Is Primary Myelofibrosis with mutated calreticulin a different entity with better prognosis? Good morning… My presentation is about Calreticulin and PMF What we know ans what is missing Juliana Hidalgo López, MD. Posdoctoral fellow Hematopathology Department Friday, January 22, 2016

Outline Case presentation Calreticulin (CALR) What, Why and Who? Correlation with other diagnostic parameters, clinical features and prognosis This is the content of the presentation: First I am going to present a case, and then I am going to talk about calreticulin Answering some questions like what, why and who Later I am going to explain the correlation with the diagnostic, clinical and prognostic parameters

Clinical History 72-year-old man, asymptomatic Splenomegaly: 15 cm below costal margin Count Normal Range Hb (g/dL) 11.4 14-18 WBC (x 109/L) 15.9 4-11 Neutrophil 56 % 42-66 % Monocyte 7 % 2-7 % Eosinophil 1 % 1-4 % Basophil 2 % 0-1 % Metamyelocyte 3 % 0% Platelets (x 109/L) 189.1 140-440 LDH (IU/L) 900 313-618 To begin with the case presentation: This a is a 72 years old male, without other medical record and asymptomatic who present an abnormal blood count in a regular medical check. At examination only splenomegaly was found. In the peripheral blood counts Mild anemia with scattered dacryocytes and occasional nucleated RBCs on peripheral blood smear were found. Also in the WBC leukocytosis, mostly of neutrophils with a mild left-shift and minimal basophilia (2%) were found. An elevation in LDH was also detected at that time Then the patient went for a bone marrow biopsy

Bone marrow As we can see here: There was an increase in megakaryocytes in dense clusters with large and atypical morphology, consistent of hyperchromatic chromatin and hyperlobulation.

Bone marrow Reticulin Trichrome Here we can see diffuse and dense increase in reticulin with extensive intersections (3 out of 4) and in In thricrome stain, we see, in blue focal bundles of collagen The grading of fibrosis at bone marrow was: 2 Reticulin Trichrome

Diagnostic parameters Molecular: Negative BCR-ABL1 fusion transcript (RT-PCR) No JAK2 V617F or MPL codon 515 Positive for calreticulin 52 bp deletion in exon 9 Karyotype: 46,XY,del(11)(q21q23)[20] At the molecular level: No BCR/ABL, JAK2 or MPL mutation was detected But calreticulin was mutated In cytogenetic a 11q23 deletion was found

Diagnosis PRIMARY MYELOFIBROSIS (MF-3) CALR type 1 mutation DIPSS-Plus score: Intermediate-2 Finally and after meeting the WHO 2008 criteria The patient was diagnosed of PMF with grade 2 fibrosis, calreticulin mutation and a DIPSS-Plus Score intermediate 2. *Dynamic International Prognostic Scoring System (2011)

Outline Case presentation Calreticulin (CALR) What, Why and Who? Correlation with other diagnostic parameter, clinical features and prognosis To continue with the presentation we are going to talk about calreticulin mutation.

What is calreticulin? Major Ca(2+)-binding in the lumen of the endoplasmic reticulum It is also found in the nucleus, suggesting that it may have a role in transcription regulation First we need to define what is calreticulin: Calreticulin is a Multifunctional protein that acts as a major Cacium binding protein in the lumen of the endoplasmic reticulum. It is also found in the nucleus, suggesting that it may have a role in transcription regulation

CALR mutations Types of CALR mutation CALR gene 19p13.3-p13.2 (50%) 52-bp deletion Type 2 (30%) 5-bp insertion Other types (20%) Its gene is located in chromosome 19p in exon 9 and the most frecuent mutations, that encompass the 80% of mutational status are: Type:1 a 52 base pair deletion, the most frequent found in 53%, and type 2: a 5 base pair gain, found in 32% of CARL mutated patients Klampfl T, NEJM 2013

How does the mutation work? Similar to other mutations (JAK2, MPL) occurring in MPN, mutations of CALR, lead to JAK-STAT target genes up-regulation Other DNA mutations (ASXL1, TET2, EZH2, IDH, SF3B1) occur in a substantial number of patients with MPN Mechanisms of activation of JAK-STAT or the cooperation with other mutations that lead to the neoplasm pathogenesis are being investigated But how it works? Similar to other mutations (JAK2, MPL) occurring in MPN, mutations of CALR, lead to JAK-STAT target genes up-regulation Also other DNA mutations occur in a substantial number of patients with MPN and PMF, but the mechanism of JAK-STAT activation or the implication of other mutations in the pathogenesis of this neoplasm is currently being investigated. Rampal R et al, Blood. 2014 Vannucchi AM et al, Leukemia. 2013

Distribution of CALR mutations in MPN Triple negative MPL mutation Polycythemia Vera Essential Thrombocythemia Primary Myelofibrosis Then we need to know why is CARL important in MPN? Well in 2013, two different groups describe CARL mutation in MPN, They found that calreticulin mutation was absence in PV patients in both studies. For the patients with Essential thrombocythemia the mutation was found in around 25% and in patients with Primary myelofibrosis calreticulin was mutated in around 35% of the cases. Calreticulin was found to be mutually exclusive for the other defining mutations like JAK2 and MPL. With this finding they identify the second most frequent mutated gen in myeloprolipherative neoplasm.   But Calreticulin is not exclusive of this type of neoplasm… CALR: 0% CALR: 25% CALR: 35% Nangalia J, NEJM 2013 Klampfl T, NEJM 2013

CALR in myeloid neoplasms Myelodysplastic syndrome: 0-8.3% Chronic myelomonocytic leukemia: 0 – 3% Atypical chronic myeloid leukemia: ~ 3.4% Refractory anemia with ring sideroblast with thrombocytosis: 0 – 12.5% Acute myeloid leukemia: 0% Chronic myelogenous leukemia: 0% But Calreticulin is not exclusive of this type of neoplasm… In Myelodisplastic syndromes, acute myelomonocytic leukemia, atypical chronic myeloid leukemia and refractory anemia with ting sideroblast and thrombocytosis this mutation was found, but as we can see here not in the same frequency as in myeloprolipherative neoplasm. Also is important to know that in both studies no mutation of calreticulin in patients with acute myeloid leukemia and chronic myeloid leukemia were identified. Nangalia J, NEJM 2013 Klampfl T, NEJM 2013

CALR and other MPN-associated alterations BCR/ABL1 and CALR-mut (case reports) Cabagnols X et al. N Engl J Med 2015 Loghavi S et al. Blood. 2015 JAK2 V617F and CALR-mut RARS-T (J Broséus et al. Leukemia, 2014) PMF (Tefferi A et al. Leukemia, 2014) ET (Lundberg P et al. Blood. 2014) (McGaffin G et al. Br J Haematol. 2014) After this, exceptions were reported: Here we can see, 2 case reports of BCR/ABL mutation associated with Calreticulin And also other reports of co-existence of JAK2 and Calreticulin in refractory anemia with ring sideroblast and thrombocytosis, Primary myelofibrosis and Essential thrombocythemia

Proposed diagnostic criteria for PMF Diagnostic: All A and at least one B criteria A1: BM biopsy showing megakaryocytic proliferation and atypia accompanied by either reticulin or collagen fibrosis grades 2 or 3 A2: Not meeting WHO criteria for CML, PV, ET, MDS or other myeloid neoplasm A3: Presence of JAK2, CALR or MPL mutation or other clonal marker (ASXL1, EZH2, TET2, IDH1/IDH2, SRSF2, SF3B1), no evidence for reactive reticulin fibrosis B: Criteria: Anemia Leukocytosis >11 x109/L Palpable splenomegaly LDH increase Leukoerythroblastosis These are the new proposed criteria for primary myelofibrosis diagnosis: The main changes came with the use of grades for fibrosis to define it and the use of Calreticulin as a clonal marker. But also in the criteria A2, calreticulin will argue against another myeloid diagnosis because its low frequency in other neoplasm T Barbui. Blood Cancer Journal 2015

Outline Case presentation Calreticulin (CALR) What, Why and Who? Correlation with clinical features, diagnostic parameters and prognosis To continue, I am going to talk about the correlation with clinical features, diagnostic parameters and prognosis

CALR and clinical features Younger age Higher platelet count Lower DIPSS-plus score Less likely to be transfusion dependent Lower risk of developing anemia, thrombocytopenia, and marked leukocytosis In these study of Tefferi in 2014, the calreticulin mutation mutation were associated with younger age, higher platelets counts and lower DIPSS-plus Score at diagnosis, also these patients were less likely to be transfusion dependent and to develop anemia, thrombocytopenia or marked leukocytosis Tefferi, A. Leukemia. 2014

CALR mutated CALR wild type Dense megakaryocytic clusters (ET) >25% megs with hyperchromatic nuclei (PMF) In diagnostic field, we can see that patients with calreticulin mutation have higher frequency of clustering and convoluted hyperchromatic nuclei when compare with wild type patient. In the morphologic review Dr. Loghavi et al fund in patient with CALR mutation higher frequency of Clusters 96% vs 80% P:0.05 Hyperchromatic nuclei 100% vs 55% P: 0.03 Convoluted nuclei 100% vs 50% P: 0.03 When campare to CALR wild type MPN There is one study not publish bur approved yet that shows the association of calreticulin expression in PMF with dense clusters of hyperchromic nuclei megakaryocytes, also in ET with the same mutational status these finding were found. higher frequency of megakaryocytes aberrancies large immature dysmorphic megakaryocytes Loghavi S et al. Am J Clin Path. 2016; In press.

Calreticulin mutation-specific IHC Novel C terminus portion of Mut Calr that is contast in all mutations The expresion was limited to megakaryocytes: Se contaron 10 ptes con ET y 10 ptes con PMF con CALR mut EL % se miro contando sobre 100MGK/slide Vannucchi AM et al. Leukemia. 2014

PMF and prognosis DIPSS score (2010) DIPSS-plus score (2011) Age >65 years Hemoglobin <10 g/dL White blood cell count >25x109/L Peripheral blood blasts ≥1% Constitutional symptoms DIPSS-plus score (2011) Unfavorable karyotype: Complex karyotype, +8, -7/7q-, i(17q), inv 3, -5/5q-, 12p-, 11q23 rearrangement Platelet count <100x109/L RBC transfusion needs And finally the prognosis DIPSS: Median survival was not reached in low-risk patients 9.8 years in intermediate-1 4.8 years in intermediate-2 2.3 years in high risk DIPSS-plus: Median survivals were 15.4 yr Low risk 6.5 yr Int 1 2.3 yr Int 2 1.3 yr High Passamonti et al, Blood. 2010 Gangat et al, J Clin Oncol 2011

CALR and prognosis Vannucchi et al. Leukemia 2013 CALR/ASXL: median survival Low CALR+/ASXL-: 10.4 yr Intermedias CALR+/+ or -/-: 5.8 yr Hish CALR-/ASXL+: 2.3 yr But we can not think only in other mutations, as I said calreticulin have 2 types of mutational status that encompass 80% of all mutated cases, then it is reasonable to look if there are difference in these two types of mutational patterns Vannucchi et al. Leukemia 2013 Nangalia J, NEJM 2013 Klampfl T, NEJM 2013 Tefferi A. Leukemia 2014

Are type 1 and 2 mutations the same? JAK2 And the answer is a big difference in survival as we can see here. Type 1 mutation (52 bp del and the most frequent one around 50%) is associated with better survival and in type 2 mutation the curve of survival is more close to JAK2 mutated patient in red. better survival independent of age, other mutation associated higher DIPSS score, other mutations, higher leukocytes count and spleen size >10cm BCM Type 2 Tefferi A. et al. Leukemia, 2014

Take home messages PMF patients with CALR-mut: Are younger and have higher platelet count Increased megakaryocytes with hyperchromatic nuclei Are associated with better overall survival The presence of calreticulin mutation argues against polycythemia vera CALR-wt and ASXL1 mutation reflects a high-risk molecular signature As a take home msn Mutations of CALR help defining MPN and are exclusive with JAK2 V617F and MPL 515 I want to highlight that: Calretioculin can Aid in the diagnosis of MPN but is not exclusive of this type of neoplasms Also that PMF patients with CALR-mutation associated have characteristic clinical features at presentation and better prognosis than CALR wild type PMF And finally the importance of the presence of ASXL-1 mutation in PMF CARL wild type patient as a high-risk molecular signature

Thank you for your attention.