The Indian Ocean Rim laboratory haematology conference in Fremantle, Western Australia showcased some of the superb and exciting research on myeloproliferative neoplasms (MPNs) being undertaken both internationally and by some of our Australian haematologists and researchers led by Professor Wendy Erber, Associate Professor Kathy Fuller and Dr Belinda Guo.
The conference was hosted by Professor Erber. It was wide ranging and included a symposium on MPNs. Jenny Myers attended on behalf of the MPN AA.*
The presentations included:
- Dr Jyoti Nangalia – myeloproliferative neoplasms- from origins to outcomes
- Professor Wendy Erber – Morphology of MPNs
- Dr Belinda Guo – Platelets in myeloproliferative neoplasms: potential as a novel disease biomarker
- Mr Aidan Yuen-Oye – GATA-1 overexpression promotes megakaryocyte hyperplasia in MPNs
Dr Jyoti Nangalia – Dr Nangalia is a clinical haematologist and scientist who focuses on using sequencing technologies to understand the evolution of haematological cancers, with an emphasis on understanding how early myeloid neoplasms originate and evolve and how this relates to normal development and ageing. She discovered the Calr mutation in MPNs.
Dr Jyoti Nangalia’s presentation had a significant focus on genomics in MPNs.
What causes myeloproliferation?
– location of JAK2 mutation produces variability of symptoms- either erythrocytosis (high red cells) or thrombocytosis (high platelets).
– location of MPL explains why MPL patients don’t get erythrocytosis but rather thrombocytosis or even fibrosis.
– CALR – due to the location of mutation has potential to be targeted (with treatment).
– Triple negative MPNs ….often 1/3 actually found to have a mutation after more sensitive screening. Sometimes have other mutations of myeloid genes. But in 2/3 of patients, nothing has yet been found as a driver. These patients tend to be young and female, and have a benign prognosis, although they can have thrombosis; but their diagnosis remains to be determined.
Disease biology and differing levels of symptoms
Degree of erythrocytosis or thrombocytosis seems to depend on thresholds, sensitivities of mutations and interactions with the different receptors:
– higher allele burdens of JAK2 lead to Polycythaemia vera (PV).
– JAK2 can be homozygous (two copies of the mutated gene) or heterozygous (one copy) but if a high allele burden is present it’s homozygous.
– JAK2 Exon 12 mutation only causes erythrocytosis.
Germ-line SNPs (single nucleotide polymorphisms) that we’re all born with can predispose patients to homozygosity. Other factors could be kidney function, erythropoietin levels, coexistent thalassaemia.
– If high platelet count -more likely to get a diagnosis of essential thrombocythaemia (ET)
– If high haemoglobin, more likely to get a diagnosis of PV.
But as these can have different disease biology, maybe a molecular classification would be more appropriate.
With myelofibrosis (MF)– the situation is a bit different as there are often additional mutations.
Classification and prediction of outcomes
The more mutations and type of mutations a patient has, suggest a higher molecular risk category- 40% of MPN patients only have one of the three driver mutations and no others. This is very rare in cancer. eg solid tumours have hundreds of thousands of mutations. Age sees increased mutations.
Patients can be grouped into various genetic categories
– JAK2 mutated patients – whether patients have ET or PV, it’s all the same – treatment just focuses on reducing thrombotic risk.
– TP 53 mutation – prognosis poorer
– Patients with additional mutations – prognosis poorer.
Perhaps myelodysplastic syndrome (MDS) and MF can sometimes be the same disease depending on how they present.
If dysplasia, diagnosis is MDS or if fibrosis, MF diagnosis – but maybe these aren’t entirely correct.
How to predict outcomes
A prognostic model for predicting individual patient outcomes is in development.
– biggest risk factor is age.
– absence of other genetic markers is important prognostically.
– splenomegaly and blood counts also relevant.
Findings from the prognostic model are consistent with those of other models which are based on blood counts, fibrosis, constitutional symptoms etc.
What are the origins of MPNs? How do we work out when mutations are acquired compared to when the patient gets the disease?
Current work with the genome is showing that MPN mutations were acquired much earlier in life than expected. For example,
– 20 year old diagnosed with ET at age 20 …this work shows mutation was acquired at age 4.
– 65 year old ET patient, this work shows the mutation was acquired by age 10.
Implications of these findings are still to be determined. This work is being analysed and written up at the moment.
- Myeloproliferation in MPNs is driven by mutations in drivers in JAK2, CALR and MPL
- The MPN a patient gets depends on the driver mutation and the amount, the germ line factors, additional mutations and patient demographics.
- MPNs can be classified genomically to build personalised outcome models
- Next steps are about determining what drives the mutation and then the subsequent triggering of the disease.
- Also future work will look at how treatment impacts prognosis.
Professor Erber is Executive Dean of the Faculty of Health and Medical Sciences at the University of Western Australia as well as a haematologist practising diagnostic haematology as well as lecturing and undertaking research.
The myeloproliferative neoplasms are a group of clonal stem cell disorders with similarities at the phenotypic and molecular level. Clinically these disorders are characterised by the overproduction of one or more mature myeloid elements and a variable tendency to develop acute myeloid leukaemia (AML). Polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (MF) overlap clinically and with their morphological features. In this presentation Professor Erber focussed on blood film appearances as well as the bone marrow morphology of some MPNs.
PV morphology is quite distinctive and has – hypercellular marrow, pan-myelosis and megakaryocytic pleomorphism, including pyknotic forms which is visible in MF but can also be sometimes seen in PV – and even in PV you can see emperipoieisis.
The marrow of PV caused by the exon 12 mutation looks completely different. Marrow is more erythroid, doesn’t show panmyelosis.
ET or pre-fibrotic myelofibrosis?
The WHO has a classification called prefibrotic MF. ET and PV are chronic phases of MPNs and are at risk of progressing to MF or AML. Professor Erber’s view is that prefibrotic MF is really the accelerated phase of ET or PV and not its own specific category of pre-fibrotic MF.
– 55-65% of ET patients have JAK2 mutation, 20-25% CALR mutation, small number with MPL mutation – all with megakaryocytic hyperplasia and some giant megakaryocytes with hyperlobation of nucleus.
– Bone marrow biopsy shows bone marrow cellularity – generally normal in ET although if JAK2 mutation, can be granulocytic with erythroid expansion.
– Some megakaryocytic expansion and clustering but rest are normal.
– Difficult to see any difference in megakaryocytes between JAK2 and a MPL mutation.
– Triple negative ET – is this ET or reactive thrombocytosis? Morphology is crucial
However the WHO believes distinguishing features of prefibrotic MF are:
– increased cellularity
– myelo-erythroid ratio increased
– tight clusters of megakaryocytes
– size of megakaryocytes: greater variability in prefibrotic MF
– megakaryocyte nuclei are hyperlobated in prefibrotic MF.
The reality however, is that it is difficult to distinguish between ET and prefibrotic MF whereas true MF is clearly distinguishable from this accelerated phase of ET and PV.
Morphology of MF (either primary or end stage PV/ET)
– New bone formation only seen at end stage MF,
– megakaryocytes near trabecular bone – very abnormal
– sinusoidal expansion and haematopoietic elements including abnormal megakaryocytes
– extensive megakaryocytic infiltration
– reduction in haematopoietic space for any haematopoeisis to take place
– reticulin stain is important in showing whether an increase in fibres or not.
Ruxolitinib is used in MF widely to give symptomatic improvement. The potential positive impact of ruxolitinib on bone marrow pathology is still uncertain but some have a view that there are improvements. However it is unclear as yet if disease pathophysiology is changing or whether the significant anti-inflammatory effect is the main reason for differences in the bone marrow.
3. Dr Belinda Guo – Platelets in myeloproliferative neoplasms: potential as a novel disease biomarker
Dr Guo is the Gunn Family National Career Development Fellow in Haematology and Lecturer at the University of Western Australia. She is undertaking a research project with Prof Erber’s team to develop a blood test for earlier detection of progression to myelofibrosis.
Myeloproliferative neoplasms (MPN) are a group of bone marrow cancers where there is an overproduction of blood cells. MPN can occur at any age and all patients have a 20% lifelong risk of progressing to bone marrow failure as a consequence of bone marrow scarring (myelofibrosis). Once scarring develops, patients have a significantly reduced quality of life, require extensive medical care and have a poor prognosis. Early recognition of progression is important for optimal management and outcome, as it would open new windows of opportunity and potential for curative treatment, e.g. allogeneic stem cell transplant.
Although the exact process responsible for fibrotic progression remains unclear, it is well established that megakaryocytes, large cells in the bone marrow that produce platelets, play a role in this process. Changes in the number, size and appearance of megakaryocytes and platelets, are hallmarks of MPN and fibrotic progression. We therefore hypothesise that specific changes in the genetic profile of platelets can enable early detection of bone marrow fibrosis before clinical symptoms are apparent.
We have developed a reproducible and stable method to analyse the genetic content (RNA) of platelets from patients and have obtained compelling preliminary data indicating that these platelets have a unique gene expression profile. Overall, we studied >20,800 genes from the platelets of 74 individuals. We analysed data from 25 MPN patients and 15 controls and identified a large number of genes which had a different pattern of expression in platelets from patients compared to controls and more than 1,000 of these were uniquely differentiated in platelets from patients with marrow fibrosis. From these, a fibrosis-associated platelet genetic signature was identified. The signature was then tested on samples from an independent cohort of 34 MPN patients and was able to discriminate between patients with and without fibrosis with 88% accuracy. This preliminary finding is promising and we are currently refining and validating this signature in a larger cohort of MPN patients. Establishing a blood-based signature will provide an objective approach to measure each patient’s risk of progression and help haematologists make informed treatment decisions before extensive and irreversible damage is done.
Mr Yuen-Oye is a medical student at the University of Western Australia who is undertaking a research project on MPNs with Prof Erber’s team to determine what causes megakaryocyte hyperplasia and progression to myelofibrosis.
Myeloproliferative neoplasms (MPN) are chronic, haematological disorders characterised by an increase in the number of megakaryocytes (hyperplasia) that look abnormal. Transformation to myelofibrosis (MF) is associated with poor prognosis in MPNs, but it is unclear what drives this progression. GATA-1 is a key transcription factor which regulates megakaryopoiesis, the process by which megakaryocytes are formed and produce platelets for normal blood clotting. A deficiency or lack of GATA-1 has been shown in mouse models to promote megakaryocyte production with paradoxical thrombocytopaenia and MF. There is little data pertaining to GATA-1 expression in human megakaryocytes and its role in MPN pathobiology. We investigated the expression of GATA-1 in megakaryocytes of MPN patients and its potential association with transformation to MF.
Methods: GATA-1 expression in megakaryocytes was evaluated in bone marrow trephine biopsy specimens of MPN (n=92) and non-MPN controls (n=39) using immunohistochemistry. The percent megakaryocytes positive for GATA-1 was assessed by microscopy. Comparisons were made between MPN and controls, by MPN subtype (i.e. polycythaemia vera (PV), essential thrombocythaemia (ET) and MF) and mutation profiles (i.e. JAK2V617F, CALR-mutated and double-negative). GATA-1 expression was also correlated with platelet count.
Results: The mean percent GATA-1 positive megakaryocytes in MPN (76.1±11.1%) was significantly higher than controls (67.6±13.7%, p<0.0001). There was no significant difference between MPN subtypes i.e. PV=74.4±11.3%, ET=76.6±11.7%, MF=76.4±10.7%, or between driver mutations (JAK2V617F 76.2±11.5%; CALR-mutated 74.8±14.3% and double-negatives 74.9±8.6%). There was a weak correlation between GATA-1 positivity and platelet count, r=0.21, p=0.01.
Conclusions: GATA-1 is upregulated in megakaryocytes of MPN patients, irrespective of the underlying disease phenotype or mutation status. Its expression also correlates with platelet production. This suggests that GATA-1 overexpression promotes megakaryocytic hyperplasia and is associated with thrombocytosis in MPN. However, it is unlikely to have a direct role in MF transformation. Nevertheless, its overexpression in human megakaryocytes may facilitate the development of an MPN and represents a potential therapeutic target.
* Jenny Myers travelled at her own expense.