1. 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

2. 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

3. 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
LDH (IU/L)
900
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

4. 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.

5. 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

6. 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

7. 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)

8. 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.

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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.

19. 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

20. 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

21. 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

22. 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

23. 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

24. Thank you for your attention.