Circulating tumor cells: advancing personalized therapy in small cell lung cancer patients

Abstract

Small cell lung cancer (SCLC) is a highly aggressive cancer with a dismal 5‐year survival of < 7%, despite the addition of immunotherapy to first‐line chemotherapy. Specific tumor biomarkers, such as delta‐like ligand 3 (DLL3) and schlafen11 (SLFN11), may enable the selection of more efficacious, novel immunomodulating targeted treatments like bispecific T‐cell engaging monoclonal antibodies (tarlatamab) and chemotherapy with PARP inhibitors. However, obtaining a tissue biopsy sample can be challenging in SCLC. Circulating tumor cells (CTCs) have the potential to provide molecular insights into a patient's cancer through a “simple” blood test. CTCs have been studied for their prognostic ability in SCLC; however, their value in guiding treatment decisions is yet to be elucidated. This review explores novel and promising targeted therapies in SCLC, summarizes current knowledge of CTCs in SCLC, and discusses how CTCs can be utilized for precision medicine.

Full text

REVIEW
Circulating tumor cells: advancing personalized therapy in
small cell lung cancer patients
Prajwol Shrestha
1,2,3
, Steven Kao
4,5
, Veronica K. Cheung
4,6
, Wendy A. Cooper
4,6,7
,
Nico van Zandwijk
4,8,9
, John E. J. Rasko
1,2,8
and Dannel Yeo
1,2,8
1 Li Ka Shing Cell and Gene Therapy Program, Faculty of Medicine and Health, University of Sydney, Camperdown, Australia
2 Precision Oncology Program, Gene and Stem Cell Therapy Program, Centenary Institute, University of Sydney, Camperdown, Australia
3 Medical Oncology, Calvary Mater Newcastle, Waratah, Australia
4 Faculty of Medicine and Health, University of Sydney, Australia
5 Medical Oncology, Chris O’Brien Lifehouse, Camperdown, Australia
6 Department of Tissue Pathology and Diagnostic Oncology, NSW Health Pathology, Royal Prince Alfred Hospital, Camperdown, Australia
7 School of Medicine, University of Western Sydney, Australia
8 Cell and Molecular Therapies, Royal Prince Alfred Hospital, Sydney Local Health District, Camperdown, Australia
9 Concord Repatriation General Hospital, Sydney Local Health District, Concord, Australia
Keywords
DLL3; liquid biopsy; schlafen11; targeted
therapies; tarlatamab; thoracic oncology
Correspondence
J. E. J. Rasko and D. Yeo, Li Ka Shing Cell
and Gene Therapy Program, Faculty of
Medicine and Health, University of Sydney,
Sydney, NSW 2050, Australia
E-mail: john.rasko@sydney.edu.au;dannel.
yeo@sydney.edu.au
(Received 20 October 2023, revised 27
March 2024, accepted 20 June 2024)
doi:10.1002/1878-0261.13696
Small cell lung cancer (SCLC) is a highly aggressive cancer with a dismal
5-year survival of <7%, despite the addition of immunotherapy to
first-line chemotherapy. Specific tumor biomarkers, such as delta-like
ligand 3 (DLL3) and schlafen11 (SLFN11), may enable the selection of
more efficacious, novel immunomodulating targeted treatments like bispeci-
fic T-cell engaging monoclonal antibodies (tarlatamab) and chemotherapy
with PARP inhibitors. However, obtaining a tissue biopsy sample can be
challenging in SCLC. Circulating tumor cells (CTCs) have the potential to
provide molecular insights into a patient’s cancer through a “simple” blood
test. CTCs have been studied for their prognostic ability in SCLC; how-
ever, their value in guiding treatment decisions is yet to be elucidated. This
review explores novel and promising targeted therapies in SCLC, summa-
rizes current knowledge of CTCs in SCLC, and discusses how CTCs can
be utilized for precision medicine.
Abbreviations
ASCL1, achaete-scute homolog 1; ATM, ataxia telangiectasia mutated; AURK, aurora kinase; Bcl2, B-cell lymphoma 2; BiTE, bispecific T-cell
engager; bTMB, blood-based tumor mutation burden; CHGA, chromogranin A; Chk1, checkpoint kinase 1; CK, cytokeratin; CPS, combined
positive score; CSC, cancer stem cells; CTC, circulating tumor cell; ctDNA, circulating tumor DNA; DAPI, 40,6-diamidino-2-phenylindole;
DLL3, delta-like ligand 3; EBUS, endobronchial ultrasound; EMT, epithelial-mesenchymal transition; EpCAM, epithelial cell adhesion
molecule; EZH2, enhancer of zeste homolog 2; HR, hazard ratio; INSM1, insulinoma-associated protein 1; LS-SCLC, limited stage SCLC;
MCL-1, myeloid cell leukemia-1; NE, neuroendocrine; NEUROD1, neurogenic differentiation factor 1; NGS, next generation sequencing;
NSCLC, non-small cell lung cancer; ORR, objective response rate; OS, overall survival; PARP, poly (ADP-ribose) polymerase; PBMC,
peripheral blood mononuclear cell; PCR, polymerase chain reaction; PD-L1, programmed cell death protein ligand-1; POU2F3, POU class 2
homeobox 3; pro-GRP, pro-gastrin-releasing-peptide; ROR1, receptor tyrosine kinase-like orphan receptor 1; Rova-T, rovalpituzumab tesirine;
SCLC, small cell lung cancer; SLFN11, schlafen-11; SYP, synaptophysin; TEP, tumor educated platelet; TMB, tumor mutational burden;
TTF1, thyroid transcription factor-1; Vim, vimentin; YAP1, Yes-associated protein 1.
Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.
1

1. Introduction
Lung cancer remains one of the most common cancers
and is the leading cause of cancer-related deaths glob-
ally [1]. Small cell lung cancer (SCLC) comprises of
15% of all lung cancers and is considered one of the
most aggressive human cancer [2]. Globally, there are
approximately 250 000 patients diagnosed with SCLC,
and at least 200 000 patients will succumb to their dis-
ease each year [3,4]. There are limited treatment
options available for SCLC patients. For many years,
combination chemotherapy comprising platinum-based
drugs and etoposide has been the standard of care.
Recently, immunotherapies atezolizumab and durvalu-
mab were approved in addition to the combinational
chemotherapies; however, survival increments were
modest (2–3 months) [5]. Classically, SCLC patients
initially show a favorable response to first-line therapy
(58–68%); however, these responses are short-lived [6].
Recent clinical trials have demonstrated promising
outcomes for novel therapeutic strategies such as bis-
pecific T-cell engager antibodies (BiTE: tarlatamab) [7]
and the synergistic combination of PARP inhibitors
with temozolomide (TMZ) chemotherapy [8]. Addi-
tionally, subtyping based on transcriptomic gene signa-
tures could select patients likely to benefit from
immunotherapy [9].
Tissue biopsies are challenging to obtain in SCLC
and are not routinely undertaken following diagnosis
[10]. As such, liquid biopsies, specifically circulating
tumor cells (CTCs) that represent bona fide tumor cells
within the blood, may provide a convenient and suit-
able alternative. This review summarizes the existing
evidence for CTCs in SCLC, including their prognostic
value, and explores their potential role in clinical treat-
ment selection and therapeutic response prediction.
CTCs in SCLC may be used to evaluate the expression
of tumor biomarkers such as delta-like ligand 3
(DLL3) and schlafen11 (SLFN11) for precision
medicine.
1.1. SCLC diagnosis
Small cell lung cancer has neuroendocrine (NE) charac-
teristics, and pathological diagnosis is based on either
(classic) histologic or cytologic appearance combined
with the expression of specific immunohistochemical
(IHC) markers. A panel of immunohistochemical
markers is required for a definitive diagnosis of SCLC.
Low-molecular-weight cytokeratins (CK; AE1/AE3
antibodies) or CK8 (cam5.2 antibody) are often
observed in SCLC, typically in a dot-like pattern. NE
markers are also used, where CD56 is the most sensitive,
followed by synaptophysin and chromogranin. How-
ever, all 3 NE markers can be negative, so
insulinoma-associated protein 1 (INSM1) may be used
in addition [11,12]. Thyroid transcription factor-1
(TTF-1) is often expressed (80–90%) regardless of pul-
monary or extrapulmonary origin [13]. For tumors lack-
ing NE markers and TTF1, it is critical to demonstrate
negative expression of squamous cell markers such as
p40 and CK5/6 to exclude basaloid squamous cell carci-
noma. For CK-negative tumors, exclusion of
non-epithelial small round cell malignancies such as
lymphoma, melanoma, or sarcoma is required. Non-
specifically, the proliferative marker Ki67 is highly
expressed (>50%) in SCLC [14]. p16 is not often used
due to non-small cell lung cancer (NSCLC) also expres-
sing p16 (40–50%).
Acquiring sufficient tissue for a pathological diagno-
sis can be challenging. The central tumor location and
the presence of tumor necrosis can frequently compli-
cate the process of obtaining a reliable diagnosis [10].
Additionally, frail patients may not be ideal candidates
for endoscopic procedures like endobronchial ultra-
sound (EBUS) [15]. In cases where patients present
with pleural effusion, thoracocentesis is a valuable
diagnostic tool, and the diagnosis can be confirmed by
examination of cells obtained from pleural fluid [16].
As SCLC is characterized by rapid progression and a
rapid tumor doubling time as short as 38 days [17,18],
the initiation of systemic therapy is commenced as
soon as possible after diagnosis. As such, a quick and
definitive pathological diagnosis in SCLC is important
and may be achieved through a liquid biopsy.
1.2. Liquid biopsy in SCLC
In recent years, liquid biopsies have gained significant
popularity as a research focus [19,20]. Liquid biopsies
may contain several types of tumor cells or their deriv-
atives, such as circulating tumor DNA (ctDNA),
CTCs, exosomes, microRNAs, tumor-educated plate-
lets (TEPs), and circulating tumor vascular endothelial
cells (CTECs) [21]. Of these, ctDNA and CTCs have
been the most extensively studied in SCLC patients.
ctDNA are fragments of tumor DNA released into the
peripheral blood, while CTCs are malignant cells that
have detached from the tumor and circulate in the
bloodstream (Fig. 1). The detection of ctDNA in
peripheral blood uses highly sensitive techniques such
as digital droplet polymerase chain reaction (ddPCR)
and next-generation sequencing (NGS). The predictive
and prognostic value of ctDNA has been explored in
2Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
Circulating tumor cells in small cell lung cancer P. Shrestha et al.

SCLC, however, has limited clinical utility due to the
lack of oncogenic driver mutations [22,23]. This is in
contrast to NSCLC, which has been found to benefit
from ctDNA monitoring [24].
CTCs have been detected in 60–90% of SCLC
patients, depending on the CTC platform used [25–27].
A recent meta-analysis found that CTCs had prognos-
tic potential in SCLC greater than NSCLC [28]. This
makes CTCs an attractive tool for disease monitoring
and molecular characterization in SCLC, serving as a
potential surrogate for tissue biopsies [29].
1.2.1. CTC detection
Accurate and reliable detection of CTCs remains a chal-
lenge, compounded by the heterogeneity of CTCs.
Recent reviews have discussed different isolation
methods for the detection of CTCs [30–32]. Briefly,
methods to detect CTCs can be done either by size, den-
sity, or expression of surface markers. Microfluidic tech-
niques like Parsortix and Clearcell use the differential
size of PBMCs to separate CTCs from other blood cells.
Alternatively, positive or negative selection methods
identify CTCs by specific surface markers, such as Cell-
Search, which captures epithelial cell adhesion molecule
(EpCAM)-positive cells [33]. Recently, high-resolution
imaging-based platforms such as high-definition single
cell assay (HDSCA) and AccuCyte-CyteFinder have
been developed without the requirement of positive or
negative selection and rely on image analysis for the
detection of positively stained CTCs using a panel of
markers.
Circulating tumor cells can undergo epithelial-
mesenchymal transition (EMT) thereby reducing the
expression of epithelial markers such as EpCAM and
CK8/18/19 and increasing the expression of mesenchymal
markerssuchasvimentin(Vim)andc-MET[34,35].As
such, epithelial markers used to detect CTCs may not
detect those undergoing EMT. For example, 27% (6/22)
of patients who had no detectable CTCs through Cell-
Search had detectable CTCs when analyzed with the
addition of Ki67 and Vim markers [36]. Combinations of
epithelial, endothelial, and mesenchymal expressions on
SCLC CTCs, detected using HDSCA, illustrate the het-
erogeneous nature of CTCs [37]. Thus, better markers for
SCLC CTCs can improve the accuracy of CTC detection
and enumeration.
2. SCLC prognosis and CTCs
The prognostic value of CTCs has been demonstrated in
several cancers, including SCLC [25–27,38–42].In
Fig. 1. Metastatic seeding in small cell lung cancer (SCLC). Primary SCLC (1) where cancer cells extravasate and undergo epithelial-
mesenchymal transition (EMT) to enter the bloodstream (2). The cancer cell/s travel through the bloodstream, termed circulating tumor cells
(CTCs) (3). Typical markers used for the detection of CTCs include cytokeratin, EpCAM, and vimentin with nuclear DAPI stain. CTCs
undergo mesenchymal-epithelial transition (MET) as they exit the bloodstream at the secondary site (4) where they grow to establish as a
metastatic tumor (5).
Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. 3
P. Shrestha et al. Circulating tumor cells in small cell lung cancer

SCLC, CTC counts at baseline and after the first cycle
of chemotherapy were prognostic, regardless of the
CTC detection method or CTC cut-off value (Table 1).
Naito et al. [25] found that a CTC baseline count of >8
correlated with poorer survival. Hilterman et al. [26]
demonstrated that the absolute CTC count after the first
cycle of chemotherapy was the strongest predictor of
survival, while Normanno et al. [27] found that a reduc-
tion of CTCs following chemotherapy had a stronger
prognostic value than the absolute CTC count. In
another study, CTC enumeration at cycle 2 was best
associated with progression-free survival (PFS) and
overall survival (OS) [43]. A meta-analysis of 16 studies
found a high CTC number pre- and post-therapy
correlated with poor OS despite significant heterogene-
ity among the studies (I
2
=81.8–87.1%) [44].
3. CTC biomarkers for SCLC
treatment
Biomarkers to identify patients for targeted therapies
are expected to become important tools in medical
oncology. However, in SCLC, there is a lack of action-
able mutations or fusions [45]. As such, this section
will discuss tissue biomarkers known in SCLC
(Table 2): PD-L1, tumor mutation burden (TMB),
DLL3, SLFN11 and explore the evidence for their use
on CTCs in SCLC and in other tumor types.
Table 1. Prognostic value of CTCs in SCLC. CK, cytokeratin; CTCs, circulating tumor cells; DAPI, 40,6-diamidino-2-phenylindole; EpCAM,
epithelial cell adhesion molecule; ES, extensive stage; LS, limited stage; LT-PCR, ligand-targeted polymerase chain reaction; OS, overall
survival; PFS, progression free survival; RT qPCR, real time quantitative PCR; SCLC, small cell lung cancer; Vim, vimentin.
Year
[reference]
SCLC
stage N
CTC detection method
(detection %) Markers
Prognostic
value
CTC cut off
(per 7.5 mL
blood)
Timepoint of CTC measurement
(CTC cut-off)
2009 [29] LS/ES 50 CellSearch (86) CK, CD56, DAPI,
CD45, M65,
M30
OS Baseline and day 22 (1)
2012 [25] LS/ES 51 CellSearch (69) EpCAM, CD45 OS ≥6 Baseline and at relapse (6)
2012 [116] LS/ES 97 CellSearch (85) EpCAM, CK,
CD45, DAPI
PFS
OS
>50 Baseline and after 1 cycle of
chemotherapy (50)
2012 [26] LS/ES 59 CellSearch (73) EpCAM, CK,
CD45, DAPI
PFS
OS
2 Baseline (2)
2013 [117] LS/ES 55 RT qPCR (78) CK19 PFS
OS
Not specified Baseline, Post cycles 1 and 5
chemotherapy (not specified)
2014 [118] LS/ES 30 TelomeScan: OBP-401
assay (95)
GFP OS 2 Baseline (2)
2014 [119] LS 112 CellSearch (78) EpCAM, CK8/18/
19, DAPI
PFS
OS
>218 (Based
on ROC
curve)
Baseline (218)
2014 [27] ES 60 CellSearch (90) Not specified OS Baseline, Post one cycle of
chemotherapy (D89%)
2017 [120] LS/ES 80 LT-PCR (84) Folate receptor PFS –Baseline
2017 [38] ES 89 CellSearch (83.3) CK, CXCR4,
CD45, DAPI
PFS
OS
≥6 Baseline and post cycle 1
chemotherapy (6)
2017 [43] LS/ES 50 CellSearch (94) EpCAM, cH2AX,
M30
PFS
OS
50 Baseline and post 1 cycle of
chemotherapy (50)
2018 [121] LS/ES 56 CellSearch (60) CK, DAPI, Vim,
Ki67, M30
PFS
OS
≥5 Baseline, 1 cycle of
chemotherapy and at disease
progression (5)
2019 [39] LS 75 CellSearch (60) EpCAM, CK,
DAPI
OS 15 Baseline (15)
2021 [21] LS/ES 33 Negative selection,
iFISH (97)
CEP8, CD44,
CD45, Vim
PFS
OS
≥12 Disease progression (12)
2022 [122] LS/ES 21 CellSearch (86) CK, EpCAM,
DAPI
PFS ≥2, ≥150 Baseline (2)
2022 [53] LS/ES 21 EpCAM positive
selection (47.8),
Parsortix (55)
CK, Vim OS ≥50 Baseline (50)
4Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
Circulating tumor cells in small cell lung cancer P. Shrestha et al.

3.1. PD-L1
Programmed cell protein-1 (PD-1) is expressed on
T-cells and, when engaged by its ligand, PD-L1, inhibits
the T-cell activation pathway [46]. PD-L1 staining on
tissues has profoundly changed the treatment manage-
ment for NSCLC, but in SCLC, it remains to be proven
[47]. As such, PD-L1 staining is not routinely under-
taken. A number of clinical trials have examined PD-L1
expression in SCLC; however, methods for measuring
and quantifying positive PD-L1 expression differ widely.
The Keynote 028 study defined PD-L1 positivity as
>1% of tumor cells, while the Keynote 158 study used a
PD-L1 combined positive score (CPS), which is the per-
centage of PD-L1 positive cells (i.e., tumor cells, lym-
phocytes, and macrophages) of the total number of
tumor cells. A pooled analysis of these studies assessing
the efficacy of pembrolizumab beyond first-line treat-
ment of SCLC demonstrated a response rate of 29.7%
(14/47) in PD-L1-positive patients compared to 3.5%
(1/28) in PD-L1-negative patients [48]. PD-L1 CPS
expression was not helpful in predicting response to
pembrolizumab with platinum doublet therapy using
the IHC22C3 pharmDx assay (Keynote 604) [49].
Impower 133 demonstrated the benefit of adding atezo-
lizumab to platinum doublet chemotherapy in
extensive-stage SCLC patients [50]. The study defined
PD-L1 expression as ≥1% and ≥5% present on either
tumor cells or tumor-infiltrating immune cells. Although
a longer median OS in the PD-L1 ≥5% group was
observed with the addition of atezolizumab, significance
was not reached due to the small numbers.
3.1.1. PD-L1 and CTCs
PD-L1 expression in CTCs has been studied in various
cancers, including SCLC [51,52]. Acheampong et al. [53]
reported PD-L-1 expression on CTCs isolated from 21
SCLC patients using the Parsortix microfluidic system
or EpCAM-positive selection. PD-L1 was found to be
positive in 7.7–9% of the total number of CTCs. Predic-
tive analysis of PD-L1 expression on CTCs was not fea-
sible due to the limited number of patients. Ilie et al. [54]
conducted a study in 18 SCLC patients and examined
PD-L1 on CTCs isolated by the ISET filtration-based
platform (Rarecells Diagnostics, Paris, France). PD-L1
Table 2. Biomarkers in SCLC CTCs. ASCL1, achaite-scute homolog 1; CHGA, chromogranin A; CK, cytokeratin; CTC, circulating tumor cell;
DAPI, 40,6-diamidino-2-phenylindole; DLL3, delta-like ligand 3; EpCAM, epithelial cell adhesion molecule; IF, immunofluorescence;
NEUROD1, neurogenic differentiation factor 1; OS, overall survival, PD-L1, programmed cell death protein ligand-1; PFS, progression free
survival; POU2F3, POU class 2 homeobox 3; SCLC, small cell lung cancer; SLFN11, schlafen-11; SYP, synaptophysin; Vim, vimentin; YAP1,
Yes-associated protein 1.
Biomarker
Year
[reference] Assay NCTC platform (CTC markers)
CTC findings
Biomarker expression %
Prognostic/predictive
relevance
PD-L1 2016 [54] IF (clone: SP142) 18 ISET system (not specified) 0 (42% on circulating
immune cells)
–
2017 [123] IF (clone: EL1L3N) 6 Epic sciences (CK) 50 OS
2021 [55] Not specified 14 CellSave (EpCAM) NA Predicted response to
platinum doublet
reintroduction
following myc
inhibitor treatment
2022 [53] IF (clone: 28.8) 21 EpCAM positibe selection and
Parsortix system (CK, CD16,
CD66b, Vim)
7.7 (EpCAM positive
selection); 9 (Parsortix)
a
No OS difference
Bcl2 2018 [107] IF (clone: 100/5) 66 CellSearch (CK, Vim, Bcl2, M30) 72.7 OS at baseline and
post cycle 1
DLL3 2019 [82] Taqman PCR 48 Parsortix System/qPCR (EpCAM,
CK19, CHGA, SYP)
8.3 OS at baseline
2019 [83] IF (clone: A45-B/B3) 108 CellSearch (CK, DLL3, Vim) 74.1 PFS at baseline and
post cycle 1
SLFN11 2022 [91,124] IF (clone: D8W1B) 42 Epic sciences (CK) 45 Predicted clinical
response
Molecular
subtyping
2022 [100] IF for ASCL1,
NEUROD1, POU2F3,
YAP-1
28 AccuCyte-CyteFinder (CK, EpCAM) 57.1 (ASCL1), 39.3
(NEUROD1), 42.9
(POU2F3), 32.1 (YAP-1)
–
a
% of total number of CTCs detected.
Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. 5
P. Shrestha et al. Circulating tumor cells in small cell lung cancer

was not found on CTCs or in the tumor cells on the
matched tissue but was observed in the tissue microenvi-
ronment and circulating immune cells. In a clinical trial
investigating a Myc inhibitor, RRx-001, PD-L1 expres-
sion was examined on CTCs before and after therapeu-
tic intervention [55]. A decrease in PD-L1 expression in
CTCs following RRx-001 treatment was observed and
was associated with a positive response when platinum
doublet therapy was reintroduced, suggesting that PD-
L1 expression could potentially aid in selecting patients
likely to respond.
3.2. Tumor mutational burden
Tumor mutational burden (TMB) has been examined in
a number of studies in SCLC patients. In studies
in patients with melanoma, NSCLC, and bladder can-
cer, TMB was found to be predictive of immunotherapy
response, leading to FDA approval for pembrolizumab
in TMB-high cancers in 2020 [56,57]. TMB is the total
number of somatic mutations in DNA measured as
mutations per megabase (Mut/Mb), where convention-
ally, ≥10 Mut/Mb is considered high [58]. It is mea-
sured in a coding area of the tumor genome and reflects
all non-synonymous coding mutations. TMB has been
studied for predicting response to immunotherapy in
SCLC; however, various TMB cut-offs and methods
have been used. In CheckMate 032, high TMB (≥248
mutations) was associated with a higher response rate in
both treatment arms: nivolumab or combination nivolu-
mab with ipilimumab; compared to tumors with the low
TMB (<144 mutations) (21.3% and 46% compared to
4.8% and 22.2%, respectively) [59–61]. The 1-year OS
was higher in the TMB high group (35% in the nivolu-
mab arm and 62% in the combination arm) compared
to TMB low group (22.1% and 23.4%, respectively).
The difference was most prominent in the combination
arm (median OS of 22 months in the TMB high group
and 3.4 months in the TMB low group). Similar results
were also found in Keynote 158, where pembrolizumab
achieved an ORR of 29% with a response duration of
4.1–32.5 months in high TMB (defined by the conven-
tional classification of ≥10Mut/Mb) [62]. In IMPower
133, TMB was measured using blood-based TMB
(bTMB), and 86% of SCLC patients had detectable
bTMB, where <10 bTMB and >16 bTMB had an OS
benefit with the addition of atezolizumab (hazard ratio
(HR) =0.69 and HR =0.73, respectively) [50,63].
3.2.1. TMB and CTCs
Tumor mutational burden has not been studied in
SCLC CTCs; however, studies have demonstrated the
feasibility of testing TMB on CTCs in other cancer
types. Rodriguez et al. [64] tested TMB in CTCs using
low-pass whole-genome sequencing in 108 patients with
prostate, breast, colorectal, bladder, and lung cancer. In
a small study, Li et al. [65] compared whole-exome
sequencing data between NSCLC primary and progres-
sive specimens of NSCLC, and an increase in TMB was
observed after cancer progression.
3.3. Delta-like ligand 3
Delta-like ligand 3 is a Notch ligand and has been
extensively studied as a biomarker and a therapeutic
target in SCLC [66–75]. Notch signaling is “oncosup-
pressive” in SCLC (Fig. 2). The Notch pathway com-
prises four receptors (Notch 1–4) and five ligands
(DLL1, 3–4, and Jagged 1–2). DLL3 is regulated by a
transcription factor, achaete-scute homolog 1 (ASH1),
and acts as an oncogenic driver in SCLC by inhibiting
Notch signaling [76]. DLL3 is highly expressed in
SCLC and is not detectable in normal tissues [76].
≥25% DLL3 tumor expression was found in 85%
(895/1050) of patients using IHC staining, with 68%
(719/1050) exhibiting high expression (≥75%) [77].A
meta-analysis of five studies involving a total of 601
SCLC patients found that high DLL3 expression in
tumor samples from extensive-stage SCLC was signifi-
cantly correlated with a poor prognosis, particularly in
the Asian population [78].
Delta-like ligand 3 targeting antibodies, rovalpituzu-
mab tesirine (Rova-T) and tarlatamab (AMG757),
have been studied in relation to DLL3 expression
[66,67]. Rova-T clinical trials did not demonstrate a
survival benefit [68,79]. DLL3 positivity (≥25%) or
high DLL3 (≥75%) tumors did not correlate with
response to Rova-T, and cell lines derived from sub-
jects that had progressed on Rova-T did not show sen-
sitivity to Rova-T [80].
Tarlatamab is a bispecific T-cell engager (BiTE) that
binds to DLL3 on cancer cells and CD3 on T-cells. This
enhances the T-cell-dependent killing of cancer cells.
The phase I study found manageable side effects and
clear signs of activity in SCLC patients [73]. In a subse-
quent study involving 107 refractory or relapsed SCLC
patients, an ORR of 23.4% was observed, including two
complete responses and 23 partial responses [7]. The
median duration of response at the time of reporting
was 12.3 months and the median PFS was 3.7 months.
Preliminary analysis suggests selecting patients with
increased DLL3 expression provides enhanced
clinical benefit. The phase II DeLLphi-301 study
(NCT05060016) involving 220 previously treated SCLC
patients found an ORR of 32–40% and an estimated 9-
6Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
Circulating tumor cells in small cell lung cancer P. Shrestha et al.

month overall survival of 66% [81]. Low DLL3 expres-
sion has been proposed as a resistance mechanism to
DLL3-targeting BiTEs [70]. Tarlatamab in combination
with other agents such as an anti-PD1 agent, AMG404
(NCT04885998), and chemotherapy and anti-PD-L1
combination (NCT05361395) are currently underway.
Several other DLL3-targeting agents, such as BI764532
[75], AMG119 [69], and HPN328 [72], are also under
clinical investigation.
3.3.1. DLL3 and CTCs
Delta-like ligand 3 expression has been evaluated in
SCLC CTCs. Obermayr et al. [82] reported the detection
of DLL3 transcripts by the TaqMan assay in CTCs iso-
lated by the Parsortix microfluidic platform as a marker
of poor survival. Similarly, Messaritakis et al. [83] found
DLL3 expression, by antibody staining, in CTCs at
baseline was significantly associated with decreased PFS
(HR =10.8) and OS (HR =28.2). 74.1% (80/108) of
patients had DLL3-positive CTCs and were not detected
in any healthy controls. Of the 14 patients with high
DLL3 tissue expression (>50% tumor cells), 85.7%
(12/14) had detectable DLL3-positive CTCs. Interest-
ingly, 83.3% (5/6) of patients with low DLL3 tissue
expression had detectable DLL3-positive CTCs, with
the authors proposing the potential of systemic migra-
tion of DLL3-positive tumor cells. The number of
DLL3-positive CTCs and the detection rate also signifi-
cantly increased with disease progression. In a small
study, 83.3% (5/6) of patients with CK-negative CTCs
were noted to have detectable DLL3-positive and Vim-
positive CTCs [80]. DLL3 expression on CTCs in
patients undergoing DLL3-targeted treatments has so
far not been examined as a predictive biomarker.
3.4. Schlafen11
SLFN11 is a DNA/RNA helicase required for chroma-
tin opening that induces irreversible DNA replication
AB
Fig. 2. Emerging therapeutic targets and biomarkers in small cell lung cancer (SCLC). (A) Delta-like ligand 3 (DLL3) belongs to the Notch
signaling pathway family. DLL3 binds to the Notch receptor (1), which triggers protease-mediated cleavage, resulting in the notch-
intracellular domain (NICD) being released into the cytoplasm (2). NICD migrates to the nucleus, where it binds to the transcription factor
CBF1-suppressor of hairless-LAG1 (CSL) and recruits the transcription co-factor mastermind-like (MAML) (3) for gene transcription of Notch
target genes (4). Targeted DLL3 therapy, tarlatamab (AMG757), is a bispecific T-cell engager (BiTE) that binds to DLL3 on cancer cells and
CD3 on T-cells, resulting in T-cell-dependent killing. (B) Schlafen11 (SLFN11) is involved in the DNA damage repair pathway. Upon DNA
damage or replication stress (a), DNA repair is initiated (b). SLFN11 binds to the replication fork (c) to prevent DNA damage repair and
induce replication block and cell death. As such, SLFN11 can sensitize cells to DNA damage-induced replication stress such as poly ADP
ribose polymerase inhibitors (PARPi) combined with chemotherapy and temozlomide (TMZ). Therapeutic agents are underlined.
Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. 7
P. Shrestha et al. Circulating tumor cells in small cell lung cancer

block by changing chromatin structure at replication
sites (Fig. 2)[84]. Fifty to sixty per cent of SCLC
patients are SLFN11-positive [8,85–87]. SLFN11
expression was detected by IHC using commercially
available antibody SLFN11 clones such as SA, D-2SC
and E-4SC [88–90]. SLFN11 is also expressed in inflam-
matory cells, which can influence tissue-based bulk
RNA sequencing. SLFN11 has been found to be down-
regulated after treatment in SCLC patients [91,92].
As SLFN11 is recruited to stalled replication forks, it
can sensitize cells to DNA damage-induced replication
stress. SLFN11 has been associated with the sensitivity
to topotecan in SCLC cell lines, suggesting its use as an
emerging predictive biomarker [93]. High SLFN11
expression has been linked to sensitivity to poly ADP-
ribose polymerase (PARP) inhibitors [86,90]. Temozolo-
mide (TMZ) chemotherapy was found to synergize with
PARP inhibitors in SCLC cell lines and mouse models
[90]. This was demonstrated in a phase II study where
the response rate was significantly higher when the
PARP inhibitor, veliparib, was combined with TMZ
compared to TMZ alone in recurrent SCLC (39% vs
14%) [8]. When archival tissue was available, SLFN11-
positive patients who received combination therapy had
significantly higher PFS (5.7 vs 3.6 months) and OS
(12.2 vs 7.5 months). SFLN11 expression has been
observed to change following treatment, potentially with
the emergence of resistance [86,94]. Downregulation of
SLFN11 has been demonstrated to be mediated by
EZH2 during treatment resistance. Using xenograft
SCLC models, the addition of EZH2 inhibitors was
found to overcome chemotherapy resistance [92].
SLFN11 expression has also been shown to correlate
with response to Topoisomerase 1 and 2 inhibitors such
as etoposide combined with alkylating agents like cis-
platin, and low SLFN11 expression has been linked to
therapy resistance in other cancers [85,95,96].
3.4.1. SLFN11 and CTCs
SLFN11 has been evaluated in CTCs in high-grade
NE cancers, including SCLC [91]. 83% (53/64) of
patients had detectable CTCs, using a high-resolution
CTC imaging platform (Epic Sciences, San Diego, CA,
USA). Of these patients, 55% (29/53) had SLFN11-
positive CTCs, and this detection rate was comparable
to the tissue. In three of these patients, longitudinal
samples were available, and overall CTC number and
SLFN11-positive CTCs were correlated with clinical
response. This suggests the potential of SLFN-11 as a
prognostic and predictive marker in SCLC patients,
especially due to its association with treatment resis-
tance; however, further studies are required.
3.5. Transcriptomic and proteomics-based SCLC
classification
Small cell lung cancer is known to have significant het-
erogeneity and variation in treatment response. Recent
molecular characterization has identified 4 distinct
SCLC subtypes based on their differential expression
of transcription factors: SCLC-A, SCLC-N, SCLC-P,
and SCLC-I, which demonstrated prognostication and
could assist in treatment selection [9,97,98]. Two NE
subtypes, expressing high expression of Chromogranin
A (CHGA) and Synaptophysin (SYP) were identified:
SCLC-A (expressing ASCL1) and SCLC-N (expressing
NEUROD1). These were the most common subtypes,
comprising 51% and 23%, respectively. The SCLC-P
subtype (expressing POU2F3) was the least common
(7%) and had the worst prognosis [9]. The fourth sub-
type, SCLC-I, did not express any of the key transcrip-
tion factors and was associated with an inflammatory
gene signature with high expression of immune check-
point molecules, such as CD274, PD-L1, PDCD1,
CTLA4, CD80, CD86, CD38, ID O1, TIGIT,
C10orf54 (Vista), ICOS, and LAG3, as well as T-cell-
attracting chemokines such as CCL5 and CXCL10.
Furthermore, the SCLC-I subtype expressed low levels
of epithelial markers such as E-cadherin and high
levels of mesenchymal markers such as Vim and Axl.
Interestingly, the previously reported subtype, SCLC-
Y, associated with Yes-associated Protein 1 (YAP1)
was not found to be a distinct subtype in this study
but was found to be expressed in both SCLC-P and
SCLC-I subtypes [99].
Importantly, there are therapeutic implications for
these molecular subtypes. The Impower 133 study
demonstrated significant OS benefit in the SCLC-I
subtype compared to other SCLC subtypes for the ate-
zolizumab combined with chemotherapy treatment
group (HR =0.566) [50]. While the SCLC-A and
SCLC-N subtypes showed a trend toward improve-
ment in median OS with the addition of atezolizumab,
there was no difference in the SCLC-P subtype.
3.5.1. Molecular subtypes and CTCs
Molecular subtyping CTCs into the four SCLC sub-
types is feasible [100]. Kopparapu et al. used a panel
of markers: ASCL1, POU2F3, NEUROD1, and YAP-
1 to characterize the CTCs detected using the high-
resolution imaging platform, AccuCyte-CyteFinder
(RareCyte, Seattle, WA, USA). SCLC-A was the most
common subtype on CTCs (80%), followed by SCLC-
P (67%), SCLC-N (55%), and SCLC-Y (50%). Inter-
estingly, the study found CTCs from the same patient
8Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
Circulating tumor cells in small cell lung cancer P. Shrestha et al.

expressed multiple subtype markers, suggesting molec-
ular intra-patient heterogeneity and/or subtype
switching.
3.6. Future perspectives
There are a number of promising biomarkers for com-
monly used therapies, such as TMZ, PARP inhibitors,
and B-cell lymphoma 2 (Bcl2) inhibitors: venetoclax
and navitoclax. In addition, there are several encour-
aging therapies for SCLC, such as ROR1 inhibitors,
aurora kinase inhibitors (AURKi), heat shock protein
90 (HSP90) inhibitors, and CDK9 inhibitors (dinaci-
clib). This section will explore these promising bio-
markers and therapies and discuss how CTCs could be
utilized for their translation.
3.6.1. TMZ and PARP inhibitors
Patients with an inflammatory gene signature, SCLC-I,
benefited from the combination of a chemotherapy,
TMZ, and a PARP inhibitor, talazoparib [101].
Absence of SCLC subtype markers (ASCL1, NEU-
ROD1, POU2F3 or NE genes, and INSM1) and
enrichment of non-NE genes have been associated with
increased EMT signatures in relapsed SCLC after first-
line platinum therapy [102], where both EMT and
ataxia telangiectasia mutated (ATM) signatures could
potentially help predict therapeutic response [86].
ATM and E-cadherin have been investigated in
SCLC xenograft models to study the activity of talazo-
parib. Low expression of ATM and checkpoint kinase
1 (CHK1) as well as high expression of SLFN11 were
associated with a response to talazoparib, with high
CHK1 expression associated with treatment resistance
[86]. In castration-resistant prostate cancer, ATM
altered tumors were associated with SLFN11-positive
CTCs [103]. So far, there are no studies examining
ATM expression in SCLC CTCs. If combined with
SLFN11 expression, CTC examination could be of
interest to determine sensitivity to PARP inhibitors.
3.6.2. Bcl2, HDAC, and ROR1 inhibitors
In the SCLC-A subtype, ASCL1 targets the antiapop-
totic regulator, Bcl2, inducing cell death through the
mitochondrial apoptotic pathway. In SCLC xenografts
and cell lines, high levels of Bcl2 were associated with
the treatment response to the Bcl2 inhibitor, navitoclax
(ABT-263) [104,105]. Navitoclax did not produce a
therapeutic benefit in SCLC patients, however, Bcl2
expression was not quantified in patients [106]. Bcl2-
positive CTCs were found in 72.6% of SCLC patients
and were significantly associated with worse PFS
(HR =4.5) and OS (HR =4.3) [107]. Bcl2 expression
has been linked to plasma pro-gastrin-releasing peptide
(pro-GRP), and this has been correlated with sensitiv-
ity to navitoclax [106].
Bcl2 inhibitors have been used in combination with
other therapies. Bcl2 inhibitors combined with a
HDAC inhibitor, vorinostat, demonstrated improved
activity including in Bcl2 resistant cell lines [108].
Another anti-apoptotic protein from the Bcl2 family,
myeloid cell leukemia-1 (MCL-1), has been shown to
be highly expressed in SCLC patients [109]. The study
found high MCL-1 expression was associated with low
Bcl-xL expression, and the combination of a MCL-1
inhibitor with navitoclax resulted in increased anti-
tumor killing. Venetoclax combined with either doxo-
rubicin or a CDK9 inhibitor, dinaciclib, demonstrated
significant anti-tumor activity in SCLC xenograft
models [110].
Bcl2 is co-expressed with receptor tyrosine kinase-
like orphan receptor 1 (ROR1) in SCLC. ROR1, an
oncofetal protein, is highly expressed in SCLC (93%
by IHC, 79% by RT-qPCR) [111]. A small-molecule
ROR1 inhibitor was found to have good anti-tumor
activity in SCLC-derived cell lines and, when com-
bined with venetoclax, demonstrated synergistic inhibi-
tion [111]. Examining MCL-1 or ROR1 on SCLC
CTCs could act as a predictive biomarker for MCL-1
or ROR1 inhibitors combined with Bcl2 inhibitors.
3.6.3. Proteomic SCLC subgroups and AURK, BCL2,
and HSP90 inhibitors
Protein biomarkers show direct pathway activation
and (protein) target expression. An analysis of plasma-
based proteins in SCLC patients found enrichment for
Myc and YAP1 [112].
Myc is a known oncoprotein overexpressed in SCLC
[96]. High levels of Myc have been found in the SCLC-
N subtype, and Myc expression resulted in sensitivity to
aurora kinase (AURK) inhibitors in SCLC cell lines
[9,113,114]. Myc and TTF-1 are known targets of the
transcription factors ASCL1 and NEUROD1 [115].
Two major SCLC proteomic subgroups were proposed
based on model-based clustering: high TTF-1/low Myc
and low TTF1/high Myc. The levels of TTF-1 or Myc
could predict treatment responses to AURK, Bcl2, or
heat shock protein 90 (Hsp90) inhibitors [113].
4. Conclusions
Considering CTCs as an easily accessible tumor surro-
gate, they are well suited to be used as a companion
Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. 9
P. Shrestha et al. Circulating tumor cells in small cell lung cancer

diagnostic for biomarker-guided therapy. However,
large prospective studies are required to confirm their
benefit before implementing for routine clinical care.
In SCLC, a critical advantage of liquid biopsies is the
ability to undertake serial sampling. This is vital, as
rapid tumor progression on therapy is characteristic of
SCLC. Several studies have demonstrated the prognos-
tic value of CTCs and shown the feasibility of evaluat-
ing the expression of therapeutic targets on CTCs such
as SLFN11 and DLL3. However, these biomarker
studies were relatively small and were retrospective. As
such, the potential for evaluating SCLC markers on
CTCs as a means for treatment selection and predict-
ing response requires further validation. A number of
biomarker-driven clinical trials are currently on-going
in SCLC: NCT03699956, NCT04334941, PRIO-
NCT04728230, and NCT04334941.
Before becoming clinically validated, CTC isolation
and biomarker evaluation should be standardized and
validated in prospective clinical trials. Several studies
have explored PD-L1 expression in tissue and CTCs,
but variation in the staining techniques and scoring is
confounding the interpretation of results. Therefore, it
is crucial to assess the practical implications of CTC-
biomarker testing (including cost) and strive toward a
general consensus on how to most effectively use CTC
detection platforms as clinical tools. The application
of CTCs for the assessment of biomarkers, in combi-
nation with transcriptomic-based subtyping and prote-
omic characterization of SCLC, presents a unique
opportunity to personalize treatment approaches to
improve SCLC patient outcomes.
Acknowledgements
Figures and graphical abstract were created with
BioRender.com (27 September 2023). This work was
supported by the Li Ka Shing Foundation (Cell &
Gene Therapy Program to JEJR); the National Health
& Medical Research Council Investigator Grant
(1177305 to JEJR); the Sydney Cancer Institute Seed
Grant (DY); the Tour de Cure Mid-Career Research
Grant (RSP-320 to DY); and philanthropic funding
(SK). PS is supported by a philanthropic PhD scholar-
ship (Chris O’Brien Lifehouse). DY is supported by
the Translational Partners Fellowship from Sydney
Cancer Partners, funded by the Cancer Institute New
South Wales (2021/CBG0002). The funders had no
role in study design, decision to publish, or prepara-
tion of the manuscript. Open access publishing is facil-
itated by the University of Sydney as part of the Wiley
—The University of Sydney agreement via the Council
of Australian University Librarians.
Conflict of interest
JEJR reports advisory roles in Gene Technology Tech-
nical Advisory Committee, Office of the Gene
Technology Regulator, Australian Government; and
Human Research Ethics Committee, Genea. JEJR also
reports honoraria speaker fees or advisory roles for
SPARK Therapeutics, Cynata, and Pfizer Inc.; Woke
Pharmaceutical (shares); Kennerton Capital (non-
executive director); AAVec Bio (co-founder); consul-
tant role for Rarecyte (stocks in lieu). SK served on
advisory boards for AstraZeneca, Pfizer, MSD, BMS,
Roche, Amgen, BeiGene, and Daiichi Sankyu. SK
received honorarium (partly to institution) from MSD,
BMS, Roche, AstraZeneca, Pfizer, Takeda, and Bei-
Gene. SK received research grant (to institution) from
AstraZeneca. All the other authors declare that the
research was conducted in the absence of any commer-
cial or financial relationships that could be construed
as a potential conflict of interest.
Author contributions
All authors (PS, SK, VKC, WAC, NZ, JEJR, and
DY) were involved in the conceptualization, writing of
the original draft, and reviewing and editing the manu-
script. PS and DY created the figures. All authors
have read and agreed to the published version of the
manuscript.
Data accessibility
Data sharing is not applicable to this article. No new
data were created or analyzed in this study.
References
1 Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre
LA, Jemal A. Global cancer statistics 2018:
GLOBOCAN estimates of incidence and mortality
worldwide for 36 cancers in 185 countries. CA Cancer
J Clin. 2018;68(6):394–424.
2 Wang Q, G€
um€
usZH, Colarossi C, Memeo L, Wang
X, Kong CY, et al. SCLC: epidemiology, risk factors,
genetic susceptibility, molecular pathology, screening,
and early detection. J Thorac Oncol. 2023;18(1):31–46.
3 Rudin CM, Brambilla E, Faivre-Finn C, Sage J. Small-
cell lung cancer. Nat Rev Dis Primers. 2021;7(1):3.
4 Surveillance, Epidemiology, and End Results (SEER)
cancer statistics [cited 2023 Feb 1]. Available from:
https://seer.cancer.gov/explorer
5 Goldman JW, Dvorkin M, Chen Y, Reinmuth N,
Hotta K, Trukhin D, et al. Durvalumab, with or
10 Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
Circulating tumor cells in small cell lung cancer P. Shrestha et al.

without tremelimumab, plus platinum-etoposide versus
platinum-etoposide alone in first-line treatment of
extensive-stage small-cell lung cancer (CASPIAN):
updated results from a randomised, controlled, open-
label, phase 3 trial. Lancet Oncol. 2021;22(1):51–65.
6 Simon GR, Turrisi A, American College of Chest
Physicians. Management of small cell lung cancer:
ACCP evidence-based clinical practice guidelines (2nd
edition). Chest. 2007;132(3 Suppl):324S–339S.
7 Paz-Ares L, Champiat S, Lai WV, Izumi H, Govindan
R, Boyer M, et al. Tarlatamab, a first-in-class DLL3-
targeted bispecific T-cell engager, in recurrent small-cell
lung cancer: an open-label, phase I study. J Clin Oncol.
2023;41(16):2893–903.
8 Pietanza MC, Waqar SN, Krug LM, Dowlati A, Hann
CL, Chiappori A, et al. Randomized, double-blind,
phase II study of temozolomide in combination with
either veliparib or placebo in patients with relapsed-
sensitive or refractory small-cell lung cancer. J Clin
Oncol. 2018;36(23):2386–94.
9 Gay CM, Stewart CA, Park EM, Diao L, Groves SM,
Heeke S, et al. Patterns of transcription factor
programs and immune pathway activation define four
major subtypes of SCLC with distinct therapeutic
vulnerabilities. Cancer Cell. 2021;39(3):346–60.e7.
10 Yung RC. Tissue diagnosis of suspected lung cancer:
selecting between bronchoscopy, transthoracic needle
aspiration, and resectional biopsy. Respir Care Clin N
Am. 2003;9(1):51–76.
11 Pelosi G, Sonzogni A, Harari S, Albini A, Bresaola E,
Marchi
o C, et al. Classification of pulmonary
neuroendocrine tumors: new insights. Transl Lung
Cancer Res. 2017;6(5):513–29.
12 Mukhopadhyay S, Dermawan JK, Lanigan CP, Farver
CF. Insulinoma-associated protein 1 (INSM1) is a
sensitive and highly specific marker of neuroendocrine
differentiation in primary lung neoplasms: an
immunohistochemical study of 345 cases, including 292
whole-tissue sections. Mod Pathol. 2019;32(1):100–9.
13 Kaufmann O, Dietel M. Expression of thyroid
transcription factor-1 in pulmonary and
extrapulmonary small cell carcinomas and other
neuroendocrine carcinomas of various primary sites.
Histopathology. 2000;36(5):415–20.
14 Pelosi G, Rindi G, Travis WD, Papotti M. Ki-67
antigen in lung neuroendocrine tumors: unraveling a
role in clinical practice. J Thorac Oncol. 2014;9(3):273–
84.
15 Rivera MP, Mehta AC, Wahidi MM. Establishing the
diagnosis of lung cancer: diagnosis and management of
lung cancer, 3rd ed: American College of Chest
Physicians evidence-based clinical practice guidelines.
Chest. 2013;143(5 Suppl):e142S–e165S.
16 Rivera MP, Mehta AC, American College of Chest
Physicians. Initial diagnosis of lung cancer: ACCP
evidence-based clinical practice guidelines (2nd
edition). Chest. 2007;132(3 Suppl):131S–148S.
17 Sone S, Nakayama T, Honda T, Tsushima K, Li F,
Haniuda M, et al. CT findings of early-stage small cell
lung cancer in a low-dose CT screening programme.
Lung Cancer. 2007;56(2):207–15.
18 Kanashiki M, Tomizawa T, Yamaguchi I, Kurishima
K, Hizawa N, Ishikawa H, et al. Volume doubling
time of lung cancers detected in a chest radiograph
mass screening program: comparison with CT
screening. Oncol Lett. 2012;4(3):513–6.
19 Shrestha P, Lee J, Kao S. Liquid biopsy in non-small
cell lung cancer: is it ready for prime time yet? Asia
Pac J Clin Oncol. 2023;19(2):e3–4.
20 Poulet G, Massias J, Taly V. Liquid biopsy: general
concepts. Acta Cytol. 2019;63(6):449–55.
21 Zhu HH, Liu YT, Feng Y, Zhang LN, Zhang TM,
Dong GL, et al. Circulating tumor cells
(CTCs)/circulating tumor endothelial cells (CTECs)
and their subtypes in small cell lung cancer: predictors
for response and prognosis. Thorac Cancer. 2021;12
(20):2749–57.
22 Devarakonda S, Sankararaman S, Herzog BH, Gold
KA, Waqar SN, Ward JP, et al. Circulating tumor
DNA profiling in small-cell lung cancer identifies
potentially targetable alterations. Clin Cancer Res.
2019;25(20):6119–26.
23 Almodovar K, Iams WT, Meador CB, Zhao Z, York
S, Horn L, et al. Longitudinal cell-free DNA analysis
in patients with small cell lung cancer reveals dynamic
insights into treatment efficacy and disease relapse. J
Thorac Oncol. 2018;13(1):112–23.
24 Li W, Liu JB, Hou LK, Yu F, Zhang J, Wu W, et al.
Liquid biopsy in lung cancer: significance in
diagnostics, prediction, and treatment monitoring. Mol
Cancer. 2022;21(1):25.
25 Naito T, Tanaka F, Ono A, Yoneda K, Takahashi T,
Murakami H, et al. Prognostic impact of circulating
tumor cells in patients with small cell lung cancer. J
Thorac Oncol. 2012;7(3):512–9.
26 Hiltermann TJN, Pore MM, van den Berg A, Timens
W, Boezen HM, Liesker JJW, et al. Circulating tumor
cells in small-cell lung cancer: a predictive and
prognostic factor. Ann Oncol. 2012;23(11):2937–42.
27 Normanno N, Rossi A, Morabito A, Signoriello S,
Bevilacqua S, di Maio M, et al. Prognostic value of
circulating tumor cells’ reduction in patients with
extensive small-cell lung cancer. Lung Cancer. 2014;85
(2):314–9.
28 Jin F, Zhu L, Shao J, Yakoub M, Schmitt L,
Reißfelder C, et al. Circulating tumour cells in patients
with lung cancer universally indicate poor prognosis.
Eur Respir Rev. 2022;31(166):220151.
29 Hou J-M, Greystoke A, Lancashire L, Cummings J,
Ward T, Board R, et al. Evaluation of circulating
Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. 11
P. Shrestha et al. Circulating tumor cells in small cell lung cancer

tumor cells and serological cell death biomarkers in
small cell lung cancer patients undergoing
chemotherapy. Am J Pathol. 2009;175(2):808–16.
30 Edd JF, Mishra A, Smith KC, Kapur R, Maheswaran
S, Haber DA, et al. Isolation of circulating tumor
cells. iScience. 2022;25(8):104696.
31 Sharma S, Zhuang R, Long M, Pavlovic M, Kang Y,
Ilyas A, et al. Circulating tumor cell isolation, culture,
and downstream molecular analysis. Biotechnol Adv.
2018;36(4):1063–78.
32 Descamps L, Le Roy D, Deman AL. Microfluidic-
based technologies for CTC isolation: a review of
10 years of intense efforts towards liquid biopsy. Int J
Mol Sci. 2022;23(4):1981.
33 Swennenhuis JF, van Dalum G, Zeune LL, Terstappen
LWMM. Improving the CellSearch

system. Expert
Rev Mol Diagn. 2016;16(12):1291–305.
34 Hou JM, Krebs M, Ward T, Sloane R, Priest L,
Hughes A, et al. Circulating tumor cells as a window
on metastasis biology in lung cancer. Am J Pathol.
2011;178(3):989–96.
35 Pore M, Meijer C, de Bock GH, Boersma-van Ek W,
Terstappen LWMM, Groen HJM, et al. Cancer stem
cells, epithelial to mesenchymal markers, and
circulating tumor cells in small cell lung cancer. Clin
Lung Cancer. 2016;17(6):535–42.
36 Messaritakis I, Politaki E, Kotsakis A, Dermitzaki
EK, Koinis F, Lagoudaki E, et al. Phenotypic
characterization of circulating tumor cells in the
peripheral blood of patients with small cell lung
cancer. PLoS One. 2017;12(7):e0181211.
37 Seo J, Kumar M, Mason J, Blackhall F, Matsumoto
N, Dive C, et al. Plasticity of circulating tumor cells in
small cell lung cancer. Sci Rep. 2023;13(1):11775.
38 Salgia R, Weaver RW, McCleod M, Stille JR, Yan
SB, Roberson S, et al. Prognostic and predictive value
of circulating tumor cells and CXCR4 expression as
biomarkers for a CXCR4 peptide antagonist in
combination with carboplatin-etoposide in small cell
lung cancer: exploratory analysis of a phase II study.
Invest New Drugs. 2017;35(3):334–44.
39 Tay RY, Fern
andez-Guti
errez F, Foy V, Burns K,
Pierce J, Morris K, et al. Prognostic value of
circulating tumour cells in limited-stage small-cell lung
cancer: analysis of the concurrent once-daily versus
twice-daily radiotherapy (CONVERT) randomised
controlled trial. Ann Oncol. 2019;30(7):1114–20.
40 Wang PP, Liu SH, Chen CT, Lv L, Li D, Liu QY,
et al. Circulating tumor cells as a new predictive and
prognostic factor in patients with small cell lung
cancer. J Cancer. 2020;11(8):2113–22.
41 Li Y, Wu G, Yang W, Wang X, Duan L, Niu L, et al.
Prognostic value of circulating tumor cells detected
with the CellSearch system in esophageal cancer
patients: a systematic review and meta-analysis. BMC
Cancer. 2020;20(1):581.
42 Yeo D, Bastian A, Strauss H, Saxena P, Grimison P,
Rasko JEJ. Exploring the clinical utility of pancreatic
cancer circulating tumor cells. Int J Mol Sci. 2022;23
(3):1671.
43 Aggarwal C, Wang X, Ranganathan A, Torigian D,
Troxel A, Evans T, et al. Circulating tumor cells as a
predictive biomarker in patients with small cell lung
cancer undergoing chemotherapy. Lung Cancer.
2017;112:118–25.
44 Jiang AM, Zheng HR, Liu N, Zhao R, Ma YY, Bai
SH, et al. Assessment of the clinical utility of
circulating tumor cells at different time points in
predicting prognosis of patients with small cell lung
cancer: a meta-analysis. Cancer Control.
2021;28:10732748211050581.
45 Denninghoff V, Russo A, de Miguel-P
erez D,
Malapelle U, Benyounes A, Gittens A, et al. Small cell
lung cancer: state of the art of the molecular and
genetic landscape and novel perspective. Cancers
(Basel). 2021;13(7):1723.
46 Pardoll DM. The blockade of immune checkpoints in
cancer immunotherapy. Nat Rev Cancer. 2012;12
(4):252–64.
47 Shklovskaya E, Rizos H. Spatial and temporal changes
in PD-L1 expression in cancer: the role of genetic
drivers, tumor microenvironment and resistance to
therapy. Int J Mol Sci. 2020;21(19):7139.
48 Chung HC, Piha-Paul SA, Lopez-Martin J, Schellens
JHM, Kao S, Miller WH Jr, et al. Pembrolizumab
after two or more lines of previous therapy in patients
with recurrent or metastatic SCLC: results from the
KEYNOTE-028 and KEYNOTE-158 studies. J Thorac
Oncol. 2020;15(4):618–27.
49 Rudin CM, Awad MM, Navarro A, Gottfried M,
Peters S, Cs}
oszi T, et al. Pembrolizumab or placebo
plus etoposide and platinum as first-line therapy for
extensive-stage small-cell lung cancer: randomized,
double-blind, phase III KEYNOTE-604 study. J Clin
Oncol. 2020;38(21):2369–79.
50 Liu SV, Reck M, Mansfield AS, Mok T, Scherpereel
A, Reinmuth N, et al. Updated overall survival and
PD-L1 subgroup analysis of patients with extensive-
stage small-cell lung cancer treated with atezolizumab,
carboplatin, and etoposide (IMpower133). J Clin
Oncol. 2021;39(6):619–30.
51 Wang Y, Kim TH, Fouladdel S, Zhang Z, Soni P, Qin
A, et al. PD-L1 expression in circulating tumor cells
increases during radio(chemo)therapy and indicates
poor prognosis in non-small cell lung cancer. Sci Rep.
2019;9(1):566.
52 Darga EP, Dolce EM, Fang F, Kidwell KM, Gersch
CL, Kregel S, et al. PD-L1 expression on circulating
12 Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
Circulating tumor cells in small cell lung cancer P. Shrestha et al.

tumor cells and platelets in patients with metastatic
breast cancer. PLoS One. 2021;16(11):e0260124.
53 Acheampong E, Abed A, Morici M, Spencer I, Beasley
AB, Bowyer S, et al. Evaluation of PD-L1 expression
on circulating tumour cells in small-cell lung cancer.
Transl Lung Cancer Res. 2022;11(3):440–51.
54 Ilie M, Hofman V, Long E, Butori C, Lalv
ee S, Selva
E, et al. Abstract 2220: PD-L1 expression in primary
tumor and circulating tumor cells in patients with
small cell lung carcinomas. Cancer Res. 2016;76(14
Suppl):2220.
55 Tomita Y, Oronsky B, Abrouk N, Cabrales P, Reid
TR, Lee MJ, et al. In small cell lung cancer patients
treated with RRx-001, a downregulator of CD47,
decreased expression of PD-L1 on circulating tumor
cells significantly correlates with clinical benefit. Transl
Lung Cancer Res. 2021;10(1):274–8.
56 McGrail DJ, Pili
e PG, Rashid NU, Voorwerk L,
Slagter M, Kok M, et al. High tumor mutation burden
fails to predict immune checkpoint blockade response
across all cancer types. Ann Oncol. 2021;32(5):661–72.
57 Marcus L, Fashoyin-Aje LA, Donoghue M, Yuan M,
Rodriguez L, Gallagher PS, et al. FDA approval
summary: pembrolizumab for the treatment of tumor
mutational burden-high solid tumors. Clin Cancer Res.
2021;27(17):4685–9.
58 Hellmann MD, Ciuleanu TE, Pluzanski A, Lee JS,
Otterson GA, Audigier-Valette C, et al. Nivolumab
plus ipilimumab in lung cancer with a high tumor
mutational burden. N Engl J Med. 2018;378(22):2093–
104.
59 Ready NE, Ott PA, Hellmann MD, Zugazagoitia J,
Hann CL, de Braud F, et al. Nivolumab
monotherapy and nivolumab plus ipilimumab in
recurrent small cell lung cancer: results from the
CheckMate 032 randomized cohort. J Thorac Oncol.
2020;15(3):426–35.
60 Boumber Y. Tumor mutational burden (TMB) as a
biomarker of response to immunotherapy in small cell
lung cancer. J Thorac Dis. 2018;10(8):4689–93.
61 Hellmann MD, Callahan MK, Awad MM, Calvo E,
Ascierto PA, Atmaca A, et al. Tumor mutational
burden and efficacy of nivolumab monotherapy and in
combination with ipilimumab in small-cell lung cancer.
Cancer Cell. 2018;33(5):853–61.e4.
62 Marabelle A, Fakih M, Lopez J, Shah M, Shapira-
Frommer R, Nakagawa K, et al. Association of
tumour mutational burden with outcomes in patients
with advanced solid tumours treated with
pembrolizumab: prospective biomarker analysis of the
multicohort, open-label, phase 2 KEYNOTE-158
study. Lancet Oncol. 2020;21(10):1353–65.
63 Horn L, Mansfield AS, Szcze
zsna A, Havel L,
Krzakowski M, Hochmair MJ, et al. First-line
atezolizumab plus chemotherapy in extensive-stage
small-cell lung cancer. N Engl J Med. 2018;379
(23):2220–9.
64 Rodriguez A, Lee J, Sutton R, Jiles R, Vansant G,
Wang Y, et al. Abstract 3616: low-pass whole-genome
sequencing of single circulating tumor cells for
detection of tumor mutation burden and chromosomal
instability across multiple cancer types. Cancer Res.
2018;78(13 Suppl):3616.
65 Li R, Wu S, Chang Y, Wang Y, Li B. Whole-exome
sequencing on circulating tumor cells explores
platinum-based chemotherapy resistance in advanced
non-small cell lung cancer. J Clin Oncol. 2021;39(15
Suppl):e21021.
66 Rudin CM, Pietanza MC, Bauer TM, Ready N,
Morgensztern D, Glisson BS, et al. Rovalpituzumab
tesirine, a DLL3-targeted antibody-drug conjugate, in
recurrent small-cell lung cancer: a first-in-human, first-
in-class, open-label, phase 1 study. Lancet Oncol.
2017;18(1):42–51.
67 Morgensztern D, Besse B, Greillier L, Santana-Davila
R, Ready N, Hann CL, et al. Efficacy and safety of
rovalpituzumab tesirine in third-line and beyond
patients with DLL3-expressing, relapsed/refractory
small-cell lung cancer: results from the phase II
TRINITY study. Clin Cancer Res. 2019;25(23):6958–
66.
68 Blackhall F, Jao K, Greillier L, Cho BC, Penkov K,
Reguart N, et al. Efficacy and safety of
rovalpituzumab tesirine compared with topotecan as
second-line therapy in DLL3-high SCLC: results from
the phase 3 TAHOE study. J Thorac Oncol. 2021;16
(9):1547–58.
69 Byers LA, Chiappori A, Smit M-AD. Phase 1 study of
AMG 119, a chimeric antigen receptor (CAR) T cell
therapy targeting DLL3, in patients with
relapsed/refractory small cell lung cancer (SCLC). J
Clin Oncol. 2019;37(15 Suppl):TPS8576.
70 Chou J, Egusa EA, Wang S, Badura ML, Lee F,
Bidkar AP, et al. Immunotherapeutic targeting and
PET imaging of DLL3 in small-cell neuroendocrine
prostate cancer. Cancer Res. 2023;83:301–15.
71 Borghaei H, Paz-Ares L, Johnson M, Champiat S,
Owonikoko T, Lai V, et al. Phase 1 updated
exploration and first expansion data for DLL3-
targeted T-cell engager tarlatamab in small cell lung
cancer. WCLC2022-Conference Abstract, 2022.
72 Johnson ML, Dy GK, Mamdani H, Dowlati A,
Schoenfeld AJ, Pacheco JM, et al. Interim results of
an ongoing phase 1/2a study of HPN328, a tri-specific,
half-life extended, DLL3-targeting, T-cell engager, in
patients with small cell lung cancer and other
neuroendocrine cancers. J Clin Oncol. 2022;40(16
Suppl):8566.
73 Owonikoko TK, Champiat S, Johnson ML, Govindan
R, Izumi H, Lai WVV, et al. Updated results from a
Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. 13
P. Shrestha et al. Circulating tumor cells in small cell lung cancer

phase 1 study of AMG 757, a half-life extended
bispecific T-cell engager (BiTE) immuno-oncology
therapy against delta-like ligand 3 (DLL3), in small
cell lung cancer (SCLC). J Clin Oncol. 2021;39(15
Suppl):8510.
74 Tully KM, Tendler S, Carter LM, Sharma SK,
Samuels ZV, Mandleywala K, et al.
Radioimmunotherapy targeting delta-like ligand 3 in
small cell lung cancer exhibits antitumor efficacy with
low toxicity. Clin Cancer Res. 2022;28(7):1391–401.
75 Wermke M, Felip E, Gambardella V, Kuboki Y,
Morgensztern D, Hamed ZO, et al. Phase I trial of the
DLL3/CD3 bispecific T-cell engager BI 764532 in
DLL3-positive small-cell lung cancer and
neuroendocrine carcinomas. Future Oncol. 2022;18
(24):2639–49.
76 Bray SJ. Notch signalling in context. Nat Rev Mol Cell
Biol. 2016;17(11):722–35.
77 Rojo F, Corassa M, Mavroudis D,
€
Oz AB, Biesma B,
Brcic L, et al. International real-world study of DLL3
expression in patients with small cell lung cancer. Lung
Cancer. 2020;147:237–43.
78 Chen B, Li H, Liu C, Wang S, Zhang F, Zhang L,
et al. Potential prognostic value of delta-like protein 3
in small cell lung cancer: a meta-analysis. World J
Surg Oncol. 2020;18(1):226.
79 Johnson ML, Zvirbule Z, Laktionov K, Helland A,
Cho BC, Gutierrez V, et al. Rovalpituzumab tesirine
as a maintenance therapy after first-line platinum-
based chemotherapy in patients with extensive-stage–
SCLC: results from the phase 3 MERU study. J
Thorac Oncol. 2021;16(9):1570–81.
80 Rath B, Plangger A, Krenbek D, Hochmair M,
Stickler S, Tretter V, et al. Rovalpituzumab tesirine
resistance: analysis of a corresponding small cell lung
cancer and circulating tumor cell line pair. Anticancer
Drugs. 2022;33(3):300–7.
81 Ahn MJ, Cho BC, Felip E, Korantzis I, Ohashi K,
Majem M, et al. Tarlatamab for patients with
previously treated small-cell lung cancer. N Engl J
Med. 2023;389(22):2063–75.
82 Obermayr E, Agreiter C, Schuster E, Fabikan H,
Weinlinger C, Baluchova K, et al. Molecular
characterization of circulating tumor cells enriched by
a microfluidic platform in patients with small-cell lung
cancer. Cells. 2019;8(8):880.
83 Messaritakis I, Nikolaou M, Koinis F, Politaki E,
Koutsopoulos A, Lagoudaki E, et al. Characterization
of DLL3-positive circulating tumor cells (CTCs) in
patients with small cell lung cancer (SCLC) and
evaluation of their clinical relevance during front-line
treatment. Lung Cancer. 2019;135:33–9.
84 Murai J, Tang SW, Leo E, Baechler SA, Redon CE,
Zhang H, et al. SLFN11 blocks stressed replication
forks independently of ATR. Mol Cell. 2018;69(3):371–
84.e6.
85 Zoppoli G, Regairaz M, Leo E, Reinhold WC, Varma
S, Ballestrero A, et al. Putative DNA/RNA helicase
Schlafen-11 (SLFN11) sensitizes cancer cells to DNA-
damaging agents. Proc Natl Acad Sci USA. 2012;109
(37):15030–5.
86 Allison Stewart C, Tong P, Cardnell RJ, Sen T, Li L,
Gay CM, et al. Dynamic variations in epithelial-to-
mesenchymal transition (EMT), ATM, and SLFN11
govern response to PARP inhibitors and cisplatin in
small cell lung cancer. Oncotarget. 2017;8(17):28575–
87.
87 Qu S, Fetsch P, Thomas A, Pommier Y, Schrump DS,
Miettinen MM, et al. Molecular subtypes of primary
SCLC tumors and their associations with
neuroendocrine and therapeutic markers. J Thorac
Oncol. 2022;17(1):141–53.
88 Takashima T, Sakamoto N, Murai J, Taniyama D,
Honma R, Ukai S, et al. Immunohistochemical
analysis of SLFN11 expression uncovers potential non-
responders to DNA-damaging agents overlooked by
tissue RNA-seq. Virchows Arch. 2021;478(3):569–79.
89 Ballestrero A, Bedognetti D, Ferraioli D, Franceschelli
P, Labidi-Galy SI, Leo E, et al. Report on the first
SLFN11 monothematic workshop: from function to
role as a biomarker in cancer. J Transl Med. 2017;15
(1):199.
90 Lok BH, Gardner EE, Schneeberger VE, Ni A,
Desmeules P, Rekhtman N, et al. PARP inhibitor
activity correlates with SLFN11 expression and
demonstrates synergy with temozolomide in small cell
lung cancer. Clin Cancer Res. 2017;23(2):523–35.
91 Zhang B, Stewart CA, Wang Q, Cardnell RJ, Rocha
P, Fujimoto J, et al. Dynamic expression of Schlafen
11 (SLFN11) in circulating tumour cells as a liquid
biomarker in small cell lung cancer. Br J Cancer.
2022;127(3):569–76.
92 Gardner EE, Lok BH, Schneeberger VE, Desmeules P,
Miles LA, Arnold PK, et al. Chemosensitive relapse in
small cell lung cancer proceeds through an EZH2-
SLFN11 axis. Cancer Cell. 2017;31(2):286–99.
93 Yin Y-P, Ma LY, Cao GZ, Hua JH, Lv XT, Lin WC.
FK228 potentiates topotecan activity against small cell
lung cancer cells via induction of SLFN11. Acta
Pharmacol Sin. 2022;43(8):2119–27.
94 Stewart CA, Gay CM, Xi Y, Sivajothi S,
Sivakamasundari V, Fujimoto J, et al. Single-cell
analyses reveal increased intratumoral heterogeneity
after the onset of therapy resistance in small-cell lung
cancer. Nat Cancer. 2020;1:423–36.
95 Zhang B, Ramkumar K, Cardnell RJ, Gay CM,
Stewart CA, Wang WL, et al. A wake-up call for
cancer DNA damage: the role of Schlafen 11
14 Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
Circulating tumor cells in small cell lung cancer P. Shrestha et al.

(SLFN11) across multiple cancers. Br J Cancer.
2021;125(10):1333–40.
96 Mao S, Chaerkady R, Yu W, D’Angelo G, Garcia A,
Chen H, et al. Resistance to pyrrolobenzodiazepine
dimers is associated with SLFN11 downregulation and
can be reversed through inhibition of ATR. Mol
Cancer Ther. 2021;20(3):541–52.
97 Schwendenwein A, Megyesfalvi Z, Barany N, Valko Z,
Bugyik E, Lang C, et al. Molecular profiles of small
cell lung cancer subtypes: therapeutic implications.
Mol Ther Oncolytics. 2021;20:470–83.
98 Rudin CM, Poirier JT, Byers LA, Dive C, Dowlati A,
George J, et al. Molecular subtypes of small cell lung
cancer: a synthesis of human and mouse model data.
Nat Rev Cancer. 2019;19(5):289–97.
99 Baine MK, Hsieh MS, Lai WV, Egger JV, Jungbluth
AA, Daneshbod Y, et al. SCLC subtypes defined by
ASCL1, NEUROD1, POU2F3, and YAP1: a
comprehensive immunohistochemical and
histopathologic characterization. J Thorac Oncol.
2020;15(12):1823–35.
100 Kopparapu PR, Duplessis M, Lo E, Yan Y, Iams
WT, Nordberg J, et al. Abstract 3375:
molecular subtyping of circulating tumor cells in
patients with small cell lung cancer. Cancer Res.
2022;82:3375.
101 Farago AF, Yeap BY, Stanzione M, Hung YP, Heist
RS, Marcoux JP, et al. Combination olaparib and
temozolomide in relapsed small-cell lung cancer.
Cancer Discov. 2019;9(10):1372–87.
102 Stewart CA, Xi Y, Wang R, Ramkumar K, Novegil
VY, Frumovitz M, et al. P2.10-01 transcriptional
diversity of emerging cell populations in refractory
small cell lung cancer biopsies and xenografts. J
Thorac Oncol. 2022;17(9 Suppl):S144–5.
103 Scher HI, Fernandez L, Cunningham K, Elphick N,
Barnett E, Lee J, et al. Schlafen 11 (SLFN11), a
putative predictive biomarker of platinum/PARPi
response, is frequently detected on circulating tumor
cells (CTCs) in advanced prostate cancer. J Clin
Oncol. 2021;39(15 Suppl):e17039.
104 Shoemaker AR, Mitten MJ, Adickes J, Ackler S,
Refici M, Ferguson D, et al. Activity of the Bcl-2
family inhibitor ABT-263 in a panel of small cell lung
cancer xenograft models. Clin Cancer Res. 2008;14
(11):3268–77.
105 Lochmann TL, Floros KV, Naseri M, Powell KM,
Cook W, March RJ, et al. Venetoclax is effective in
small-cell lung cancers with high BCL-2 expression.
Clin Cancer Res. 2018;24(2):360–9.
106 Rudin CM, Hann CL, Garon EB, Ribeiro de Oliveira
M, Bonomi PD, Camidge DR, et al. Phase II study of
single-agent navitoclax (ABT-263) and biomarker
correlates in patients with relapsed small cell lung
cancer. Clin Cancer Res. 2012;18(11):3163–9.
107 Messaritakis I, Nikolaou M, Politaki E, Koinis F,
Lagoudaki E, Koutsopoulos A, et al. Bcl-2 expression
in circulating tumor cells (CTCs) of patients with
small cell lung cancer (SCLC) receiving front-line
treatment. Lung Cancer. 2018;124:270–8.
108 Nakajima W, Sharma K, Hicks MA, Le N, Brown R,
Krystal GW, et al. Combination with vorinostat
overcomes ABT-263 (navitoclax) resistance of small
cell lung cancer. Cancer Biol Ther. 2016;17(1):27–35.
109 Yasuda Y, Ozasa H, Kim YH, Yamazoe M, Ajimizu
H, Yamamoto Funazo T, et al. MCL1 inhibition is
effective against a subset of small-cell lung cancer with
high MCL1 and low BCL-XL expression. Cell Death
Dis. 2020;11(3):177.
110 Inoue-Yamauchi A, Jeng PS, Kim K, Chen HC, Han
S, Ganesan YT, et al. Targeting the differential
addiction to anti-apoptotic BCL-2 family for cancer
therapy. Nat Commun. 2017;8(1):16078.
111 Wang WZ, Shilo K, Amann JM, Shulman A, Hojjat-
Farsangi M, Mellstedt H, et al. Predicting ROR1/BCL2
combination targeted therapy of small cell carcinoma of
the lung. Cell Death Dis. 2021;12(6):577.
112 Fahrmann JF, Katayama H, Irajizad E, Chakraborty
A, Kato T, Mao X, et al. Plasma based protein
signatures associated with small cell lung cancer.
Cancers (Basel). 2021;13(16):3972.
113 Cardnell RJ, Li L, Sen T, Bara R, Tong P, Fujimoto
J, et al. Protein expression of TTF1 and cMYC define
distinct molecular subgroups of small cell lung cancer
with unique vulnerabilities to aurora kinase inhibition,
DLL3 targeting, and other targeted therapies.
Oncotarget. 2017;8(43):73419–32.
114 Gay CM, Tong P, Cardnell RJ, Sen T, Su X, Ma J,
et al. Differential sensitivity analysis for resistant
malignancies (DISARM) identifies common candidate
therapies across platinum-resistant cancers. Clin
Cancer Res. 2019;25(1):346–57.
115 Borromeo MD, Savage TK, Kollipara RK, He M,
Augustyn A, Osborne JK, et al. ASCL1 and
NEUROD1 reveal heterogeneity in pulmonary
neuroendocrine tumors and regulate distinct genetic
programs. Cell Rep. 2016;16(5):1259–72.
116 Hou JM, Krebs MG, Lancashire L, Sloane R, Backen
A, Swain RK, et al. Clinical significance and
molecular characteristics of circulating tumor cells
and circulating tumor microemboli in patients with
small-cell lung cancer. J Clin Oncol. 2012;30(5):525–32.
117 Shi WL, Li J, Du YJ, Zhu WF, Wu Y, Hu YM, et al.
CK-19 mRNA-positive cells in peripheral blood
predict treatment efficacy and survival in small-cell
lung cancer patients. Med Oncol. 2013;30(4):755.
118 Igawa S, Gohda K, Fukui T, Ryuge S, Otani S,
Masago A, et al. Circulating tumor cells as a
prognostic factor in patients with small cell lung
cancer. Oncol Lett. 2014;7(5):1469–73.
Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. 15
P. Shrestha et al. Circulating tumor cells in small cell lung cancer

119 Fu L, Liu F, Fu H, Liu L, Yuan S, Gao Y, et al.
Circulating tumor cells correlate with recurrence in
stage III small-cell lung cancer after systemic
chemoradiotherapy and prophylactic
cranial irradiation. Jpn J Clin Oncol. 2014;44(10):
948–55.
120 Shen J, Zhao J, Jiang T, Li X, Zhao C, Su C, et al.
Predictive and prognostic value of folate receptor-
positive circulating tumor cells in small cell lung
cancer patients treated with first-line chemotherapy.
Oncotarget. 2017;8(30):49044–52.
121 Messaritakis I, Politaki E, Koinis F, Stoltidis D,
Apostolaki S, Plataki M, et al. Dynamic changes of
phenotypically different circulating tumor cells sub-
populations in patients with recurrent/refractory small
cell lung cancer treated with pazopanib. Sci Rep.
2018;8(1):2238.
122 Mondelo-Mac
ıa P, Garc
ıa-Gonz
alez J, Abalo A,
Mosquera-Presedo M, Agu
ın S, Mateos M, et al.
Plasma cell-free DNA and circulating tumor cells as
prognostic biomarkers in small cell lung cancer
patients. Transl Lung Cancer Res. 2022;11(10):1995–
2009.
123 Boffa DJ, Graf RP, Salazar MC, Hoag J, Lu D,
Krupa R, et al. Cellular expression of PD-L1 in the
peripheral blood of lung cancer patients is associated
with worse survival. Cancer Epidemiol Biomarkers
Prev. 2017;26(7):1139–45.
124 Zhang B, Stewart CA, Gay CM, Wang Q, Cardnell R,
Fujimoto J, et al. Abstract 384: detection of DNA
replication blocker SLFN11 in tumor tissue and
circulating tumor cells to predict platinum response in
small cell lung cancer. Cancer Res. 2021;81(13
Suppl):384.
16 Molecular Oncology (2024) ª2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
Circulating tumor cells in small cell lung cancer P. Shrestha et al.