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\begin{document}

\title{SARS-COV2 VACCINATION RESPONSE IN PEDIATRIC ONCOLOGY PATIENTS}



\author[1]{Amber D'Souza}%
\author[1]{Thomas M}%
\author[1]{Manu Gnanamony}%
\author[1]{Nguyen TH}%
\author[1]{Pedro A. de Alarcon}%
\affil[1]{University of Illinois Chicago College of Medicine at Peoria Department of Internal Medicine}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\selectlanguage{english}
\begin{abstract}
\textbf{Background:} There remains limited knowledge about the immune
response to severe acute respiratory syndrome coronavirus 2 (SARS-COV2)
vaccination in pediatric oncology patients, which is essential to
provide counseling and risk adaptation in this vulnerable population.
The goal of this study was to understand immunogenicity after
vaccination in pediatric oncology patients and determine if certain
clinical factors impacted response. \textbf{Methods:} Patients 0-25
years of age with a diagnosis of cancer and actively receiving therapy
were enrolled on study. We excluded patients who were completely
vaccinated prior to their cancer diagnosis. Blood samples were collected
pre-vaccination, as well as 2, 4-6, and 8-12 weeks after vaccination.
Healthy children who were fully vaccinated enrolled as controls.
Clinical data and complete blood counts around time of vaccination were
collected. To study B and T cell immunity, we measured neutralizing
antibodies by enzyme-linked immunoassay and interferon gamma secretion
by enzyme-linked immunospot, respectively. \textbf{Results:} 26 patients
enrolled on study, for which 11 were evaluable oncology patients and 7
were healthy controls. Adequate B cell response was seen in 36.4\% of
patients and adequate T cell response in 77.8\% of patients. Numbers
were too small to detect differences based on malignancy type. There was
no differences in immunity based on absolute lymphocyte count (ALC) or
intensity of therapy. \textbf{Conclusion:} Pediatric oncology patients
have a suboptimal immune response to SARS-COV2 vaccination. Booster
doses will be imperative to provide optimal protection against COVID-19,
however blood counts may not be a useful guide to optimize the time of
administration.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

The burden from severe acute respiratory syndrome coronavirus 2
(SARS-CoV2) has significantly impacted the healthcare system, and
vaccination remains our best protection against this virus. Children
with cancer have a higher morbidity and mortality from SARS-CoV2
infection compared to healthy children.\textsuperscript{1,2} This
includes need for oxygen administration, pleural effusions,
pneumothorax, pulmonary arterial hypertension, bronchiolitis, diffuse
alveolar hemorrhage, and septic shock.\textsuperscript{1} Additionally,
infection has been associated with postponement and modification of
chemotherapy, which may impact outcome.

Unfortunately, immunocompromised patients were excluded from original
vaccine studies, leaving the oncology community to speculate the level
of protection achieved with vaccination. A few recent studies from
Europe have identified an impaired immune response in pediatric oncology
patients after the BNT162b2 COVID-19 vaccination, especially those with
hematologic malignancies or those undergoing intensive
therapy.\textsuperscript{3,4} In the adult oncology literature,
vaccination has high rates of seroconversion, with hematologic
malignancies showing lower immunogenicity.\textsuperscript{5--7} In
solid organ transplant recipients, a third dose of an mRNA vaccine was
needed to achieve a substantial immune response.\textsuperscript{8,9}The
primary goal of this study was to understand immunogenicity after
vaccination with BNT162b2 or mRNA-1273 in pediatric oncology patients
and determine if certain clinical factors impacted response.

\textbf{Methods:}

After institutional review board (IRB) approval, a prospective study
analyzing immune response to COVID-19 vaccination in pediatric cancer
patients was undertaken. Eligibility criteria included cancer patients
aged 0-25 years of age actively receiving therapy, including
chemotherapy, immunotherapy, and targeted therapy. We excluded patients
who were completely vaccinated prior to their cancer diagnosis. Patients
may have received any of the vaccines approved for emergency use by the
U.S. Food and Drug Administration. This includes BNT162b2, developed by
Pfizer and BioNTech, mRNA-1273, developed by Moderna. For the remainder
of this paper, these vaccines will be referred to as Pfizer and Moderna.
Blood samples were collected pre-vaccination, as well as 2, 4-6, and
8-12 weeks after completion of COVID vaccination. Completion was defined
as completion of the two dose series. As a control group, we enrolled
patients in our clinic aged 0-25 years who were not receiving cancer
directed therapy, followed for a benign hematology issue, or were more
than 6 months after completion of therapy for their cancer diagnosis.
These participants had one sample drawn between 2 weeks and 6 months of
completion of vaccination.

Demographic data was collected from the medical record, including age,
gender, ethnicity, cancer diagnosis, personal history of COVID, current
therapy, type of vaccine received, and dates of vaccination. Therapy
details included if it was chemotherapy, immunotherapy, targeted
therapy, or combination therapy. We also collected if therapy was
intravenous, oral, or both. If available, we also collected complete
blood count (CBC) prior to vaccination and CBCs within 4 days of all
sample time points.

Once blood samples were obtained in clinic, they were transported to the
lab and processed within two hours. In order to maximize T cell yield
for analysis in patients who may have low lymphocyte counts due to
therapy, participants blood samples were collected in Vacutainer\selectlanguage{ngerman}® CPT\selectlanguage{english}
Cell Preparation Tube (BD) and processed according to the manufacturer's
instructions. The cells were then re-suspended in 1ml of freezing medium
(RPMI containing 40\% FBS and 12.5\% DMSO) and stored in liquid
nitrogen.

To measure immunogenicity, we performed two assays. First, we measured
neutralizing IgG antibodies, which has been shown to be highly
predictive of immune protection from symptomatic SARS-CoV2
infection.\textsuperscript{10} This was done using the
SARS-CoV2-Surrogate Virus Neutralization Test Kit (GenScript) according
to the manufacturer's instructions. All the samples were run in
duplicates. The quantitative detection range of this assay is between 47
to 185U/ml. Titers \selectlanguage{english}[?]60.8 U/ml (lower 10\% of the quantifiable detection
range) was taken as inadequate B-cell response. Titers \selectlanguage{english}[?]60.8U/ml was
taken as adequate B-cell response. The second assay measures interferon
gamma (IFN\selectlanguage{greek}γ\selectlanguage{english}) secretion, which is a marker of T cell response to viral
antigens and another established method of measuring cellular immune
response after vaccination.\textsuperscript{11} This is done using an
enzyme-linked immunosorbent spot (ELISPOT) kit for IFN\selectlanguage{greek}γ \selectlanguage{english}(Mabtech).
Briefly, frozen peripheral blood mononuclear cells were thawed and then
seeded onto anti-human IFN\selectlanguage{greek}γ \selectlanguage{english}monoclonal antibody pre-coated plates at a
concentration of 250,000 cells/well. These cells were then stimulated
with SARS --CoV-2 spike peptide pools at a concentration of 2\selectlanguage{greek}µ\selectlanguage{english}g/ml by
incubating for 36 hours in 5\% CO incubator and sent to Mabtech, Inc.
Ohio, USA, for quantitation. Results were reported as Spot forming units
per million cells (SFU/10\textsuperscript{6}cells). SFU values [?] Mean-
standard deviation (i.e. [?] 659 SFU/10\textsuperscript{6} cells) of the
control sample values were defined as an inadequate T-cell response.

When analyzing what clinical features may impact vaccine response, we
utilized B cell response by neutralizing IgG antibodies to define our
adequate and inadequate response groups. This was because this assay
allowed for replicates to strengthen the validity of our results as
compared to the T cell assay, which could be only done once. Statistical
analysis was done using Graphpad Prism 8.0. When appropriate, unpaired t
tests were obtained to evaluate for statistical significance, which was
defined as p value \textless{}0.05.

\textbf{Results}

A total of 19 cancer patients enrolled on study. Of these patients, one
died of disease prior to vaccination, 4 did not complete the series, and
three only had blood draws after boosters, leaving 11 evaluable
patients. Table 1 highlights the demographic and clinical
characteristics of these patients. There were 7 control patients,
including 5 who were \textgreater{} 6 months from cancer diagnosis and 2
followed for a benign hematology issue. Within the evaluable cancer
patients, the number of samples for each time-point were as follows: 6
pre-vaccination, 8 two weeks post vaccination, 8 four-six weeks post
vaccination, and 11 eight to twelve weeks after vaccination.

Among the 11 cancer patients analyzed, only 36.4\% (n=4) had an adequate
B cell response (Figure 1). Of these four patients, one had Ewing
sarcoma on oral chemotherapy and another had Hodgkin lymphoma on
chemoimmunotherapy. The other two had leukemia, including a patient with
chronic myelogenous leukemia (CML) on an oral tyrosine kinase inhibitor
and one with acute lymphoblastic leukemia (ALL) on maintenance therapy.
It's important to note that two of these four patients may have had
subclinical COVID infection prior to vaccination based on their
neutralizing antibodies. Patient 23 (with Hodgkin lymphoma), maintained
very elevated neutralizing antibodies despite extremely low blood
counts, however because we were unable to obtain a pre-vaccination
sample, it's uncertain if he started with high antibody levels. The
patient with CML (Patient 3) had elevated neutralizing antibodies prior
to vaccination, suggestive of subclinical COVID infection. However his
titers continued to decrease over the 8-12 week period after
vaccination, suggestive of a waning response. The other seven subjects
had an inadequate B cell response, including one patient with Ewing
sarcoma on myelosuppressive intravenous chemotherapy. All other poor
responders were ALL patients in various phases of therapy, including
escalated IV methotrexate, delayed intensification, and oral maintenance
chemotherapy. Additionally, one of those patients (Patient 26) had
documented COVID infection in the middle of the vaccination series, and
despite this, still had an inadequate response.

Next, we measured T cell response after vaccination in pediatric
oncology patients. Of note, 9 patients had a sample for this analysis at
8-12 weeks. Adequate T cell response was seen in 77.8\% of patients (n =
7). This includes Patients 3 and 21 with suspected subclinical COVID
infection and both solid tumor patients. Two leukemia patients with an
inadequate T cell response were in highly myelosuppressive phases of
therapy (Patient 2 and 16).

In order to understand what clinical factors may impact response to
vaccination, we analyzed vaccine response (by neutralizing antibody
levels) based on cancer diagnosis (hematologic vs solid tumor), absolute
lymphocyte count (ALC), and therapy regimen (Figure 2). Because the
majority of patients had leukemia, it was not possible to determine if
there were alterations in response based on cancer type. However, we did
note that the majority of patients with leukemia had an inferior B cell
response compared to healthy controls (Figure 2A). As previously
mentioned, the two patient who maintained similar levels of neutralizing
antibodies as the healthy controls (Patient 3 and 23) may have had
subclinical COVID prior to vaccination. There was no correlation seen
between ALC and B cell response, as ALC varied widely among both groups
(Figure 2B). In order to evaluate the relationship between chemotherapy
regimen and vaccine response, we reviewed the phase of therapy patients
were on at the time of vaccination and divided them into highly
myelosuppressive or less myelosuppressive (Figure 2C). We also focused
on the patients with a hematologic malignancy since there were only two
patients with solid tumors. Patients with highly myelosuppressive
therapy included those receiving escalated methotrexate, delayed
intensification, or early Continuation. Less myelosuppressive therapy
included oral targeted therapy and maintenance chemotherapy. There was
no difference in neutralizing antibody levels at any time point between
the two groups, suggesting that leukemia therapy in general was
associated with poor immune response to vaccination.

\textbf{Discussion}

Vaccination in oncology patients is typically limited to diseases that
are highly prevalent with high risk for morbidity and mortality. This is
largely because we know that efficacy of vaccination in
immunocompromised patients is poor.\textsuperscript{1} The analysis of
our pediatric oncology cohort, albeit small, reiterates the findings
seen in pediatric and adult oncology and transplant patients.
Specifically, our cohort showed an inadequate immune response to
SARS-COV2 vaccination compared to healthy controls. There was a notable
difference between B cell response and T cell response, with less
patients having an adequate B cell response. No patients received
targeted B cell directed therapy, but we do not have lymphocyte subset
data to quantitate B vs T lymphocytes. Another limiting factor is our
ability to only complete the T cell assay once, which may impact the
validity of that data compared to the B cell data which was repeated
multiple times. Lastly, while no patients received steroids during
within one week of SARS-COV2 vaccination, many of them had steroids in
that phase of therapy, which may have had a greater impact on T cell
immunity.\textsuperscript{12}

While previous studies have demonstrated worse immune response in
patients with leukemia/lymphoma as compared to solid tumor
malignancy,\textsuperscript{4--7} our numbers were too small to detect
differences between these two cohorts. Similarly, we did not find
significant differences in immune response based on ALC or degree of
myelosuppressive therapy, suggesting that ALC or phase of therapy may
not be useful in guiding timing of vaccination or boosters.

There were two other studies to date that have also looked at immunity
after SARS-COV2 vaccination in pediatric oncology patients. Both of
these studies were from European countries, where only the BNT162b2
(Pfizer) vaccine was available. One study out of Germany included 21
pediatric oncology patients after receiving three doses (the two dose
series and a booster).\textsuperscript{4} The majority of these patients
elicited both B and T cell immunity, which was stronger in patients with
a solid tumor malignancy and in maintenance phase of therapy. The other
study by a group in the Netherlands analyzed 73 patients who received
either 2 or 3 doses and also included patients who received a
hematopoietic stem cell transplant or CAR T-cell
therapy.\textsuperscript{3} This study demonstrated that time between
last treatment and start of the vaccination series impacted immunity,
with improved vaccination response in patients who were \textgreater{} 6
weeks from last treatment. Similar to the German study, they showed that
three dose series was effective in increasing the humoral immune
response. There are a few key differences between these studies and
ours. First, there are differences in treatment regimens between North
America and Europe that may impact immune response during therapy.
Additionally, these other two studies did not include healthy patients
as a control, but rather used cutoffs defined by the assays to determine
response. Lastly, all of our patients only received a two vaccine
series, with very few patients receiving boosters. Our data, taken in
context of these other two studies, reiterate the value of booster shots
in immunocompromised patients.

The major limitation of our study is a small cohort size, which impacted
our ability to analyze how different clinical factors may impact
response. Similarly, our minimal enrollment of patients with solid tumor
malignancy did not allow us to assess how different malignancies may
impact response to vaccination. The Children's Oncology Group has a
study currently open to evaluate immunologic response to COVID-19
vaccination in pediatric oncology patients. This study should shed
additional light on how immunocompromised children responds to
vaccination, what factors may impact that response, and most
importantly, how we should counsel our patients and families regarding
risks of infection, importance and timing of boosters, and risk for
serious sequelae.

\textbf{Conclusions}

Pediatric oncology patients have suboptimal response to COVID-19
vaccination compared to healthy controls. Booster shots should be
encouraged to improve immunity and reduce risk of severe disease.
Further work in larger cohorts of patients is needed to understand how
malignancy type, blood counts, and intensity of therapy may impact
immune response.

\textbf{Conflict of Interest Statement} : The authors have no conflicts
of interest to discolose.

\textbf{Acknowledgements} : none

\textbf{References}

1. Meena JP, Kumar Gupta A, Tanwar P, Ram Jat K, Mohan Pandey R, Seth R.
Clinical presentations and outcomes of children with cancer and
COVID-19: A systematic review. \emph{Pediatr Blood Cancer} .
2021;68(6):e29005. doi:10.1002/pbc.29005

2. Madhusoodhan PP, Pierro J, Musante J, et al. Characterization of
COVID-19 disease in pediatric oncology patients: The New York-New Jersey
regional experience. \emph{Pediatr Blood Cancer} . 2021;68(3):e28843.
doi:10.1002/pbc.28843

3. Schmidt KLJ, Dautzenberg NMM, Hoogerbrugge PM, et al. Immune Response
following BNT162b2 mRNA COVID-19 Vaccination in Pediatric Cancer
Patients. \emph{Cancers (Basel)} . 2023;15(9):2562.
doi:10.3390/cancers15092562

4. Lehrnbecher T, Sack U, Speckmann C, et al. Longitudinal Immune
Response to 3 Doses of Messenger RNA Vaccine Against Coronavirus Disease
2019 (COVID-19) in Pediatric Patients Receiving Chemotherapy for
Cancer.\emph{Clin Infect Dis} . 2023;76(3):e510-e513.
doi:10.1093/cid/ciac570

5. Addeo A, Shah PK, Bordry N, et al. Immunogenicity of SARS-CoV-2
messenger RNA vaccines in patients with cancer. \emph{Cancer Cell} .
2021;39(8):1091-1098.e2. doi:10.1016/j.ccell.2021.06.009

6. Thakkar A, Gonzalez-Lugo JD, Goradia N, et al. Seroconversion rates
following COVID-19 vaccination among patients with cancer. \emph{Cancer
Cell} . 2021;39(8):1081-1090.e2. doi:10.1016/j.ccell.2021.06.002

7. Agha M, Blake M, Chilleo C, Wells A, Haidar G. Suboptimal response to
COVID-19 mRNA vaccines in hematologic malignancies
patients.\emph{medRxiv} . Published online April 7,
2021:2021.04.06.21254949. doi:10.1101/2021.04.06.21254949

8. Boyarsky BJ, Werbel WA, Avery RK, et al. Antibody Response to 2-Dose
SARS-CoV-2 mRNA Vaccine Series in Solid Organ Transplant
Recipients.\emph{JAMA} . 2021;325(21):2204-2206.
doi:10.1001/jama.2021.7489

9. Hall VG, Ferreira VH, Ku T, et al. Randomized Trial of a Third Dose
of mRNA-1273 Vaccine in Transplant Recipients. \emph{N Engl J Med} .
2021;385(13):1244-1246. doi:10.1056/NEJMc2111462

10. Khoury DS, Cromer D, Reynaldi A, et al. Neutralizing antibody levels
are highly predictive of immune protection from symptomatic SARS-CoV-2
infection. \emph{Nat Med} . 2021;27(7):1205-1211.
doi:10.1038/s41591-021-01377-8

11. Sewell HF, Agius RM, Kendrick D, Stewart M. Covid-19 vaccines:
delivering protective immunity. \emph{BMJ} . 2020;371:m4838.
doi:10.1136/bmj.m4838

12. Fauci AS. Mechanisms of corticosteroid action on lymphocyte
subpopulations. II. Differential effects of in vivo hydrocortisone,
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function. \emph{Clin Exp Immunol} . 1976;24(1):54-62.

\textbf{Figures Legends} :

\textbf{Figure 1: B cell response by Neutralizing IgG Antibodies in
Pediatric Cancer Patients compared to Healthy Controls.} Quantitative
neutralizing antibody response at each time point after vaccination
compared to healthy controls. Please note that healthy controls only had
one time point and for optimal visualization, is presented in a linear
fashion over each time point (red line). \textbf{A.} Pediatric cancer
patients with adequate B cell response. \textbf{B.} Pediatric cancer
patients with inadequate B cell response.

\textbf{Figure 2: Evaluation of clinical factors that may response to
SARS-COV2 vaccination} . \textbf{A} . B cell response after vaccination
in leukemia/lymphoma patients (upper) and solid tumor patients
(lower\textbf{). B.} Comparison of absolute lymphocyte counts prior to
vaccination (0 week) and 8-12 weeks post vaccination in the adequate vs
inadequate response groups. There was no statistical significance
between any of the groups (Adequate response 0 wk and inadequate
response 0 wk, p = 0.86; Adequate response 8 wk and inadequate response
8 wk, p = 0.99; adequate response 0 wk and adequate response 8 wk, p =
0.75; inadequate response 0 wk and adequate response 8 wk, p =
0.79).\textbf{C.} B cell response after vaccination in patients with
highly myelosuppressive (upper) and less myelosuppressive (lower)
chemotherapy. There was no statistical difference in neutralizing
antibodies between both therapy groups (2 wk, p = 0.859; 4-6 wk, p =
0.740, 8-12 wk, p = 0.758).\selectlanguage{english}
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