Bone marrow failure Flashcards
Define PNH + pathogenesis - Paroxysmal nocturnal hemoglobinuria
- Acquired clonal disorder, often associated with aplastic anaemia and MDS
- Acquired mutation in PIGA gene (X linked)
- Leads to partial/absolute deficiency of all GPI linked proteins in that clone of HSCs
- This leads to complement mediated destruction of red cells
- Also causes intravascular haemolysis, propensity to thrombosis and marrow dysplasia.
- CD59 (Membrane Inhibitor of reactive lysis) is GPI-linked and loss of this protein specifically leads to the haemolysis seen in PNH
- CD55 also GPI-linked and while loss is not causative of haemolysis, detection of loss of CD55 is specific to PNH
PNH association in AA
40% of PNH evolves from AA
The loss of the GPI-anchor proteins on PNH stem cells confers a survival advantage compared to normal stem cells; PNH clones can evolve from AA
Hence serial monitoring of the PNH clone is important in AA patients; there is no role for serial monitoring in MDS patients
Frequency of screening
No detectable clone: q6m-q1y
Detectable clone: q3m until shown to be stable for 2 years
Differentiation of erythrocytes in PNH
type I, II and III depending on their sensitivity to complement-mediated lysis
Type I: normal erythrocytes
Type II: modest hypersensitivity to complement-mediated lysis; corresponds to partial GPI deficiency by flow cytometry
Type III: pronounced hypersensitivity to complement-mediated lysis; corresponds to complete GPI deficiency by flow cytometry
White cells are differentiated into GPI-deficient versus GPI-present
Clinical manifestations of PNH
Haemolysis
Thrombosis!!!
Vasospasm
Pancytopenia - BM failure
Management of PNH
Folate (give in all haemolysis)
Iron supplements if needed
Supportive transfusions
Eculuzimab -Target C5 (stop MAC formation)
Ravulizumab - Longer acting , targets C5
Pegcetoplan - anaemia can still continue due to accumulation of unopposed C3b –> extravascular haemolysos.
Pegcetoplan is superior to eculuzimab in improving haemoglobin and normalzing haemolytic markers . Blocks both intravascular and extravascular haemolysis (loss of C3b fragments and reduced RC oponisation)
AlloSCT
Flow cytometry PNH
- Flow cytometry
- EDTA sample
- SS and 45 to gate out dead cells and debris
- development of FLAER, a fluorochrome-conjugated non-lysing, mutated form of proaerolysin (a bacterial protein which binds to the glycan portion of the GPI anchor) has increased the sensitivity of PNH flow cytometry
- more sensitive than CD59
PNH flow
.Neutrophils and Monocytes:
- CD15 is used to gate neutrophils
- CD64 is used to gate monocytes
GPI-Linked antibodies:
- CD 24 & FLAER (granulocytes)
- CD14 & FLAER (monocytes)
50,000 events
Red Cells
- CD235a is used to gate RBCs
- GPI linked antibody is CD59
100,000 events
A PNH clone can be identified by the loss of FLAER and either CD24 (granulocytes) or CD14 (monocytes) or CD59 for RBC
Deficiency of two GPI linked molecules should be demonstrated in leucocyte lineages for
diagnosis.
Limitations of PNH flow
Eculizumab treatment usually display expanded RBC clones due to the increased lifespan of the GPI-deficient red blood cells.
- Sample age
- If low numbers of neuts/monocytes
- Inadequate washing of RBC
- Basophils, NK cells and blasts captured in the granulocuyte gate may stain dim for FLAER
Insufficient cell events
Pure red cell aplasia - definition
Rare group of syndromes characterized by severe normocytic normochromic anemia with normal WCC and platelets.
Severe reticulopenia (retic count <1%) and marked reduction of erythroid precursors in a normocellular BM (<0.5% mature erythroblasts).
No significant abnormalities in other lineages.
PRCA can be either constitutional or acquired and either acute or chronic.
Differentials diagnosis of PRCA
Children
- Diamond Blackfan anaemia (usually <1 y.o)
- Transient erythroblastopenia of childhood (usually >1y.o) due to HHV6, EBV, parvovirusB19
- Other systemic illness (coeliac, kawasaki)
Adult
- Parvovirus B19 (patients have pre-existing red cell defects/haemolytic disorders ie, HS, sickle and can cause aplastic crisis)
- Primary acquired PRCA (autoimmune)
- Secondary acquired PRCA (often assoc with thymoma, HL, NHl, T-LGL, CLL)
- Drugs, inc recombinant EPO (rare)
- MDS
Protein-calorie malnutirion (kwashiorkor)
- ABOi alloSCT
- rarely pregnancy (resolevs on dleivery)
PRCA Morphology
PB
- normocytic, normochromic anaemia w absence of polychromasia
- DB - macrocytosis +/- neutropenia, thrombocytosis
- TEB - mild neutropenia and mild thrombocytosis
-PB19 - features of underlying defect.
BM
- reduced maturing erythroid cells
proerythroblasts may be present in normal or increased numbers
- DB - minimal maturation of proerythroblasts. Increased haematagones/lymphocytes
- TEB - left shifted granulopoiesis. May have maturation arrest at myelocyte stage if assoc neutropenia
-PB19 - giant proerythroblasts with prominent nucleoli and nuclear inclusions (many times the size of normal proerythroblasts). IHC to show parvovirus antigens
Investigations of PRCA
- FBE, film, reticulocytes
- EPO level
- DAT
- Nutritional screen (B12, iron, folate)
-ADA and genetic testins (DBA) - Flow cytometry (LGL)
- Autoimmune screen
- SPEP,SRFLC, immunoglobulins
- Viral serology - Parvovirus, viral hepatitis, HIV, EBV, CMV
- Drug history
PB19 Serology not adequate as most patients who have chronic parvo infection are immunocompromised –> PCR for parvo DNA
Additional tests including lymphoid flow, CG (MDS)
Management of PRCA
Treatment of specific causes
- MDS, LPD, thymoma resection
Transient causes - observe 3-4weeks
- PB19, drugs, pregnancy, ABOi SCT
Consider immunosuppression if >1 month
- prednisolone –> consider ciclospoirn
- PB19 –> IVIg
Diagnostic approach to IBMF
Congential disorders that result in failure of BM to produce adequate haematopoesis > all or a combination of cell lines
Clinical history
> discuss with referring clinical about concerns
Family history
Pt personal history
Abnormal congenital malformations and facies, absent radi, thumb abnormalities
History of bleeding / infection/clotting
History of malabsorbtopn
Birth and neonatal history and complications
Pregnancy history
Patients paramets
FBE > cytopenias may indicate type of syndrome concerns.
(Monocytopenia GATA2)
Film > diagnostic changes
Evidence of ineffective haematopoeiss > pappenheimer, basophilic stippling
Degree of dysplasia
Presence of thrombocytopenia or size of platelets
Ancillary tests
Infective screen
PNH screen > inherited rarely have PNH clone > this is a feature of acquired disorder
Faecal elastase > SDS with malabsorption
FLOW/FISH telomere testing > telemore disorders
Chromosomal fragility > fanconi anaemia
Red cell ADA > diamond blackfan > also HbF raised
SCN
Consider infective serolgy that may confound
Transient erthroblastopenia
Parvovirus
Bone marrow biopsy
- Assess cellularity
- Check for dysplasia
- Megakaryocyte atypia
Cytogenetics (monosomy 7)
IBMF specific NGS panel > offered at reference laboratory
Look at fanconi genes etc.
DDX41, GATA1, RUNX1
Dedicated genetic testing and sequencing
Bone marrow failures - Ddx
Acquired: AA, MDS
Inherited: germline predisposition to myeloid malignancy, IBMF syndromes
Autoimmune: HLH, SLE
Infection: viruses, sepsis
Malignancy: solid and haematologic
Drugs/toxins/nutritional: B12, folate, Cu, Zn, anorexia, alcohol, medications
Summary slide for IBMF
Fanconi Anaemia
Most common IBMFD
Pathogenesis:
- >23 genes involved
- defective DNA repair leading to genomic instability
-biallelic mutations –> inability to repair and apoptosis
-single allele mutations –> reduced gene product (increased risk of breast/ovarian Ca in the case of FANCD1 = BRCA2 and FANCS = BRCA1)
Lab findings:
- Macrocytosis
- Inc HbF
- BM Aplasia (80% BMF by 20)
- predisposition to MDS/AML
Clinical features:
- short stature/microcephaly
- triangular facies
Investigations:
- PB lymphocytes/skin cells for chromosomal breakage analysis
> DNA cultures with T cell mitogen to stimulate lymphocyte division. Stress the DNA and looking for breaks and rearrangements
>Done in Royal Brisbane
> reported as % of cells with abberations
- Cytogenetics: monosomy 7, Gain 1q, Gain 3q
- Molecular: IBMF panel.
FANC gene or FANCB, FANCR
TREATMENT
- BM failure w androgens (Danazol)
SCT only curative option
- Outcomes better if BEFORE onset of leukemia.
Diamond Blackfan Anaemia
Ribosomopathy caused by mutations in ribosomal proteins
Most AD with variable penetrance
Presentation:
Usually < 1.yo
Elevated Red cell ADA (can be diagnostic)
Elevated HbF
Macrocytic anaemia, mild other cytopenias
reticulocytopenia
BMAT - normocellular but reduced erythroids
Molecular - RPS19, Other RPL genes
Clinical fx:
- half may have no congenital abnormalities
- thumb and radius defects
- heart defects
- increased no-haem cancer risk
TREATMENT:
Steroids improve anaemia in majority of patients
Supportive care (transfusion) preferred until the age of 12/12 to reduce exposure (growth) and allow for vaccine responses
-40% remain dependent on transfusion
AlloHSCT curative
-best performed in young patients without iron overload
-however need to balance with potential for spontaneous remission
Schwachman Diamond Syndrome
Ribsosomal biogenesis disorder caused by mutations in SBDS (7q11) .
SBDS crucial role in joining 40S and 60S ribosomal units to form 80S ribosome.
90% biallelic
Autosomal recessive
Others: DNAJC21, ELF1
Haem presentation
- variable neutropenia
- anaemia or macrocytosis
- thrombocytopenia
- BMA - hypocellular, leukemia
Other clinical features:
- GI insufficiency - FAECAL ELASTASE
- skeletal deformities
- immune/cognitive/endocrine dysfunction
Molecular:
- close haem f/u and cytogentic surveillance
- isochrome 7q and del20q
- tp53 commonly mutated
TREATMENT:
- supportive with oral enzyme replacement, GCSF, transfusion
AlloSCT only curative option
Telomere BIOLOGY disorders
Ends of DNA are short or damaged - these ends (telomeres) protect the DNA from damage
Pathogenesis :
- long TTAGGG repeats at chromosome ends, shorten with each cell division.
Many pathways maintain telomere length
- Abnormal ageing of BM stem cells through defective telomere maintenance
Genetics
DKC1 –> 40% of cases
-XLR (x linked recessive)
-encodes dyskerin
-telomere maintenance, ribosome biogenesis
-associated with early childhood onset, multisystem disease
TERT –> up to 7%
-AD or AR
-enzymatic component of telomerase complex
TERC –> 5%
-AD
-internal RNA template strand
Haem presentation:
- BMF –> MDS/AML
Clinical presentation:
- Triad - nail dystrophy, leucoplakia, abnormal skin pigmentation/rash
Organs - pulmonary fibrosis, liver cirrhosis, hepatopulmonary/syndrome
Ix:
Telomere length testing with telomere flow FISH (Westmead, not rebatable)
- reduced length <1st centile in lymphocytes.
- peptide nucleic acid probes to telomeres (for –> flow (analysis by signal strength and subset markers)
TREATMENT
- androgens (danazol/oxymetholone)
- alloSCT
- Poor prognosis with mean survival of 30 years
GATA2 deficiency
Transcription factor important for early haematopoiesis
Germline heterozygous mutations –> distinct clinical syndrome
Acquired AA
Haem:
Cytopenias presenting with assoc infection - MONOCYTOPENIA
PB: monocytes/B cells and NK cells reduced
- Hypocellular marrow with meg dysplasia and absent B haematogones
- AML 90% risk
Cytogenetics:
monosomy 7
trisomy 8
(risk MDS to AML)
Myeloid NGS:
acquired ASXL1 most common
GATA2 sequencing
Treatment
- alloSCT
SAMD9 & SAMD9L
tumors suppressor gene
7q - Gain of function heterozygous mutations
7q Suppression of haematopoiesis
Preferential loss of 7q when evolves –> protects from growth restriction but clonal evolution to MDS
Very common cause of monosomy 7 MDS
MIRAGE Syndrome
-Myelodysplasia
-Infections
-Restrictions of Growth
-Adrenal hypoplasia
-Genital Abnormalities
-Enteropathy
SAMD9L
BM failure/cytopenias similar to GATA2
-B/NK deficiency
-progression to MDS with del7q
Plus ataxia (cerebellar dysfunction)
Must check NON-HAEM sample for SAMD9/SAMD9L in del7q MDS
-loss of germline abnormality (7q) due to preferential loss
Myeloid neoplasms with germline predisposition
And new ones
SAMD9/SAMD9L (with pre existing organ dysfunction)
and TP53 (without pre existing plt disorder or organ dysfunction)!
DDX41
Located on 5q
Encodes a DEAD box RNA helicase
Likely to be involved in splicing of pre-mRNAs
Role in malignancy and immunity
What are myeloid neoplasms assoc with germline predisposition
Myeloid neoplasms associated with germline predisposition include AML, MDS, MPNs and MDS/MPN that arise in individuals with genetic conditions associated with increased risk of myeloid malignancies.
Heterogenous group of disease caused by deleterious germline pathogenic variants in genes that affect critical biological pathways.
They are divided into categories - those with/without pre existing platelet disorder or organ dysfunction
Severe Congenital Neutropenia
3-8/million prevalence
Impaired differentiation of neutrophils
Neuts <0.5
BMAT -> maturation arrest at promyelocyte stage
Rx: G-CSF to get neut >1
