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\begin{document}

\title{Anti-PD-1 monoclonal antibody-resistant esophageal squamous cell
carcinoma showing the abscopal effect: A case report with T-cell
receptor/B-cell receptor repertoire analysis}



\author[1]{Yuka Takehara}%
\author[1]{Yoshiyuki Suzuki}%
\author[2]{Kousaku Mimura}%
\author[1]{Yuya Yoshimoto}%
\author[1]{Yohei Watanabe}%
\author[1]{Zenichiro Saze}%
\author[1]{Hisashi Sato}%
\author[1]{Tomoaki Tamaki}%
\author[1]{Koji Kono}%
\affil[1]{Fukushima Medical University School of Medicine}%
\affil[2]{Fukushima Medical University}%


\vspace{-1em}



  
  \date{May 18, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background Several good results of clinical trial of nivolumab or
involving nivolumab in advanced esophageal squamous cell carcinoma were
reported. However, the response rate was still poor. A rare phenomenon
called the ``abscopal effect'' refers to the regression of not only the
irradiated tumor but also non-irradiated distant tumors after local
radiotherapy. The mechanism is not completely clear, but it is thought
that the activation of anti-tumor immunity induced by radiotherapy is
the main factor. Case A 66-year-old man with recurred and nivolumab
resistant esophageal squamous cell carcinoma in left-side cervical and
abdominal para-aortal lymph node metastasis was treated with a total of
40 Gy (10 fractions) of radiotherapy to the left-side cervical lymph
node metastasis which caused neck pain as a palliative treatment.
Nivolumab was resumed the day after completion of radiotherapy. At 3
months after radiotherapy showed that the irradiated lesion in the left
neck had regressed to a scar-like appearance. Notably, the abdominal
para-aortal lymph nodes outside the irradiation area, which had
previously tended to progress, had also shrunk (abscopal effect). The T
cell receptor and B cell receptor (TCR/BCR) repertoire analysis before
and after radiotherapy revealed that radiotherapy caused the changes in
the TCR/BCR repertoire. Conclusion Changes in the TCR/BCR receptor
repertoire repertoires were assumed to be a part of the mechanism of the
abscopal effect. The findings in this patient suggest that combination
of immune checkpoint inhibitors and radiotherapy can be a promising
treatment approach, even for patients with immune checkpoint inhibitors
resistant cancer.%
\end{abstract}%



\sloppy


\emph{Case Report}

\emph{Title}

Anti-PD-1 monoclonal antibody-resistant esophageal squamous cell
carcinoma showing the abscopal effect: A case report with T-cell
receptor/B-cell receptor repertoire analysis

\emph{Autohrs}

Yuka Takehara\textsuperscript{1}, Kosaku Mimura\textsuperscript{2,3},
Yoshiyuki Suzuki\textsuperscript{1}, Yohei Watanabe\textsuperscript{2},
\begin{CJK}{UTF8}{gbsn}　　　　　　\end{CJK}\selectlanguage{english}Yuya Yoshimoto\textsuperscript{1}, Zenichiro
Saze\textsuperscript{2}, Hisashi Sato\textsuperscript{1}, Tomoaki
Tamaki\textsuperscript{1}, Koji Kono\textsuperscript{2}

\emph{Institurional affiliations}

1. Department of Radiation Oncology, Fukushima Medical University School
of Medicine, Fukushima, Japan

2. Department of Gastrointestinal Tract Surgery, Fukushima Medical
University School of Medicine, Fukushima, Japan

3. Department of Blood Transfusion and Transplantation Immunology,
Fukushima Medical University School of Medicine, Fukushima, Japan

\emph{Corresponding author information}

Yoshiyuki Suzuki, MD, PhD

Department of Radiation Oncology, Fukushima Medical University, School
of Medicine

Hikarigaoka 1, Fukushima-City, Fukushima 960-1295, Japan

Tel: +81-24-547-1630; Fax: +81-24-547-1631

E-mail:\emph{ysuzu@fmu.ac.jp}

\emph{}

\emph{Abstract}

\emph{Background}

Several good results of clinical trial of nivolumab or involving
nivolumab in advanced esophageal squamous cell carcinoma were reported.
However, the response rate was still poor. A rare phenomenon called the
``abscopal effect'' refers to the regression of not only the irradiated
tumor but also non-irradiated distant tumors after local radiotherapy.
The mechanism is not completely clear, but it is thought that the
activation of anti-tumor immunity induced by radiotherapy is the main
factor.

\emph{Case}

A 66-year-old man with recurred and nivolumab resistant esophageal
squamous cell carcinoma in left-side cervical and abdominal para-aortal
lymph node metastasis was treated with a total of 40 Gy (10 fractions)
of radiotherapy to the left-side cervical lymph node metastasis which
caused neck pain as a palliative treatment. Nivolumab was resumed the
day after completion of radiotherapy. At 3 months after radiotherapy
showed that the irradiated lesion in the left neck had regressed to a
scar-like appearance. Notably, the abdominal para-aortal lymph nodes
outside the irradiation area, which had previously tended to progress,
had also shrunk (abscopal effect). The T cell receptor and B cell
receptor (TCR/BCR) repertoire analysis before and after radiotherapy
revealed that radiotherapy caused the changes in the TCR/BCR repertoire.

\emph{Conclusion}

Changes in the TCR/BCR receptor repertoire repertoires were assumed to
be a part of the mechanism of the abscopal effect. The findings in this
patient suggest that combination of immune checkpoint inhibitors and
radiotherapy can be a promising treatment approach, even for patients
with immune checkpoint inhibitors resistant cancer.

\emph{Keywords}

esophageal squamous cell carcinoma, abscopal effect, radiation therapy,
anti-PD-1 monoclonal antibody, B-cell receptor repertoire

\par\null

\emph{Main text}

\emph{Introduction}

Despite the fact that esophageal cancer is one of the deadliest cancers
in the world, there are fewer drugs that have been proven to be
effective against esophageal cancer than against cancers at other sites.
In the United States and Europe, adenocarcinoma is the most common
histological type, and chemotherapy regimens are often selected in a
manner similar to that for gastric cancer. On the other hand, squamous
cell carcinoma is the main histological type in Asia, and the
combination of cisplatin and 5-fluorouracil has been used as a key
approach in Japan.

With the clinical success of immune checkpoint inhibitors (ICIs), such
as anti-PD-1 and anti-CTLA-4 antibodies, tumor immunity has attracted
much attention in cancer treatment. In the 2000s, it became clear that
immunity against tumors plays an important role in not only suppressing
cancer development but also cell-killing anticancer therapies, such as
radiotherapy (RT) and chemotherapy.

The ATTRACTION-3 study is a phase III clinical trial with unresectable
advanced or recurrent esophageal cancer that is refractory or intolerant
to combination chemotherapy (1). Nivolumab showed a significant increase
in survival compared with chemotherapy (taxane), and then, nivolumab was
approved as a second-line treatment for unresectable advanced,
recurrent, or metastatic esophageal squamous cell carcinoma (ESCC) by
the Food and Drug Administration. and for unresectable advanced,
recurrent, or metastatic esophageal cancer by the Pharmaceuticals and
Medical Devices Agency, Japan, in 2020. However, the response rate was
only 19\%. The combination of nivolumab with other therapies to enhance
its efficacy is being actively studied worldwide. Recently, the results
of the CheckMate 648 Trial, a phase III clinical trial involving
nivolumab combination therapy as a first-line treatment in advanced ESCC
were reported (2). Nivolumab plus chemotherapy and nivolumab plus
ipilimumab resulted in significantly longer overall survival than
chemotherapy alone, with no new safety signals identified. However, the
median overall survival period was 13.7--15.4 months, and further
improvement in the treatment protocol is still needed.

A rare phenomenon called the ``abscopal effect'' refers to the
regression of not only the irradiated tumor but also non-irradiated
distant tumors after local RT. The mechanism is not completely clear,
but it is thought that the activation of anti-tumor immunity induced by
RT is the main factor (3,4). Recently, the ``abscopal effect'' was
reported in patients treated with a combination of ICIs and RT (ICI+RT)
(5,6). Though the abscopal effect is exceedingly rare in esophageal
cancer, some reports were recently published (7,8).

Here, we report a patient with anti-PD-1 monoclonal antibody
(mAb)-resistant ESCC showing the abscopal effect (systemic response),
and present the findings of T-cell receptor (TCR) and B-cell receptor
(BCR) repertoire analysis before and after RT.

\emph{Case}

A 66-year-old man was diagnosed with advanced ESCC (Mt-Lt, cT3N2M1,
stage IVb). After neoadjuvant chemotherapy (1 \selectlanguage{ngerman}× 5-fluorouracil+cisplatin
and 1 × docetaxel+5-fluorouracil+cisplatin), he underwent esophagostomy
with regional lymph node dissection. Tegafur/gimeracil/oteracil (S-1)
was started as postoperative adjuvant chemotherapy from 2 months after
the esophagostomy. Six months after esophagostomy, left-side cervical
and abdominal para-aortal lymph node metastasis was detected on computed
tomography (CT) (shown in Fig. 1A). Then, S-1 was discontinued and
nivolumab 240 mg per body was administered as second-line treatment 6
times every 2 weeks. Two months later, a follow-up CT scan showed that
all metastatic lymph nodes progressed, especially the left cervical
lymph node (shown in Fig. 1B). In other words, these tumors were
nivolumab resistant. To treat left-side neck pain caused by the
progressed metastatic lymph nodes, as a palliative treatment, a total of
40 Gy (10 fractions) of RT was administered using the
intensity-modulated, image-guided technique (shown in Fig. 1C).

Nivolumab was resumed the day after completion of RT. A CT scan at 3
months after RT showed that the irradiated lesion in the left neck had
regressed to a scar-like appearance. Notably, the abdominal para-aortal
lymph nodes outside the irradiation area, which had previously tended to
progress, had also shrunk (shown in Fig. 1D). Adverse events were grade
1 oral mucositis, dry skin, pruritus, and fatigue, and no grade 2 or
higher adverse events have been observed in combination with RT to date.
All the adverse events were associated with nivolumab and not with RT.

The TCR and BCR repertoire analysis was outsourced to Repertoire Genesis
Inc. (Tokyo, Japan). The diversity indexes of the TCR and BCR repertoire
analysis before and after RT are shown in table 1 and 2. All the
diversity indexes in both the TCR and BCR repertoires decreased after
RT. The number of unique clones accounting for \textgreater{}0.01\% and
\textgreater{}0.1\% of total TCRs/BCRs before and after RT are shown in
Figure 2A, and the top 10 clones of TCRs/BCRs after RT compared with
before RT are shown in Figure 2B. There were decreases in the number of
unique clones accounting for \textgreater{}0.01\% of total TCRs/BCRs
before and after RT (TCR: 1795--884, BCR: 1792--1212) and large
increases in that of unique clones accounting for \textgreater{}0.1\% of
total TCRs/BCRs before and after RT (TCR: 60--179, BCR: 130--241). The
top 10 clones were mostly similar in the TCR repertoire (8 of the top 10
clones before RT were still in the top 10 clones after RT), but there
were some changes (the percentages of the 1st, 2nd, and 4th clones
increased more than 0.5\%). In the BCR repertoire, all the top 10 clones
before RT were replaced after RT. Furthermore, 9 of the 10 clones were
newly detected after RT.

Thereafter, the irradiated tumors showed complete response (for 11
months). However, re-progression of the abdominal para-aortal lymph
nodes was detected at 7 months after RT, and they were irradiated.
Nivolumab was stopped due to nivolumab-related skin rash (out of the
irradiated area) at 10 months after the first RT (3 months after the
second RT). At 11 months after the first RT, new lymph node metastases
(inguinal and axillary) were detected, and the patient is living with
the disease.

\emph{Discussion}

Chen et al. reported that RT plays a role in antigen spread, which leads
to T-cell immune activation in the cancer immunity cycle (9). Moreover,
according to the review by Sharabi et al., the roles of RT in cancer
immunology are as follows: 1) radiation induces changes to the tumor
cell immunophenotype, 2) radiation enhances cross-presentation of tumor
antigens, and 3) radiation combined with an ICI increases tumor cell
susceptibility to immune-mediated cell death (2). We have reported that
RT induced cancer/testis antigen-specific cytotoxic T-cell activation in
ESCC patients treated with chemo-RT, and multi-antigen-specific T-cell
responses were observed in some patients (4).

Already, the concept of ICI+RT (so-called immunoradiotherapy) has been
clinically proven in non-small-cell lung cancer (NSCLC) (10,11). The
anti-PD-L1 antibody durvalumab administered every 2 weeks for 12 months
after radical chemo-RT for stage 3 NSCLC (PACIFIC trial) improved the
progression-free survival rate by about 20\% at 1 year (10) and the
overall survival rate by about 15\% at 4 years (11). This ICI+RT regimen
has already become the standard therapy for stage 3 NSCLC. At present,
many clinical trials of ICI+RT are underway for cancers at other sites,
including esophageal cancer (12).

Regarding the adverse effects of ICI+RT, severe (grade 3 or higher)
adverse effects were not increased in the PACIFIC trial. Furthermore,
Sha et al. performed a systemic review and meta-analysis, and reported
comparable grade 3--4 toxicity using ICI+RT compared with ICI alone in
CNS melanoma, NSCLC, and prostate cancer, and they concluded that ICI+RT
was safe (13).

A change in the TCR/BCR repertoire after treatment is proof of a
treatment-induced immunoresponse. Changes in the TCR and BCR repertoires
by treatment have already been reported (14,15). Moreover, changes in
the TCR repertoire by RT and chemo-RT have been reported in myeloma
(14), and head and neck cancer (15). The usefulness of analyzing the TCR
repertoire to detect the efficacy of anti-PD-1 antibody (clonality {[}1
-- Pielou's evenness{]} (16) and frequency of the top 30 most frequent
clonotypes: 17)) and the BCR repertoire to detect the efficacy of
autologous cellular immunotherapy (clonality (18)) has been reported.
Analysis of the TCR and BCR repertoires may facilitate the dissection
and understanding of the immune response in human cancer patients and
may be useful as a biomarker of RT or ICI+RT. In our patient, the BCR
repertoire ranking greatly changed with RT and the TCR repertoire also
changed. All diversity indexes for both the TCR and BCR repertoires
decreased after RT. These changes in the TCR and BCR repertoires may be
associated with RT-induced tumor-specific anti-tumor immunity, resulting
in the abscopal effect in the patient with nivolumab-resistance. In
other words, it is possible that further antigen spread by RT may
retrieve the effect of nivolumab in patients with nivolumab-resistance.
Since each metastatic tumor is not the same (homogenous), RT to another
metastatic tumor may induce new or more immune responses (19).

In our patient, RT was performed as a palliative local treatment after
disease progression during nivolumab treatment, and the abscopal effect
was observed. Changes in the TCR and BCR repertoires were observed after
RT, and these were assumed to be a part of the mechanism of the abscopal
effect. The findings in this patient suggest that ICI+RT can be a
promising treatment approach, even for patients with ICI-resistant
cancer.

\emph{Ethical Statement}

\emph{Patient consent} The authors declare that they have obtained
written informed consent from the patient.

\emph{Data Availability Statement}

Data available on request due to privacy/ethical restrictions.

\emph{Acknowledgment}

\emph{Funding Sources}

This work was partially supported by Japan Society for the Promotion of
Science (JSPS) KAKENHI Grant Number 18K07685.

\emph{Conflict of Interest}

The authors have no conflicts of interest to declare.

\emph{Author's Contributions}

Yuka Takehara: resources(equal), data curation(equal),
visualization(equal), writing-original draft(lead), writing-review and
editing(equal)

Kosaku Mimura: conceptualization(equal), resources(equal), data
curation(equal), writing-review and editing(equal)

Yoshiyuki Suzuki: conceptualization(equal), data curation(equal),
visualization(equal), supervision(equal), writing-review and
editing(equal), Funding Acquisition(lead)

Yohei Watanabe: resources(equal), writing-review and editing(equal)

Yuya Yoshimoto: resources(equal), writing-review and editing(equal)

Zenichiro Saze: resources(equal), writing-review and editing(equal)

Hisashi Sato: resources(equal), dwriting-review and editing(equal)

Tomoaki Tamaki: resources(equal), writing-review and editing(equal)

Koji Kono: supervision(equal), writing-review and editing(equal)

\emph{References}

1. Kato K, Cho BC, Takahashi M, et al. Nivolumab versus chemotherapy in
patients with advanced oesophageal squamous cell carcinoma refractory or
intolerant to previous chemotherapy (ATTRACTION-3). \emph{Lancet Oncol}
. 2019;20(11):1506-1517.

2. Doki Y, Ajani JA, Kato K, et al. Nivolumab combination therapy in
advanced esophageal squamous-cell carcinoma. \emph{N Engl J
Med.}2022;386(5):449-462.

3. Sharabi AB, Lim M, DeWeese TL, Drake CG. Radiation and checkpoint
blockade immunotherapy: radiosensitisation and potential mechanisms of
synergy. \emph{Lancet Oncol.} 2015;16(13):e498-509.

4. Suzuki Y, Mimura K, Yoshimoto Y, et al. Immunogenic tumor cell death
induced by chemoradiotherapy in patients with esophageal squamous cell
carcinoma. \emph{Cancer Res.} 2012;72(16):3967-3976.

5. Postow MA, Callahan MK, Barker CA, et al. Immunologic correlates of
the abscopal effect in a patient with melanoma. \emph{N Engl J
Med.}2012;366(10):925-931.

6. Grimaldi AM, Simeone E, Giannarelli D, et al. Abscopal effects of
radiotherapy on advanced melanoma patients who progressed after
ipilimumab immunotherapy. \emph{Oncoimmunology.} 2014;3:e28780.

7. Du D, Song T, Dai H, Zhao J, Chen P, Wu S. Stereotactic body
radiation therapy and thymosin alpha-1-induced anti-tumor effects in
heavily pretreated, metastatic esophageal squamous cell carcinoma
patients. \emph{Oncoimmunology.} 2018;7(8):e1450128.

8. Zhao X, Kang J, Zhao R. Abscopal effect of radiation on lymph node
metastasis in esophageal carcinoma. \emph{Oncol
Lett.}2018;16(3):3555-3560.

9. Chen DS, Mellman I. Oncology meets immunology.
\emph{Immunity.}2013;39(1):1-10.

10. Antonia SJ, Villegas A, Daniel D, et al. Durvalumab after
chemoradiotherapy in stage III non-small-cell lung cancer. \emph{N Engl
J Med.} 2017;377(20):1919-1929.

11. Faivre-Finn C, Vicente D, Kurata T, et al. Four-year survival with
durvalumab after chemoradiotherapy in stage III NSCLC-an update from the
PACIFIC trial. \emph{J Thorac Oncol.} 2021;16(5):860-867.

12. https://clinicaltrials.gov/

13. Sha CM, Lehrer EJ, Hwang C, et al. Toxicity in combination immune
checkpoint inhibitor and radiation therapy. \emph{Radiother
Oncol.}2020;151:141-148.

14. Smith EL, Mailankody S, Staehr M, et al. BCMA-targeted CAR T-cell
therapy plus radiotherapy for the treatment of refractory myeloma
reveals potential synergy. \emph{Cancer Immunol
Res.}2019;7(7):1047-1053.

15. Sridharan V, Margalit DN, Lynch SA, et al. Definitive chemoradiation
alters the immunologic landscape and immune checkpoints in head and neck
cancer. \emph{Br J Cancer} . 2016;115(2):252-260.

16. Tumeh PC, Harview CL, Yearley JH, et al. PD-1 blockade induces
responses by inhibiting adaptive immune resistance.
\emph{Nature.}2014;515(7528):568-571.

17. Gros A, Robbins PF, Yao X, et al. PD-1 identifies the
patient-specific CD8\selectlanguage{english}+ tumor-reactive repertoire infiltrating human
tumors. \emph{J Clin Invest.} 2014;124(5):2246-2259.

18. Zhang L, Kandadi H, Yang H, et al. Long-term sculpting of the B-cell
repertoire following cancer immunotherapy in patients treated with
sipuleucel-T. \emph{Cancer Immunol Res.} 2020;8(12):1496-1507.

19. Brooks ED, Chang JY. Time to abandon single-site irradiation for
inducing abscopal effects. \emph{Nat Rev Clin Oncol.}2019;16(2):123-135.

\emph{Figure Legends}

Figure 1.

Results of diagnostic and radiotherapy simulation imaging throughout the
disease course. Axial computed tomography (CT) images are shown,
corresponding to the timeline showing therapy. White arrows indicate
left-neck lymph node metastasis, and red circles indicate abdominal
para-aortal lymph node metastasis. (A) Representative figures before
treatment with nivolumab. (B) Figures showing enlargement of the
left-neck lymph node and abdominal para-aortal lymph node metastases.
(C) Figure showing the CT simulation image for radiotherapy planning.
The isodose paint represents total doses of \textgreater{}40 Gy (red),
20--36 Gy (green), and 12--20 Gy (blue). (D) Figures showing findings 3
months after radiotherapy. The irradiated left-neck lymph node
metastasis regressed to a scar-like appearance. Furthermore, disease
response outside of the radiation field was seen.

Figure 2.

(A) Enrichment of the T-cell receptor (TCR)/B-cell receptor (BCR) pool
in clones. There are slight increases in the percentage of unique clones
accounting for \textgreater{}0.01\% of total TCRs/BCRs before and after
radiotherapy, and large increases in that of unique clones accounting
for \textgreater{}0.1\% of total TCRs/BCRs. (B) Top 10 TCR/BCR clones
after radiotherapy. In the TCR repertoire, the top 10 clones are mostly
similar to that before radiotherapy, but there are some changes (the
percentages of the 1st, 2nd, and 4th clones increased more than 0.5\%).
In the BCR repertoire, all the top 10 clones before radiotherapy are
replaced after radiotherapy. Furthermore, 9 of the 10 clones are newly
detected after radiotherapy.

#: before radiotherapy; #: after radiotherapy; *: not detected

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