JAK/STAT INHIBITION AND BEYOND IN Ph-NEGATIVE MPNs

Molecular prognostication in Ph-negative

MPNs in 2022

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Alessandro Maria Vannucchi and Paola Guglielmelli
CRIMM, Center Research and Innovation of Myeloproliferative Neoplasms, University of Florence, Azienda Ospedaliero­Universitaria Careggi,
Florence, Italy

The application of genomic techniques, including cytogenetics and DNA sequencing, to decipher the molecular land-
scape of patients with myeloproliferative neoplasms (MPNs) has radically modified diagnostic approach and man-
agement through improved risk stratification. Three driver mutated genes (JAK2, MPL, CALR) are variably harbored
by >80% of patients and associated with clinical characteristics, as well as major disease-related complications and
different survival outcomes. Therefore, JAK2 V617F mutation is included in the revised International Prognosis Score
of Thrombosis for Essential Thrombocythemia score for prediction of thrombosis in patients with essential throm-
bocythemia and prefibrotic primary myelofibrosis, while a CALR type 1 mutated genotype constitutes a favorable
variable for survival in patients with myelofibrosis (MF). Novel, integrated clinical and cytogenetic/mutation scores
(Mutation-Enhanced International Prognostic Score System for Transplantation-Age Patients with Primary Myelofi-
brosis [MIPSS70/v2], genetically inspired prognostic scoring system [GIPSS], Myelofibrosis Secondary to PV and ET-
Prognostic Model [MYSEC-PM]) have been devised that guide selection of stem cell transplantation candidates with
MF or help predict the risk associated with the transplant procedure (Myelofibrosis Transplant Scoring System), with
greater performance compared with conventional scores based on hematologic and clinical variables only. On the
other hand, several clinical needs remain unmet despite the great amount of molecular information available nowa-
days. These include the prediction of evolution to acute leukemia in a clinically actionable time frame, the identifi-
cation of patients most likely to derive durable benefits from target agents, in primis JAK inhibitors, and, conversely,
the significance of molecular responses that develop in patients receiving interferon or some novel agents. Here, we
discuss briefly the significance and the role of genomic analysis for prognostication in patients with MPNs from a clini-
cian’s point of view, with the intent to provide how-to-use hints.

LEARNING OBJECTIVES
• To appreciate the variety of abnormalities in the cytogenetic and mutation profiles of patients with myeloprolifera­
tive neoplasms (MPNs)
• To be aware of the central role of molecular tests in the modern management of MPNs
• To learn how to use at best molecular information for risk­stratifying patients with MPNs
• To acknowledge the major clinical needs remaining unmet

Deep characterization of genomic abnormalities is essen­
tial for modern management of chronic myeloprolifera­
tive neoplasms (MPNs), including polycythemia vera (PV),
essential thrombocythemia (ET), and primary myelofibrosis
(PMF), as well as post­PV and post­ET myelofibrosis (MF)
(collectively, secondary myelofibrosis [sMF]). PMF includes
an early/prefibrotic stage (pre­PMF) as well as an overt
fibrotic stage, as 2 distinct diagnostic entities.1 Genom­
ics informs diagnosis and risk assessment and supports
therapy decision­making. Herein, we refer to genomic
abnormalities in MPNs to include only changes in chro­
mosomes/DNA sequence that deserve diagnostic and
prognostic significance and can be identified through the
application of methods available in specialized clinical lab­
oratories. Therefore, this article is by no way an exhaustive
review of genomics in MPNs, nor does it discuss mechanis­
tic implications of genomic abnormalities.
(A few) Technical tips to know
Conventional methods to assess chromosomal abnormal­
ities in MPNs are chromosomal banding (numerical and
structural changes) and fluorescence in situ hybridization

Molecular prognostication in MPN | 225

(that has the poten­tial to dis­cover also cyto­ge­net­i­cally cryp­tic
abnor­mal­i­ties), pref­er­a­bly using bone mar­row (BM) or, if unavail­
able, periph­ eral blood (PB) cells. Driver muta­ tions are rou­
tinely assessed in PB sam­ples (pref­er­a­bly on gra­di­ent-puri­fied
granulocytes, which rep­ re­
sent the variably involved mye­ loid
clone and allow more repro­duc­ible mea­sure­ment of muta­tion
var­i­ant allele fre­quency [VAF] by elim­i­nat­ing con­tam­i­na­tion of
lym­phoid cells) by using dif­fer­ent poly­mer­ase chain reac­tion–
based tech­niques that, par­tic­u­larly for JAK2 V617F muta­tion,
allow pre­cise cal­cu­la­tion of the ratio of mutated vs wild-type
allele VAF; recommended assay sen­si­tiv­ity is ≤1%, in order to
iden­tify early dis­ease pre­sen­ta­tion and, on the other side, to
accu­rately mon­i­tor response to treat­ment (stem cell trans­plan­
Chromosomal abnor­mal­i­ties in MPNs
Chromosomal alter­ations occur at vary­ing fre­quen­cies and have
var­i­able prog­nos­tic sig­nif­i­cance depending on the under­ly­ing
dis­ease (Table 1). Cytogenetic abnor­ mal­i­
ties in PV and ET at
diag­no­sis are rare (5%-15%) but increase with dis­ease pro­gres­
sion to sMF (up to 80%).3 Most com­mon are dele­tion of the long
arm of chro­mo­some 20, tri­somy 8, or tri­somy 9. In PV, an inter­
me­di­ate- and high-risk kar­yo­type cat­e­gory was iden­ti­fied (Table
1) influ­enc­ing over­all sur­vival (OS) and leu­ke­mia-free sur­vival.3,4
In ET, cyto­ge­netic abnor­mal­i­ties are infre­quent (7%) and asso­ci­
ated with shorter sur­vival (10-12 years vs 21 years with­out abnor­
mal­i­ties).5 However, abnor­mal kar­yo­type is not included in any
inte­grated prog­nos­tic model for ET, while in asso­ci­a­tion with

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ta­tion [SCT], inter­feron). Quantification of MPL and CALR VAF is
not rou­tinely performed owing to tech­ni­cal con­straints and lack
of stan­dard­ized assays; how­ever, dis­tinc­tion of type 1 and type 2
CALR muta­tion should be rou­tinely reported since it is prognos­
tically infor­ma­tive. Next-gen­er­a­tion sequenc­ing (NGS)–based
meth­ods are cur­rently incor­po­rated in the workup of selected
patients with MPNs. Usually, they are panel based, inter­ro­gat­ing
20 to 100+ most fre­quently mutated genes, with a con­ven­tional
detec­tion limit of 2% to 5%. Although NGS data can high­light
struc­tural changes and copy num­ber var­i­a­tions, for clin­i­cal pur­
poses, only sin­gle-nucle­o­tide var­i­ants and inser­tions/dele­tions
(indels) are reported. Ideally, attri­bu­tion of a sin­gle-nucle­o­tide
var­i­ant to a somatic change requires paired anal­y­sis of a germ­line
DNA source, which in clin­i­cal prac­tice is unfea­si­ble and expen­
sive and is reserved for selected cases; there­fore, somatic def­i­
ni­tion of a var­i­ant is based on com­par­a­tive inter­ro­ga­tion of large
pop­u­la­tion data­bases to fil­ter out known pop­u­la­tion poly­mor­
phisms. A sec­ond level of com­plex­ity in interpreting NGS data
con­cerns the attri­bu­tion of path­o­ge­nic­ity to the reported var­i­
ant (ie, var­i­ant anno­ta­tion), which requires bioinformatic exper­
tise and should be performed in accor­ dance with published
guide­lines.2
advanced age, leu­ko­cy­to­sis, and venous throm­bo­sis, as well as
advanced age and leu­ko­cy­to­sis, abnor­mal kar­yo­type is prognos­
tically infor­ma­tive for OS and leu­ke­mic trans­for­ma­tion, respec­
tively, in PV.6
Conversely, spe­cific chro­mo­somal abnor­mal­i­ties are an inte­
gral com­po­nent of prog­nos­tic scores for PMF, such as the DIPSS-
plus, genetically inspired prognostic scoring system (GIPSS),
and Mutation-Enhanced International Prognostic Score System
for Transplantation-Age Patients with Primary Myelofibrosis
(MIPSS70/v2) (discussed below). Patients with pre-PMF and overt
PMF have a sim­i­lar inci­dence and type of cyto­ge­netic abnor­
mal­i­ties.7 An iso­lated 13q−, 20q−, and 9+ abnor­mal­ity deserves
favor­able prog­nos­tic value, while unfa­vor­able abnor­mal­i­ties con­
sist of a com­plex kar­yo­type (>3 abnor­mal­i­ties) or a sin­gle or 2
abnor­mal­i­ties, includ­ing +8, −7/7q−, i(17)q, −5/5q−, 12p−, inv(3),
or 11q23 rearrangements or a monosomal kar­yo­type (MK). More
recently, the prog­nos­tic value of cyto­ge­netic abnor­mal­i­ties was
refined in a series of 1002 patients, resulting in 3 risk categories8:
very high risk (VHR), favor­able, and unfa­vor­able kar­yo­type (see
Table 1 for details), with a median sur­vival of 1.2 years (haz­ard
ratio [HR], 3.8; 95% con­fid ­ ence inter­val [CI], 2.9-4.9), 2.9 years
(HR, 1.7; 95% CI, 1.4-2.0), and 4.4 years (ref­er­ence), respec­tively.

Table 1. Prognostically infor­ma­tive cyto­ge­netic abnor­mal­i­ties most com­monly encoun­tered in patients with MPNs

Essential Blast phase

Polycythemia vera Primary mye­lo­fi­bro­sis Post-PV/post–ET-MF
thrombocythemia post-MPNs
Prevalence, % 1-7 10-20 35-50 40-80 80-90
Chromosome • “Intermediate risk”: • “Unfavorable karyo”: com­plex • “Unfavorable” karyo: • Complex
abnor­mal­i­ties - Sole del(20q) or sin­gle or 2 abnor­mal­i­ties, abnor­mal­i­ties other karyo
with prog­nos­tic - Double abnor­mal­i­ties, includ­ing +8, −7/7q−, i(17)q, than iso­lated 13q− includ­ing
sig­nif­i­cance includ­ing +1q (median OS, −5/5q−, 12p−, inv(3), or 11q23 and 20q−, mono­somy more
86 months) rear (included in DIPSS-plus) fre­quently
• “High risk”: • “Favorable” karyo: nor­mal kar­ −5/del(5q),
- Complex kar­yo­type yo­type, sole abnor­mal­i­ties −7/del(7q),
(median OS, 9 months) of 20q−, 13q−, +9, chr 1 trans­ −17/del(17p)/
lo­ca­tion/dupli­ca­tion, or sex i(17q), and −18
chro­mo­some abnor­mal­ity
includ­ing −Y
• “VHR” karyo: sin­gle or mul­ti­ple
abnor­mal­i­ties of −7, inv (3),
I (17q), 12p−, 11q−, and
auto­so­mal tri­so­mies other
than +8 or +9
• “High-risk” karyo: all­ abnor­mal­i­
ties that are not VHR and favor­
able (included in MIPSS70/plus
v2.0 and GIPSS scores)

226 | Hematology 2022 | ASH Education Program

The unfa­vor­able and VHR kar­yo­type con­ferred a 2.0- and 4.4-fold
increased risk to prog­ress to blast phase (BP), respec­tively.
An abnor­mal kar­yo­type was found in 34% of 376 patients with
sMF and had neg­at­ ive impact on median sur­vival: 10.1 years (95%
CI, 8.1-not reached) com­pared with 6.1 years (95% CI, 4.8-not
reached) for nor­mal kar­yo­type.3 Shortened sur­vival was fur­ther
affected by the pres­ence of a com­plex kar­yo­type (2.7 years),
com­plex kar­yo­type with­out MK (3.4 years), and MK (2.1 years).
However, when added to the Myelofibrosis Secondary to PV and
ET-Prognostic Model (MYSEC-PM) strata (see below), abnor­mal
kar­yo­type did not add to sur­vival pre­dic­tion.
Cytogenetic abnor­mal­i­ties con­sti­tute an impor­tant risk fac­tor
in the pro­gres­sion of MPNs to the accel­er­ated phase (AP)/BP and
V617F–neg­at­ive PV; muta­tions of the thrombopoietin recep­tor
(MPL W515L/K/A) in 5% to 8% of patients with ET and PMF, includ­
ing rare S505N; and exon 9 calreticulin muta­tions (CALR) in 20% to
25% of ET and PMF. There are 2 pro­to­type CALR muta­tions: type
1 (52-bp dele­tion) and type 2 (5-bp inser­tion), with sev­eral other
type 1–like and type 2–like var­i­ants. Driver muta­tions rep­re­sent
major diag­nos­tic cri­te­ria in the lat­est World Health Organization
(WHO) clas­si­fi­ca­tion,1 as well as in the fifth edi­tion of the WHO
clas­si­fi­ca­tion12 and the International Consensus Classification of
Myeloid Neoplasms and Acute Leukemias.13 However, a driver
muta­tion is miss­ing in occa­sional patients with PV and 10% to
15% of those with ET and PMF (“tri­ple neg­a­tive”); this even­tu­al­ity
is acknowl­edged in cur­rent diag­nos­tic sys­tems that rec­om­mend

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are reported in up to 90% of patients.9 The chro­mo­somal defects
most sig­nif­i­cantly asso­ci­ated with AP/BP are the com­plex kar­
yo­type (includ­ing −5/del(5q), −7/del(7q), −17/del(17p)/i(17q), and
−18), gains of chro­mo­some 1q (MDM4 gene locus, encoding a
neg­a­tive reg­u­la­tor of TP53), or MK.10 Most patients with AP/BP
show loss of 17p13, which leads to a dele­tion of TP53.11
Mutations asso­ci­ated with MPNs
MPN-asso­ci­ated driver muta­tions include JAK2 V617F muta­tion in
>95% of patients with PV and 60% with ET and PMF; JAK2 exon 12
indels (the most fre­quent is N542_E543del) in 1% to 3% of JAK2
NGS to search uncom­mon (“noncanonical”) somatic var­i­ants in
JAK2 and MPL14 or to iden­tify a clonal marker. NGS may also dis­
cover rare JAK2/MPL germline var­i­ants that under­lie hered­i­tary
erythrocytosis or thrombocytosis mim­ick­ing MPNs.15,16
Similar to those with acute leu­ke­mia and myelodysplastic
syn­dromes, patients with MPN may har­bor muta­tions in a vari­
ety of so-called mye­loid genes that include mainly epi­ge­netic
reg­u­la­tors, spliceosome com­po­nents, and onco­genes (Table 2
lists the most fre­ quent).17 These muta­ tions may pre­ date or
fol­low the acqui­si­tion of the driver muta­tion and affect dis­ease
phe­no­type.18 Not all­muta­tions have uni­form prog­nos­tic sig­nif­
i­cance. A set of so-called high muta­tion risk (HMR) genes,19,20

Table 2. Prognostically infor­ma­tive muta­tions of most fre­quent detec­tion in patients with MPN

Gene Polycythemia vera Essential thrombocythemia Primary mye­lo­fi­bro­sis “Secondary” mye­lo­fi­bro­sis

JAK2 • Higher risk of throm­bo­sis • Intermediate prog­no­sis and
V617F (included in revised IPSET) higher risk of throm­bo­sis
com­pared to patients with
CALR muta­tion
CALR • Lower risk of throm­bo­sis com­ • CALR type 1/like: improved • Improved OS com­pared
pared to JAK2 mutated sur­vival com­pared to JAK2 to JAK2 V617F and “tri­ple
V617F and “tri­ple neg­a­tive” neg­a­tive”
• Improved out­come after SCT • Improved out­come after SCT
• Absence of CALR type 1/like • Absence of CALR muta­tion
included in MIPSS70/plus v2.0 included in MYSEC-PM and
• Absence of CALR muta­tion MTSS scores
included in MYSEC-PM and
MTSS scores
MPL W515 • Intermediate prog­no­sis and
higher risk of throm­bo­sis
com­pared to patients with
CALR muta­tion
“Triple • Inferior OS and LFS com­pared
neg­a­tive” to JAK2/CALR muta­tion (espe­
cially in pre-PMF)
JAK2 • Similar rates of throm­bo­sis,
exon 12 evo­lu­tion to post–PV-MF
and BP, and sur­vival to
JAK2 V617F
ASXL1 • “Adverse var­i­ant” asso­ci­ • “HMR var­i­ant” asso­ci­ated with
ated with infe­rior OS and infe­rior OS/LFS, lower PFS fol­
MFS low­ing SCT
• Included in MIPSS-PV • Included in MIPSS70/plus v2.0
and GIPSS
EZH2 • “Adverse var­i­ant” asso­ci­ated • “HMR var­i­ant” asso­ci­ated with
with infe­rior OS/LFS infe­rior OS
• Included in MIPSS70/plus v2.0

Molecular prog­nos­ti­ca­tion in MPN | 227

Table 2. Prognostically infor­ma­tive muta­tions of most fre­quent detec­tion in patients with MPN (Continued )

Gene Polycythemia vera Essential thrombocythemia Primary mye­lo­fi­bro­sis “Secondary” mye­lo­fi­bro­sis

IDH1 • IDH1: “adverse var­i­ant” • IDH2: “adverse var­i­ant” asso­ci­ • “HMR var­i­ants” asso­ci­ated with • “Adverse var­i­ant” asso­ci­ated
IDH2 asso­ci­ated with infe­rior OS ated with infe­rior OS infe­rior OS/LFS and as lower with lower PFS fol­low­ing SCT
and LFS PFS fol­low­ing SCT
• Included in MIPSS70/plus v2.0
SRSF2 • “Adverse var­i­ant” asso­ci­ • “Adverse var­i­ant” asso­ci­ated • “HMR var­i­ant” asso­ci­ated with
ated with infe­rior OS with infe­rior OS/LFS infe­rior OS/LFS
• Included in MIPSS-PV • Included in MIPSS-ET • Included in MIPSS70/plus v2.0
and GIPSS
U2AF1 • “HMR var­i­ant” asso­ci­ated infe­
Q157 rior OS
• Included in MIPSS70/plus v2.0

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and GIPSS
U2AF1 or • U2AF1: “adverse var­i­ant” asso­ • “Adverse var­i­ant” asso­ci­ated • “Adverse var­i­ant” asso­ci­ated
DNMT3A ci­ated with infe­rior OS/MFS with worse OS in SCT with worse OS in SCT
or CBL • Included in MIPSS-ET
RUNX1 • “Adverse var­i­ant” asso­ci­ • “Adverse var­i­ant” asso­ci­ated
ated with infe­rior OS and with infe­rior OS/LFS
LFS
TP53 • “Adverse var­i­ant” asso­ci­ • “Adverse var­i­ant” asso­ci­ated • “Adverse var­i­ant” asso­ci­ated
ated with infe­rior LFS with infe­rior OS/LFS with leu­ke­mic trans­for­ma­tion
• Included in MIPSS-ET
LNK • “Adverse var­i­ant” asso­ci­ated
(SH2B3) with infe­rior OS
SF3B1 • “Adverse var­i­ant” asso­ci­ated
with infe­rior OS and MFS
• Included in MIPSS-ET
RAS • “Adverse variant” associated
with inferior OS
• Associated with JAKi-refrac­to­
ri­ness
LFS, leu­ke­mia-free sur­vival; MFS, myelofibrosis-free survival; PFS, progression-free survival.

includ­ing ASXL1, EZH2, SRSF2, IDH1, IDH2, and U2AF1Q517, pre­
dict infe­rior sur­vival in PMF, inde­pen­dent of each other and
other risk fac­tors, with >1 mutated gene har­bor­ing addi­tional
neg­a­tive weight.21
CLINICAL CASE
A 42-year-old man presented to an out­pa­tient clinic fol­low­ing
the ser­en­dip­i­tous dis­cov­ery of thrombocytosis (800 × 109/L)
in rou­ tine blood cell tests. Hemoglobin was in the lower
range (13.2  g/dL with nor­mal indexes), and leu­ko­cytes were
10.3 × 109/L, with no imma­ ture cells in the blood smear. All
other rou­tine tests were nor­mal. He was asymp­tom­atic, had no
referred famil­ial his­tory for hema­to­logic malig­nan­cies, and had
no known generic car­dio­vas­cu­lar risk fac­tor; phys­i­cal exam­i­na­
tion was unre­mark­able, and spleen was not pal­pa­ble. An ultra­
sound scan con­firmed nor­mal spleen vol­ume and also ruled out
splanch­nic vein throm­bo­sis. The patient under­went BM biopsy,
which revealed hypercellularity with gran­u­lo­cytic pro­lif­er­a­tion
with­out atypia or blast increase; marked expan­sion of the mega­
kar­yo­cytic lin­e­age with mega­kar­yo­cytes of var­i­able size and
nuclei atypia, in tight clus­ters; and slightly reduced eryth­ro­poi­
e­sis. Reticulin fibro­sis was grade 0/1. Cytogenetics showed a
nor­mal male kar­yo­type. A JAK2 V617F muta­tion with a VAF of
34% was detected in PB granulocytes. On the basis of those
find­ings, the patient received a WHO 2016 diag­no­sis of prefi­
brotic MF. The patient had a score of 2 according to the MPN-10
total symp­tom score (MPN-10).22
Molecular prog­nos­ti­ca­tion sys­tems to address the risk
of vas­cu­lar events
Cardiovascular events are the main rea­son for mor­bid­ity and
mor­tal­ity in PV and ET; in pre-PMF, the rate of throm­bo­sis is sim­i­
lar to ET, esti­mated at 1.99% patients/year. Patients with PV and
ET are con­ven­tion­ally risk-strat­i­fied for throm­bo­sis based on
2 clin­i­cal cri­te­ria: age ≥60 years and throm­bo­sis his­tory.23 How­
ever, the dis­cov­ery that patients with ET who have a CALR muta­
tion have a sig­nif­i­cantly reduced rate of vas­cu­lar events led to the
devel­op­ment of an inte­grated score, the International Prognosis
Score of Thrombosis for Essential Thrombocythemia (IPSET),
includ­ing the cur­rently recommended revised ver­sion (Table 3).24
The pos­i­tiv­ity of a JAK2 V617F muta­tion, in the absence of the

228 | Hematology 2022 | ASH Education Program

Table 3. Molecularly inte­grated risk scores in patients with ET and PV

Essential thrombocythemia Polycythemia vera

Characteristic
Revised IPSET MIPSS-ETa MIPSS-PVa
Age, y >60 >60 (4) >67 (2)
History of throm­bo­sis Yes — Yes (1)
JAK2 V617F Yes — —
White blood cell count — ≥11×109/L [1] ≥15×109/L (1)
Adverse muta­tion — SRSF2, SF3B1, U2AF1, TP53 [2] SRSF2 [3]
Male sex — Yes [1] —
Thrombosis risk group (rate % patients/y) Risk group [score]: median sur­vival (y)

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Categories Very low: no throm­bo­sis his­tory, age ≤60y, JAK2WT Low [0-1]: 34.4 Low [0-1]: 24.0
Low: no throm­bo­sis his­tory, age ≤60y, JAK2 V617F Intermediate [2-5]: 14.1 Intermediate [2-3]: 13.1
Intermediate: no throm­bo­sis his­tory, age >60y, JAK2WT High [≥6]: 7.9 High [≥4]: 3.2
High: throm­bo­sis his­tory or age >60y with JAK2 V617F
Values within brack­ets indi­cate the var­i­able’s rank.
a

2 afore­men­tioned clin­i­cal cri­te­ria, qualifies a patient as low risk,
with aspi­rin as suggested treat­ment. IPSET predicted throm­bo­
sis risk also in prefibrotic MF.25 The role of addi­tional muta­tions
for throm­bo­sis in ET is largely unset­tled.26 In PV, no com­pel­ling
evi­dence for driver/addi­tional muta­tions as being infor­ma­tive
for throm­ bo­sis risk has been reported yet; the dem­ on­
stra­
tion that a JAK2 V617F VAF ≥50% has an inde­pen­dent HR of 3.8
(95% CI, 1.7-8.6) for venous throm­bo­sis27 might fos­ter devel­op­
ment of inte­grated scores.
Molecular prog­nos­ti­ca­tion sys­tems to address the risk
of dying
According to a series of 3023 patients with MPNs, median OS is
around 20, 15, and 5 years, respec­tively, for ET, PV, and PMF.28 In
ET, the con­ven­tional IPSET score uses age >60 years, leu­ko­cytes
>11 × 109/L, and throm­bo­sis his­tory to dif­fer­en­ti­ate low-, inter­
me­di­ate-, and high-risk patients with respec­tive median sur­
vival not reached, 24.5 years, and 13.8 years.29 For PV, the
International Working Group sur­vival model delin­eated 3 risk
groups with median sur­ viv­als of 10.9, 18.9, and 27.8 years,
based on older age, leu­ko­cy­to­sis, and venous throm­bo­sis.6
More recently, a muta­tion-enhanced inter­na­tional prog­nos­tic
sys­tem (MIPSS) for ET and PV was devised.4 Spliceosome muta­
tions adversely affected OS (SF3B1, SRSF2 in ET and SRSF2
in PV) and mye­lo­fi­bro­sis-free sur­vival (U2AF1, SF3B1 in ET);
TP53 muta­tions predicted BP in ET. These adverse muta­tions
occurred in 10% and 2% of patients with ET and PV, respec­
tively. The inte­grated clin­i­cal-molec­u­lar model iden­ti­fied 3 risk
categories with a median sur­vival of 8.3 to 34.3 years in ET and
4.6 years to not reached in PV4 (Table 3). However, now­a­days,
these scores are not used in clin­ i­
cal prac­
tice for ther­ apy
deci­sion-mak­ing.
Conversely, prog­nos­tic scores deserve a cen­tral role in the
man­age­ment of patients with MF spe­cif­i­cally concerning the
indi­ca­tion to SCT, the only poten­tially cura­tive option; how­
ever, the not neg­ li­
gi­
ble risk asso­ ci­ated with the trans­ plant
man­dates care­ful selec­tion of patients to make the pro­ce­dure
risk-effec­tive.30 Conventional clin­i­cal-only scores, such as Inter­
national Prognostic Scoring System (IPSS) and Dynamic-IPSS
(DIPSS), or the cyto­ge­net­ics-inte­grated DIPSS-plus con­tinue to
be largely used in prac­tice and, nota­bly, still are used for selec­
tion of patients in clin­i­cal tri­als. Furthermore, the IPSS and DIPSS
scores are rou­ tinely applied also to patients with pre-PMF,
although it was dem­on­strated that they poorly dis­crim­i­nate
the inter­me­di­ate 1 and 2 risk categories.7,31 Such short­com­ings
might be addressed by more recently devel­oped, more infor­
ma­tive, molec­u­lar-inte­grated scores.32
The abovementioned HMR genes con­fig­ure an adverse var­
i­able in inte­grated risk scores for PMF such as the Mutation-
Enhanced International Prognostic Score System for Trans­
plantation-Age Patients With Primary Myelofibrosis (MIPSS70)31
and MIPSS70v2.0, enriched with sex-adjusted hemo­ glo­bin
and revised kar­yo­type clas­si­fi­ca­tion.33 MIPSS70, orig­i­nally
devel­ oped for patients of trans­ plant age but infor­ ma­tive
age-inde­ pen­dently as well, includes 9 var­ i­
ables, 3 genetic
(HMR muta­tion, >1 HMR muta­tion, and absence of CALR type
1/like muta­tion), 5 hema­to­logic-clin­i­cal fac­tors, and bone
mar­row fibro­sis grade ≥2 (Table 4). Based on unique var­ia ­ ble-
spe­cific HR weighted scores, 3-tiered MIPSS70 low-, inter­me­
di­ate-, and high-risk categories, with cor­re­spond­ing median
sur­vival ranges of 27.7 years to “not reached,” 6.3 to 7.1 years,
and 2.3 to 3.1 years, were iden­ti­fied and val­i­dated in 2 inde­
pen­dent cohorts. Conversely, MIPSSv2 includes 5 risk cate­
gories: VHR (median sur­vival, 1.8 years), high risk (4.1 years),
inter­me­di­ate risk (7.7 years), low risk (16.4 years), and very low
risk (median not reached) (Table 4). Improved per­for­mance of
MIPSS/v2 com­pared with con­ven­tional IPSS/DIPSS was dem­
on­strated, with up to 40% of the patients being upgraded.
Of note, the MIPSS score was devel­oped for all­patients with
PMF and includes fibro­sis grade (0-1 vs 2-3 cat­e­gory) as an
embed­ ded var­ i­able addressing the pre-PMF and overt-PMF
cat­e­gory. A sep­a­rate model based only on molec­u­lar fac­tors,
GIPSS, incor­ po­ rated the 3-tiered kar­ yo­
type categories and
4 muta­tions (ASXL1, SRSF2, and U2AF1Q157, plus absence of type
1/like CALR muta­tion) as inde­pen­dent risk fac­tors for sur­vival;
risk categories were low (median sur­vival, 26.4 years), inter­me­
di­ate 1 (8.0 years), inter­me­di­ate 2 (4.2 years), and high (2 years

Molecular prog­nos­ti­ca­tion in MPN | 229

Table 4. Molecularly inte­grated risk scores in patients with PMF and sMF

MIPSS70/plus
DIPSS-plus MIPSS70 GIPSS MYSEC-PM MTSS
v2.0
Prognostic var­i­ables (points)
Age, y >65 (1) — — — 0.15 point/y ≥57 (1)
Constitutional Present (1) Present (1) Present (2) — Present (1) —
symp­toms
Hemoglobin, g/dL <10.0 (2) <10 (1) 8-9.9 F; 9-10.9M — <11 (2) —
(1)
<8 F, <9M (2)
Leukocyte >25.0 (1) >25.0 (2) — — >25.0 (1)

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count,×109/L
Circulating blast ≥1% (1) ≥2% (1) ≥2% (1) — ≥3% (2) —
cells, %
Platelet <100 (1) <100 (2) — — <150 (1) <150 (1)
count,×109/L
RBC trans­fu­sion Yes (1) — — — — —
need
Karyotype Unfavorable (1) — High risk (3) High risk (1) — —
Very high risk (4) Very high risk (2)
BM fibro­sis grade — ≥MF2 (1) — — — —
Absence of CALR — Yes (1) Yes (2) Yes (1) Yes (2) Yes/absence of
MPL (2)
Molecular pro­file — HMR 1 mut (1) HMR+U2AF1 Q157 ASXL1 (1) — ASXL1 (1)
HMR ≥2 mut (2) 1 mut (2) SRSF2 (1)
HMR+U2AF1 U2AF1 Q157 (1)
Q157≥2 mut (3)
Karnofsky PS — — — — — < 90% (1)
HLA donor — — — — — MUD (2)
Risk group (score): Overall sur­vival in years
Categories Low (0):15y Low (0-1): 27.7y Very low (0): not Low (0): 26.4y Low (<11): not Low (0-2): 90%*
Intermediate 1 (1): Intermediate (2-4): reached Intermediate 1 (1): reached Intermediate (3-
6.7y 7.1y Low (1-2): 10.3y 8.0y Intermediate 1 4): 77%*
Intermediate 2 High (≥5): 2.3y Intermediate (3- Intermediate 2 (11-14): 9.3y High (5): 50%*
(2-3): 2.9y 4): 7.0y (2): 4.2y Intermediate 2 Very high (6-9):
High (≥4): 1.3y High (5-8): 3.5y High (3-6): 2.0y (14-16): 4.4y 34%*
Very high (≥9): High (≥16): 2.0y
1.8y
When/for whom During fol­low-up; At diag­no­sis; PMF At diag­no­sis; PMF At diag­no­sis; PMF At diag­no­sis; sMF MF patients
PMF patients patients patients patients planned for SCT
Website tool https:​­/​­/qxmd​ www​­.mipss70score​­.it www​ NA www​­.mysec-pm​ NA
.­ com​­/calculate​ ­.mipss70score​­.it ­.eu
­/calculator_315​­/
dipss​­-plus​­-score​
­-for​­-prognosis​­-in​
­-myelofibrosis
Molecular pro­file descrip­tion Karyotype descrip­tion
DIPSS-plus Unfavorable: com­plex kar­yo­type or sin­gle or 2 abnor­mal­i­ties, includ­ing +8,
−7/7q−, i(17q), −5/5q−, 12p−, inv(3), or 11q23 rearrangement
MIPSS70 Absence of CALR type 1 Not included
HRM: muta­tion in at least 1 of these
mutated genes: ASXL1, SRSF2, IDH1, IDH2,
EZH2
MIPSS70/plus v2.0 Absence of CALR type 1 High risk (HR): all­the abnor­mal­i­ties that are not VHR and favor­able (nor­mal kar­
HMR: muta­tion in at least 1 of these yo­type or sole abnor­mal­i­ties of 20q−, 13q−, +9, chro­mo­some 1 trans­lo­ca­tion/
mutated genes: ASXL1, SRSF2, IDH1, IDH2, dupli­ca­tion, or sex chro­mo­some abnor­mal­ity includ­ing −Y)
EZH2, U2AF1 Q157 Very high risk (VHR): includes sin­gle or mul­ti­ple abnor­mal­i­ties of −7, inv (3),
I (17q), 12p−, 11q−, and auto­so­mal tri­so­mies other than +8 or +9

230 | Hematology 2022 | ASH Education Program

Table 4. Molecularly inte­grated risk scores in patients with PMF and sMF (Continued )

MIPSS70/plus
DIPSS-plus MIPSS70 GIPSS MYSEC-PM MTSS
v2.0
Prognostic var­i­ables (points)
GIPSS Mutation in ASXL1, SRSF2, U2AF1 Q157 Same as MIPPS70/plus v2.0
MYSEC-PM Absence of CALR (all­types) Not included
MTSS Absence of CALR (all­types)/MPL Not included
Mutation in ASXL1
According to National Comprehensive Cancer Network guide­lines, in patients with MF, a lower-risk cat­e­gory includes the fol­low­ing com­bi­na­tion:
MIPSS70≤3, MIPSS70v2≤3, DIPSS-plus ≤1, DIPSS ≤2, and MYSEC-PM <14, while a higher-risk cat­e­gory includes MIPSS70≥4, MIPSS70v2≥4, DIPSS-plus >1,
DIPSS >2, and MYSEC-PM ≥14.

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*5-year overall survival (learning-cohort).
HLA, human leukocyte antigen; NA, not available; PS, performance status; RBC, red blood cell.

[≥3 points]).34 For patients with sMF, the MYSEC-PM includes
5 clin­i­cal fac­tors plus an unmutated CALR geno­type, while the
role of “mye­loid” muta­tions is still debated.35 The sim­pli­fied
National Comprehensive Cancer Network guide­ lines strat­ i­
fi­ca­tion cri­te­ria include a lower and a higher risk cat­eg ­ ory,
based on var­i­able scores of MIPSS and MIPSSv2, if geno­mics is
avail­­able, or DIPSS-plus and MYSEC-PM if molec­u­lar test­ing is
not avail­­able.36
Mutations are included also in the Myelofibrosis Transplant
Scoring System (MTSS), an inte­grated score devel­oped for pre­
dicting prog­no­sis after SCT for PMF and sMF that includes age,
Karnofsky per­for­mance sta­tus, throm­bo­cy­to­pe­nia, leu­ko­cy­to­sis,
human leukocyte antigen (HLA)-mismatched unre­lated donor,
ASXL1 muta­tion, and non-CALR/MPL driver muta­tion geno­type.
The 5-year sur­vival was 83% (95% CI, 71%-95%), 64% (53%-75%),
37% (17%-57%), and 22% (4%-39%), respec­ tively, in the low-,
inter­me­di­ate-, high-, and very high-risk categories37 (Table 4).
evo­lu­tion of pre-PMF to overt PM was made. The dis­ease was
strat­i­fied as fol­lows:
DIPPS-plus: inter­me­di­ate 1 risk, due to con­sti­tu­tional symp­
toms and >1% PB blasts; the esti­mated sur­vival is about 7 years.
MIPSSv2: very high risk, due to BM fibro­sis ≥G2, con­sti­tu­
tional symp­toms, absence of CALR type 1 muta­tion, and HMR
cat­e­gory with ≥2 HMR mutated genes; the esti­mated sur­vival is
<2 years with <5% like­li­hood of sur­vival at 10 years.
The patient had become highly symp­tom­atic and had an
MPN-10 score of 44. An indi­ca­tion to hema­to­poi­etic SCT was
posed after thor­ ough dis­
cus­sion with the patient, based
on MIPSSv2. He had a fully HLA-matched sib­ling donor. The
MTSS score yielded an inter­me­di­ate risk cat­e­gory, due to the
absence of a CALR/MPL muta­tion and the pres­ence of an ASXL1
muta­tion; the esti­mated 5-year sur­vival was 77%. After SCT, the
patient cleared the JAK2 V617F muta­tion at 4 months and HMR
muta­tions (assayed at 1 year). He is alive, with no evi­dence of
dis­ease, after 3 years.

CLINICAL CASE (Con­t in­u ed)
The patient was cat­e­go­rized at diag­no­sis as IPSS low risk, with
projected OS >10 years. No cytoreductive ther­apy was insti­
tuted, except low-dose aspi­ rin since, based on the revised
IPSET score, he belonged to the low-risk cat­e­gory. For the next
5 years, the dis­ease course was unevent­ful. Then, his hemo­
glo­bin level began to down­trend (11.4 g/dL), mild leu­ko­cy­to­
sis (18 × 109/L) appeared steadily, plate­lets were 550 × 1012/L, 1%
blasts and leukoerythroblastosis were detected in the blood
smear, and lactate dehydrogenase was increased 1.8-fold
above nor­mal. Spleen was pal­pa­ble at 4 cm from the left cos­
tal mar­gin, and there was a recent onset of night sweats. The
patient consulted another insti­tu­tion, where a new diag­nos­tic
pro­ce­dure was performed. The bone mar­row biopsy spec­i­men
revealed slightly reduced age-adjusted cel­lu­lar­ity, with marked
increase of atyp­ i­
cal mega­ kar­
yo­cytes in paratrabecular clus­
ters; CD34+ cells were around 3%. Fibrosis was G2, with sparse
areas of G3 and ini­tial focal collagenization. PB blasts were 2%.
Cytogenetics showed iso­lated tri­somy 9. Targeted NGS panel
iden­ti­fied muta­tions in ASXL1 (VAF, 24%) and SRSF2 (VAF, 33%).
The VAF of JAK2 V617F had increased to 75%. A diag­no­sis of
How we use molec­u­lar tests in patients with MPNs
Our approach to the use of molec­u­lar tests for prog­nos­ti­ca­
tion pur­poses in MPNs is depicted in Figure 1. Driver muta­tions
are step­wise inter­ro­gated in all­patients since, beyond being
required for diag­no­sis, they are prognostically infor­ma­tive in ET
and pre-PMF for throm­bo­sis (revised IPSET) and in MF for sur­
vival (favor­able impact of CALR type 1/like). We use quan­ti­ta­tive
assays for mea­sur­ing JAK2 V617F VAF at diag­no­sis and even­tu­
ally doc­u­ment its increase at the time of evo­lu­tion to post-PV
and post-ET MF. Furthermore, serial mea­sure­ment of JAK2 V617F
might be infor­ma­tive in patients with PV receiv­ing ropeginter­
feron to doc­um ­ ent changes in the clone of mutated cells.38,39
Owing to the lim­ited appli­ca­bil­ity of sur­vival prog­nos­tic mod­
els for deci­sion-mak­ing, together with finan­cial con­sid­er­ations,
we do not cur­rently sup­port rou­tine appli­ca­tion of NGS to inter­
ro­gate mye­loid muta­tions in patients with PV and ET, while we
per­form NGS rou­tinely in all­trans­plant-age patients with PMF
and sMF at diag­no­sis. Although most expe­ri­ence was obtained
until now in overt PMF, we cur­rently per­form NGS also in youn­ger
patients with pre-PMF at diag­no­sis to cal­cu­late the MIPSS score,
as the presented clin­i­cal case would have advo­cated. Results

Molecular prog­nos­ti­ca­tion in MPN | 231

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Figure 1. Flowchart on how we use genomic assays for diagnosis (A) and for prognostication in patients (B) with an established
diagnosis of MPN. Selected information (cytogenetics, JAK2 V617F and CALR mutations) collected at the time of diagnosis may also
inform risk scores at step B. NGS analysis for myeloid genes, if not available at the time of original investigation, is recommended in
patients with PMF and sMF who are transplant eligible. PET, post-ET; PPV, post-PV.

could be even­ tu­ally used also for assessing the trans­ plant-
related risk (MTSS). There is still no indi­ca­tion if, and at what
inter­vals, NGS should be repeated dur­ing the course of dis­ease;
in prac­tice, now­ad ­ ays NGS is usu­ally performed coin­ci­dent with
some clin­i­cal and hema­to­logic evi­dence of dis­ease pro­gres­sion.
In SCT-inel­i­gi­ble patients, inte­grated scores do not pro­vide clin­
i­cally action­able infor­ma­tion, and we con­sider them not man­da­
tory if cost con­sid­er­ations limit their use. Cytogenetics should
be ide­ally obtained in all­patients with MPNs at diag­no­sis and
dur­ing the clin­i­cal course, if indi­cated, to exclude com­plex kar­
yo­
type and/or TP53 involve­ ment, although we acknowl­ edge
that results of the kar­yo­type inform clin­i­cal deci­sions only in
patients with PMF.
Conclusions
The past 10 years have witnessed incred­i­ble advance­ments in
molec­u­lar-based prog­nos­ti­ca­tion of MPNs. Yet, we are left with
a num­ber of clin­i­cally rel­e­vant unmet needs, includ­ing the pre­
dic­tion of acute leu­ke­mia in a clin­i­cally action­able time frame,
the pre­dic­tion of response to JAKi (which might be adversely

232 | Hematology 2022 | ASH Education Program

asso­ci­ated with RAS/CBL muta­tions),40 and the short- and long-
term prog­nos­tic sig­nif­i­cance of molec­u­lar responses under
treat­ment.38,41,42 However, “Nothing hap­pens quite by chance.
It’s a ques­tion of accre­tion of infor­ma­tion and expe­ri­ence” (Jonas
Salk). So, it may be expected that dis­cov­ery of new muta­tions,
epi­ge­netic mark­ers, and altered RNA expres­sion pro­file; appli­
ca­tion of a sin­ gle-cell geno­type; and exploi­ ta­
tion of arti­fi­
cial
intel­li­gence to inter­pret geno­mic data might grad­u­ally result in
novel, improved prog­nos­tic scores with a stron­ger impact on
clin­i­cal deci­sion-mak­ing for patients with MPNs.
Conflict-of-inter­est dis­clo­sure
Alessandro Maria Vannucchi has no rel­e­vant con­flict of inter­est.
Paola Guglielmellihas has no rel­e­vant con­flict of inter­est.
Off-label drug use
Alessandro Maria Vannucchi: nothing to disclose.
Paola Guglielmelli: nothing to disclose.
Correspondence
Alessandro Maria Vannucchi, CRIMM, Center Research and Inno­
vation of Myeloproliferative Neoplasms, University of Florence,
Azienda Ospedaliero-Universitaria Careggi, 50134 Florence, Italy;
e-mail: amvannucchi@unifi​­.it.
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© 2022 by The Amer­i­can Society of Hematology
DOI 10.1182/hema­tol­ogy.2022000339
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