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\begin{document}

\title{Programmed Death-Ligand 1 (PD-L1) Expression in Patients with Primary or
Secondary Myelofibrosis}



\author[1]{Francisco Socola}%
\author[2]{Moayed Ibrahim}%
\author[2]{Catherine Murphree}%
\author[3]{Kirtesh Patel}%
\author[3]{Matthew Mastrodomenico}%
\author[2]{Nakhle S. Saba}%
\author[2]{Hana Safah}%
\author[4]{Janet Schmid}%
\affil[1]{AdventHealth Orlando}%
\affil[2]{Tulane University John W Deming Department of Medicine}%
\affil[3]{Delta Pathology Group}%
\affil[4]{Pathology Department at Tulane University}%


\vspace{-1em}



  
  \date{May 30, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
It has been described in mice models that Primary Myelofibrosis (PMF)
with JAK2-V617F mutation has an increased expression of programmed
death-ligand 1 (PD-L1) in megakaryocytes leading to cancer immune
evasion by inhibiting the T-lymphocytes. To prove this hypothesis, we
quantified PD-L1 expression on 29 bone marrow (BM) biopsies. We created
a scoring system to quantify PD-L1 expression in megakaryocytes. We
obtained 14 BM with JAK2 positive PMF, 5 JAK2 negative PMF and 10
patients with normal BM biopsies. PD-L1 expression was higher in the
JAK2 positive group compared to the control group with a score of 212.6
vs 121.1 (t-value 2.05,p-value 0.025). In addition, the score was higher
in the PMF group regardless of JAK2 mutational status when compared to
the control group with score of 205.9 vs 121.1(t-value 2.12,p-value
0.021). There was no difference in the PD-L1 score between the JAK2
negative vs the control group 187.2 vs 121.1 (t-value 1.02,p-value
0.162). These findings suggest that PMF patients with a JAK2 mutation
have a higher PD-L1 expression in megakaryocytes compared to the control
group. We postulate that the combination of checkpoint and JAK2
inhibitors may be an active treatment option in JAK2 mutated PMF given
the higher PD-L1 expression.%
\end{abstract}%



\sloppy


\textbf{Article title}

Programmed Death-Ligand 1 (PD-L1) Expression in Patients with Primary or
Secondary Myelofibrosis

\textbf{Short title}

PD-L1 Expression in Myelofibrosis

\textbf{Author affiliations}

Moayed Ibrahim, MD\textsuperscript{1}, Catherine Murphree,
MD\textsuperscript{1}, Kirtesh Patel MD\textsuperscript{2}, Matthew
Mastrodomenico, MD\textsuperscript{2}, Nakhle S. Saba,
MD\textsuperscript{1}, Hana Safah, MD\textsuperscript{1}, Janet
Schmid\textsuperscript{3}, Francisco Socola, MD\textsuperscript{4}

\textsuperscript{1}Section of Hematology and Medical Oncology, Deming
Department of Medicine, Tulane University, New Orleans, LA, USA

\textsuperscript{2}Delta Pathology Group, Shreveport, LA, USA

\textsuperscript{3}Pathology Department at Tulane University, New
Orleans, USA

\textsuperscript{4}Hematology and Bone Marrow Transplant , AdventHealth
Hospital Orlando, Orlando, FL, USA

MI: mibrahim1@tulane.edu, MM: Matthew.Mastrodomenico@lcmchealth.org, KP:
Kirtesh.Patel@lcmchealth.org, CM: cmurphree@tulane.edu, NS:
Nsaba@tulane.edu, HS: Hsafah@tulane.edu, JS: jschmid@tulane.edu, FS:
francisco.socola@adventhealth.com

\textbf{Corresponding author}

Francisco Socola , MD

Attending Physician

Hematology and Bone Marrow Transplantation

AdventHealth Hospital Orlando

2415 N. Orange Ave., Ste. 601 Orlando, FL 32804

tel: 407.303.5932 \textbar{} fax 407.303.0586 \textbar{}
E:~francisco.socola@adventhealth.com

\textbf{Word count}

Text: 2656, Abstract: 200

\textbf{Figue/table count}

Figures: 5, tables: 2

\textbf{Reference Count:} 11

\textbf{Key points}

PMF patients with a JAK2 mutation have a higher PD-L1 expression than
patients with JAK2-unmutated PMF or control groups.

A lower PD-L1 expression in PMF resulted in longer overall survival
compared to patients with a higher PD-L1 expression score.

\textbf{Abstract}

It has been described in mice models that Primary Myelofibrosis (PMF)
with JAK2-V617F mutation has an increased expression of programmed
death-ligand 1 (PD-L1) in megakaryocytes leading to cancer immune
evasion by inhibiting the T-lymphocytes. To prove this hypothesis, we
quantified PD-L1 expression on 29 bone marrow (BM) biopsies. We created
a scoring system to quantify PD-L1 expression in megakaryocytes. We
obtained 14 BM with JAK2 positive PMF, 5 JAK2 negative PMF and 10
patients with normal BM biopsies. PD-L1 expression was higher in the
JAK2 positive group compared to the control group with a score of 212.6
vs 121.1 (t-value 2.05,p-value 0.025). In addition, the score was higher
in the PMF group regardless of JAK2 mutational status when compared to
the control group with score of 205.9 vs 121.1(t-value 2.12,p-value
0.021). There was no difference in the PD-L1 score between the JAK2
negative vs the control group 187.2 vs 121.1 (t-value 1.02,p-value
0.162). These findings suggest that PMF patients with a JAK2 mutation
have a higher PD-L1 expression in megakaryocytes compared to the control
group. We postulate that the combination of checkpoint and JAK2
inhibitors may be an active treatment option in JAK2 mutated PMF given
the higher PD-L1 expression.

\textbf{Keywords}

Primary myelofibrosis, secondary myelofibrosis, PD-L1, immunotherapy,
JAK/STAT pathway



\textbf{Introduction}

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN)
characterized by constitutional activation of the JAK-STAT signaling
pathway and bone marrow (BM) fibrosis which leads to decreased
peripheral blood counts, pro-inflammatory state, and a potential for
transformation to acute myeloid leukemia (AML). The median age at
presentation is 65 years {[}2,3{]}. JAK2-V617F is the most common
mutation in PMF and is found in 50-60\% of patients {[}4{]}. The only
curative treatment option currently available is allogeneic stem cell
transplant. However, most patients are ineligible because of advanced
age and comorbidities {[}5{]}. Ruxolitinib and fedratinib, JAK2
inhibitors, are the only FDA approved treatment for intermediate/high
risk PMF patients {[}6,7{]}. However, those drugs have their limitations
and only improve symptoms and decrease splenomegaly, without an overall
survival benefit {[}6,7{]}. Therefore, there is a significant unmet need
for treatment options in this patient population.

Prestipino et al. discovered that mice models with JAK2-V617F mutated
MPN generally have an increased expression of PD-L1 that leads to cancer
immune evasion by inhibiting the antitumor effect of the T lymphocytes
against cancer cells {[}1{]}. Checkpoint inhibitors are monoclonal
antibodies that block the interaction between PD-L1 and its receptor,
allowing the immune system to fight cancer cells with an enhanced
antitumor response. Pembrolizumab was the first checkpoint inhibitor
approved by the FDA for patients with metastatic non-small-cell lung
cancer with \textgreater{}50\% PD-L1 expression in tumor cells by
immunohistochemistry~stain. In this subset of patients, pembrolizumab
was more effective than systemic chemotherapy {[}9{]}.

In this paper, we compared the PD-L1 expression among patients with
JAK2-mutated PMF versus JAK2-unmutated PMF patients versus normal
controls without no PMF or JAK2 mutation.



\textbf{Patient and methods}

\textbf{Study population}

We collected bone marrow biopsies of patients with PMF done at Tulane
Medical Center from 1990 to 2019. All these patients had a known JAK2
status and a well-preserved specimen for adequate PD-L1 staining. We
only used the initial bone marrow biopsy obtained to diagnose patients
with PMF. We collected 10 additional samples of patients that came to
clinic with transient cytopenias from benign hematologic conditions.
These patients had normal karyotype, FISH panel for myelodysplastic
syndrome (MDS), and a negative 64-gene next generation sequencing (NGS)
myeloid mutation panel. An institutional review board approval was
obtained before collecting these bone marrow samples and all
participants gave informed consent. The authors analysed the data to
which all authors had access. All relevant participant data was
deidentified and shared as appropriate in the text.

\textbf{PD-L1 immunohistochemistry staining}

We used the FDA approved test PD-L1 IHC 28-8 pharmDx to assess the PD-L1
expression on the bone marrow biopsies. This is a qualitative
immunohistochemical assay which uses monoclonal mouse anti-PD-L1 Clone
22C3 intended for use in the detection of PD-L1 protein in
formalin-fixed, paraffin-embedded tissues of different cancers such as
non-small cell lung cancer and gastric/gastroesophageal junction
adenocarcinoma. This test is used to identify patients who may be
treated with pembrolizumab.

Bone marrow sections of 4-5 \selectlanguage{greek}µ\selectlanguage{english}m were made with tissues mounted on
microscope slides then placed in a 58 \selectlanguage{ngerman}± 2 °C oven for 1 hour. The slides
were stained with the PD-L1 IHC 22C3 pharmDx reagent and the samples
were incubated. Finally, all the slides were numbered and labeled with
codes.

\textbf{Interpretation of PD-L1 expression}

The slides were reviewed and scored independently by two clinical
pathologists specialized in in interpreting PD-L1 expression in solid
tumors. PD-L1 was scored according to the quantity and intensity in the
megakaryocytic lineage of cells. The quantity of PD-L1 expression was
graded from 0 to 100\% while the intensity was graded from 1+ to 3+,
according to the level of intensity. The result was a PD-L1 score
calculated by multiplying percentage by intensity of PD-L1 in the bone
marrow megakaryocytes. This score will be detailed further in the
statistical analysis section.

\textbf{JAK2 mutation and NGS myeloid mutation panel}

Patients with PMF diagnosis had a JAK2-V617F mutation status known while
the patients diagnosed with PMF after 2018 also had a myeloid NGS done.
The controls had the myeloid NGS done en sure they did not have any
clonal diseases. The genes included in our inhouse myeloid NGS mutation
panel are: ABL1, ASXL1, ATM, ATRX, BCOR, BCORL1, BRAF, CALR, CBL, CBLB,
CDKN2A, CEBPA, CSF3R, DAXX, DNMT3A, EED, EGFR, ETV6, EZH2, FBXW7, FLT3,
GATA1, GNAS, HRAS, IDH1, IDH2, IKZF1, JAK1, JAK2, JAK3, KAT6A, KIT,
KMT2A, KRAS, MPL, NF1, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PRPF40B, PTEN,
PTPN11, RAD21, RB1, RUNX1, SETBP1, SF1, SF3A1, SF3B1, SH2B3, SMARCB1,
SMC1A, SMC3, SRSF2, STAG2, SUZ12, TET2, TP53, U2AF1, U2AF2, WT1 and
ZRSR2.

\textbf{Results}

We studied the bone marrow biopsies of 29 patients in total; 14 patients
had JAK2-mutated PMF, 5 JAK2-unmutated PMF, and 10 controls with
negative bone marrow biopsies. The median age of the whole group was 57
years, with 34\% males and 66\% females. The main clinical
characteristics of the myelofibrosis patients is described in
the\textbf{table 1.} PD-L1 expression and intensity is described
in\textbf{table 2.} We have a few examples how the PD-L1 expression and
intensity was quantified in the bone marrow biopsies, shown
in\textbf{figures 1-4} .

The average PD-L1 expression score for the JAK2-mutated PMF group was
212.57, the JAK2-unmutated PMF group 187.2, and the control group 121.1.
There was a statistically significant difference between the PD-L1 score
between the JAK2-mutated PMF group vs the control group (t-value 2.05
and p-value of 0.025) and when we compared the PMF group regardless of
the JAK2 status vs the control group (t-value 2.12 and p-value of
0.021). However, there was not a statistically significant difference
when we compared the PD-L1 expression between the JAK2 negative vs the
control group (t-value 1.02 and p-value 0.162).

Myelofibrosis patients with a PD-L1 score \textless{}250 had a median
overall survival of 130 months vs PD-L1 score \textgreater{}250 of 64
months (hazard ratio 2.63: 95\% CI, 0.82 to 8.4; P=0.09), it was
numerically longer but not statistically significant as shown
in\textbf{figure 5} .

\textbf{Discussion}

The presented results show that PD-L1 score was higher in the PMF group
vs the control group regardless of JAK2 mutation status, which turned
out to be statistically significant. This helps build on the data
reported by Prestipino et al that showed oncogenic JAK2 activity led to
STAT phosphorylation which in turn enhanced PD-L1 promoter activity and
PD-L1 protein expression in JAK2 mutant cells {[}1{]}. In addition,
PD-L1 expression was higher on primary cells isolated from patients with
JAK2-mutated MPNs as compared to healthy individuals and declines upon
JAK2 inhibition {[}1{]}. Moreover, Lee et al were able to demonstrate
that PD-L1 expression was significantly associated with overt
myelofibrosis and JAK2 mutational status {[}9{]}. Moreover, in the
previously mentioned study, there were 4 patients who were found to have
a particularly high PD-L1 expression that also harbored the JAK2
mutation {[}9{]}.

This supports further that PD-L1 may play a more important role than
previously realized in MPNs and should perhaps be a future target in our
current small armamentarium of viable drugs. To the best of our
knowledge, there have only been 2 small phase II trials in which the
investigators tested the utility of PD-L1 inhibition (pembrolizumab and
nivolumab) in patients with PMF. Hobbs, et al conducted a phase 2,
single arm study of pembrolizumab in patients with Dynamic International
Prognostic Scoring System (DIPSS) intermediate-2 or greater, primary, or
secondary, post essential thrombocythemia or post polycythemia vera MF
who were ineligible for or previously treated with ruxolitinib {[}9{]}.
This study had 10 patients, 5 with JAK2 mutation who were treated with
pembrolizumab without objective clinical responses. However, an
important takeaway from this data showed that flow cytometry, T-Cell
receptor (TCR) sequence and proteomics demonstrated changes in the
immune makeup of patients, suggesting improved T cell responses
{[}10{]}. Although this study was terminated early as no objective
clinical responses were seen, the latter changes mentioned suggest that
perhaps PD-L1 inhibition is not enough to elicit a clinical response and
combination therapy may be more effective.

Another study, in which Dalle et al investigated the efficacy and safety
of single agent nivolumab in 8 adult patients with myelofibrosis, was
also terminated early due to failure to meet predetermined efficacy
endpoint (primary endpoint was objective response rate (ORR) defined as
complete response (CR), partial response (PR) and clinical improvement
(CI) after 8 doses) {[}11{]}. The median duration of enrolled patients
on the study was 5.4 months with a median number of cycles of 3.
Unfortunately, in this study, none of the patients responded to
nivolumab therapy. These patients showed more advanced disease including
intermediate 2 and high risk DIPSS score with 5 patients failing
ruxolitinib and 7 with clonal evolution, i.e., progressive disease
{[}11{]}.

It is important to note that these two described studies had very small
sample sizes, with most patients in the high risk DIPSS category,
multiple previous lines of therapy, and complex mutational status or
clonal evolution. However, they were able to characterize changes in the
patient's immune milieu after administration of PD-L1 blockade
{[}10,11{]}. It's noteworthy to the authors that both studies employed
PD-L1 blocked only after patients had undergone multiple lines of
therapy, in a relapsed or refractory setting, raising the question that
perhaps this is not the right setting to use this line of therapy. In
addition, it would be interesting to expand further on the hypothesis
that perhaps PD-L1 blockade is not enough and combination therapy with
ruxolitinib may be more effective in patients with PMF.

\textbf{Conclusions}

We found that the PD-L1 expression in the bone marrow megakaryocytes of
JAK2-mutated PMF patients was higher than the control group. This may
confirm the hypothesis that the oncogenic JAK2 mutation enhances the
PD-L1 promoter activity and its expression in JAK2 positive patients. We
hypothesize that patients with high PD-L1 expression may benefit from
checkpoint inhibitors, in combination with JAK2 inhibitors, in the
upfront setting of therapy.

\textbf{Author contributions}

FS, MM, KP, CM,and MI contributed substantially to original draft
writing while HS, NS and FS contributed significantly to reviewing and
editing.

\textbf{Acknowledgements}

We thank the patients for the permission to conduct this research.

\textbf{Funding}

Carol Lavin Bernick Faculty Grant Program at Tulane University School of
Medicine.

\textbf{Declarations of interest}

The authors have no competing interests to declare.

\textbf{Figue/table Captions}

Visual abstract: Myelofibrosis patients with a PD-L1 score
\textless{}250 had a median overall survival of 130 months vs PD-L1
score \textgreater{}250 of 64 months, P=0.09.

Figure 1.76 y/o woman with JAK2 positive PMF (Expression 100\%,
intensity +3)

Figure 2. 28 y/o woman with anemia and normal BM BX (Expression 0\%,
intensity 0)

Figure 3. 73 y/o woman with JAK2 negative PMF (Expression 78\%,
intensity +2)

Figure 4. 60 y/o man with JAK2 negative PMF (Expression 100\%, intensity
+3)

Table 1.- Main characteristics of patients with myelofibrosis.

Table 2.- Level of expression and intensity of PD-L1 on the 29 bone
marrow biopsies

Figure 5.- Median Overall Survival according to a PD-L1 socre
\textgreater{}250 vs \textless{}250.

\textbf{References:}

\begin{enumerate}
\tightlist
\item
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\end{enumerate}\selectlanguage{english}
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