Contributions
Abstract: PB2276
Type: Publication Only
Background
Philadelphia negative myeloproliferative neoplasms pose a significant diagnostic challenge. About 30-45% patients of Primary Myelofibrosis (PMF) and Essential Thrombocythemia(ET) are also negative for JAK2 mutation. In recent times calreticulin (CALR) mutations have been identified which can characterise about 50-80% of such patients. However, the heterogeneity of calreticulin mutations demand extensive molecular testing which are not widely available, labour intensive, costly and time consuming. To overcome these limitations immunostaining by immunohistochemistry(IHC) was developed to detect calreticulin mutations.
Aims
1. Detection of calreticulin mutation by allele specific oligonucleotide (ASO) PCR and IHC in patients of JAK2 negative primary myelofibrosis and essential thrombocythemia
2. To determine the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IHC when compared to PCR.
Methods
This is a prospective observational study. We included all suspected cases of PMF and ET both clinically and on bone marrow examination. Cases with positive BCR-ABL and JAK2 V617F mutation were excluded.
Eligible cases were screened for CALR mutation by ASO-PCR for type 1 and type 2 mutations. Interpretation was done by comparing bands on gel electrophoresis to the expected product size (wild type CALR: 357bp, CALR type 1 mutation: 302bp and CALR type 2 mutation: 272 bp). For all these cases IHC was simultaneously done on the bone marrow biopsy section using monoclonal CAL2 antibody. Those cases with more than 90% strongly labelled megakaryocytes were taken as positive.
Results
A total of 61 patients were suspected to have PMF or ET on clinical and bone marrow examination. All these patients were negative for BCR-ABL fusion transcript. On further analysis 36 patients were found to have JAK2V617F mutation and were excluded from the study. Remaining 25 cases were analysed for CALR mutation by PCR and IHC.
Amongst the 14 cases with positive CALR mutation, 12 were positive by both PCR and IHC. However, two patients who were CALR positive by PCR showed negative immunostaining by IHC. Remaining 11 cases reported negative for CALR mutation by both PCR and IHC.
On statistical analysis of IHC performance, we found a sensitivity of 85.71% and specificity of 100% at 95% confidence interval. The positive predictive value (PPV) and negative predictive value (NPV) were 100% and 84.62% respectively.
Conclusion
IHC is a readily available and less labour-intensive method to identify CALR mutations in patients with PMF and ET with high sensitivity and specificity. In resource limited settings, immunostaining with CAL2 monoclonal antibody may serve as a valuable tool for diagnosing patients with PMF and ET.
Limitation of study: The two cases with positive PCR result and negative IHC need to be evaluated by sanger sequencing to exclude the possibility of false positive PCR.
Session topic: 16. Myeloproliferative neoplasms - Clinical
Keyword(s): Essential Thrombocytemia, Immunohistochemistry, Myelofibrosis, Myeloproliferative disorder
Abstract: PB2276
Type: Publication Only
Background
Philadelphia negative myeloproliferative neoplasms pose a significant diagnostic challenge. About 30-45% patients of Primary Myelofibrosis (PMF) and Essential Thrombocythemia(ET) are also negative for JAK2 mutation. In recent times calreticulin (CALR) mutations have been identified which can characterise about 50-80% of such patients. However, the heterogeneity of calreticulin mutations demand extensive molecular testing which are not widely available, labour intensive, costly and time consuming. To overcome these limitations immunostaining by immunohistochemistry(IHC) was developed to detect calreticulin mutations.
Aims
1. Detection of calreticulin mutation by allele specific oligonucleotide (ASO) PCR and IHC in patients of JAK2 negative primary myelofibrosis and essential thrombocythemia
2. To determine the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IHC when compared to PCR.
Methods
This is a prospective observational study. We included all suspected cases of PMF and ET both clinically and on bone marrow examination. Cases with positive BCR-ABL and JAK2 V617F mutation were excluded.
Eligible cases were screened for CALR mutation by ASO-PCR for type 1 and type 2 mutations. Interpretation was done by comparing bands on gel electrophoresis to the expected product size (wild type CALR: 357bp, CALR type 1 mutation: 302bp and CALR type 2 mutation: 272 bp). For all these cases IHC was simultaneously done on the bone marrow biopsy section using monoclonal CAL2 antibody. Those cases with more than 90% strongly labelled megakaryocytes were taken as positive.
Results
A total of 61 patients were suspected to have PMF or ET on clinical and bone marrow examination. All these patients were negative for BCR-ABL fusion transcript. On further analysis 36 patients were found to have JAK2V617F mutation and were excluded from the study. Remaining 25 cases were analysed for CALR mutation by PCR and IHC.
Amongst the 14 cases with positive CALR mutation, 12 were positive by both PCR and IHC. However, two patients who were CALR positive by PCR showed negative immunostaining by IHC. Remaining 11 cases reported negative for CALR mutation by both PCR and IHC.
On statistical analysis of IHC performance, we found a sensitivity of 85.71% and specificity of 100% at 95% confidence interval. The positive predictive value (PPV) and negative predictive value (NPV) were 100% and 84.62% respectively.
Conclusion
IHC is a readily available and less labour-intensive method to identify CALR mutations in patients with PMF and ET with high sensitivity and specificity. In resource limited settings, immunostaining with CAL2 monoclonal antibody may serve as a valuable tool for diagnosing patients with PMF and ET.
Limitation of study: The two cases with positive PCR result and negative IHC need to be evaluated by sanger sequencing to exclude the possibility of false positive PCR.
Session topic: 16. Myeloproliferative neoplasms - Clinical
Keyword(s): Essential Thrombocytemia, Immunohistochemistry, Myelofibrosis, Myeloproliferative disorder