Background
The myeloproliferative neoplasms (MPN) are clonal disorders of hematopoietic progenitors that includes the classical chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), chronic eosinophilic leukemia (CEL), chronic myelomonocytic leukemia (CMML), and systemic mastocytosis [
1]. Three distinct phenotypes of PV, ET and PMF mainly constitute to Philadelphia-negative myeloproliferative syndromes [
2].
Genetic alterations in both Janus Kinase 2 (
Jak2) and thrombopoietin receptor myeloproliferative leukemic (
MPL) virus oncogenes serve as molecular targets in the diagnosis of MPN [
3]. Approximately, 60% of patients with MPN carry a non-synonymous substitution (V617F) in exon14 of the
Jak2 [
4]. Among patients with distinct clinical phenotypes, 90% with PV and 60% with ET and PMF also carry these discrete gene mutations. Likewise, other
Jak2 variants in exon 10 and 12 [
4,
5] and non-synonymous MPL variant in exon 10 (W515 L and W515K) [
5,
6] were also reported to occur among 5% of patients with MPN.
Albeit,
Jak2/MPL genetic variants essentially contribute in the diagnoses of MPN, a significant number of MPN patients can be missed in genetic screening due to the absence of these mutations. Nevertheless, 80–85% of
Jak2 V617F negative MPN patients can be diagnosed with a recently discovered distinct frame-shift mutation in exon 9 of the calreticulin (
CALR) gene [
7,
8] which weakens Ca2 + binding affinity of CALR [
9,
10]. Since its discovery,
CALR domain serve as valuable molecular target for the diagnosis of clonal MPNs and since then World Health Organization (WHO) has revised the diagnoses algorithm for patients with Philadelphia-negative MPNs [
1,
11].
Until date, several
CALR mutations had been described. However, two of those namely, type − 1 (52 bp deletion) and a type-2 (5 bp insertion) mutations account for 85% of
Jak2 V617F Philadelphia-double negative MPNs [
7,
8]. Until date, consensus has yet not been established for use of a single given methodology for
CALR genotyping [
12‐
14]. Current
CALR genetic mutation screening methods include high resolution melting curve analysis (HRMA), direct Sanger sequencing and real-time PCR based techniques that utilize allele specific probes. All current available assays in the detection of
CALR mutations are arguable, either on cost, sensitivity and/or specificity.
This study aimed to establish simultaneous detection of CALR type-1 and CALR type-2 mutations following a PCR based method to differentiate the alleles based on amplicon size on a normal agarose gel. This established PCR based amplicon length differentiation assay (PCR-ALDA) was further validated and evaluated with other available assays for their diagnostic performance in a cohort of Vietnamese patients with MPN devoid of JAK2 V617F mutation.
Discussion
Accurate detection of disease-specific mutations facilitates precise diagnostics and better treatment regimens. In the scope of MPN, the best example is the
BCR-ABL associated chronic myeloid leukemia (
BCR-ABL positive CML) [
11]. No other genetic lesions in MPN acquire a similar degree of diagnostic accuracy or therapeutic relevance like that of
BCR-ABL positive CML. However,
Jak2 mutations are detected in significant portions of Philadelphia-negative MPN patients including PV, ET, PMF and in a minor part of the patients carrying
MPL mutations. Therefore, both
Jak2 or
MPL mutations are used as clonal markers in establishing the diagnosis of MPN [
1,
11]. The fact is that not all MPN patients possess
Jak2 mutations and only minor proportion of MPN patients carries
MPL mutations, indicating that the diagnostic value of
Jak2 or
MPL mutations is limited by suboptimal sensitivity and specificity. The molecular diagnostic gap in
Jak2/MPL-unmutated MPN patients is complimented by the recent discovery of
CALR mutations in the majority of such cases [
7,
8]. However, the described genetic lesions of
CALR gene are very heterogenous that make a technical difficulty to gain a consensus approach for routine diagnosis of
CALR mutations in clinical settings.
In this study, we used an ARMS-PCR based Jak2 V617F method to screen for the presence or absence of the
Jak2 V617F mutation in BCR-ABL transcript negative MPN patients. This assay is very clinically relevant in the context of acquiring an acceptable limit of detection (LOD) at 0.5%, which is comparable to the levels developed by other groups [
19,
20]. At this level of LOD, we are able to identify 63 Jak2 V617F positive cases (60%) from 105 recruited MPN samples. The distribution of Jak2 mutation reported in this study was expectable as it falls in range similar to that of previously published data [
3,
21]. The identification of
Jak2 V617F split our study cohort into two different subgroups:
Jak2 V617F positive group (Jak-pos) which is found in elder patients and characterized by higher levels of WBC, RBC and PLT but lower level of Hb compared to that of
Jak2 V617F negative (Jak-neg) group. This observation is similar to previous studies [
22,
23]. Additionally, our
Jak2 V617F screening assay works very simple, without special requirement of equipment or personnel. However, we have not yet validated this assay for the purposes of quantitative monitoring like previous studies [
19,
20], therefore, we only recommend to use our in-house assay for identification of
Jak2 V617F but not for quantification of
Jak2 mutant alleles in clinical practice.
As mentioned, the molecular diagnostic gap in
JAK2/MPL-unmutated ET/PMF patients can be complemented by the identification of
CALR gene mutations [
7,
8]. Therefore, we proposed the PCR-ALDA approach for surveillance of these mutations. Our data revealed that PCR-ALDA approach is obviously acquired a limit of detection (LOD) at 1%. This quantitative level is not stronger than that of Zinke‘s [
13] or the other approach [
14]. Nevertheless, as the PCR-ALDA, Zinke’s approach and Sanger sequencing were comparatively co-applied to the same studied cohort, the PCR-ALDA assay has demonstrated a better diagnostic performance over that gained by Zinke’s approach. PCR-ALDA assay and Sanger sequencing could coherently identify 12 CALR mutant samples but the Zinke’s Real-time PCR method detected only 10 positive samples out of Jak2 V617F negative cases. With the single use of
Jak2 mutation screening assay (tetra primer assay), only 60% (63/105) of MPN patients were identified, but with the combinatory uses of
Jak2 mutation screening and PCR-ALDA assays, 71.4% (75/105) of patients were diagnosed during hospitalization.
We consider that PCR- ALDA assay might not be able to resolve mutant versus the wild-type alleles of
CALR if the mutant amplicons differ at less 5 bps from that of wild-type. However,
CALR mutations that possess deletion or insertion stretches shorter than 5 bps are very infrequent [
14]. Thus, we believe that it is seldom for our PCR-ALDA to leak the diagnostics of
CALR mutations if the clinical samples contain more than 1% of mutant alleles. We also be aware that it is needed to have methodologies that can precisely quantify the burden of
CALR mutant alleles allowing the physicians to indirectly monitor treatment responses to specific therapies like situations in which the quantitative levels of
Jak2 V617F allele burden were used as the predictive marker for treatment response in PV patients [
15,
24,
25]. Nonetheless, in this current study, the quantification of
CALR mutant alleles has not yet validated therefore, we recommend to utilize the assay for identification of
CALR mutations but not for quantification of
CALR mutant alleles.