Haematology Flashcards
Examples of primary haematological disorders
Primary disorders usually arise from DNA mutation(s)
inherited (germline gene mutated):
FIX: deficiency > haemophilia B, Excess > FIX Padua (gene therapy use)
Erythrocytes: Deficiency> B globin gene mutation > Hb S, Excess > High affinity Hb mutation >Erythrocytosis
acquired (somatic gene mutated) BM rapid turnover system! (more common in blood cancers)
Erythrocytes: Excess> JAK2 > Polycythaemia vera, Deficiency> PIG A >PNH paroxysmal nocturnal haemoglobinuria
Soluble factors No acquired DNA mutations because not rapidly dividing cells (hepatocytes/endothelial cells)
Examples of secondary haematological disorders
Secondary disorders are changes in haematological parameters in response to a non-haematological disease or scenario. eg
Factor VIII:
Excess > inflammatory response/pregnancy
Deficiency>2ndry to anti-FVIII auto antibodies (acquired haemophilia A)
Systemic (non-haematological) conditions causing haematological abnormalities
Chronic Inflammation:
raised FVIII levels> increased Thrombosis risk.
Erythrocyte (Hb) count
Raised {altitude/hypoxia or Epo secreting tumour}
Reduced - BM infiltration or deficiency {Vit B12, or Fe} disease, Shortened survival {Haemolytic anaemia}
Platelet count
Raised {Bleeding, Inflammation, splenectomy}
Reduced - BM infiltration or deficiency disease {Vit B12 }, Shortened survival {ITP, TTP}
Leucocytes
Raised {Infection, Inflammation, corticosteroids}
Reduced - BM infiltration or deficiency disease {Vit B12 }
Anaemia; malignancy or systemic disease
may be first sign of systemic disease or occult malignancy eg.
Folate deficiency and Howell Jolly bodies > Coeliac
Fe deficiency > bowel or gastric cancer, peptic ulcer, IBD, renal cell carcinoma, blood cancer (urinary tract ones more rare)
Leucoerythroblastic anaemia > primary infiltration in bone marrow; blood cancer leukaemia/lymphoma/myeloma), metastatic breast/lung/prostrate cancer, could also be miliary TB or severe fungal infection, if massive splenomegaly -> myelofibrosis
Haemolytic anaemia > acquired immune and non-immune
Anaemia of Inflammation (chronic disease)
Leuco-erythroblastic anaemia
= red cell and white cell precursor anaemia
peripheral bloodfilm features: teardrop RBCs, nucleated RBCs, immature myeloid cells (myelocytes) - these features are present normally but in bone marrow not peripheral blood
Haemolytic anaemia lab results
anaemia
reticulocytosis (may cause modest elevation of MCV [upper limit of normal])
bilirubinaemia (unconjugated/pre-hepatic cause)
raised LDH
reduced haptoglobins (binding protein for Hb)
Immune haemolytic anaemia (acquired)
spherocytes
positive direct antiglobulin test
associated with systemic diseases:
immunological disorders (lymphoma, chronic lymphocytic leukaemia)
infection (mycoplasma)
idiopathic
Non-immune haemolytic anaemia (acquired)
DAT negative
infection of erythrocytes (malaria)
Micro-angiopathic haemolytic anaemia (MAHA) = acquired associated with systemic disease -> underlying adenocarcinoma or haemolytic uraemic syndrome
MAHA
bloodfilm features: RBC fragments, thrombocytopenia
Adenocarcinomas, low grade DIC
Platelet activation
Fibrin deposition and degradation
Red cell fragmentation (microangiopathy)
Bleeding (low platelets and coag factor consumption)
Types of white blood cells
Bone marrow:
blasts (myeloid and lymphoid) (usually <5% of BM cells if excess consider leukaemia)
promyelocytes
myelocytes
Peripheral blood:
Phagocytes
Granulocytes: Neutrophils, Eosinophils, basophils
Monocytes
immunocytes
T lymphocytes
B lymphocytes
NK cells
high WCC with blasts in peripheral blood
acute myeloid leukaemia
high WCC with myelocytes in peripheral blood
chronic myeloid leukaemia
Reactive neutrophilia causes
pyogenic infection
corticosteroids
underlying neoplasia
tissue inflammation (eg. colitis, pancreatitis, myocarditis, MI)
neutrophils + toxic granulation, no immature cells
Malignant neutrophil abnormalities
Neutrophilia plus basophilia & myelocytes. Suggestive of chronic myeloid leukaemia (CML)
Neutropenia plus myeloblasts suggests acute myeloid leukaemia (AML)
Reactive Eosinophilia
Parasitic infestation
Allergic diseases e.g. asthma, rheumatoid, polyarteritis, pulmonary eosinophilia.
Neoplasms, esp. Hodgkin’s, T-cell NHL (reactive eosinophilia)
Drugs (reaction erythema multiforme)
Chronic eosinophilic leukaemia
Eosinophils part of the “clone”
FIP1L1-PDGFRa Fusion gene
Monocytosis
Rare but seen in certain chronic infections and primary haematological disorders:
TB, brucella, typhoid
Viral; CMV, varicella zoster
sarcoidosis
chronic myelomonocytic leukaemia (MDS)
Raised lymphocyte count secondary/reactive causes
EBV, CMV, Toxoplasma (Infectious mononucleosis IM)
infectious hepatitis, rubella, herpes infections
autoimmune disorders
Sarcoidosis
Reduced lymphocyte count secondary/reactive causes
Infection: HIV
Auto immune disorders
Inherited immune deficiency syndromes
Drugs (chemotherapy)
Lymphocytosis in peripheral blood morphology
Mature lymphocytes (PB):
- reactive/atypical lymphocytes (IM)
- small lymphocytes and smear cells (CLL/NHL)
Immature Lymphoid cells in PB:
Lymphoblasts; Acute Lymphoblastic Leukaemia (ALL)
Determining clonality in b-cell lymphocytosis using light chain restriction
polyclonal
Kappa &
Lambda -> 60:40 -> reactive
Monoclonal
kappa only or
lambda only -> 99:1 -> malignant
Haemato-oncology diagnosis
Morphology: architecture of tumour, cytology, cytochemsitry
Immunophenotype: flow cytometry, immunohistochemsitry
cytogenetics: conventional karyotyiping, fluorescent in-situ hybridisation (interphase or metaphase FISH)
molecular genetics: mutation detection (direct and pyrosequencing), PCR analysis, gene expression profiling, whole genome sequencing
Morphology
malignant cells; large or small, mature or immature?
Lymph node architecture (diffuse invasion or forming follicles?)
Immunophenotype (flow cytometry or Immunohistology)
myeloid or lymphoid?
T or B lineage?
stage of maturation (precursor or mature? )
Cytogenetics (translocations: 1) fusion gene or 2)deregulated oncogene )
confirm morphological diagnosis eg
Philadelphia Chromosome > CML.
t(8;14) activates c-myc oncogene in Burkitt Lymphoma
Prognostic information eg 17p del in CLL
Molecular genetics (PCR, pyro-sequencing)
JAK2 mutation in suspected polycythemia vera
BCR ABL cDNA detection and quantification
Common blood cancer presentations and problems
Lympho-haemopoietic failure (a dispersed organ!):
Bone marrow (myeloid): anaemia, bacterial infection (neutrophils) bleeding (platelets)
Immune system (lymphoid): recurrent viral or fungal infection
Excess of malignant cells:
Erythrocytes or leucocytes: impair blood flow >stroke or TIA
Massively enlarged lymph nodes (lymphoma)> compress hollow tubes: bowel, vena cava, ureters, bronchus.
Infiltrate and impair other organ function:
CNS lymphoma
Skin lymphoma
Kidney failure (light chain deposition from myeloma)
Miscellaneous problems:
hyepercalcaemia
hypermetabolism
Lymphomas
Definition:
The term ‘lymphoma’ means a neoplastic (malignant) tumour of lymphoid cells.
Lymphomas usually found in:
lymph nodes, bone marrow and/or blood (the lymphatic system)
lymphoid organs; spleen or the gut-associated lymphoid tissue
Skin (often T cell disease)
Rarely “anywhere” (breast kidney){*Immune privilege sites CNS, occular, testes}
Incidence:
There are approximately 200 new cases per year for every million of the population (around 10,000 new cases a year in the UK).
Non-Hodgkin’s Lymphomas 80%
Hodgkin Lymphoma 20%
Types of lymphoid malignancies
Precursor malignancies:
B or T cell lineage
Mature B cell malignancies:
Non Hodgkin Lymphoma (NHL)
Or
Hodgkin Lymphoma
Mature T cell malignancies:
T cell or NK cell Non Hodgkin
Lymphoma (NHL)
Mechanisms of risk factors for NHL subtypes
Constant antigenic stimulation:
Bacteria infection (chronic)
Auto immune disorders
Viral Infection
(direct viral integration of lymphocytes)
Loss of T cell function and EBV infection plus EBV infections of B cells:
Loss of T cells (HIV infection untreated )
Iatrogenic immunosuppression
Chronic antigen stimulation and NHL
(Bacterial or auto immune antigenic drive)
B cell Non Hodgkin Lymphoma Marginal zone sub type (MZL):
- H.Pylori : Gastric MALT (mucosa associated lymphoid tissue) (MZL of stomach)
- Sjogren syndrome : MZL of
- Hashimoto’s : MZL of thyroid
Enteropathy associated T-Cell Non Hodgkin lymphoma (EATL):
- Coeliac disease/Gluten: small intestine EATL
Direct viral integration and NHL
HTLV1 retrovirus infects T cells by vertical transmission
Caribbean, Japan (and world wide) endemic infection
Risk of Adult T cell leukaemia lymphoma (ATLL) is 2.5% at 70 years
ATLL is a subtype of T cell Non Hodgkin Lymphoma
Loss of T cell function and EBV-driven NHL
EBV infection:
- EBV infects B lymphocytes, healthy carrier state post glandular fever.
- EBV driven proliferation of B cells is associated with surface expression of EBV antigens. Proliferating B cells targeted and killed by EBV specific cytotoxic T cell response
Loss of T cell function:
- HIV (in uncontrolled infection there is x60 increased incidence of B NHL )
- Iatrogenic (transplant immunosuppression)
PTLD (post transplant lymphoproliferative disorder)
Loss of cytotoxic T cell function can cause failure to eliminate EBV driven proliferation of B cells
The lymphoreticular system
3 types of tissue
Generative LR tissue:
- Bone marrow and thymus
- Function - generation/maturation of lymphoid cells
Reactive LR tissue:
- Lymph nodes and spleen
- Function - development of immune reaction
Acquired LR tissue:
- Extranodal lymphoid tissue
E.g. Skin, stomach, lung
- Function - development of local immune reaction
Cells of the lymphoreticular system
2 categories:
Lymphocytes:
Classified according to function.
B lymphocytes
- Express surface immunoglobulin
- Antibody production
T lymphocytes
- Express surface T cell receptor
- Regulation of B cell and macrophage function
- Cytotoxic function
Accessory cells:
Antigen presenting cells
Macrophages
Connective tissue cells
B cell area in lymphoid tissue
Paracortical Tcell zone
Lymphoid follicle:
Mantle zone- Naïve unstimulated B cells
Germinal center - B cells and Antigen presenting cells
This is where B cells which bind antigen epitopes are selected and activated
T cell area in lymphoid tissue
Comprises:
T cells
Antigen presenting cells
High endothelial vessels
This is where T cells which bind antigen epitopes are selected and activated
Lymphoma pathogenesis
Neoplastic proliferation of lymphoid cells – clonal
Mutation in genes to allow uncontrolled cell growth
- Normal lymphocytes undergo controlled genomic “instability” of lymphoid cells - mistakes in this process produce neoplastic mutations
- Inherited disorders – inherited disorder resulting in increased/abnormal genomic instability
- Viral agents – EBV, HTLV-1
- Environmental agents – mutagens, chronic immune stimulation (e.g H pylori)
- Iatrogenic causes – radiotherapy, chemotherapy
Immunosuppression predisposes to development of lymphoma
- Infection
- Loss of surveillance
WHO lymphoma classification
HODGKIN LYMPHOMA
Classical
Lymphocyte predominant
NON-HODGKIN LYMPHOMA
B cell:
- Precursor B cell neoplasms
- Peripheral B cell neoplasms (Low and High grade)
T cell:
- Precursor T cell neoplasms
- Peripheral T cell neoplasms
Key cell surface markers for T and B cells
T = CD3, CD5
B = CD20
these normal cell markers may be decreased in lymphoma
abnormal markers may be expressed in lymphoma eg. cyclin D1 (not present normally)
Common B cell NHLs
Low grade: (small lymphocytes)
- Follicular lymphoma
- Small lymphocytic lymphoma/chronic lymphocytic leukaemia
- Marginal zone lymphoma
High grade: (big lymphocytes)
- Diffuse large B cell lymphoma
- Burkitt’s lymphoma
Aggressive:
- Mantle cell lymphoma
Follicular lymphoma
Clinical:
- Lymphadenopathy MA/elderly
Histopathology:
- Follicular pattern
- Germinal centre cell origin CD10, bcl-6+
Molecular:
- 14;18 translocation involving bcl-2 gene
Indolent but can transform to high grade lymphoma (incurable, median survival 12-15 years, may require 2-3 cycles of chemotherapy during this time)
SLL/CLL
Clinical:
- MA/elderly; nodes (SLL) or blood (CLL)
Histopathology:
- Small lymphocytes, Naïve or post-germinal centre memory B cell
- CD5, CD23 + (CLL)
Molecular:
- Multiple genetic abnormalities
Indolent, but can transform to high grade lymphoma (Richter transformation)
Marginal zone/ MALT lymphoma
Arise mainly at extranodal sites (many sites, e.g. gut, lung, spleen)
Thought to arise in response to chronic antigen stimulation (e.g. by Helicobacter in stomach)
Post germinal centre memory B cell
Indolent but can transform to high grade lymphoma
Can treat low grade disease with non-chemotheraputic modalities - i.e. remove antigen
- E.g Helicobacter eradication
Mantle cell lymphoma
Clinical:
- MA male predominence
- Lymph nodes, GI tract
- Disseminated disease at presentation
Histopathology:
- Located in mantle zone
- Pre-germinal centre cell
- Aberrant CD5, cyclin D1 expression
Molecular:
- 11;14 translocation
- Cyclin D1 over expression
Median SR 3-5 yrs
Burkitt’s lymphoma
Clinical:
- Jaw or abdominal mass children/young adults (endemic, sporadic, immunodeficiency)
- EBV associated
Histopathology:
- Germinal center cell origin
- “starry-sky” appearance
- very high Ki-67 index
Molecular:
- C-myc translocation (8:14, 2:8, 8;22)
Aggressive disease (fastest proliferating malignancy)
NB: Tumour lysis syndrome can occur during treatment but even before treatment
Diffuse large B cell lymphoma
Clinical:
- MA/elderly
- Lymphadenopathy
Histopathology:
- Germinal center (CD10) or post-germinal center B cell
- Sheets of large lymphoid cells
- Germinal center phenotype = good prognosis
- p53 positive, high proliferation fraction = poor prognosis
Peripheral T cell lymphomas
MA/elderly
Lymphadenopathy and extranodal sites
Large T lymphocytes
Often with associated reactive cell population, esp eosinophils
Aggressive
Special forms of T cell lymphomas
Adult T cell leukaemia/lymphoma:
- Caribbean and Japan
- Associated with HTLV-1 infection
- arises from CD4 positive T cells (also express CD25)
- acute and chronic presentations
- aggressive esp in acute form
Enteropathy associated T cell lymphoma:
- Some patients with long standing coeliac disease
- arises from intra-epithelial CD8 positive T cells
- T cells form mass in bowel
- very aggressive
- treatment: gluten avoidance
Cutaneous T cell lymphomas:
E.g. mycosis fungoides
- begins as long-standing patches/plaques on skin in sun-protected areas and progresses to nodules and tumours
- arises from CD4 positive T cells
Anaplastic large cell lymphoma
- CD3 and CD2 cell markers but may have null cell marker phenotype
Anaplastic large cell lymphoma
Clinical:
- Children/young adults
- Lymphadenopathy
Histopathology:
- Large “epithelioid” lymphocytes
- T cell or null phenotype
Molecular:
- 2;5 translocation
- Alk-1 protein expression
Aggressive
Alk-1 positive better prognosis
Hodgkin’s lymphoma (HL)
More often localised to a single nodal site
Spreads contiguously to adjacent lymph nodes
Classical:
(Several subtypes)
- Nodular sclerosing
- Mixed cellularity
- Lymphocyte rich and lymphocyte depleted
Lymphocyte predominant:
- Some relationship to non-Hodgkin’s lymphoma
Classical HL
Clinical:
- Young and MA
- Often involves just single lymph node group (painless lymphadenopathy)
- may cause obstructive symptoms
- constitutional symptoms (B symptoms/FLAWS, rare: pruritus and alcohol-induced painful lymphadenopathy)
Thought to be germinal center/post germinal center B cell origin
EBV associated
CD30 +, CD15+, CD20-
Histopathology:
- Sclerosis, mixed cell population in which scattered Reed-Sternberg and Hodgkin cells with eosinophils
Moderately aggressive
Nodular lymphocyte predominant HL
Clinical: Isolated lymphadenopathy
Germinal centre B cell (positive for some germinal centre B cell markers)
No association with EBV
CD20+, CD30-, CD15-
Histopathology: B cell rich nodules with scattered L&H cells
Indolent
Can transform to high grade B cell lymphoma
Lymphoma presentations
Painless progressive lymphadenopathy
Palpable node
Extrinsic compression of any “tube”
Eg Ureter, Bile duct, large blood vessel, bowel, trachea, oesophagus
Infiltrate/impair an organ system
E.g. skin rash, ocular&CNS, liver failure
Recurrent infections
Constitutional symptoms (FLAWS)
Coincidental e.g. FBC, Imaging
HL classification
Classical HL:
Nodular sclerosing 80% Good prognosis (causes the peak incidence in young women)
Mixed cellularity 17% Good prognosis
Lymphocyte rich (rare) Good prognosis
Lymphocyte depleted (rare) Poor Prognosis
Nodular Lymphocyte predominant HL 5% (disorder of the elderly multiple recurrences)
HL staging
(using lymph node biopsy and PET)
Stage:
I; one group of nodes
II; >1 group of nodes same side of the diaphragm
III; nodes above and below the diaphragm
IV; extra nodal spread
Suffix A if none of below, B if any of below:
Fever
Unexplained Weight loss >10% in 6 months
Night sweats
cHL nodular sclerosing subtype
Young women(>men) 20-29 years
Neck nodes and mediastinal mass(may be massive and compress SVC or trachea)
May have B symptoms
Needs a Tissue diagnosis
Combination chemotherapy for cHL
ABVD:
Adriamycin
Bleomycin
Vinblastine
DTIC
given at 4-weekly intervals.
ABVD is Effective treatment
Preserves fertility (unlike MOPP the original chemo)
Can cause (long term): Pulmonary fibrosis, cardiomyopathy
Chemotherapy (essential for cure)
ABVD 2-6 cycles (depends:stage&interim response)
PET CT
Interim: After x2 cycles, response assessment
End of Treatment: Guides need for additional radiotherapy
+/- Radiotherapy (see next slide)
Relapse {salvage chemotherapy}
High dose chemotherapy + Autologous PB stem cell transplant as support
Radiotherapy for HL
Modern practice involved field only
Adv: Low/negligible risk of relapse within field
Disadv:
Risk of damage to normal tissue (collateral damage)
Ca breast (risk 1:4 after 25 years)
Leukaemia/mds (3%@10years)
Lung or skin cancer
Combined modality (chemo + radio) greatest risk of 2o malignancy
Diffuse large B cell NHL Treatment
Treated by x 6-8 cycles of R-CHOP (Rituximab-CHOP)
combination chemotherapy using a mixture of drugs usually including an anthracycline (e.g. doxorubicin).
Combination drug regimens e.g. CHOP
Cyclophosphamide 750 mg/m2 i.v. D1
Adriamycin 50 mg/m2 i.v. D1
Vincristine 1.4 mg/m2 i.v. D1
Prednisolone 40 mg/m2 p.o. D1‑D5
R is Immunotherapy using the anti CD20 monoclonal antibody Rituximab
Aim of therapy is curative (overall approx 50%)
Relapse:
High dose chemotherapy and Auto stem cell transplant
CAR-T cell therapy (Chimeric antigen receptor)
Follicular lymphoma treatment
At presentation Watch and wait only treat “if clinically indicated”:
Nodal extrinsic compression: bowel, Bile duct, ureter, vena cava,
Massive painful nodes, recurrent infections
Treatment:
combination Immuno-chemotherapy R-COP or R-CHOP
Treatment is not curative, may require 2nd or 3rd line
Median survival 13 years (wide range)
Chronic lymphocytic leukaemia
Proliferation of mature B-lymphocytes
Commonest leukaemia in the western world
Caucasian
UK incidence 4.2/100,000/year
Age at presentation median 72 (10% aged <55yrs)
Relatives x7 increased incidence
(cancer primarily of bone marrow and blood, whereas lymphoma typically present with lymphadenopathy initially)
CLL lab findings
Lymphocytosis between 5 and 300 x 109/l
Smear cells
Normocytic normochromic anaemia
Thrombocytopenia
Bone marrow lymphocytic replacement of normal marrow elements
B lymphocytes that are CD5 and CD19 positive (usually don’t express CD5 as this is T cell marker) - will also have other normal b cell markers
CLL prognosis
Highly variable natural history:
Initially 5-10 years good health until progression to a 2-3 year terminal phase
Rapid progression to death within 2-3 years
In a disorder of elderly:
1/3 never progress
1/3 Progress, respond to CLL Rx (death from unrelated disorder)
1/3 Progress, require multiple lines of Rx, refractory disease, death from CLL
Cell based prognostic factors:
IgHV mutation status
CLL FISH cytogenetic panel
TP53 mutation status (Chromosome 17p del and/or TP53 point mutation)
Clinical staging systems:
Binet or Rai (clinical staging)
CLL IPI score
CLL clinical issues
Increased risk of infection
bone marrow failure
lymphadenopathy, splenomegaly, lymphocytosis
transform to high grade lymphoma
autoimmune complications (eg. immune haemolytic anaemia)
CLL supportive care
Sino-pulmonary infections:
Early Rx with antibiotics
Pneumocystis prophylaxis (may also require zoster ppx)
Recurrent infection + IgG < 5g/l > IVIG replacement therapy
Vaccinations:
Pneumococcal
Covid19
Seasonal flu
Avoid live vaccines
CLL indications to treat
Watch and wait unless:
Progressive lymphocytosis
>50% increase over 2 months
lymphocyte doubling time <6 months
Progressive marrow failure
Hb < 100, platelets <100, neutrophils <1
Massive or progressive lymphadenopathy/splenomegaly
Systemic symptoms (B symptoms)
Autoimmune cytopenias (treat with immunosuppression not chemotherapy)
CLL therapy:
Combination Immuno-chemotherapy (being superseded by targeted Rx)
Targeted Therapy (BTK inhibitor eg ibrutinib, BCL2 inhibitor [allowing apoptosis] eg. venetoclax)
Cellular therapy only for relapsed high risk cases (Allogeneic SCT, CAR-T therapy)
NB: risk of tumour lysis syndrome on initiating venetoclax treatment
Raised Hb causes
Polycythaemia = raised Hb concentration and Haematocrit %
- Relative (lack of plasma) (non-malignant)
- True (excess erythrocytes)
- Secondary (non-malignant)
- Primary (myeloproliferative neoplasm)
Myeloproliferative neoplasms (MPDs):
- Ph (Philadelphia Chromosome)negative
- Polycythaemia vera (PV)
- Essential Thrombocythaemia (ET)
- Primary Myelofibrosis (PMF)
- Ph positive:
- Chronic myeloid leukaemia (CML)
Causes of secondary polycythaemia (non-malignant)
(raised EPO)
Appropriate:
High altitude
Hypoxic lung disease
Cyanotic heart disease
High affinity haemoglobin
Inappropriate:
Renal disease (cysts, tumours inflammation)
uterine myoma
other tumours (liver, lung)
treatment often not indicated
Haematological malignancies
Myeloid:
Acute myeloid leukaemia (blasts >20%
Myelodysplasia (blasts 5-19%)
Myeloproliferative disorders
-Essential thrombocythaemia (megakaryocyte)
-Polycythemia vera (erythroid)
-Primary myeofibrosis
Chronic myeloid leukaemia
Lymphoid:
Precursor cell malignancy
-Acute lymphoblastic leukaemia (B & T)
Mature cell malignancy
-Chronic Lymphocytic leukaemia
-Multiple myeloma
-Lymphoma (Hodgkin & Non Hodgkin)
What processes are disrupted by mutation?
Impair/block cell differentiation (type 2)
Cellular proliferation (type 1)
Prolong cell survival (anti-apoptosis)
Mutation mechanisms:
DNA point mutations
Chromosomal translocations
- Creation of novel Fusion gene
- Disruption of proto-oncogene
Tyrosine kinase signalling in haemotopoeisis
Normal Tyrosine kinases:
Transmit cell growth signals from surface receptors to nucleus
Activated by transferring phosphate groups to self and downstream proteins
Normally held tightly in inactive state
Promote cell growth do not block maturation
Activating Tyrosine kinase mutations:
expansion increase in mature/end cells
Red cells; polycythaemia
Platelets; essential thrombocythaemia
Granulocytes; chronic myeloid leukaemia
Diagnosing philadelphia negative myeloproliferative disorders (MPD)
Based on combination of :
Clinical features
- Symptoms
- splenomegaly
FBC +/- Bone marrow biopsy
Erythropoietin level (epo)
Mutation testing (eg. JAK2)
Phenotype linked to acquired mutation
Polycythaemia Vera
Incidental diagnosis routine FBC (median Hb 184g/l, Hct 0.55)
Symptoms of increased hyper viscosity:
Headaches, light-headedness, stroke
Visual disturbances
Fatigue, dyspnoea
Increased histamine release:
- Aquagenic pruritus
Peptic ulceration
Test for JAK2 V617F mutation
Treatment:
Aim to reduce HCT : target HCT <45%
- Venesection
- Cytoreductive therapy hydroxycarbamide
Aim to reduce risks of thrombosis
- Control HCT
- Aspirin
- Keep platelets below 400x109/l (see treatment of ET)
Essential Thrombocythaemia
Chronic MPN mainly involving megakaryocytic lineage
Incidental finding on FBC (50% cases)
Thrombosis: arterial or venous
CVA, gangrene, TIA
DVT or PE
Bleeding: mucous membrane and cutaneous
Headaches, dizziness visual disturbances
Splenomegaly (modest)
Treatment:
Aspirin: to prevent thrombosis
Hydroxycarbamide: antimetabolite. Suppression of other cells as well.
Anagrelide: specific inhibition of platelet formation, side effects include palpitations and flushing
Prognosis:
Normal life span may not be changed in many patients.
Leukaemic transformation in about 5% after >10 years
Myelofibrosis also uncommon, unless there is fibrosis at the beginning
Primary Myelofibrosis
A clonal myeloproliferative disease associated with reactive bone marrow fibrosis
Extramedullary haematopoieisis
Primary presentation:
Incidence 0.5-1.5 /100000
Males=females
7th decade. Less common in younger patients
Other MPDs (ET & PV) may transform to PMF
Incidental diagnosis in 30%
Presentations related to:
Cytopenias: anaemia or thrombocytopenia
Thrombocytosis
Splenomegaly: may be massive
Budd-Chiari syndrome
Hepatomegaly
Hypermetabolic state:
Weight loss
Fatigue and dyspnoea
Night sweats
Hyperuricaemia
Primary myelofibrosis haematological findings
Blood film:
Leucoerythroblastic picture
Tear drop poikilocytes
Giant platelets
Circulating megakaryocytes
Liver and spleen
Extramedullary haemopoiesis in spleen and liver
DNA : JAK2 or CALR mutation
Bone marrow:
‘Dry tap’
Trephine:
Increased reticulin or collagen fibrosis
Prominent megakaryocyte hyperplasia and clustering with abnormalities
New bone formation
Primary myelofibrosis prognosis
Median 3-5 years but very variable
Bad prognostic signs:
- Severe anaemia <100g/L
- Thrombocytopenia <100x109/l
- Massive splenomegaly
Prognostic scoring system (DIPPS)
Score 0 – median survival 15years
Score 4-6– median survival 1.3 years
Primary myelofibrosis treatment
Supportive: RBC and platelet transfusion often ineffective because of splenomegaly
Cytoreductive therapy: hydroxycarbamide (for thrombocytosis, may worsen anaemia)
Ruxolotinib: JAK2 inhibitor (high prognostic score cases)
Allogeneic SCT (potentially curative reserved for high risk eligible cases)
Splenectomy for symptomatic relief: hazardous and often followed by worsening of condition
Chronic myeloid leukaemia (CML)
Incidence 1-2/100,000
M:F 1.4:1
40-60 years @ presentation
Radiation exposure risk factor (Hiroshima & Nagasaki)
History:
Lethargy/ hypermetabolism/ thrombotic event : monocular blindness CVA, bruising bleeding
Exam:
Massive splenomegaly +/- hepatomegaly
FBC:
Hb and platelets well preserved or raised
Massive leucocytosis 50-200x109/L
Blood film :
Neutrophils and myelocytes (not blasts if chronic phase)
Basophilia
Treatment:
Imatinib (Abl tyrosine kinase inhibitor)
CML lab features
Leucocytosis between 50 – 500x109/l
Mature myeloid cells
Bi phasic peak Neutrophils and myelocytes
Basophils
No excess (<5%) myeloblasts
Platelet count raised/upper normal
(contrast acute leuk)
Philadelphia chromosome
Translocation of part of the long arm (q) of chromosome 22 to chromosome 9
And reciprocal translocation of part of chromosome 9, which includes the ABL oncogene to a specific breakpoint cluster region (BCR) of chromosome 22
A fusion gene results on the derived chromosome 22
This leads to the synthesis of an abnormal protein with TK activity greater than the normal ABL protein
Detection:
Conventional Karyotyping
FISH metaphase or interphase karyotyping
RT-PCR amplification and detection
Diagnosing and monitoring CML
FBC and measure leucocyte count
Cytogenetics and detection of Philadelphia chromosome
RT-PCR of BCR-ABL fusion transcript which can be quantified by RQ-PCR to determine response to therapy
Haematological response
Complete Haematological Response WBC<10x109/l
Cytogenetic response (on 20 metaphases)
Partial 1-35% Philadelphia positive
Complete 0% Ph positive
Molecular ( reduction in % BCR-ABL transcripts)
BCR-ABL transcripts reduce 100% > 10% > 1% > 0.1%
Major Molecular response (MMR) <0.1% (3 log reduction)
CML clinical course
Commence on oral TKI 1st generation
Monitor response FBC, Cytogenetics, RQ-PCR
CCyR at 12mo 97% FFP at 6 years (Fail to achieve CCyr 80%)
Average 95% 5 year survival
Annual mortality 2%
Reasons for not working:
Failure to achieve CCyR
Non compliance
Side effects; fluid retention pleural effusions
Loss of MMR
Acquiring abl point mutations leading to resistance
Evolution to blast crisis
Treatment algorithm CML
Chronic phase Tyrosine kinase Inhibitor (TKI)
Imatinib (1Gen,) Dasatanib Nilotonib (2G) Bosutinib (3G)
Failure (1) > switch to 2Gen or 3G TKI
No complete cytogenetic response @ 1year
Respond but acquire resistance
Failure (2) > consider allogeneic SCT
Inadequate response or intolerant of 2G TKIs
Progression to accelerated or blast phase
Myelodysplastic syndromes (MDS)
Biologically heterogeneous group of acquired haemopoietic stem cell disorders (~ 4 per 100,000 persons)
Characterised by:
The development of a clone of marrow stem cells with abnormal maturation resulting in -
functionally defective blood cells AND
a numerical reduction
This results in:
i. Cytopenia(s).
ii. Qualitative (i.e. functional) abnormalities of erythroid, myeloid and megakaryocyte maturation.
iii. Increased risk of transformation to leukaemia
Typically a disorder of the elderly.
Symptoms/signs are those of general marrow failure
Develops over weeks & months
MDS bone marrow and blood film features
Pelger-Huet anomaly (bilobed neutrophils)
Dysganulopoieses of neutrophils
Dyserythropoiesis of red cells (ringed sideroblasts on iron [prussian blue] staining)
Dysplastic megakaryocytes – e.g. micro-megakaryocytes
Increased proportion of blast cells in marrow (normal < 5%)
Diagnosis of MDS
requires:
- cytopenia of at least on blood cell line
- <20% blasts in blood and bone marrow
- Either:
- characteristic cytogenetic or molecular findings without evidence of an alternate cause of these findings
OR
- morphological dysplasia in >/ 10% of nucleated cells of at least one cell lineage
WHO classification of MDS
Number of dysplastic lineages
Percentage of blasts in bone marrow and peripheral blood
Cytogenetic findings
Percentage of ringed sideroblasts
Number of cytopenias (based on criteria from the International Prognostic Scoring System - IPSS)
Hb < 100 g/L
Platelets < 100 x 10^9/L
Neutrophils < 1.8 x10^9/L
Monocytes < 1.0 x 10^9/L (if > 1.0 x 10^9/L then diagnosis is CMML)
Gene mutations in MDS
Driver mutations in MDS - carry prognostic significance:
TP53, EZH2, ETV6, RUNX1, ASXL1
Others: SF3B1, TET2, DNMT3A
Majority of common mutations are found more frequently in high risk MDS than in low risk MDS
MDS evolution
- Deterioration of blood counts
* Worsening consequences of marrow failure - Development of acute myeloid leukaemia
– Develops in 5-50%< 1 year (depends on subtype)
– Some cases of MDS are much slower to evolve
– AML from MDS has an extremely poor prognosis and is usually not curable - As a rule of thumb
* 1/3 die from infection
* 1/3 die from bleeding
* 1/3 die from acute leukaemia
MDS treatment
At present, the only two treatments that can prolong survival are:
1. allogeneic stem cell transplantation (SCT)
2. intensive chemotherapy
but only a minority of MDS patients can really benefit from them
Majority:
- Supportive care:
Blood product support
Antimicrobial therapy
Growth factors (Epo, G-CSF, TPO-Receptor Agonist) - Biological Modifiers:
Immunosuppressive therapy (Azacytidine )
Hypomethylating agents (Decitabine)
Lenalidomide (for del(5q) variant)
Bone marrow failure
- PRIMARY (primarily in children)
Congenital: Fanconi’s anaemia (multipotent stem cell)
Diamond-Blackfan anaemia (red cell progenitors)
Kostmann’s syndrome (neutrophil progenitors)
Acquired: Idiopathic aplastic anaemia (multipotent stem cell) - SECONDARY (much more common)
Marrow infiltration:
Haematological (leukaemia, lymphoma, myelofibrosis)
Non-haematological (Solid tumours)
Radiation
Drugs
Chemicals (benzene)
Autoimmune
Infection (Parvovirus, Viral hepatitis)
