DR TANJINA AFRIN

FCPS PART-II

COURSE TRAINEE

Presenter:

Presenter:

DR TANJINA AFRIN

FCPS PARTII COURSE TRAINEE

Hayeong Rho,¹ *Richard A Wells^1,2

Faculty of Medicine, University of Toronto, Toronto, Canada ,2018

A Game of Clones: �The Complex Interplay of Aplastic Anaemia, �Myelodysplastic Syndrome and �Paroxysmal Nocturnal Haemoglobinuria

• Hayeong Rho,¹ *Richard A Wells^1,2
• Faculty of Medicine, University of Toronto, Toronto, Canada ,2018

A Game of Clones:

The Complex Interplay of Aplastic Anaemia , Myelodysplastic Syndrome and

Paroxysmal Nocturnal Haemoglobinuria

INTRODUCTION

• Bone marrow failure (BMF) occurs when the production of blood cells of one or more lineages by the bone marrow (BM) diminishes to such an extent that cytopenia ensues.
• Severe BMF is life- threatening, owing to the risks of infection and haemorrhage.
• Failure of haematopoiesis can result from a variety of causes.
• Three acquired BMF syndromes: aplastic anaemia, myelodysplastic syndrome, and paroxysmal nocturnal haemoglobinuria (PNH). These syndromes appear, on the surface, to be distinct disorders, but have been revealed to be closely interconnected.

​

PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA, APLASTIC ANAEMIA AND MYELODYSPLASTIC SYNDROME: �DISORDERS OF �BONE MARROW FAILURE WITH DISTINCT PATHOBIOLOGY

​

PNH

• Approximately 1–2 people per million per year are diagnosed with PNH, a rare clonal disorder of haematopoietic stem cells (HSC).

​

• PNH is characterised by intravascular haemolysis, pancytopenia, and thrombosis, and, although it is considered a benign disease, can be life-threatening.

​

​

Two kinds of membrane proteins: transmembrane and glycosyl phosphatidyl inositol (GPI)-linked. The latter are anchored to cell membranes through a covalent attachment to a glycosyl phosphatidyl inositol moiety. In PNH, GPI cannot be synthesized, leading to a global deficiency of GPI-linked membrane proteins

Three-step model for the development of paroxysmal nocturnal hemoglobinuria (PNH).

Step 1, Somatic mutation of PIG-A in a hematopoietic stem cell.

Step 2, Immunologic attack to hematopoietic stem cells decreases the number of stem cells, resulting in expansion of the PIG-A mutant clone.

Step 3, The second somatic mutation occurs in the PIG-A mutant, leading to further expansion and generation of a large number of glycosylphosphatidylinositol (GPI)-anchor–deficient blood cells. RBC indicates red blood cells; PMN, polymorphonuclear leukocytes

PNH

• In PNH, somatic mutation of PIGA occurs in a single HSC, resulting in failure of biosynthesis of the glycophosphatidylinositol (GPI) anchor. This structure serves as the attachment point for diverse cell-surface proteins, including the complement defence proteins CD55 and CD59.
• PNH clone can be detected by highly reliable flow cytometry assays in peripheral blood.

​

PNH

• Expansion of the PIGA-mutant HSC clone leads to production of blood cells that lack CD55 and CD59 and are, therefore, very sensitive to lysis by complement proteins, resulting in intravascular destruction of RBC and
• depletion of nitric oxide, because this compound is scavenged by free haemoglobin.
• Reduction of the plasma nitric oxide concentration, combined with direct interactions between the complement and coagulation pathways, lead to the clinical complications of PNH: fatigue, dysphagia, abdominal pain, pulmonary hypertension, kidney injury, and a virulent hypercoagulable state.

​

PNH

• Two hypotheses exist as to how PNH HSC clones survive and expand within the BM:
1. Relative growth advantage (intrinsic)

and

• Immune escape (extrinsic)hypotheses.

​

Aplastic anemia (AA) is defined by pancytopenia with a hypocellular bone marrow in the absence of an abnormal infiltrate and with no increase in reticulin.

ACQUIRED CAUSES

• In acquired AA, it is proposed that an inciting event, such as a virus or drug provoke aberrant immune response, triggering an oligoclonal expansion of cytotoxic T-cells that destroy haemopoietic stem cells.

INHERITED CAUSES

PATHOPHYSIOLOGY

Normal bone marrow micro environment

Pathogenesis: Immune mediated

Increased production of interleukin-2 leads to polyclonal expansion of T cells. Activation of Fas receptor by the Fas ligand leads to apoptosis of target cells.

MYELODYSPLASTIC SYNDROME

• Myelodysplastic syndrome (MDS) is a heterogeneous group of malignant clonal BM disorders characterised by dysplasia of haematopoietic cells in the BM and peripheral blood, and ineffective haematopoiesis, leading to BMF and cytopenias, and progressing to acute myelogenous leukaemia (AML) in 25% of patients.
• MDS is more common than PNH and AA, with an incidence of 50 persons per million per year.

​

MYELODYSPLASTIC SYNDROME

• Although MDS is typically associated with BM hypercellularity, in ˜10% of cases the BM is hypocellular. This variant, known as hypocellular MDS (hypoMDS), is clinically and pathologically similar to AA (with the exception of the presence of dysplasia) and responds similarly to immunosuppressive treatment.

​

INTER-RELATIONSHIP BETWEEN PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA, APLASTIC ANAEMIA, AND MYELODYSPLASTIC SYNDROME: COMMON ASSOCIATIONS AMONG RARE DISEASES

Aplastic Anaemia and Paroxysmal Nocturnal Haemoglobinuria

• AA is strongly associated with PNH. When highly sensitive flow cytometry protocols are used, PNH cells are detectable in 40–59% of AA patients.
• No correlation is seen between the presence or absence of PNH cells and the severity of AA.
• However, the presence of PNH cells is predictive of both response to immunosuppressive therapy and overall survival.

​

The behaviour of PNH clones in AA patients who undergo immunosuppressive therapy is;

• Because of the risk of development of clinically important PNH, as well as the predictive and prognostic value of the detection of a PNH clone, flow cytometric analysis for the presence of PNH cells is recommended for all patients diagnosed with AA.

​

Myelodysplastic Syndrome and Paroxysmal Nocturnal Haemoglobinuria

• A strong association also exists between PNH and MDS,
• The proportion of MDS cases in which a PNH clone is detectable is much smaller (at ˜2-5%); as for AA, PNH clones found in association with MDS tend to be small (<1% in 45% of cases).

​

Myelodysplastic Syndrome and Paroxysmal Nocturnal Haemoglobinuria

• The strength of this association is stronger for the hypocellular variant of MDS. While hypoMDS accounts for 10% of MDS, and a PNH clone can be detected in 40% of cases of hypoMDS.¹⁵
• As for AA, the presence of a PNH clone in MDS has been reported to be predictive of response to IST, although this association has not been evident in all studies.

​

​

​

• Although AA is an autoimmune disease, it has long been understood that it possesses some clonal characteristics. Clonal cytogenetic abnormalities are observed in 10–15% of AA patients, and in 15% of AA cases patients go on to develop the indisputably clonal diseases MDS or AML.

Myelodysplastic Syndrome and Paroxysmal Nocturnal Haemoglobinuria

DYNAMICS OF PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA IN BONE MARROW FAILURE:�A GAME OF CLONES

​

• For the persistence and expansion of PNH clones, two hypotheses have arisen: the ‘intrinsic growth advantage’ hypothesis and the ‘immune escape’ hypothesis.
• Consequently, the immune escape hypothesis gained favour. This hypothesis (Figure 1A) postulates that, in the context of T cell-mediated autoimmune attack on normal HSC, PIGA-mutant HSC gain a competitive advantage because they are less vulnerable to immune attack owing to loss of the GPI and its associated proteins.

​

• Recent data favours the intrinsic growth advantage hypothesis. Of 60 patients with a diagnosis of classical PNH, 24 were found to have somatic mutation of genes other than PIGA. These data supported an analysis of the clonal dynamics of PNH wherein additional mutations are present, of which three categories were defined.
• 1. In the majority of cases, a PIGA mutation arose as a secondary event, creating a PNH subclone of an ancestral HSC clone
• 2. In other cases, the PIGA mutation was the founding event, and acquisition of myeloid mutations led to clonal evolusion.

• 3. or the PIGA and myeloid-mutant clones were independent of one another.
• These observations bolster the intrinsic growth advantage hypothesis by pointing to the possibility that PNH clones could gain a growth advantage by hitchhiking with certain myeloid mutations

​

​

The immune escape hypothesis is consistent with the coexistence of PNH clonal haematopoiesis in AA but can not favour the expansion of PNH clone size that is frequently seen following treatment of AA with IST.

This can be explained by intrinsic growth advantage hypothesis.

​

• New data from a study reveal Clone expansion was seen only in patients who had detectable PNH cells prior to IST and was less frequent among patients treated with ATG than those treated with less intensive immune suppression.

​

• This supports a model in which successful and complete immunosuppression results in the disappearance of AA-associated PNH clones, but partially effective treatment supports their outgrowth. It should be noted that these data are consistent with the expansion of a residual HSC possessing both a PIGA mutation and a myeloid mutation, such as the common TET2 mutation, which confers a clonal advantage.

​

• MDS is a disease characterised by clonal dominance but also, particularly in its hypocellular variant, is in part immune-mediated.

​

• The persistence of small PNH clones in MDS may largely be sustained by immune escape, a notion supported by the observation that the presence of a PNH clone predicts response to IST.

​

• An MDS clone that already possesses an intrinsic growth advantage acquires a PIGA mutation, resulting in a persistent PNH subclone

TET2 and PIGA mutations

Figure 1: Depictions of the immune escape and intrinsic growth advantage hypotheses for the persistence and expansion of paroxysmal nocturnal haemoglobinuria clones.

A) The immune escape hypothesis. A GPI-dependent T cell-mediated autoimmune attack applies selective pressure on the HSC population. Lacking GPI and its associated proteins, rare PIGA-mutant HSC are relatively resistant to the attack and undergo clonal expansion.

​

​

TET2 and PIGA mutations

​

. B) The intrinsic growth advantage hypothesis. An HSC that already carries a myeloid mutation that confers a growth advantage acquires a PIGA mutation, resulting in the creation of a PNH subclone.

GPI: glycophosphatidylinositol; HSC: haematopoietic stem cells; MDS: myelodysplastic syndrome; PNH: paroxysmal nocturnal haemoglobinuria.

Figure 2: The interplay between the intrinsic and extrinsic influences on paroxysmal nocturnal haemoglobinuria clonal expansion.�

• In AA the dominant influence is T cell-mediated autoimmune attack, which permits the outgrowth of relatively resistant GPI-negative HSC. This mechanism may also apply in MDS, particularly in the hypocellular variant, which is clinically and pathologically very similar to AA. In MDS the presence of myeloid mutations that confer clonal advantage (e.g., by enhancing HSC self-renewal) also allow concurrent PNH mutations to be fixed and expand in the HSC population. Clonal haematopoiesis of indeterminate potential or subclinical autoimmune HSC attack may also provide support through the same mechanisms for expansion of PNH clones, leading to classical PNH, in which PNH clones expand in the absence of clinicopathological evidence of AA or MDS.

​

AA: Aplastic anaemia; CHIP: clonal haematopoiesis of indeterminate potential; GPI: glycophosphatidylinositol;

HSC: haematopoietic stem cells; MDS: myelodysplastic syndrome; PNH: paroxysmal nocturnal haemoglobinuria.

These thoughts may also be applied to classical PNH that arises in the absence of any clinically apparent BMF (Figure 2). Here, the PNH clone must, in order to persist and expand, also gain a competitive advantage over normal HSC.

• In such cases, which account for most cases of PNH, there is always either a concurrent subclinical autoimmune suppression of normal haematopoiesis or clonal haematopoiesis of indeterminate potential/age-related clonal haematopoiesis. In the former scenario, the PNH clone expands via immune escape and in the latter by intrinsic growth advantage.

​

• The presence of an intrinsic or extrinsic competitive advantage over normal HSC is a necessary condition for the persistence and expansion of PNH clones, then

​

​

• The application of advanced diagnostic techniques might support refined or novel management approaches in classical PNH.
• For example, patients who are shown by genomic analysis to have a PNH clone that also carry myeloid mutations would be followed more closely for the development of MDS or AML and may be advanced as candidates for curative treatment with allogeneic stem cell transplantation.
• Conversely, for patients with classical PNH in whom no myeloid mutations were found, it might be deduced that an underlying T cell-mediated

• Conversely, for patients with classical PNH in whom no myeloid mutations were found, it might be deduced that an underlying T cell-mediated autoimmune attack on HSC had supported the expansion of the PNH clone. In such patients, it may be predicted that aggressive IST could lead to the eradication of the condition supporting the persistence and expansion of the PNH clone by removing it, thus potentially curing the disease and removing the need for lifelong anticomplement therapy.

​

Scenario 1

• A 33-year-old man, with a history of severe aplastic anaemia diagnosed 8 years ago
• successfully treated with IST,
• Now presents with dark urine, dyspnoea, and severe anaemia.
• Initial investigations reveal pulmonary embolism and iliac vein thrombosis.
• His lactate dehydrogenase is elevated 9-times the upper limit of the normal range.
• Flow cytometry for PNH reveals GPI-deficient white blood cells (WBC) (FLAER-/CD157-): CD15+ neutrophils: 54.7%; CD64+ monocytes: 76.3%. Total GPI-deficient RBC (CD235a+/CD59-): 22.5% (Type III RBC: 14.4%; Type II RBC: 8.1%).

​

Scenario 1

• What is the importance of testing for the presence of PNH cells in all patients diagnosed with aplastic anaemia?

�Scenario 2

• A 50-year-old man who is otherwise healthy and takes no medication presents with pancytopenia.
• Investigations, including a BM examination, lead to a diagnosis of severe aplastic anaemia.
• Flow cytometry for PNH reveals the presence of a small clone GPI-deficient WBC (FLAER-/CD157-): CD15+ neutrophils: 1.3%; CD64+ monocytes: 1.5%. Total GPI-deficient RBC (CD235a+/CD59-): 0.01% (Type III RBC: <0.01%; Type II RBC: <0.01%).

​

Scenario 2

• What will be the best first-line therapy for this patient ??

Scenario 3

• A 57-year-old woman presents with pancytopenia (red cell transfusion dependence, platelets <20x10⁹/L and neutrophils <0.5 x10⁹/L).
• BM examination reveals multilineage dysplasia with markedly decreased BM cellularity (10%) and normal karyotype.
• The International Scoring System risk category is intermediate-1, and the Revised International Scoring System risk category is intermediate.
• Flow cytometry testing for PNH shows GPI-deficient WBC (FLAER-/CD157-): CD15+ neutrophils: 2.5%, CD64+ monocytes: 2.8%. Total GPI-deficient RBC (CD235a+/CD59-): 1.04% (Type III RBC: 1.03%; Type II RBC: 0.01%).

​

Scenario 3

• Patients with lower risk MDS who experience multiple cytopenias what will be the treatment options??

Conclusion

The detection of a PNH clone in AA or MDS patients is of established clinical value, since the presence of even minor populations of PNH cells in patients with AA or MDS is an important predictor of a higher rate of response to IST and superior overall survival.

This is important in lower-risk MDS patients, in whom active therapeutic options are limited.

In addition, PNH clones in AA may expand and cause life-threatening PNH. Therefore, identification and surveillance of PNH clones is recommended in all patients diagnosed with AA

THANK YOU