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IMMUNOHISTOCHEMICAL ANALYSIS OF CALRETICULIN MUTATIONS IN PRIMARY MYELOFIBROSIS (PRMF) PATIENTS: RESULTS OF OUR INSTITUTION IN THE JAK2V617F MUTATED AND JAK2V617F WILD TYPE PRMF PATIENTS.
Author(s):
Süheyla Uyar Bozkurt
,
Süheyla Uyar Bozkurt
Affiliations:
pathology,marmara university,istanbul,Turkey
Fatma Geçgel
,
Fatma Geçgel
Affiliations:
Hematology,Marmara university,istanbul,Turkey
Işık Atagündüz
,
Işık Atagündüz
Affiliations:
hematology,marmara university,istanbul,Turkey
Yeşim Ağyol
,
Yeşim Ağyol
Affiliations:
Internal Medicine,Marmara university,istanbul,Turkey
Kemal Türköz
,
Kemal Türköz
Affiliations:
Pathology,Marmara university,istanbul,Turkey
Tulin Tuğlular
Tulin Tuğlular
Affiliations:
Hematology,Marmara university,istanbul,Turkey
(Abstract release date: 05/18/17) EHA Library. Uyar Bozkurt S. 05/18/17; 182741; PB2027
Süheyla Uyar Bozkurt
Süheyla Uyar Bozkurt
Contributions
PDF Abstract
Abstract

Abstract: PB2027

Type: Publication Only

Background
Myeloproliferative neoplasms (MPNs) are a group of chronic myeloid cancer characterized by overproduction of mature hematopoetic cells. Mutations in one of three genes; Janus kinase 2 (JAK 2), myeloproliferative leukemia protein (MPL) and calreticulin (CALR), have been found in most patients with BCR-Abl negative MPNs. JAK2 mutations are present virtually all cases of Polycythaemia Vera and 50-60% of prMF and Essential Thrombocythemia (ET). Recently, mutations in CALR gene were found in 50-80% of JAK2 and MPL mutation negative ET and prMF patients.

Aims
To evaluate immunohistochemical results of CALR gene mutation in the bone marrow samples of the JAK2V617F mutated and JAK2V617F wild type Primary Myelofibrosis (prMF) patients.

Methods
Material: Bone marrow biopsy samples from 32 patients previously diagnosed as primary myelofibrosis with known JAK V617F mutation status were obtained from archives of Marmara University Pathology Laboratory. Bone marrow samples of two patients were already known as CALR mutated by PCR analysis. Bone marrow samples of three JAK2 wild type and CALR mutated ET, two JAK2 wild type, CALR mutated prMF patients and two CALR wild type ET patients were used as positive and negative control tissues for CALR immunohistochemistry.

Immunohistochemistry: 4-µm unstained sections of each bone marrow biopsy specimens were cut onto electrostatically charged glass slides. Immunohistochemistry was performed on an automated immunostainer (Ventana BenchMark Ultra; Ventana Medical Systems, Inc). CALR antibody (clone CAL2, Dianova, Germany) staining used a 1:100 dilution.Any cytoplasmic staining of the cells with CAL2 antibody was considered positive immunostaining.

Results
We studied 32 bone marrow specimens of primary myelofibrosis with 15 (47%) of them having JAK2 V617F mutation and 17(53%) of them lacking JAK2 V617Fmutation. CALR immunoreactivity was seen in 8 (25%) of all pr MF patients. CALR immunoreactivity was seen in 8 (47%) of patients with PMF myelofibrosis who are negative for JAK2V617F mutation. CALR immunoreactivity was not seen in patients with PMF myelofibrosis who are positive for JAK2V617F mutation. CALR immunoreactivity was seen in 3 (100%) of patients with ET and 2 (100%) of patients with known CALR mutation. CALR immunoreactivity was not seen in patients with CALR wild type ET patients.

We observed that CAL2 immunostaining was seen mainly in the cytoplasm of the small and large megakaryocytes, and atypical megakaryocytes as found in fibrotic PrMF. Pale immunostaining was seen in myeloid and erytoid cell precursors. This immunostain also stained some small cells appearing as micromegakaryocytes.

Conclusion
An immunohistochemical stain easily detects the CALR mutation by staining of megakaryocytes in formalin-fixed bone marrow biopsy specimens . This method would be a easy, rapid, and cost effective way to detect CALR mutations in daily routine hematopathology biopsy evaluation of the myeloproliferative patients.

Session topic: 15. Myeloproliferative neoplasms - Biology

Keyword(s): Myelofibrosis, Mutation status, Immunohistochemistry

Abstract: PB2027

Type: Publication Only

Background
Myeloproliferative neoplasms (MPNs) are a group of chronic myeloid cancer characterized by overproduction of mature hematopoetic cells. Mutations in one of three genes; Janus kinase 2 (JAK 2), myeloproliferative leukemia protein (MPL) and calreticulin (CALR), have been found in most patients with BCR-Abl negative MPNs. JAK2 mutations are present virtually all cases of Polycythaemia Vera and 50-60% of prMF and Essential Thrombocythemia (ET). Recently, mutations in CALR gene were found in 50-80% of JAK2 and MPL mutation negative ET and prMF patients.

Aims
To evaluate immunohistochemical results of CALR gene mutation in the bone marrow samples of the JAK2V617F mutated and JAK2V617F wild type Primary Myelofibrosis (prMF) patients.

Methods
Material: Bone marrow biopsy samples from 32 patients previously diagnosed as primary myelofibrosis with known JAK V617F mutation status were obtained from archives of Marmara University Pathology Laboratory. Bone marrow samples of two patients were already known as CALR mutated by PCR analysis. Bone marrow samples of three JAK2 wild type and CALR mutated ET, two JAK2 wild type, CALR mutated prMF patients and two CALR wild type ET patients were used as positive and negative control tissues for CALR immunohistochemistry.

Immunohistochemistry: 4-µm unstained sections of each bone marrow biopsy specimens were cut onto electrostatically charged glass slides. Immunohistochemistry was performed on an automated immunostainer (Ventana BenchMark Ultra; Ventana Medical Systems, Inc). CALR antibody (clone CAL2, Dianova, Germany) staining used a 1:100 dilution.Any cytoplasmic staining of the cells with CAL2 antibody was considered positive immunostaining.

Results
We studied 32 bone marrow specimens of primary myelofibrosis with 15 (47%) of them having JAK2 V617F mutation and 17(53%) of them lacking JAK2 V617Fmutation. CALR immunoreactivity was seen in 8 (25%) of all pr MF patients. CALR immunoreactivity was seen in 8 (47%) of patients with PMF myelofibrosis who are negative for JAK2V617F mutation. CALR immunoreactivity was not seen in patients with PMF myelofibrosis who are positive for JAK2V617F mutation. CALR immunoreactivity was seen in 3 (100%) of patients with ET and 2 (100%) of patients with known CALR mutation. CALR immunoreactivity was not seen in patients with CALR wild type ET patients.

We observed that CAL2 immunostaining was seen mainly in the cytoplasm of the small and large megakaryocytes, and atypical megakaryocytes as found in fibrotic PrMF. Pale immunostaining was seen in myeloid and erytoid cell precursors. This immunostain also stained some small cells appearing as micromegakaryocytes.

Conclusion
An immunohistochemical stain easily detects the CALR mutation by staining of megakaryocytes in formalin-fixed bone marrow biopsy specimens . This method would be a easy, rapid, and cost effective way to detect CALR mutations in daily routine hematopathology biopsy evaluation of the myeloproliferative patients.

Session topic: 15. Myeloproliferative neoplasms - Biology

Keyword(s): Myelofibrosis, Mutation status, Immunohistochemistry

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