Poster Presentation

1. Spectrum of mutations in a cohort
of Indian patients with
myeloproliferative neoplasms
Spectrum of mutations in a cohort
of Indian patients with
myeloproliferative neoplasms
Nikhil Rabade, Shruti Chaudhary, Swapnali Joshi, Prashant
Tembhare, Russel Mascarenhas, Sumeet Gujral,
P.G Subramanian, Nikhil Patkar
Tata Memorial Centre, Hematopathology Laboratory,
Mumbai, India
E mail for correspondence: nvpatkar@gmail.com
Nikhil Rabade, Shruti Chaudhary, Swapnali Joshi, Prashant
Tembhare, Russel Mascarenhas, Sumeet Gujral,
P.G Subramanian, Nikhil Patkar
Tata Memorial Centre, Hematopathology Laboratory,
Mumbai, India
E mail for correspondence: nvpatkar@gmail.com

2. Introduction
• The classical BCR-ABL1 negative myeloproliferative neoplasms (MPN) comprise
polycythemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis
(PMF)
• The JAK2 V617F mutation has been observed in up to 98% of patients with PV and 50–
60% of patients with ET and PMF
• Pathogenetic mutations affecting codons S505 or W515 within exon 10 of the
thrombopoietin receptor, MPL, have been identified in up to 10% ET and PMF patients
• High frequencies of mutations in exon 9 of Calreticulin gene with diagnostic and
prognostic significance have been described in JAK2/MPL- unmutated ET and PMF
• ASXL1 mutations are described in 20 -35% cases of PMF and are associated with an
aggressive disease and a poor overall survival
• The classical BCR-ABL1 negative myeloproliferative neoplasms (MPN) comprise
polycythemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis
(PMF)
• The JAK2 V617F mutation has been observed in up to 98% of patients with PV and 50–
60% of patients with ET and PMF
• Pathogenetic mutations affecting codons S505 or W515 within exon 10 of the
thrombopoietin receptor, MPL, have been identified in up to 10% ET and PMF patients
• High frequencies of mutations in exon 9 of Calreticulin gene with diagnostic and
prognostic significance have been described in JAK2/MPL- unmutated ET and PMF
• ASXL1 mutations are described in 20 -35% cases of PMF and are associated with an
aggressive disease and a poor overall survival
• The classical BCR-ABL1 negative myeloproliferative neoplasms (MPN) comprise
polycythemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis
(PMF)
• The JAK2 V617F mutation has been observed in up to 98% of patients with PV and 50–
60% of patients with ET and PMF
• Pathogenetic mutations affecting codons S505 or W515 within exon 10 of the
thrombopoietin receptor, MPL, have been identified in up to 10% ET and PMF patients
• High frequencies of mutations in exon 9 of Calreticulin gene with diagnostic and
prognostic significance have been described in JAK2/MPL- unmutated ET and PMF
• ASXL1 mutations are described in 20 -35% cases of PMF and are associated with an
aggressive disease and a poor overall survival

3. Materials and Methods
• 130 cases diagnosed and classified based on WHO 2008 criteria.
• Genomic DNA was extracted using Qiagen Blood Mini DNA kit
• DNA QC check was done on NanoDrop 2000 spectrophotometer
• JAK2 and MPL mutations were detected by high sensitivity allele specific
polymerase chain reaction (PCR) using fluorescent labelled primers and capillary
electrophoresis
• CALR exon 9 mutations were assessed using fluorescent labelled primers followed
by capillary electrophoresis on ABI3500 genetic analyser and fragment length
analysis
• Exon 12 of ASXL1 was amplified with overlapping primer sets followed by
fragment length analysis and Sanger sequencing
• A positive control, negative control and non template control was included in
each run
• 130 cases diagnosed and classified based on WHO 2008 criteria.
• Genomic DNA was extracted using Qiagen Blood Mini DNA kit
• DNA QC check was done on NanoDrop 2000 spectrophotometer
• JAK2 and MPL mutations were detected by high sensitivity allele specific
polymerase chain reaction (PCR) using fluorescent labelled primers and capillary
electrophoresis
• CALR exon 9 mutations were assessed using fluorescent labelled primers followed
by capillary electrophoresis on ABI3500 genetic analyser and fragment length
analysis
• Exon 12 of ASXL1 was amplified with overlapping primer sets followed by
fragment length analysis and Sanger sequencing
• A positive control, negative control and non template control was included in
each run
• 130 cases diagnosed and classified based on WHO 2008 criteria.
• Genomic DNA was extracted using Qiagen Blood Mini DNA kit
• DNA QC check was done on NanoDrop 2000 spectrophotometer
• JAK2 and MPL mutations were detected by high sensitivity allele specific
polymerase chain reaction (PCR) using fluorescent labelled primers and capillary
electrophoresis
• CALR exon 9 mutations were assessed using fluorescent labelled primers followed
by capillary electrophoresis on ABI3500 genetic analyser and fragment length
analysis
• Exon 12 of ASXL1 was amplified with overlapping primer sets followed by
fragment length analysis and Sanger sequencing
• A positive control, negative control and non template control was included in
each run

4. Results
• 130 cases of MPN with a median age of 50 years (range 17 – 83 years) and
included 82 males and 48 females (M:F 1.7:1)
• 20 cases of PV, 34 cases of ET and 59 of MF
• Seven patients presented with past or present history of thrombotic
events such as Budd Chiari syndrome, cerebrovascular accidents, portal
vein thrombosis and two additional ones has chronic non healing ankle
ulcers.
• Patients with history of thrombosis included four PV and three ET cases
• All the 7 were JAK2p.V617F positive
• 130 cases of MPN with a median age of 50 years (range 17 – 83 years) and
included 82 males and 48 females (M:F 1.7:1)
• 20 cases of PV, 34 cases of ET and 59 of MF
• Seven patients presented with past or present history of thrombotic
events such as Budd Chiari syndrome, cerebrovascular accidents, portal
vein thrombosis and two additional ones has chronic non healing ankle
ulcers.
• Patients with history of thrombosis included four PV and three ET cases
• All the 7 were JAK2p.V617F positive
• 130 cases of MPN with a median age of 50 years (range 17 – 83 years) and
included 82 males and 48 females (M:F 1.7:1)
• 20 cases of PV, 34 cases of ET and 59 of MF
• Seven patients presented with past or present history of thrombotic
events such as Budd Chiari syndrome, cerebrovascular accidents, portal
vein thrombosis and two additional ones has chronic non healing ankle
ulcers.
• Patients with history of thrombosis included four PV and three ET cases
• All the 7 were JAK2p.V617F positive

5. Results
Table I Laboratory findings of MPN patients
Median values All cases (n = 130) PV (n = 20) ET (n= 34) MF (n=59)
Age (years)
50 (17 - 83) 50 (31 -83) 46 (21 - 76)
53 (16 - 81)
54/36, 60 14/6, 70 19/14, 57.5 38/21, 64.4
50 (17 - 83) 50 (31 -83) 46 (21 - 76)
Male/Female, % male
54/36, 60 14/6, 70 19/14, 57.5 38/21, 64.4
Hemoglobin (g/dl)
11.9
(4.2 - 22.3)
18.2
(13.5 - 22-3)
13.8
(7.8 - 16.1)
9.6
(4.2 - 17.7)
Total WBC count (×10^9/L)
12.9
(1.5 - 97)
11.2
(8.4 - 40)
10.6
(2.1 - 46.4)
17.5
(1.5 - 97)
Platelet count (×10^9/L)
344
(15 - 1754)
320
(153 - 560)
810
(450 - 1754)
263
(15 - 816)
Table II Distribution of mutations in MPNTable II Distribution of mutations in MPN
JAK2V617F CALR MPL Triple Negative
PV (n=20) 20 (100%) - - -
MF (n = 59) 34 (57.6%) 14(23.7%) 02 (3.4%) 09 (15.3%)
ET (n = 34) 21 (61.7%) 05 (15.1%) 03 (9.1%) 05 (15.2%)
MPN-U (n = 17) 03 (17.6%) 12 (70.6%) 02 (11.8%) -

6. Results
Table III. Characteristics of ET patients according to their
mutation profiles
JAK2
(n = 21)
CALR
(n=5)
MPL
(n = 3)
Triple
Negative
(n = 5)
Table IV. Characteristics of MF patients according to their
mutation profiles
JAK2
positive
(n = 34)
CALR
positive
(n = 14)
MPL
positive
(n=2)
Triple negative
(n = 9)
Age (years) 57
(35 -81)
48
(16 - 71)
53
(48 – 59)
40
(20 - 58)
Triple
Negative
(n = 5)
Age (years) 48
(21 – 76)
41
(33-63)
45
(42-60)
33
(21-56)
M/F, % male 13/8,
61.9
4/1, 80 2/1, 66.6 1/5, 20
Hemoglobin
(g/dl)
14.7
(7.8-16.1)
13.3
(8 -13.3)
15.6 12.3
(10.1 -15.7)
Total WBC count
(×10^9/L)
13.6
(7 – 46.4)
9.8
(4.1 -12.6)
6.7 7.1
(2.1 – 9.3)
Age (years) 57
(35 -81)
48
(16 - 71)
53
(48 – 59)
40
(20 - 58)
M/F, % male 22/12, 64.7 10/4, 71.4 0/2 6/3, 66.6
Hemoglobin
(g/dl)
10.6
(4.2 - 17.7)
9.8
(6.1 -12.3)
6.9 8.5
(6.2 - 11.9)
Total WBC
count
(×10^9/L)
20.6
( 2.6 - 97)
14.8
(1.5 - 40)
6.8 12.5
(2.9 - 50.9)
Platelet
count
(×10^9/L)
238
(64 - 816)
195
(21 -652)
500 309
(15 - 619)
Total WBC count
(×10^9/L)
13.6
(7 – 46.4)
9.8
(4.1 -12.6)
7.1
(2.1 – 9.3)
Platelet count
(×10^9/L)
781
(450 –
1635)
1350
(1000 –
1670)
617
(546 –
648)
1005
(850 –
1754)
Platelet
count
(×10^9/L)
238
(64 - 816)
195
(21 -652)
309
(15 - 619)
Grade of
fibrosis
2 3 3 2
Spleen size
(cm)
8 (2-25) 22(6-28) - 11(2-20)

7. JAK2V617F
CALR type I
CALR type II
MPL W515L
Wild type
Wild type
JAK2V617F 5 bp Insertion
Control Band
CALR type I MPL W515L
Wild type
Wild type52 bp Deletion
W515L
ASXL1ASXL1
23bp
del

8. CALR mutations
• A total of 23.8% (31 of 130) patients (21 males and 10 females) with a median age
of 46 years (range 21-60 years) harboured CALR mutations.
• CALR exon 9 mutations were detected in 60.8% (14 of 23) and 50% (5 of 10) of JAK2
and MPL negative MF and ET cases respectively.
• Ten different types of CALR indels (8 deletions and 2 insertions) were detected
• Type I and type II mutations were the most common, occurring at a frequency of
45.1% (14 of 31) and 22.5% (7 of 31) respectively
• Eight of the 14(57.1%)CALR positive MF cases had type I whereas 3 (21.4%) had
type II mutations.
• Two of the five (40%)CALR mutations in ET were type I and only one showed type II
mutation.
• A total of 23.8% (31 of 130) patients (21 males and 10 females) with a median age
of 46 years (range 21-60 years) harboured CALR mutations.
• CALR exon 9 mutations were detected in 60.8% (14 of 23) and 50% (5 of 10) of JAK2
and MPL negative MF and ET cases respectively.
• Ten different types of CALR indels (8 deletions and 2 insertions) were detected
• Type I and type II mutations were the most common, occurring at a frequency of
45.1% (14 of 31) and 22.5% (7 of 31) respectively
• Eight of the 14(57.1%)CALR positive MF cases had type I whereas 3 (21.4%) had
type II mutations.
• Two of the five (40%)CALR mutations in ET were type I and only one showed type II
mutation.
• A total of 23.8% (31 of 130) patients (21 males and 10 females) with a median age
of 46 years (range 21-60 years) harboured CALR mutations.
• CALR exon 9 mutations were detected in 60.8% (14 of 23) and 50% (5 of 10) of JAK2
and MPL negative MF and ET cases respectively.
• Ten different types of CALR indels (8 deletions and 2 insertions) were detected
• Type I and type II mutations were the most common, occurring at a frequency of
45.1% (14 of 31) and 22.5% (7 of 31) respectively
• Eight of the 14(57.1%)CALR positive MF cases had type I whereas 3 (21.4%) had
type II mutations.
• Two of the five (40%)CALR mutations in ET were type I and only one showed type II
mutation.

9. ASXL1 mutations
• ASXL1 mutations were detected in 27.7% (10 of 36) MF but none of the
ET(n=11) patients tested
• Five indels and one point mutation
Table VI. Types of ASXL1 mutations
p.R634Kfsx15 c.1900_1922del23
04
p.G646WfsX12 c.1934dupG
• ASXL1 mutations were detected in 27.7% (10 of 36) MF but none of the
ET(n=11) patients tested
• Five indels and one point mutation
Table V. Frequency of ASXL1 mutations in MF
Mutations JAK2
(n=16)
MPL
(n=2)
CALR
(n=13)
Triple
negative
(n=5)
Frequency(%) 12.5
(2 of 16)
50
(1 of 2)
38.4
(5 of 13)
40
(2 of 5)
p.G646WfsX12 c.1934dupG
03
p.P808LfsX10 c.2423delC
01
p.F862DfsX18 c.2583insA
01
p.P750Vfs* c.2249CGTT>GTG 01
p.R693X c.2077C>T
01
• ASXL1 mutations were detected in 27.7% (10 of 36) MF but none of the
ET(n=11) patients tested
• Five indels and one point mutation
p.P808fs*
c.2423delC

10. Discussion and Conclusion
• JAK2, CALR and MPL mutations were mutually exclusive of each other
• CALR mutations have changed the diagnostic algorithm of myeloproliferative
neoplasm
• The detection of these mutations has not only diagnostic but also prognostic
relevance
• Presence of ASXL1 mutations is associated with a poor prognosis especially in the
absence of CALR mutations
• We report frequencies of JAK2V617F, MPL exon 10, CALR and ASXL1 mutations in
130 patients similar to those reported in western literature
• This is the first report of MPL and ASXL1 mutations in myeloproliferative
neoplasms from India
• JAK2, CALR and MPL mutations were mutually exclusive of each other
• CALR mutations have changed the diagnostic algorithm of myeloproliferative
neoplasm
• The detection of these mutations has not only diagnostic but also prognostic
relevance
• Presence of ASXL1 mutations is associated with a poor prognosis especially in the
absence of CALR mutations
• We report frequencies of JAK2V617F, MPL exon 10, CALR and ASXL1 mutations in
130 patients similar to those reported in western literature
• This is the first report of MPL and ASXL1 mutations in myeloproliferative
neoplasms from India
• JAK2, CALR and MPL mutations were mutually exclusive of each other
• CALR mutations have changed the diagnostic algorithm of myeloproliferative
neoplasm
• The detection of these mutations has not only diagnostic but also prognostic
relevance
• Presence of ASXL1 mutations is associated with a poor prognosis especially in the
absence of CALR mutations
• We report frequencies of JAK2V617F, MPL exon 10, CALR and ASXL1 mutations in
130 patients similar to those reported in western literature
• This is the first report of MPL and ASXL1 mutations in myeloproliferative
neoplasms from India