Validating assay in JAK2 wild type ET/PMF samples

4.6.1 Demographic details and diagnoses

A critical aspect of validation was to confirm that the assay would discriminate mutant CALR products from wild type. To achieve this, the assay needed to be performed on samples with varying CALR mutations. As no suitable reference material was available, the published data was used to identify a group of samples which would be most likely to have mutations present. The literature indicated that CALR mutations were found in a significant proportion of patients with JAK2 V617F wild-type ET or PMF (Nangalia et al., 2013) and so a group of patients with these diagnoses were identified from laboratory records.

Sample group 1 (Figure 3-6 and section 3.5.1) was used to validate the assays ability to detect mutations in CALR. As described in 3.5.1, this group consisted of a cohort

of 48 patients with a confirmed diagnosis of ET or PMF, but where no demonstrable mutation in JAK2 V617F or MPL had been identified. In the absence of such a mutation, this diagnosis could only be made by morphological assessment of bone marrow and therefore, archived DNA from bone marrow was available for testing. In addition, all 48 cases also had DNA stored from a peripheral blood sample

received alongside the bone marrow sample. The patient characteristics are shown in Table 4-1 .

ET PMF

Number 42 6

Median age (years) 64.6 71

Male 23 (52.3%) 5 (83.3%)

Table 4-1. Summary of patients in sample group 1 - for the validation of CALR mutation screening assay.

4.6.2 Identification of CALR mutations

DNA from all samples was amplified with both CALR and JAK2 primers in a single PCR reaction. The fluorescent PCR products were analysed using Genemapper fragment analysis software (ABI, ThermoFisher). CALR mutations were identified in a total of 24 patients (50%). The characteristics of these mutations are shown in Table 4-2, with corresponding electrophoretograms shown in Figure 4-2. Of note, the most frequently seen mutation was the 52bp deletion, followed by the 5bp

insertion – which is in keeping with published data (Chen et al., 2014; Klampfl et al., 2013b; Nangalia et al., 2013).

Complete concordance was seen between peripheral blood and bone marrow samples for the same patient. The majority of referrals for the investigation of a suspected MPN are peripheral blood samples and JAK2 V617F screening has been routinely performed on peripheral blood since its introduction. The ability to perform CALR

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mutation screening on the same specimen was a key aspect of incorporating the assay into routine laboratory use.

Figure 4-2 Results of fluorescent fragment analysis obtained during assay validation. Each panel showing a distinct mutation, the wild-type CALR peak is shown in the highlighted box. The second green peak shows the mutated CALR PCR product and its relative size (number identified shown in brackets).

(A) 5bp insertion (n=8), (B) 10bp deletion (n=1), (C) 20bp deletion (n=1), (D) 22bp deletion (n=1), (E) 32bp deletion (n=1), (F) 52bp deletion (n=12).

Mutation Number

of cases

Diagnosis Gender Median age

(years) ET PMF M F 5bp insertion 8 7 1 4 4 64.54 10 bp deletion 1 1 0 1 0 64.30 20 bp deletion 1 1 0 1 0 62.06 22 bp deletion 1 0 1 1 0 63.02 32 bp deletion 1 0 1 1 0 47.63 52 bp deletion 12 11 0 8 4 67.78

Table 4-2. Characteristics of CALR mutations in the validation group (sample group 1).

4.6.3 Increased sensitivity of JAK2 V617F assay

This innovative approach showed a higher level of sensitivity over the existing assay. All samples used in the validation assay had been originally reported as JAK2 wild type. Following analysis using the fluorescent fragment analysis approach, one case showed the presence of a JAK2 V617F mutation in the DNA from both the peripheral blood and bone marrow sample. The sensitivity of agarose gel electrophoresis had been previously estimated to be 2-3% allelic burden in our hands, whereas fluorescent fragment analysis is estimated to be capable of

identifying a much lower level of mutated DNA, ~1% allelic burden. The increased sensitivity of the fluorescent assay would account for this discordant result and as it was expected that clinicians would want existing patients to be investigated for CALR mutations, this would be taken into consideration when analysing and reporting assay results.

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4.6.4 Inter-laboratory validation

HMDS has long standing collaborations with several groups within the UK, including one with Professor N Cross of the Wessex Regional Genetics Service, Salisbury, UK. A series of 322 DNA samples from JAK2 V617F wild type MPN patients diagnosed by HMDS had been provided by for Genome Wide Association Studies (GWAS) in Salisbury. As the group performing this analysis had also characterised the CALR mutation (Nangalia et al., 2013) the samples provided by HMDS had also been analysed for the presence of CALR mutations. This included 30/48 patients selected for test validation. The results of mutational screening in HMDS was 100% concordant with the findings from the GWAS.

A sample exchange was requested by Dr I Carter from Nottingham City Hospital who was in the process of setting up an assay using the same CALR primers and a similar approach. DNA samples from 20 patients were exchanged between our two centres. Concordance was 97.5% (39/40 cases), with Dr Carter detecting a 5bp insertion in one sample which was not detected using the assay developed in this work. Following discussions, it was found that there was a small difference between our techniques. In Nottingham, the JAK2 V617F and CALR amplifications were being performed in two separate PCR reactions, whereas I had amplified both targets in a single multiplex reaction. It was hypothesised that a multiplex approach may have a reduced sensitivity compared with single target amplification. The mutation was detectable when the two targets were reamplified separately and mixed prior to fragment analysis and therefore, this approach was adopted for all future testing.