yet have limited GVHD capacity.

After expansion, CIK cells have antitumor capabilities primarily through NKG2D-mediated mechanisms, as well as other cytolytic functional receptor ligand interactions.

278
Q

What is the role CART in HSCT?

A

CAR-T cells are the subject of intense investigation as a strategy of a bridge to transplant or instead of transplantation.

These cells have been used in a small number of patients from bone marrow donors and shown to have limited capacity for GVHD induction but exert an antitumor effect.

FRCPath MPAD Part 1 (6 decks)

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