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\begin{document}

\title{Late-onset lymphopenia during radiation is associated with an increased
risk of tumor recurrence in newly-diagnosed pediatric medulloblastoma}



\author[1]{Holly B. Lindsay}%
\author[2]{Michael Scheurer}%
\author[3]{Anthony K. Allam}%
\author[4]{Susan McGovern}%
\author[2]{Murali Chintagumpala}%
\author[4]{Arnold Paulino}%
\affil[1]{University of Colorado Anschutz Medical Campus}%
\affil[2]{Texas Children's Cancer Center and Hematology Centers}%
\affil[3]{Baylor College of Medicine}%
\affil[4]{The University of Texas MD Anderson Cancer Center Division of Radiation Oncology}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Background:} Recent data found a correlation between lymphopenia
occurring early during craniospinal irradiation (CSI) and risk of
disease recurrence in newly-diagnosed childhood medulloblastoma.
\textsuperscript{1} However, the population included patients that
received myelosuppressive chemotherapy prior to or during RT. Here we
investigate the effect of lymphopenia during RT in patients with
newly-diagnosed pediatric medulloblastoma who did not receive
myelosuppressive chemotherapy with RT. \textbf{Procedure:} We analyzed
54 patients with newly-diagnosed medulloblastoma (ages 2-21 years)
treated between 1997-2013 with CSI. Log-rank tests were used to
determine survival differences, and Cox proportional hazards regression
was used to assess associations between patient characteristics and
lymphopenia with disease recurrence risk. \textbf{Results:} 78\% of
patients (40/51) had grade [?]3 lymphopenia by RT week 3; 49\% (23/47)
improved to grade [?]2 lymphopenia by week 5. Similarly, the lowest median
absolute lymphocyte count (ALC) occurred during RT week 3. Sixteen of 54
(30\%) patients recurred an average of 30.2 months post-diagnosis. There
was higher risk of disease recurrence in patients with grade [?]3
lymphopenia during weeks 4 (log-rank p=0.015; Cox p=0.03) and 5
(log-rank p=0.0009; Cox p=0.004) of RT. Recurrence-free survival was
lower in patients with ALC Conclusions: Lymphopenia during RT weeks 4
and 5 correlates with increased risk of tumor recurrence in pediatric
patients with newly-diagnosed medulloblastoma. Future studies should
correlate baseline numbers of tumor-infiltrating lymphocytes with risks
of lymphopenia during RT and tumor recurrence.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Lymphocytes are the most radiosensitive peripheral blood cells due to
their inefficient DNA repair mechanisms. Lymphopenia secondary to
radiation therapy (RT) is associated with poorer progression-free
survival (PFS) and overall survival (OS) in patients with tumors of
breast, brain, gastrointestinal tract, head and neck, lung, and
pancreas.\textsuperscript{2-6} The poorer outcome in patients with
RT-induced lymphopenia is hypothesized to be secondary to a decrease in
tumor-infiltrating lymphocytes (TIL). It is known that medulloblastoma
tumors have low numbers of TIL even prior to the initiation of
RT.\textsuperscript{7-10} In patients with childhood medulloblastoma, a
recent publication demonstrated a correlation between peripheral
lymphopenia occurring during weeks 1 and 2 of craniospinal irradiation
(CSI) and risk of disease recurrence.\textsuperscript{1} However, many
patients in this study received myelosuppressive chemotherapy prior to
and/or concomitantly with RT; consequently, lymphopenia cannot be
entirely attributed to RT alone.

The purpose of this study is to examine the effect of lymphopenia during
RT in an independent population of patients with newly-diagnosed
pediatric medulloblastoma, none of whom received myelosuppressive
chemotherapy concurrently with RT.

\textbf{METHODS}

This retrospective study was approved by our Institutional Review Board
(IRB) prior to data abstraction or analysis. A waiver of informed
consent was granted by the IRB as the study was entirely retrospective.
The study population included patients 2 to 21 years old at tumor
diagnosis treated for newly-diagnosed medulloblastoma at our tertiary
children's hospital between 1997-2013 with therapy that included CSI.
Patients treated on clinical research studies SJMB96 (NCT00003211) or
SJMB03 (NCT00085202)\textsuperscript{11,12}, and those treated for
medulloblastoma outside of the context of a clinical trial were eligible
for inclusion in our analyses. All patients treated prior to 2007
received photon beam RT while those treated in or after 2007 received
proton beam RT.

Patients were classified as having high-risk disease, prior to the
advent of molecular risk stratification, if they had metastatic disease
(M stage) or post-operative residual disease \textgreater{}1.5
cm\textsuperscript{2}; patients not meeting these criteria were
classified as having clinically standard-risk disease.

The following data were abstracted from the medical record for each
eligible patient: name, date of birth, gender, date of tumor diagnosis,
age at diagnosis, tumor pathology, date and extent of surgical
resection, M stage, start and end dates of RT, dose and modality (photon
or proton beam) of RT, complete blood count (CBC) results at the start
of/during/immediately after RT, post-RT chemotherapy regimen used, date
of first recurrence (to allow calculation of recurrence-free survival
(RFS), and survival status (alive or dead).

Weekly lymphocyte counts were analyzed both as a continuous variable and
graded per the National Cancer Institute's Common Terminology Criteria
for Adverse Events (CTCAE) version 5.0: grade 1---absolute lymphocyte
count (ALC) \textless{} lower limit of normal -
0.8x10\textsuperscript{9}/L, grade 2---ALC \textless{} 0.8 -
0.5x10\textsuperscript{9}/L, grade 3---ALC \textless{} 0. 5-
0.2x10\textsuperscript{9}/L, and grade 4---ALC \textless{}
0.2x10\textsuperscript{9}/L.

Descriptive statistics were used to characterize the study population.
Kaplan-Meier survival curves were constructed, and the log-rank test was
used to determine differences in survival curves by key clinical
variables. Cox proportional hazards regression was used to assess the
associations (hazards ratio and 95\% confidence intervals) between
patient characteristics and lymphopenia during RT with risk of disease
recurrence, unadjusted and adjusted for risk group. A p-value of 0.05
was used as the cut-off for statistical significance. All analyses were
conducted in Stata (SE version 17; Stata Corp, College Station, TX).

\textbf{RESULTS}

\textbf{Clinical Characteristics of Cohort}

Fifty-four patients diagnosed between 1997 and 2013 met our inclusion
criteria; the clinical characteristics of the cohort are listed
in\textbf{Table 1} . Seventy-four percent of patients in our cohort
(40/54) had M0 disease. Two of the 54 patients (4\%) received
chemotherapy prior to RT: 1 of these patients was 2 years old at
diagnosis and received chemotherapy treatment according to the
Children's Oncology Group ACNS0334 for patients with high risk infant
medulloblastoma until he reached the age of 3 years at which time CSI
was initiated, and the 2\textsuperscript{nd} patient received 2 cycles
of topotecan prior to starting RT.

All patients initiated RT within 35 days of tumor resection. Patients
with standard-risk medulloblastoma received 23.4 Gray (Gy) CSI, while
those with high-risk disease received 36-39.6 Gy CSI; both populations
then received a boost to the tumor bed to a total dose of 54-55.8 Gy.
Eighty percent of patients (43/54) received proton beam RT, while the
remaining 11 patients received photon beam RT. For patients that were
treated with proton therapy, the entire vertebral body was covered with
the CSI prescription dose for skeletally immature patients, while for
those who had achieved skeletal maturity, only the posterior portion of
the vertebral body was treated during CSI.

One patient (2\%) received concurrent chemotherapy during RT; however,
the chemotherapy was single agent vincristine, which is not
myelosuppressive. Following completion of RT, 82\% of patients (44/54)
received chemotherapy either enrolled on or treated according to
clinical trials SJMB96 or SJMB03 with vincristine, cisplatin, and
cyclophosphamide.

\textbf{Lymphopenia During RT}

The number of patients with grade 3 or 4 lymphopenia during RT as well
as the median ALC of all patients during RT is shown in \textbf{Figs.
1}and \textbf{2} and \textbf{Supplemental Table 1} . Seventy-eight
percent of patients (40/51) had at least grade 3 lymphopenia by week 3
of RT, but 51\% (24/47) had improved to grade 2 or lower lymphopenia by
week 5. Similarly, the lowest median ALC occurred during week 3 of RT.
Of note, not all 54 patients in our cohort had lymphocyte counts checked
each week (see \textbf{Supplemental} \textbf{Table 1} ). The degree of
lymphopenia was greater in those patients who received a higher
craniospinal dose (\textbf{Figs. 1} and \textbf{2} ).

\textbf{Impact of Clinical Factors and RT-induced Lymphopenia on Risk of
Disease Recurrence}

Sixteen of 54 (30\%) patients developed a recurrence at an average of
30.2 months after diagnosis. As expected, patients with M+ disease had a
significantly increased risk of disease recurrence (log-rank p=0.0004;
Cox p=0.001). \textbf{Table 2} demonstrates the impact of clinical
factors and RT-induced lymphopenia on the risk of disease recurrence.
There was a higher risk of disease recurrence (lower RFS) in patients
with grade 3 or lymphopenia during weeks 4 (log-rank p=0.015; Cox
p=0.03) and 5 (log-rank p=0.0009; Cox p=0.004) of RT (\textbf{Fig. 3} ).
When considering the RFS of patients with ALC above or below the median
during weeks 4 and 5 of RT, there was a significant difference during
week 5 (log-rank p=0.0026; Cox p=0.002) (\textbf{Fig. 4B} ) but not
during week 4 (log-rank p=0.123; Cox p=0.22) (\textbf{Fig. 4A} ). These
results all held true when adjusted for risk group (\textbf{Table 2} ).
No correlation was found between risk of tumor recurrence and early
lymphopenia (RT weeks 0-3) either when measured as CTCAE grade or as
median ALC. Additionally, there was not a statistically significant
difference between RFS in patients treated with proton compared to
photon RT (Cox p=0.09) (\textbf{Supplemental Fig. 1} ) or based on sex
(Cox p=0.810).

\textbf{DISCUSSION}

Almost all patients in our study were treated uniformly, with only 2/54
(4\%) receiving chemotherapy prior to RT and 1/54 (2\%) receiving
non-myelosuppressive chemotherapy (vincristine) during RT. The majority
of the patients (80\%, 43/54) also received proton beam RT. Typically,
for patients receiving 23.4 Gy CSI, the CSI portion of the RT course is
completed by the end of week 3, with CSI extended through week 4 for
those receiving [?]36 Gy; by week 5, CSI is normally complete, and the RT
field shrinks to cover only the tumor bed. Consequently, late
lymphopenia during weeks 4 and 5 may have more clinical significance
than lymphopenia that develops earlier in the RT course, as the exposure
to CSI is completed at the end of week 3 or later.

As previously mentioned, RT-induced lymphopenia is associated with worse
PFS and OS in numerous adult tumors, a phenomenon presumed secondary to
RT causing a decrease in TIL.\textsuperscript{2-6,13-15} Among CNS
tumors, higher numbers of both CD3+ T cells generally and specifically
CD8+ cytotoxic TIL at the time of tumor diagnosis are associated in the
majority of studies with improved prognosis in adult high grade glioma
and atypical meningioma.\textsuperscript{13,14,16-19}

Unfortunately, medulloblastoma tumor samples characteristically have low
numbers of TIL even prior to the start of
RT.\textsuperscript{7-10,20,21} These findings suggest that
medulloblastoma has an immunosuppressive tumor microenvironment.
Overall, CD8+ T cells are the largest component of the TIL in
medulloblastoma; additionally, there are differences in numbers of CD8+
T cells and TIL in general among medulloblastoma molecular groups,
subgroups, and even down to the subtype
level.\textsuperscript{8,9,20,22-24} The few published investigations
assessing for a correlation between survival and TIL/CD8+ T cell numbers
in pediatric medulloblastoma have had inconsistent results,
demonstrating that larger studies are clearly
needed.\textsuperscript{23,24}

Our data showed no statistically significant difference in RFS between
patients treated with protons vs. photons and in fact showed a trend
toward lower RFS in patients that received proton RT. However, there is
increasing awareness that proton beam RT may induce less lymphopenia
than photons.\textsuperscript{25-27} When proton vertebral body sparing
techniques are utilized for CSI, which in our cohort occurred for
skeletally mature patients, data shows that lymphopenia is
lessened\textsuperscript{28,29}, as the vertebral bodies contain
\textgreater{}25\% of the total bone marrow.\textsuperscript{30} The
majority of our patients had their entire vertebral body covered by the
CSI dose due to receiving photon beam RT or being skeletally immature
during proton beam RT.

To our knowledge, there are no published data regarding the association
of molecular group with lymphopenia during RT. It is of value to control
for molecular group when assessing for an association between risk of
recurrence and lymphopenia over the course of RT which unfortunately we
were unable to do in our study as the vast majority were diagnosed prior
to the routine use of molecular grouping as standard diagnostic
practice.

While our study shows the influence of lymphopenia during week 4 and 5
of RT on risk of medulloblastoma, other groups have shown that early
lymphopenia (weeks 1 and 2 of RT) is what impacts the risk of recurrence
in medulloblastoma \textsuperscript{1} or that there is no effect at all
of lymphopenia on RFS in childhood medulloblastoma \textsuperscript{31}.
More data are clearly needed from additional patients with
newly-diagnosed childhood medulloblastoma and ideally should be
collected in a prospective fashion. Future studies should consider
analyzing lymphocyte subsets throughout the RT course to characterize
changes in specific lymphocyte populations over time. It would also be
worthwhile to correlate the baseline number of TIL from the initial
tumor resection with the risk both of lymphopenia throughout the RT
course and tumor recurrence.

In summary, lymphopenia during weeks 4 and 5 of RT measured as CTCAE
grade 3-4 and during week 5 of RT measured as below median ALC
correlates with an increased risk of tumor recurrence in patients with
newly-diagnosed childhood medulloblastoma.

\textbf{CONFLICT OF INTEREST STATEMENT:} SLG serves on the advisory
board for Chimerix, which has no relationship to this manuscript. HBL,
MES, AKA, MC, and ACP have no conflicts of interest to disclose.

\textbf{ACKNOWLEDGEMENTS:} None. No funding was utilized for this work.

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\textbf{Hosted file}

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\textbf{LEGENDS}

\textbf{FIGURE 1} Percent of patients that received 23.4 Gray
(\textbf{A} ) or 36-39.6 Gray (\textbf{B} ) craniospinal irradiation who
developed grades 2-4 lymphopenia during radiation therapy

\textbf{FIGURE 2} Absolute lymphocyte count during radiation therapy in
patients that received 23.4 Gray (\textbf{A} ) or 36-39.6 Gray
craniospinal irradiation (\textbf{B} )

\textbf{FIGURE 3} Higher risk of disease recurrence (lower
recurrence-free survival) with grade 3 or 4 lymphopenia in weeks 4
(\textbf{A} ) and 5 (\textbf{B} ) radiation therapy (log-rank p=0.015
and Cox p=0.03 for week 4; log-rank p=0.0009 and Cox p=0.004 for week 5)

\textbf{FIGURE 4} Low absolute lymphocyte count (ALC) during week 5 of
radiation therapy is associated with lower recurrence-free survival
(higher risk of disease recurrence) (\textbf{B} ); this association was
significant during week 4 of radiation (\textbf{A} ) (log-rank p=0.123
and Cox p=0.22 for week 4; log-rank p=0.0026 and Cox p=0.002 for week 5)
(*low/high split on median ALC value of 0.4368 for week 4 and 0.4905 for
week 5)

\textbf{SUPPLEMENTAL FIGURE 1} No difference between recurrence-free
survival in patients treated with proton vs. photon radiation therapy
(hazard ratio=0.40, p=0.09)\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

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\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

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\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}

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\verb`Table 1.docx`  available at \url{https://authorea.com/users/674857/articles/673017-late-onset-lymphopenia-during-radiation-is-associated-with-an-increased-risk-of-tumor-recurrence-in-newly-diagnosed-pediatric-medulloblastoma}

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\verb`Table 2.docx`  available at \url{https://authorea.com/users/674857/articles/673017-late-onset-lymphopenia-during-radiation-is-associated-with-an-increased-risk-of-tumor-recurrence-in-newly-diagnosed-pediatric-medulloblastoma}

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