Changes to the molecular diagnostic pathway for MPN patients
Whole exome analysis has recently identified mutations affecting exon 9 of the calreticulin gene
(CALR) as high frequency events in patients with essential thrombocythaemia and myelofibrosis.
The identification of recurrent indels in this region has the potential to improve the molecular
diagnostic pathway for essential thrombocythaemia and myelofibrosis, with up to 70% of patients
having mutations identified in JAK2, MPL or CALR.
In light of these recent findings the molecular haematology department at RIE has introduced a
new capillary fragment analysis method for detecting insertion and deletions affecting exon 9 of
CALR and this has now been validated for use as a clinical assay. This is in keeping with the
recommendation by the Molecular Pathology Evaluation Panel (Scotland) that CALR screening be
introduced to the clinical diagnostic pathway for MPNs.
In addition to the new CALR exon 9 mutation screening, we have implemented a new, more
sensitive real time PCR assay for detecting JAK2 V617F; we also now have an in-house high
resolution melt curve analysis for detecting JAK2 exon 12 mutations and will soon be able to offer
MPL exon 10 screening using the same technology. Further details of these new assays, including
sensitivity and reportable results are included on pages 3 and 4 of this document.
It has been agreed that patients with putative MPN will be entered into an ‘MPN screening panel’
pathway when samples are referred for molecular investigation. Patients will be fully investigated
for the clinically relevant mutations, including JAK2 V617F and exon 12 mutations for
?polycythaemia vera patients; and JAK2 V617F, CALR exon 9 and MPL exon 10 mutations for
?essential thrombocythaemia or ?myelofibrosis patients. Patients will be fully investigated
through the appropriate pathway (see Figure 1 on page 2) and a final report will be issued with the
combined results at the end of the investigative pathway.
In order for this new system to function effectively only patients with clinical information clearly
stating that they are being referred for molecular investigation of a MPN and with phenotypic
details (i.e. ?ET/?MF or ?PV and indication if EPO levels are available or awaited) will be entered
into the pathway. Those who are referred with incomplete information, for other reasons (e.g.
abdominal thrombus) or who have not had reactive causes excluded will only have JAK2 V617F
mutation screening and the DNA will be stored should more clinical information highlight a reason
for further testing.
To communicate regarding patient cases and queries regarding the service please direct
enquiries to our new email address – Molecular.MPN@nhslothian.scot.nhs.uk and a member of
specialist staff will respond.
Figure 1. Molecular MPN investigative pathway at RIE
JAK2 V617F Assay
As of 1st April 2014, the Molecular Haematology laboratory at RIE changed the method of
detecting JAK2 V617F mutation from an allele-specific PCR (based on Baxter et al, 2005) to a realtime PCR based method (based on Larsen et al, 2010).
This new method has been extensively validated and is more sensitive, with a reportable
sensitivity of approximately 1% allele burden in this department. This means we can accurately
and consistently detect abnormal clones to this level.
There will be three possible reportable outcomes from this new assay:
“JAK2 V617F Positive” – Detectable amplification of the mutant V617F allele above 1%.
“JAK2 V617F Not detected” – No mutation detected at the reportable level of sensitivity of this
assay.
“JAK2 V617F Equivocal result” – Low level detection of the mutant allele below the level of
sensitivity for this assay which may represent a true low level abnormal clone. Repeat analysis is
advised in 3-6 months. As described in the guidelines for molecular diagnosis of the
myeloproliferative neoplasms (Bench et al, 2012) “a result of <1-3% V617F should be interpreted
in the context of clinical, morphological, haematological and other laboratory findings”.
Where appropriate, ?PV patients who are JAK2 V617F negative will be referred for JAK2 exon 12
analysis prior to issuing the final report and ?ET/?MF patients will be referred for CALR exon 9 and
MPL exon 10 screening prior to issuing the final report. We will be moving to a combined report
form which will show results from all molecular investigations undertaken within the Molecular
Haematology laboratory at RIE. This means that cumulative JAK2 V617F results will no longer be
printed. In instances where there is a change in reported mutation status this will be commented
upon in the new report.
CALR exon 9 mutation screening
A molecular method for detecting insertions and deletions in exon 9 of the CALR gene has been
validated for clinical use and involves capillary fragment analysis using fluorescent primers, based
on published methods by Klampfl and colleagues (2013). This method will accurately detect
clones of ≥2% in a background of wild type DNA. Mutation positive samples will be confirmed
using direct DNA sequence analysis, where possible. CALR exon 9 mutation ‘not detected’ patients
will automatically be referred for MPL exon 10 analysis prior to issuing the final report.
JAK2 exon 12 and MPL exon 10 mutation screening
An in-house method has been developed and validated using High Resolution Melt analysis to
screen for mutations in exon 12 of JAK2 based on the work of Ugo et al (2010). Any samples which
are suggestive of the presence of a mutation are confirmed and classified using direct sequencing
where possible. The same approach is currently being validated for investigation of MPL exon 10
in patients with ?ET/PMF (based on Boyd et al, 2010) where no JAK2 V617F or CALR mutations
have been identified. The sensitivity of these assays is approximately 5-10% dependent on the
type of mutation present.
References
Baxter, E. et al (2005). Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative
disorders. Lancet: 365; 1054-61
Larsen T.S. et al (2006). The JAK2 V617F mutation involves B and T lymphocyte lineages in a
subgroup of patients with Philidelphia chromosome negative chronic myeloproliferative disorders.
BJH 136;745-751
Bench A. J. et al (2012). Molecular diagnosis of the myeloproliferative neoplasms: UK guidelines
for the detection of JAK2 V617F and other relevant mutations. BJH 160;25-34
Klampfl T. et al (2013). Somatic mutations of calreticulin in myeloproliferative neoplasms. NEJM
369;2379-90
Ugo V. et al (2010). Interlaboratory development and validation of a HRM method applied to the
detection of JAK2 exon 12 mutations in polycythaemia vera patients. PLos one, 5(1)ppe8893
Boyd E. M. et al (2010). Clinical utility of routine MPL exon 10 analysis in the diagnosis of essential
thrombocythaemia and primary myelofibrosis. BJH 149;250-257