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Lymphopenia and radiation dose to circulating lymphocyte with neoadjuvant
chemoradiation in esophageal squamous cell carcinoma
Tsz Him So, FRCR, Sik Kwan Chan, BSC, Wing Lok Chan, FRCR, Horace Choi,
PhD, Chi Leung Chiang, FRCR, Victor Lee, MD, Tai Chung Lam, FRCR, Ian Wong,
FRCS, Simon Law, MS, Dora Kwong, MD, Feng Ming (Spring) Kong, FASTRO,
JianYue Jin, PhD, Ka On Lam, FRCR
PII: S2452-1094(20)30079-8
DOI: https://doi.org/10.1016/j.adro.2020.03.021
Reference: ADRO 444
To appear in: Advances in Radiation Oncology
Received Date: 19 January 2020
Revised Date: 25 March 2020
Accepted Date: 31 March 2020
Please cite this article as: So TH, Chan SK, Chan WL, Choi H, Chiang CL, Lee V, Lam TC, Wong I,
Law S, Kwong D, Ming (Spring) Kong F, Jin J, Lam KO, Lymphopenia and radiation dose to circulating
lymphocyte with neoadjuvant chemoradiation in esophageal squamous cell carcinoma, Advances in
Radiation Oncology (2020), doi: https://doi.org/10.1016/j.adro.2020.03.021.
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Title page
Lymphopenia and radiation dose to circulating lymphocyte with neoadjuvant
chemoradiation in esophageal squamous cell carcinoma
Short Title: Radiotherapy and Lymphopenia in CA Esophagus
Tsz Him So,
#
FRCR, Sik Kwan Chan
#
, BSC, Wing Lok Chan,
#
FRCR, Horace Choi,
#
PhD, Chi
Leung Chiang
#
, FRCR, Victor Lee,
#
MD, Tai Chung Lam
#
, FRCR, Ian Wong, * FRCS ,Simon
Law,* MS, Dora Kwong,
#
MD, Feng Ming (Spring) Kong,
#
FASTRO, JianYue Jin, PhD, Ka On
Lam,
#
FRCR
Department of
#
Clinical Oncology and * Surgery, the University of Hong Kong, Hong Kong
&
Department of Radiation Oncology, University Hospitals/Seidman Cancer Centre and Case
Comprehensive Cancer Centre, the United States
Declarations of interest: none
Source of Financial Support/Funding Statement: No external funding received
*Corresponding author
Dr. Ka-on Lam MBBS, FRCR, FHKCR, FHKAM
Specialist in Clinical Oncology and Clinical Assistant Professor
Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, The University of Hong
Kong; 1/F, Professorial Block, Queen Mary Hospital, Hong Kong; Email: lamkaon@hku.hk; Tel:
+852 22554352; Fax: +852 28726426
Summary
In advanced squamous cell oesophageal cancer (ESCC) treated with trimodality therapy according to the
Dutch CROSS trial regimen, we found that radiation induced lymphopenia can be predicted by the
effective dose to the circulating immune cells (EDIC). EDIC was found to be highly correlated with
lymphocyte nadir (Spearman coefficient = -0.505; P < 0.01) which was found to significantly
independently associated with shorter OS (Hazard ratio [HR] = 0.63; P < 0.001).
1
Title
Lymphopenia and radiation dose to circulating lymphocyte with neoadjuvant
chemoradiation in esophageal squamous cell carcinoma
Abstract
Background: We hypothesized that radiation-induced lymphopenia could be predicted
by the effective dose to the circulating immune cells (EDIC) in advanced esophageal
squamous cell carcinoma (ESCC) treated with trimodality therapy according to the
Dutch CROSS trial regimen. To test this hypothesis, we examined the effect of EDIC
on the degree of lymphocyte drop (lymphocyte nadir).
Methods: Patients with advanced non-metastatic ESCC treated in a single tertiary
cancer centre from 2012–2018 were eligible for this study. All patients must have a
radiotherapy plan available for EDIC computation and received neoadjuvant
chemoradiation according to the Dutch CROSS trial regimen before radical
esophagectomy. The EDIC was calculated as a function of integral doses to the lung,
heart and total body with a verified mathematical model. The association between
EDIC and lymphocyte nadir was studied, and the relationships of overall survival (OS)
2
with lymphocyte nadir and EDIC were assessed using multivariable Cox regression
model.
Results: This analysis included 92 eligible consecutive patients with 77 males and 15
females. The mean EDIC was 2.8 Gy (range 0.6–4.4). EDIC was significantly
correlated with lymphocyte nadir (Spearman coefficient = -0.505; P < 0.01) and
lymphocyte nadir was a significant independent factor for shorter OS (Hazard ratio
[HR] = 0.63; P < 0.001). Lymphocyte nadir was also the most significant factor in
determining OS among other clinical parameters. Exploratory analysis showed
significant OS differences between EDIC groups (< 2 Gy, 2–4 Gy and > 4 Gy). The 2–
year OS rates were 66.7%, 42.7% and 16.7% for EDIC < 2 Gy, 2–4 Gy and > 4 Gy,
respectively.
Conclusions: There was significant correlation between radiation dose to circulating
immune cells and lymphocyte nadir which in turn impacted overall survival in patients
with advanced non–metastatic ESCC treated by trimodality therapy.
Key words: Esophageal Squamous Cell Carcinoma (ESCC), lymphopenia, radiation
dose, lymphocyte, CROSS Regimen
3
4
Background
Radiation is well-known to have a potent lymphocyte-killing effect. Radiation can
destroy mature circulating lymphocyte at as low as 1 Gy [1].
The importance of this
has been overlooked for decades until recently when immunotherapy emerges as a
standard treatment in many cancers. Since lymphocytes play a major role in exerting
anti-tumour immunity, many clinical studies have demonstrated the correlation
between absolute lymphocyte counts (ALCs) and survival [2-4].
Radiation–induced lymphopenia has been reported to be adversely associated with
overall survival and recurrence–free survival in various cancers including
glioblastoma, non–small cell lung cancer (NSCLC), pancreatic cancer, and head and
neck cancer [5-9]. One of the possible mechanisms of lymphopenia in radiotherapy is
the large volume of low dose bath that kills a vast number of circulating lymphocytes
in both systemic and pulmonary circulation [7]. Yovino et al [10]
first reported that a
single fraction of typical RT plan (60Gy in 30 fractions) for glioblastoma patients
resulted in 0.5 Gy exposure to 5% of circulating cells. Ninety-nine percent of
circulating lymphocytes would be exposed to at least 0.5 Gy after the whole course of
5
30 fractions.
Tang et al [7] further reported significant correlation between low dose
lung dosimetry V1–V5 with the degree of lymphopenia in NSCLC patients. They
hypothesised that as all cardiac output must go through pulmonary circulation, the
large volume low dose bath radiation to pulmonary vasculature would kill most
circulating lymphocytes, contributing to worse outcomes in patients with higher V5
value. XXXX et al [11] further studied the contributing factors and calculated the
dose to circulating lymphocyte in lung cancer patients. They developed a model to
estimate the effective dose to circulating immune cells (EDIC) and found that higher
EDIC was correlated with poorer survival in lung cancer patients in the RTOG 0617
trial.
Esophagus is an organ mainly confined in the thorax. The majority of entry and exit
radiation beams have to pass through lung tissue during treatment of esophageal
cancer with either conformal 3D or intensity modulated radiotherapy (IMRT) photon
technique. Recently published studies [12-14] in the US suggested higher lymphocyte
nadir predicts better pathological and survival outcomes for esophageal cancer
patients. However, these studies included a majority of adenocarcinoma patients
treated with radical chemoradiation and there was heterogeneity regarding
6
radiotherapy doses and chemotherapy regimens. Therefore, it is worth studying the
dosimetry and clinical parameters that cause lymphopenia in esophageal squamous
cell cancer (ESCC) during neoadjuvant chemoradiation with the CROSS regimen [15],
a standard of care employed in Europe and Asia.
As immunotherapy is increasingly incorporated to the systemic treatment of various
cancer, it is important to devise a better lymphocyte–sparing radiation technique. For
example, stereotactic body radiation therapy (SBRT) is associated with significantly
less radiation–induced lymphopenia than fractionated radiotherapy in locally
advanced pancreatic cancer [16]. Lymphocyte–sparing radiation technique may be
crucial for subsequent immunotherapy if there were metastases later. For instance,
there are reported phases II and III studies on the use of immunotherapy including
checkpoint inhibitors in advanced ESCC. The landmark phase III Keynote 181
study[17] demonstrated improved clinical outcomes with second line pembrolizumab
in advanced esophageal cancer with PDL1 combined proportional score ≥ 10. Thus,
studying factors in preserving lymphocytes during initial radiotherapy for esophageal
cancer may have far-reaching importance in subsequent metastatic settings.
7
Method
Hypothesis and objectives
We performed two analyses to test two related hypotheses. Firstly, radiation–induced
lymphopenia can be predicted by the EDIC that was computed by modelling the doses
to the total body, lung and heart. Secondly, EDIC had an impact on survival in
patients with ESCC treated with trimodality therapy.
The primary objective of our study was to validate the EDIC model in ESCC and to
study the correlation of EDIC with lymphocyte nadir (lowest absolute lymphocyte
count). Secondary objectives were to explore the relationships between EDIC,
lymphocyte nadir and overall survival.
Study population
This was a retrospective study in patients with ESCC treated with neoadjuvant
chemoradiation therapy according to the CROSS regimen. After approval by our
institutional review board, clinical and dosimetric data of all ESCC patients at our
hospital from June 2012 to February 2018 were retrospectively reviewed. Patient
8
confidentiality was maintained according to our institution regulations.
Included subjects must have a histological diagnosis of ESCC and they must have
received neoadjuvant chemoradiation with the CROSS regime [15]
(Five weekly
carboplatin AUC = 2 and paclitaxel 50 mg/m
2
, radiation dose = 41.4 Gy in 23 daily
fractions) and subsequent surgery. All subjects must have records of weekly blood test
available during chemoradiation and at 2 months after the completion of
chemoradiation, and a complete dosimetry data available for EDIC computation.
Subjects with distant metastasis or dropouts before the end of chemoradiation were
excluded.
Data collection and dosimetric computation
For each eligible patient, results of blood tests including total white blood cell count,
neutrophil count, lymphocyte count and platelet count during neoadjuvant
chemoradiation were retrospectively retrieved. Lymphocyte nadir was defined as the
lowest absolute value of lymphocyte count among the set of lymphocyte values
during chemoradiation and at 2 months after the completion of chemoradiation.
9
Demographic and clinical data were retrieved from a prospectively–maintained
database. Dosimetry data was obtained from our planning system.
The EDIC was computed as a function of mean heart dose, mean lung dose, integral
dose (ITD), and number of fractions according to the method reported by XXXX et al
[11]. In this model, estimated dose to the immune system was computed by using
the dose to the circulating immune cells as a surrogate. The model assumed that the
radiation dose was uniformly delivered to all rapidly circulating immune cells in the
heart, lung and great vessels but also to the slowly circulating immune cells within the
irradiated volumes. With regard to chemoradiation for ESCC, major organs to
consider include the lung, heart, large vessels and other organs. ITD of the total body
was used to approximate the mean organ dose (MOD) for large vessels and other
organs. The blood dose contributions of the heart (mean heart dose MHD) and lungs
(mean lung dose MLD) were derived from the respective MODs and the estimated
percentage of cardiac output and blood volume they received. Then the equivalent
uniform dose (EUD) was calculated from the DVH. EDIC is the sum of EUDs of all
organs in the irradiated volume. EUD could be estimated by a simple function of
MOD of the above 4 organs.
10
In summary, EDIC was calculated using the following equation[11]:
=.∗+.∗+.+.∗.∗
/
∗
∗
where n is the fraction number (23), k =45.
Statistical consideration
For primary endpoint of lymphocyte nadir, study variables included patient
demographics, tumour factors and treatment factors such as radiation dosimetry to the
major organs at risk (OAR). Spearman’s correlation coefficient between EDIC and
lymphocyte nadir was analysed.
Further univariable and multivariable linear regression analyses were used to correlate
other variables with lymphocyte nadirs. PTV was converted into log(PTV) to obtain
normal distribution.[7] Parameters with p-values <0.1 in univariable model were
considered for multivariable analysis. Normal distribution assumption was tested
11
using using Shapiro-Wilk normality test. The significance of low dose lung dosimetry
with lymphocyte nadirs was similarly analysed.
For secondary endpoint of survival, Cox regression was used to study the prognostic
factors (including lymphocyte nadir) of overall survival (OS) and recurrence–free
survival (RFS). Similar to linear regression, the multivariable analysis included
parameters with p-values <0.1 in univariable models. We also performed survival
analysis in different lymphocyte nadir and EDIC subgroups. Statistical analyses were
conducted by Statistical Package for the Social Sciences (SPSS) version 25.0 (IBM)
and R version 3.5.1. P less than 0.05 was considered significant.
Results
Patient characteristics
Ninety–two patients were eligible for final analysis. Baseline demographics, tumour
and treatment characteristics of these patients are listed in Table 1. All patients were
ethnic Chinese. Around half of the patients (54.3%) were of age 65 or above, and 84%
were male. All 92 patients had squamous cell carcinoma, which reflected the disease
pattern in XXX as opposed to the Western population where adenocarcinoma
12
predominates. Seventy-nine patients (86%) had normal baseline lymphocyte counts
before chemoradiation. All patients but one (99%) had T3 disease and all of them had
at least one regional lymph node involvement (N1: 50%, N2 42.4% and N3 7.6%).
Only 60.8% of patients finished the planned 5 cycles of weekly chemotherapy (4
cycles = 30.4%; 3 cycles = 7.6%; 2 cycles = 0%; 1 cycle = 1.1%) and the commonest
reason for non-compliance was suboptimal marrow tolerance. More patients were
treated with 3D conformal technique than with IMRT technique (56.5% vs 40%).
EDIC and lymphocyte nadir
Univariable and multivariable linear regressions demonstrated that larger PTV volume
was significantly associated with lower lymphocyte nadirs (Table 2), in which
normality was not rejected for the Studentized residuals of the multiple linear
regression with p=0.44 using Shapiro-Wilk normality test. In addition, baseline
lymphocyte count was also associated with lower lymphocyte nadir. No other
demographic and clinical parameter correlated significantly with lymphocyte nadir.
Both radiotherapy technique and the number of courses of chemotherapy did not
affect lymphocyte nadirs.
13
The result of primary outcome was shown in Table 3. The estimated EDIC, which is a
function of mean heart dose, mean lung dose and mean doses to large vessels and
other thoracic organs (approximated by mean total body dose), had a significantly
negative correlation with the lymphocyte nadir (Spearman Coefficient = -0.505; P <
0.01). Individual mean doses to the lung, heart and total body were also highly
significant in predicting lymphocyte nadirs (Spearman coefficients = -0.34, -0.502
and -0.36 respectively; P < 0.01).
Lymphocyte nadir and low dose bath in lung
Previous study has established the correlation between low dose bath to lung and
lymphocyte nadir in non-small cell lung cancer [11]. We performed a similar analysis
to see if this relationship also exists in our patients with ESCC. Spearman’s
correlation coefficients (R) between lymphocyte nadir and different lung dose-volume
histogram (DVH) parameters, namely, V1 to V40, were shown in Figure 1. The
coefficients were highly significant at low dose lung DVH zone from V1 to V25
(Coefficient from -0.47 to -0.27; P < 0.05). The coefficients lost statistical
significance at V30 level onward (P > 0.05). The absolute values of correlation
coefficients decreased together with incremental increase in P value from low dose
14
zone level (V1) to high dose zone level (V40).
Lymphocyte nadir, EDIC and survivals
Our secondary outcome was the survival impact of lymphocyte nadir and EDIC.
Median overall survival (OS) for the whole population was 17.5 months (95% CI
9.30–25.77 months) after a median follow up of 16.9 months (range 1.3–79.3).
Univariable and multivariable Cox regression results of clinical variables on OS and
recurrence–free survival (RFS) are shown in Table 4. Multicollinearity was not found
with variance inflation factor (VIF) ≤1.18 in either OS and PFS. Multivariable models
suggested that lymphocyte nadir was the only variable with statistical significance
(p<0.05) in both OS and RFS. Higher lymphocyte nadir was found to have lower risk
on both OS and RFS (hazard ratio [HR] = 0.75 per 10
8
cells/L; P =0.003, and 0.78 per
10
8
cells/L; P =0.022 respectively) after adjustment in the multivariable Cox
regression models. log PTV, in contrast to common belief, did not reach statistically
significant effect on both OS and RFS in both univariable and multivariable models.
Nadirs of total white cell count, age, Sex, radiotherapy techniques, number of courses
of chemotherapy given, and N stage did not have impact on survival.
15
We then performed further analysis to explore relationships between overall survival
with different groups of lymphocyte nadir and EDIC. Following previously reported
studies [5]
we used lymphocyte count 0.5 (10
9
cells/L) as cut-off as reported The
Kaplan Meier (KM) curve is shown in Figure 2a. There is a trend of better survival for
the group with lymphocyte nadir ≥ 0.5 than the group < 0.5 (P = 0.192).
EDIC of our 92 patients ranged from 0.64 to 4.39 Gy. We divided the patients into
three groups according to EDIC value, namely <2 Gy, 2 – <4 Gy and ≥4 Gy. There
was significant difference in overall survival with different EDIC groups, with higher
EDIC predicting poorer survival (P = 0.01; Figure 2b). In addition, higher EDIC also
predicted lower probability of 2–year overall survival.
Discussion
Our study demonstrated the importance of lymphocyte count in determining the
outcomes of locally advanced ESCC and advised additional dose constraints in RT
planning. Lower lymphocyte nadir was associated with poorer OS and RFS in this
group of patients. Similar results have been shown in other solid tumours including
lung, pancreatic, head and neck and glioblastoma [5]. In our study, the HR of
lymphocyte nadir on OS is 0.63 per 10
5
lymphocytes/mL (or 10
8
cells/L). In other
16
words, there is 37% relative reduction in death per 10
5
lymphocytes/mL (or 10
8
cells/L) increment in lymphocyte nadir. The size of the effect is comparable to similar
study on NSCLC reported by Tang et al [7], in which the corresponding HR is 0.51
per 10
3
lymphocytes/mL (P = 0.01) on univariate analysis. Contrary to previous
reports, PTV was not a predictor of survivals with statistical significant after adjusting
of lymphocyte nadirs in the multivariable in Table 4. log PTV has large HR (>6) but
not statistically significant (p=0080 / 0.10 in uni-/multi-variable for OS). Similarly in
RFS, log PTV was close to statistically significant with p values =0.086 and 0.15,
respectively. The findings suggested that log PTV might have a trend in clinical
significance with high HR but cannot reach the statistical significance. We believe that
PTV effect on survivals may likely be indirect and PTV might exert its effect on
survivals through its contribution to lymphocyte nadirs.
Interestingly, the large scale study INT–0123 published in 2002 proved that there was
no survival benefit and local regional control of oesophageal cancer by escalating the
radiation dose above 50.4Gy [18]
. Despite controversies on the trial design and RT
techniques adopted in that study, our results may uncover the missing link between
RT dose and survival. Higher radiation dose might have detrimental effect on OS due
to lymphocyte killing. A recent analysis of radiation dose escalation in oesophageal
17
cancer [19] concluded that while local control might benefit from dose escalation,
there was no benefit in OS. Similar result was also shown in the dose escalation study
RTOG 0617 for NSCLC. The study [20]
showed an unexpected detrimental effect on
OS for higher dose of 74Gy compared to the control arm of 60Gy. This was
postulated to be due to lower lymphocyte nadir in higher dose arm by XXXX et al
[11]. As both Gross Tumour Volumes (GTV) and Planning Target Volumes (PTV) of
oesophageal cancer and NSCLC are mainly confined to the thorax, and lymphopenia
was shown to affect the outcomes in both cases, it is reasonable to hypothesize that
the lack of survival benefit with dose escalation in ESCC might be due to higher dose
to immune cells.
Low dose parameters of lung DVH (namely, V1–V25) are relevant to the survival of
circulating lymphocyte as almost all the circulating blood go through the pulmonary
circulation. Lung V5 has been shown to be correlated with survival in NSCLC [7].
The low dose bath could have effectively killed off a large number of circulating
lymphocytes. Similarly, PTV was shown to correlate with lymphocyte nadir. This was
likely caused by a larger radiation field resulting in more circulating immune cells
being exposed to radiation. We postulate that it might impair anti–tumour immunity
18
and decrease the ability to mount immunity response to infection. Thus, it might
explain a poorer survival in subjects with lower lymphocyte nadir.
Besides the lung DVH, dose to other previously undefined organs and tissue might
also contribute to the lymphocyte drop. For instance, the heart, large vessels and the
thoracic duct, which house the return of lymphocytes into systemic circulation, are all
in the thorax. In addition, the atrophic thymus in adult could have retained some
lymphatic function as well. Validated mathematical model is needed to account for
dose received by these organs in the thorax.
Therefore, we adopted the formula of predicting EDIC from our co–authors (JJ and
SK) in their study on lymphopenia and survival in NSCLC in RTOG 0617 [11]. We
found that EDIC strongly correlated with the lymphocyte nadir with Spearman
coefficient of -0.505 (P < 0.01). In our study, EDIC ranged from 0.64 to 4.39Gy
which was significantly lower than that reported in the RTOG 0617 cohort (2.05–
12.20Gy), this can be explained by a much lower dose of 41.4Gy prescribed in our
study according to the Dutch CROSS trial. Despite the difference of EDIC range, both
studies demonstrated the importance of EDIC in determining the lymphocyte nadir.
19
Besides, Ladbury et al[21] published their results in adopting the EDIC formula on
stage III non-small cell lung cancer patients in the International Journal of Radiation
Oncology·Biology·Physics. They retrospectively reviewed 117 patients and calculated
their EDIC. They found that EDIC was independently associated with overall survival
(HR 1.17, P = 0.03), local progression-free survival (HR 1.17, P = 0.02), and
disease-free survival (HR 1.15, P = 0.04). As both lung cancers and esophageal
cancers are mainly located within the thoracic, we believe that EDIC formula would
also be a valid model in esophageal cancer too. On the other hand, Saito et al[22]
found that splenic dose-volumes but not bone marrow dose-volumes were predictive
in treatment-related lymphopenia during chemoradiotherapy for esophageal cancer.
We believe future studies can incorporate dose to the spleen as a better estimate of
dose to EDIC.
While lymphocyte nadir has been proven to affect OS in multivariable Cox regression,
the Kaplan Meir survival curve only showed a trend of better survival in lymphocyte
nadir ≥ 0.5 compared with that of < 0.5. We think that given the 0.5 cut-off as
reported by other studies is arbitrary, a significant KM curve may be shown with other
20
lymphocyte cut-offs or with larger sample size. Furthermore, we were able to show
significant difference in 2–year survival probability in different EDIC subgroups. This
was in line with the RTOG0617 cohort.
Similar studies have been reported by Fang et al [13]
from the MD Anderson Cancer
Centre. In their cohort of 313 oesophageal cancer patients who received neoadjuvant
chemoradiotherapy, a higher lymphocyte nadir correlated with a higher pathological
complete response rate (OR = 1.82, P <0.003). They also found that mean body dose
was inversely related to high ALC nadir (OR = 0.77 per Gy, P <0.001). Although this
is consistent with our finding, there are substantial differences between the two
studies. Ninety-five percent of patients were adenocarcinoma in their study, and there
was significant heterogeneity in terms of radiotherapy dose, use of neoadjuvant
chemotherapy and chemotherapy regimens in their concurrent chemoradiotherapy
phase. In our study, all patients had squamous cell carcinoma, and all received the
same chemotherapy and radiotherapy dose.
While lower lymphocyte count predicts worse survival outcomes in other solid
tumours, there have been contradicting reports in the literature for oesophageal cancer.
21
One study [2] in the United States showed that in stage I–III oesophageal cancer
patients (85% adenocarcinoma), the lymphopenia group (defined as lymphocyte < 0.5
x 10
9
/L has a higher risk of death (HR for death 1.6; P = 0.027) compared with the
non–lymphopenia group. This is supported by another study [23] comparing proton
beam therapy with IMRT in 448 stage I-IVA oesophageal cancer patients, which
showed improved survival with higher lymphocyte count (HR for survival = 1.551, P
= 0.01 per 1 unit of lymphocyte). However, another study [24] showed that in 395
stage I–III oesophageal cancer patients, 5–year OS was not significantly different
between patients with grade 4 and non–grade 4 lymphopenia (34% vs 41%; P = 0.47).
As from the Cox regression model in our study, lymphocyte nadir strongly predicts
survivals. We believe the inconsistencies were mostly due to the arbitrary cut-off
value of lymphocyte count and statistical power of respective sample sizes [24].
Some other groups have proposed special radiation techniques to preserve lymphocyte
in other solid tumours. It involves the use of stereotactic body irradiation (SBRT) [16]
to minimize the number of fractions and the PTV, in order to reduce the low dose bath
effect to circulating lymphocyte. Use of proton therapy instead of photon has also
be suggested [23]. As there is minimal exit dose beyond the Bragg Peak, the total
22
integral dose is reduced in proton therapy. This has been shown in the MD Anderson
patient cohort in which proton therapy was a predictor of higher lymphocyte nadir
(compared with IMRT, OR = 4.18; P <0.001). However, neither of these was standard
for radiotherapy in esophageal cancer. Drug therapy in preserving circulating
lymphocyte is also an attractive option. However, there is no well-established therapy
at the time of writing.
Our study was unique that the tumour histology of ESCC and treatment regimens of
the Dutch CROSS were homogenous, in contrast to many other studies in the US in
which adenocarcinoma predominated. Our hospital is the major tertiary referral centre
for esophageal cancer patients in XXX which has a population of over 7 million and
we have included all locally advanced ESCC treated during the period. We believe the
results could be generalized to Asian patients in whom ESCC predominates.
Our study shared the intrinsic weakness of all retrospective studies. We minimized the
selection bias by including only patients with ESCC treated by the Dutch CROSS
regimen. Another limitation was that the lymphocyte count obtained was total
lymphocyte count. Lymphocyte subsets such as CD4+ve and CD8+ve cell counts
23
were not available. Future prospective trial shall address this issue by collecting
weekly cell counts of lymphocyte subsets.
Conclusion
Higher EDIC is associated with lower lymphocyte nadir and lymphocyte nadir
predicts OS. These findings shall be confirmed with larger scale prospective data in
future studies. Perhaps, the retrospective analysis of dosimetry data and lymphocyte
nadirs in INT–0123 trial [18] and CROSS trial [15] might be able to validate our
finding.
List of abbreviations
ALC - absolute lymphocyte counts
DVH - dose volume histogram
EDIC - effective dose to circulating immune cells
ESCC - oesophageal squamous cell carcinoma
24
EUD - equivalent uniform dose
GTV - Gross Tumour Volume
IMRT - Intensity modulated radiotherapy
ITD - Integral dose
NSCLC - non–small cell lung cancer
OAR - organs at risk
OS - overall survival
PTV - Planning Target Volume
RFS - recurrence–free survival
25
SBRT - stereotactic body radiation therapy
Declarations
Ethics approval and consent to participate
Approval was obtained by the Institutional Review Board of the University
of XXX
Consent was obtained from each subject
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Figure legends.
Figure 1. Lung Dose-Volume Histogram and correlation with lymphocyte nadir
Lymphocyte nadir (primary outcome) was correlated negatively with low dose lung
dosimetry only – reflecting dose to circulating lymphocyte. Significance lost above
V25.
Figure 2 Overall survival relationship with (a) lymphocyte nadir and (b) EDIC
groups
32
Table 1 – Baseline characteristics
Table 2
Univariable and multivariable linear regression associating baseline variables
with lymphocyte nadirs (10
9
cells/L) during radiation treatment
Table 3: OAR Dosimetry and EDIC versus lymphocyte nadir
Table 4 Cox regression association with outcomes during radiation treatment
Table 1 – Baseline characteristics
Characteristics N = 92 (%)
Age at diagnosis
Under 65 42 (45.7%)
65 and above 50 (54.3%)
Sex
Male 77 (83.7%)
Female 15 (16.3%)
Baseline lymphocyte level
Normal 79 (85.9%)
Low 13 (14.1%)
RT technique
3DCRT 52 (56.5%)
IMRT 40 (43.5%)
T-staging
T1 0 (0%)
T2 1(1.1%)
T3 91 (98.9%)
T4 0 (0%)
N-staging
N0 0 (0)
N1 46 (50%)
N2 39(42.4%)
N3 7 (7.6%)
RT, radiation therapy; 3DCRT, 3-dimensional conformal radiation
therapy; IMRT, intensity-modulated radiation therapy
Number of chemotherapy courses
5 56 (60.8%)
4 28 (30.4%)
3 7 (12.5%)
2 0 (0%)
1 1 (1.1%)
EDIC group
<2 Gy 18 (19.6%)
2 - <4 Gy 68 (73.9%)
>- 4Gy 6 (6.5%)
Table 3: OAR Dosimetry and EDIC versus Lymphocyte nadir
EDIC= effective dose to the circulating immune cells
Spearman coefficient P
mean total body dose -0.36 < 0.001
mean lung dose -0.34 0.01
mean heart dose -0.502 < 0.001
EDIC -0.505 < 0.01
Table 2
Univariable and multivariable linear regression associating baseline variables with lymphocyte nadirs (10
9
cells/L)
during radiation treatment
Univariable analysis Multivariable analysis
Characteristic
Regression
coefficient 95% CI P
Regression
coefficient 95% CI P
Male (vs female) 0.048 -0.039 to 0.136 0.278
NI
Age (1 y) -0.001 -0.005 to 0.002 0.537
NI
Stage
N3 (vs N2 and N1) -0.085 -0.206 to 0.037 0.168
NI
Baseline lymphocyte
(10
9
cells/L) 0.112 0.068 to 0.156 <0.001 0.098 0.060 to 0.135 <0.001
Chemotherapy
5 courses
(vs fewer than 5 courses) 0.060 -0.005 to -0.126 0.071 0.044 –0.005 to 0.093 0.081
Radiation therapy
log PTV -0.393 -0.629 to -0.158 0.001
–0.205
–0.399 to –0.011
0.038
EDIC -0.085 -0.115 to -0.055 <0.001 –0.061
–0.089 to –0.034
<0.001
IMRT (vs 3DCRT) 0.019 -0.047 to 0.084 0.567
NI
Abbreviations: 3DCRT, 3-dimensional conformal radiotherapy; CI, confidence interval; IMRT, intensity-modulated radiotherapy; NI, not
included; PTV, planning target volume.
Table 4
Cox regression association baseline variables with outcomes during radiation treatment
Overall survival Univariable Multivariable
Variables HR 95% CI P HR 95% CI P
Lymphocyte nadir (10
8
cells/L) 0.72 0.58 to 0.89 0.003 0.75 0.58 to 0.96 0.022
Age (1 y) 0.99 0.97 to 1.02 0.68 NI
WCC nadir (10
9
cells/L) 1.02 0.83 to 1.27 0.84 NI
Male (vs female) 1.002 0.51 to 1.98 0.995 NI
Stage
N3 (vs N2 and N1) 0.66 0.24 to 1.83 0.43 NI
Baseline lymphocyte 0.56 0.36 to 0.87 0.010 0.67 0.41 to 1.11 0.12
Chemotherapy
≥5 courses (vs <5 courses) 0.66 0.40 to 1.10 0.11 NI
Radiation therapy
log
10
PTV 6.41 0.80 to 51.16 0.080 5.32 0.56 to 50.51 0.15
EDIC 1.18 0.89 to 1.57 0.25 NI
IMRT (vs 3DCRT) 0.74 0.44 to 1.24 0.25 NI
Recurrence-free survival Univariable Multivariable
Variables HR 95% CI P HR 95% CI P
Lymphocyte nadir (10
8
cells/L) 0.74 0.60 to 0.91 0.004 0.78 0.62 to 0.99 0.043
Age (1 y) 0.99 0.96 to 1.01 0.30 NI
WCC nadir (10
9
cells/L) 1.01 0.82 to 1.23 0.96 NI
Male (vs female) 1.14 0.58 to 2.24 0.71 NI
Stage
N3 (vs N2 and N1) 0.60 0.22 to 1.64 0.32 NI
Baseline lymphocyte 0.52 0.33 to 0.82 0.005 0.63 0.37 to 1.06 0.079
Chemotherapy
≥5 courses (vs <5 courses) 0.72 0.44 to 1.19 0.20 NI
Radiation therapy
log
10
PTV 6.07 0.76 to 47.45 0.086 4.79 0.52 to 44.47 0.17
EDIC 1.19 0.91 to 1.56 0.21 NI
IMRT (vs 3DCRT) 0.70 0.43 to 1.18 0.19 NI
Abbreviations: 3DCRT, three-dimensional conformal radiation therapy; CI, confidence interval; EDIC, effective dose to the circulating immune
cells; IMRT, intensity-modulated radiation therapy; NI, not included; PTV, planning target volume; WCC, white cell count.
Figure 1 Lung Dose Volume Histogram and correlation with lymphocyte nadir
Lymphocyte nadir(primary outcome) is correlated negatively with low dose lung
dosimetry only – reflecting dose to circulating lymphocyte. Significance lost above
V25
† P < 0.001, ** P < 0.01, * P < 0.05
This is the correlation coefficient plot showing decreasing coefficient value at higher dose
lung DVH and lost statistical significance after 25 Gy.
†
†
**
** **
**
-0.5
-0.45
-0.4
-0.35
-0.3
-0.25
-0.2
-0.15
-0.1
-0.05
0
0 10 20 30 40 50
Spearman's Correlation coefficient
Percent Lung Dose (Gy)
Figure 2 Exploratory Analysis
To determine overall survival relationship with lymphocyte nadir and EDIC groups
Figure 2a
OS difference between two groups (lymphocyte nadir < 0.5 or ≥ 0.5)
Log rank P = 0.192
Figure 2b
OS difference between high and lower EDIC
Log rank P = 0.010
(EDIC range 0.64 to 4.39 Gy)
EDIC= effective dose to the circulating immune cells
