Discussion - 鈣網蛋白突變在骨髓增生性腫瘤的角色

The identification of CALR mutations is important in the molecular diagnosis of MPN especially in JAK2/MPL-unmutated patients. In addition, CALR mutational status was

found to be one of the most significant risk factor for survival in PMF.³³ Sanger sequencing has been used to detect CALR exon 9 mutations in many studies, but it is rarely sensitive below a 10% mutant allele burden as illustrated in Figure 2B and D. Fragment analysis assay was also used and the sensitivity was estimated to be 5% or less for CALR exon 9 mutations.²⁷ Although fragment analysis assay is able to detect most indel mutations in CALR, it cannot discriminate point mutation from wild-type sequence.

Recently, Bilbao-Sieyro et al. showed that HRMA is a feasible method for the detection of CALR mutations using the LightCycler 480 platform.²⁸ The amplicon size of their primer sequences was 265 bp, and the limit of detection for CALR type 2 (K385fs*47) mutant was of 3%. However, the ideal amplicon length for HRMA is usually less than 250 bp. In this study, the HRMA primers with an amplicon size of 134 bp were designed and are capable of detecting common CALR exon 9 mutations in myeloid neoplasms with satisfactory sensitivity.

Based on the dilution studies using patients’ genomic DNA, the maximal sensitivity of our HRMA using CFX Connect real-time system for both CALR type 1 (L367fs*46) and type 2 (K385fs*47) mutants was of 2.5%. In addition to 16 CALR mutated samples that could be detected by both HRMA and Sanger sequencing, we were able to identify another 5 patients with low CALR mutant allele burden only by HRMA. In this situation, we used TA-cloning followed by Sanger sequencing to confirm the mutation suspected.

Alternatively, fragmented analysis may be used for mutation detection because it also has a better sensitivity than Sanger sequencing. We were not able to detect CALR mutation in 1 of the 6 patients after screening for 100 clones. It is likely that this patient might still have low allele burden CALR mutation which, by chance was missed by random selection of clones (Figure 5A). However, we counted the result as a possible 3% false positive rate to

avoid overestimation of our data. Importantly, no false negative was found in our HRMA system and this is critical in regard to its role as a screening tool.

HRMA developed in this study can be utilized for rapid, sensitive and reliable detection of CALR mutations. Although a total of 5 SNPs (rs201971744, rs143880510, rs370029737, rs374121178 and rs150264068) are reported in the region covered by our amplicon, the minor allele frequency of 3 of them is reported to be less than 0.01%. Therefore, the influence of these 5 SNPs to our HRMA system will likely be very small. Nevertheless, one limitation to this HRMA methodology is that it will not be able to identify the 2 CALR exon 9 point mutations reported in follicular lymphoma (E403X and E405Q) because they are not covered by our 134 bp amplicon. The frequency and significance of these 2 CALR point mutations in follicular lymphoma are currently not yet clear. Therefore, our HRMA methodology is suitable for use in patients suspected to have myeloid neoplasms especially ET and PMF. By using HRMA, we detected a total of 6 different types of CALR mutations in ET patients. All the CALR mutations detected in this study resulted in a +1 base-pair shifting in the reading frame and generated the characteristic novel peptide sequence in the C-terminus. All the CALR exon 9 indel mutations likely contribute to a similar, yet not clearly understood molecular pathogenesis in ET and PMF. In addition, the number of female patients was slightly higher than male patients (57% vs 43%) in our 21 ET patients with CALR mutations, and this has also been observed in other study.⁷²

HRMA, a close-tube method, is not only rapid as it is conducted immediately after PCR amplification, but is also cost effective because it can reduce the use of Sanger sequencing.

By using HRMA, a medium-throughput screening for CALR mutations is also possible.

Based on these advantages, our results clearly illustrated that HRMA is a more suitable and

sensitive method over Sanger sequencing for the screening of CALR mutations in both clinical and research settings. Nevertheless, in samples with distinct melting curves, complimentary Sanger sequencing is still required to determine their exact genotypes because the pattern of melting curves does not correlate with specific CALR mutational types.

In conclusion, we have shown that HRMA is a rapid, sensitive, reliable and cost effective method for the detection of CALR mutations. Because CALR mutations have important diagnostic and prognostic significance in ET and PMF, HRMA can be a useful screening method for the identification of common CALR mutations.

Chapter 3 The clinical and prognostic significance of CALR mutations and JAK2/CALR co-mutations in Taiwanese ET patients

1. Summary

Frequent CALR mutations have been discovered in patients with JAK2/MPL-unmutated essential thrombocythemia (ET) and primary myelofibrosis. We sought to screen for CALR exon 9 alterations with high-resolution melting analysis (HRMA) in 92 adult ET patients, and to determine the clinical and molecular correlates. In this cohort, 59 (64%) patients harbored JAK2 V617F mutation and one (1%) harbored MPL W515K mutation. By HRMA followed by TA-cloning, we identified classic CALR indel mutations in 21 (22.8%) patients. Eleven (12%) patients were triple-negative. The 59 JAK2-mutated patients were also screened for CALR exon 9 alterations by HRMA, and 16 (27.1%) samples were found to have distinct melting curves from wild-type. In 2 of these 16 samples, one CALR type 3 mutation and one single nucleotide polymorphism (rs143880510) were detected by Sanger sequencing. Although the remaining 14 patients were wild-type by Sanger sequencing, CALR alterations were detected in 12 (85.7%) patients after TA-cloning: 3 harbored classic CALR indel mutations, 5 (8.5%) harbored 4 types of 3 bp inframe deletions, and 5 (8.5%) harbored 5 types of point mutations. Overall, various CALR exon 9 alterations were detected in 13 (22%) of 59 JAK2-mutated ET patients. In comparable to previous reports, CALR mutations were associated with younger age (p=0.025), higher platelet count (p<0.001) and lower hemoglobin level (p=0.016). JAK2-mutated ET patients with concomitant CALR alterations were associated with oldest age (p=0.025), higher thrombotic events after diagnosis (p=0.048), higher major arterial thrombotic events after diagnosis (p=0.022) and more patients being high risk group for thrombo-hemorrhagic

complications (p=0.023). Frequent CALR exon 9 alterations in JAK2-mutated ET patients define a specific subgroup of patients with increased risk of thrombotic events.

2. Introduction

Essential thrombocythemia (ET) is a clonal hematopoietic stem cell neoplasm and one of the classic BCL-ABL1-negative chronic myeloproliferative neoplasm (MPN), which also includes polycythemia vera (PV) and primary myelofibrosis (PMF).² JAK2 V617F mutation can be detected in more than 95% PV patients, and 50% to 60% of ET and PMF patients. MPL mutations at codon 515 are found in 3% to 5% of JAK2-unmutated ET and PMF patients. The 2008 World Health Organization (WHO) classification has incorporated JAK2 V617F and MPL mutations into the diagnostic criteria of MPN. Both JAK2 V617F and MPL mutations cause activation of the Janus kinase/signal transducer and activator of transcription (STAT) signaling pathway leading to the development of JAK inhibitor therapy in MPN.

Recently, two seminal studies discovered a high frequency of somatic calreticulin (CALR) mutations in patients with JAK2/MPL-unmutated ET and PMF.^18,19 The pattern of most CALR mutations in MPN is heterozygous indels in exon 9 causing one base pair (bp) reading frameshift. CALR mutations have been shown to have important diagnostic and prognostic significance in ET and PMF patients,^18,19,29 and will likely be incorporated into the WHO diagnostic criteria for MPN. In vitro studies on the molecular pathogenesis of CALR mutations in MPN have shown controversial results in regard to the involvement and/or activation of the JAK/STAT signaling pathway,^18,19,38 and the exact pathogenesis of

Several techniques such as Sanger sequencing and polymerase chain reaction (PCR) followed by fragment analysis have been used to detect CALR mutations.10,18,19,27

High-resolution melting analysis (HRMA) is a well-established method for the screening of mutations, and we have developed a rapid and sensitive HRMA for the detection of CALR exon 9 mutations.⁷³ In this study, we sought to screen a cohort of 92 Taiwanese ET patients for CALR exon 9 mutations with HRMA and Sanger sequencing independently, and to determine the clinical and molecular correlates.

3. Patients and Methods 3.1 Patients

The institutional review board of Mackay Memorial Hospital has approved the screening for mutations. All patients provided written informed consent. Diagnosis of ET was established based on the 2008 WHO criteria. The clinical and laboratory characteristics at the time of diagnosis or referral were collected. Genomic DNA derived from bone marrow, peripheral blood, and peripheral blood granulocytes and/or mononuclear cells were used for mutation screening.

3.2 Screening for CALR mutations

CALR mutations were screened by Sanger sequencing on an ABI 3730 sequencer as preciously described.¹⁹ CALR exon 9 mutations were independently screened by HRMA using a CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA) as previously described with a maximal sensitivity of 2.5% for both CALR type 1 and type 2 mutants.⁷³ Briefly, a pair of oligonucleotide primers were used to amplify a 134 bp amplicon [GenBank: NM_004343] which flanked all CALR exon 9 variants reported in

MPN. All samples with distinguished melting curves from wild-type were confirmed by duplicate studies. Peripheral blood samples from 78 healthy adults were also used to validate the specificity of our HRMA. JAK2 V617F mutation was screened by allele-specific PCR with an analytic sensitivity of 5% and MPL exon 10 mutation by Sanger sequencing as previously described.^7,9

3.3 TA-cloning

TA-cloning was performed by pGEM-T easy vector system (Promega, Madison, CA, USA) as previously described.⁷³ At least 10 clones in each individual were randomly selected for the screening of CALR exon 9 alterations by Sanger sequencing. All novel single nucleotide variant that was only detected once was treated as artifact and excluded.

3.4 Statistical analysis

The correlation between clinical characteristics and mutational status was calculated by the chi-square test or Fisher’s exact test. The comparison between continuous and categorical variables was performed by the Mann-Whitney U test or Kruskal-Wallis H test. SPSS Statistics software (IBM, New York, USA) was used for all calculations. P values <0.05 were considered significant.

4. Results

4.1 CALR exon 9 mutations

Among the 92 ET patients (median age 53 years; 58% females), 59 (64%) patients harbored JAK2 V617F mutation and one (1%) patient harbored MPL W515K mutation. 32 JAK2/MPL-unmutated ET patients were utilized for the development of our HRMA

wild-type. In 16 of these 22 samples, Sanger sequencing confirmed the presence of 6 types of CALR mutations: 5 type 1 (p.L367fs*46), 6 type 2 (p.K385fs*47), 1 type 3 (p.L367fs*48), 2 type 34 (p.K385fs*47), and 2 other types (p.L367fs*43 and p.E369fs*50).

The other 6 samples were wild-type by sequencing, and CALR type 2 mutations were detected in 5 of 6 patients after TA-cloning indicating the presence of low allele burden CALR mutants in them. By using our HRMA platform, we identified CALR mutations in 21 (22.8% overall and 65.6% in JAK2/MPL-unmutated) ET patients and this frequency is comparable to other studies.^18,19,29 11 (12%) ET patients were negative for JAK2, CALR and MPL mutations. In the 78 healthy adults, 2 samples were found by HRMA to have distinct melting curves from wild-type. One single nucleotide polymorphism (SNP, rs143880510) and one wild-type were found after Sanger sequencing in these 2 samples.

Therefore, the false positive rate of our HRMA system was 1.3%.

4.2 CALR exon 9 alterations

After screening the 59 JAK2 V617F-mutated ET patients for CALR alterations by HRMA, 16 (27.1%) samples were found to have distinct melting curves from wild-type (Figure 6).

In 2 of these 16 samples, one CALR type 3 mutation (p.L367fs*48) and one SNP (rs143880510) were detected by Sanger sequencing. All the other 14 samples were wild-type by sequencing. Surprisingly, we detected a high frequency of CALR exon 9 alterations in 12 (85.7%) of these 14 patients after TA-cloning (Table 2). Three patients harbored the classic CALR indel mutations: one each of type 2 p.K385fs*47, p.E370fs*60 and p.E371fs*59. Hence, 4 (6.8%) ET patients had classic CALR indel and JAK2 V617F co-mutations in this cohort. Five patients (8.5%) including the aforementioned patient (P520) with type 2 CALR mutation harbored 4 types of 3 bp inframe deletions all resulted in the deletion of a single amino acid of glutamic acid: two p.E381del, and one each of

p.E371del, p.E378del and p.E396del (Figure 7). Another five patients (8.5%) harbored 5 types of point mutations: one each of p.E374X, p.E380X, p.K391X, p.E372G and p.E380G.

The latter p.E380G has been reported as a SNP but might be a low allele burden somatic mutation in this patient because it was only detected after TA-cloning and not by Sanger sequencing on patient’s genomic DNA. The remaining two patients were found to have wild-type CALR exon 9 after screening for 100 independent clones, and were counted as CALR wild-type. Overall, various CALR exon 9 alterations were detected in 13 (22%) of 59 JAK2 V617F-mutated ET patients.

4.3 Clinical and molecular correlates

We then examined the clinical and molecular correlates in 91 ET patients excluding the one MPL-mutated patient (Table 3). JAK2-mutated ET patients with concomitant CALR alterations were associated with oldest age (p=0.025), higher thrombotic events after diagnosis (p=0.048), higher major arterial thrombotic events after diagnosis (p=0.022) and more patients being high risk group for thrombo-hemorrhagic complications (p=0.023). In comparable to previous reports, CALR mutations were associated with younger age (p=0.025), higher platelet count (p<0.001) and lower hemoglobin level (p=0.016). JAK2 V617F-mutation was associated with leukocytosis (p=0.046).

5. Discussion

After the discovery of CALR mutations, it has been proposed to be mutually exclusive with JAK2 and MPL mutations in MPN. However, CALR and JAK2 V617F co-mutations have been reported in a few MPN cases across different ethnic groups and the frequency is usually below 1%.10,32,74-76

In contrast to these reports, we detected a higher frequency of

6.8% CALR indel and JAK2 co-mutations in ET patients. Interestingly, 3 of these CALR mutations were low allele burden mutants not detected by Sanger sequencing. Nevertheless, the use of a sensitive HRMA technique has enabled us to detect these low allele burden CALR mutants in both JAK2-mutated and JAK2/MPL-unmutated ET patients.

In addition, we also detected several CALR exon 9 point mutations and inframe deletions in JAK2-mutated ET patients, but none in our JAK2/MPL-unmutated ET patients. Recently, point mutations in CALR were also reported in follicular lymphoma (E403X and E405Q), PMF (E379D) and chronic neutrophilic leukemia (E398D).²² Two rare inframe deletions in CALR exon 9 (p.E393_E395del and p.E405del) have been reported in the National Heart, Lung, and Blood Institute Grand Opportunity Exome Sequencing Project with undetermined significance. All the 5 inframe deletions we detected were 3 bp deletions similar to the latter one. Although the possibility of low allele burden germline sequence variations cannot be completely excluded, these 3 bp inframe deletions detected by HRMA were more likely to be low allele burden somatic mutations not detected by Sanger sequencing in our patients.

Interestingly, CALR point mutations (E381A and D373M) and inframe deletions (E381_A382>A, D397_D400>D, D400_K401>D and E405_V409>V) were also detected in patients with suspected MPN and JAK2-mutated MPN in another study albeit with a lower frequency.⁷⁷ These CALR alterations were also found to co-occur with MPL, CSF3R, ASXL1 and ZRSR2. Currently, the role of these CALR point mutations and inframe deletions in the molecular pathogenesis of MPN is not yet clear. Because they frequently

co-occurred with mutations involving the JAK-STAT pathway and affected disease phenotype in JAK2-mutated ET patients, these non-classic CALR mutant proteins are suspected to play a contributory role in the pathogenesis of MPN.⁷⁷ The frequency of these non-classic CALR mutations in PMF and other MPN requires further study.

In conclusion, we have detected a high frequency of both classic and non-classic CALR exon 9 alterations in JAK2-mutated ET patients by HRMA. The presence of CALR alterations in JAK2-mutated ET defines a specific subgroup of patients requiring careful follow-up and management for their increased risk of thrombotic events. Because our study is limited by small patient number, larger study is warranted to confirm our observation.

Chapter 4 B cell immune profiles in CALR mutated ET patients 1. Summary

Essential thrombocythemia (ET) is a BCL-ABL1-negative myeloproliferative neoplasm.

We have reported that increased activated B cells can facilitate platelet production mediated by cytokines regardless JAK2 mutational status in ET. Recently, calreticulin (CALR) mutations were discovered in ~30% JAK2/MPL-unmutated ET and primary myelofibrosis. Here we sought to screen for CALR mutations and to evaluate B cell immune profiles in a cohort of adult Taiwanese ET patients. B cell populations, granulocytes/monocytes membrane-bound B cell-activating factor (mBAFF) levels, B cells toll-like receptor 4 (TLR4) expression and intracellular levels of interleukin (IL)-1β/IL-6 and the expression of CD69, CD80, and CD86 were quantified by flow cytometry. Serum BAFF concentration was measured by ELISA. 48 healthy adults were used for comparison.

19 (35.2%) of 54 ET patients harbored 8 types of CALR exon 9 mutations including 4 (7.4%) patients with concomitant JAK2V617F mutations. Compared to JAK2V617F mutation, CALR mutations correlated with younger age at diagnosis (p=0.04), higher platelet count (p=0.004), lower hemoglobin level (p=0.013) and lower leukocyte count (p=0.013). Multivariate analysis adjusted for age, sex, follow-up period and hematological parameters confirmed that increased activated B cells were universally present in JAK2-mutated, CALR-mutated and triple-negative ET patients when compared to healthy adults. JAK2- and CALR-mutated ET have significantly higher fraction of B cells with TLR4 expression when compared to triple-negative ET (p=0.019 and 0.02, respectively).

CALR-mutated ET had significantly higher number of CD69-positive activated B cells when compared to triple-negative ET (p=0.035). In conclusion, increased B cell activation is present in ET patients across different mutational subgroups.

2. Introduction

Essential thrombocythemia (ET) is a BCL-ABL1-negative myeloproliferative neoplasm (MPN), and is characterized by increased number of mature megakaryocytes (MKs) in the bone marrow and sustained thrombocytosis in the peripheral blood.² ET is associated with an increased risk of hemorrhagic and thrombotic complications and leukemic transformation.² Most ET patients can have a normal life expectancy but some may encounter serious events during their disease course. In 2005, the JAK2V617F mutation was discovered in MPNs including 50-60% patients with ET and primary myelofibrosis (PMF).^78-81 JAK2V617F mutation plays an important role in cytokine-independent hematopoietic stem cells (HSCs) proliferation in MPNs. Also, hypersensitivity of hematopoietic cells to cytokines stimulation is noted in MPNs through the interaction between JAK2V617F mutation and various cytokine receptors.⁸² Recently, a high frequency of calreticulin (CALR) mutations was discovered in JAK2/MPL-unmutated ET and PMF.^18,19,65 We and others have reported that CALR mutations are associated with distinct clinical characteristics including higher platelet counts, lower leukocyte counts and hemoglobin levels, and a lower thrombosis risk when compared to JAK2-mutated ET patients.18,19,29,65,83

Using in vitro and/or in vivo models, we and others have recently reported that mutant CALR can activate JAK-STAT signaling pathway through an MPL-dependent mechanism to mediate pathogenic thrombopoiesis.39-43,84,85

CALR is a 46-kDa Ca2+ binding chaperone protein located in the endoplasmic reticulum, but it can also localize to cell surface and accumulate in extracellular compartments.¹⁴ In addition to ensuring proper protein and glycoprotein folding within the lumen of

endoplasmic reticulum, CALR was also found to involve the immune response to pre-apoptotic cancer cells, and early cell surface exposure of CALR was followed by expression and release of heat-shock proteins (e.g. HSP70), and high-mobility group I (HMGB1) protein.¹⁵ Recombinant CALR fragment was shown to exhibit potent stimulatory activities against B cells.^16,17 Recently, we reported that activated B cells are increased in ET patients, and can facilitate platelet production mediated by cytokines, such as interleukin (IL)-1β and IL-6 regardless JAK2V617F mutational status.⁶² We found that increased production of B cell-activating factor (BAFF) by granulocytes and monocytes up-regulates toll-like receptor 4 (TLR4) expression on B cells and promotes B cell activation in ET patients. Consequently, these activated B cells play a pathogenic role in augmenting thrombocytosis by producing IL-1β and IL-6 in ET patients through cytokine-dependent thrombopoiesis in the bone marrow. However, ET with CALR mutations was not included in our previous study because CALR mutations have not yet been discovered in MPNs when we conducted our study in 2013. The discovery of CALR mutations in JAK2/MPL-unmutated ET patients in December 2013 have prompted us to ask the question that whether increased B cell activation can also be found in ET with CALR mutations similar to that in JAK2V617F-mutated ET.^18,19,65 Hence, we sought to

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