Accepted Manuscript
Early Detection of Recurrence in Patients with Locally Advanced Non-small Cell Lung
Cancer via Circulating Tumor Cell Analysis
Chimbu Chinniah, BA, Louise Aguarin, MS, Phillip Cheng, BA, Cristina Decesaris,
MD, Alicia Cutillo, MD, Abigail T. Berman, MD, Melissa Frick, BA, Abigail Doucette,
MS, Keith A. Cengel, MD, PhD, William Levin, MD, Stephen Hahn, MD, Jay F.
Dorsey, MD, PhD, Charles B. Simone, II, MD, Gary Kao, MD, PhD
PII:

S1525-7304(19)30102-0

DOI:

https://doi.org/10.1016/j.cllc.2019.04.011

Reference:

CLLC 955

To appear in:

Clinical Lung Cancer

Received Date: 17 January 2019
Revised Date:

29 March 2019

Accepted Date: 19 April 2019

Please cite this article as: Chinniah C, Aguarin L, Cheng P, Decesaris C, Cutillo A, Berman AT, Frick
M, Doucette A, Cengel KA, Levin W, Hahn S, Dorsey JF, Simone II CB, Kao G, Early Detection of
Recurrence in Patients with Locally Advanced Non-small Cell Lung Cancer via Circulating Tumor Cell
Analysis, Clinical Lung Cancer (2019), doi: https://doi.org/10.1016/j.cllc.2019.04.011.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and alllegal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

RIPT

Early Detection of Recurrence in Patients with Locally Advanced Non-small Cell
Lung Cancer via Circulating Tumor Cell Analysis

SC

Chimbu Chinniah1, BA; Louise Aguarin1, MS; Phillip Cheng1, BA; Cristina Decesaris1,
MD; Alicia Cutillo1, MD; Abigail T. Berman1, MD; Melissa Frick1, BA; Abigail Doucette1,
MS; Keith A Cengel1, MD, PhD; William Levin1, MD; Stephen Hahn2, MD; Jay F.
Dorsey1, MD, PhD; Charles B. Simone, II3, MD; Gary Kao1, MD, PhD,

TE
D

M
AN
U

1= Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA
2= MD Anderson Cancer Center, Houston, TX
3= New York Proton Center, New York, NY

Corresponding Author: Gary Kao, MD, PhD, Gary.Kao@uphs.upenn.edu, 215-5732285, Perelman Center for Advanced Medicine, 3400 Civic Center Boulevard,
Philadelphia, PA, 19108

AC
C

EP

Co-Corresponding Author: Charles B. Simone, II, MD, csimone@nyproton.com ,New York Proton Center, 225 East 126th Street, New York, NY 10035

pg. 1

ACCEPTED MANUSCRIPT

MicroAbstract
We investigated the potential usefulness of sequential circulating tumor cell (CTC)
analysis for patients treated for locally advanced non-small cell lung cancer (LA-RI
PT

NSCLC). We found CTC rise gave median and mean lead-time notice of progression of
disease of about 6 months ahead of radiographic evidence. This telomerase-based
CTC assay may thus complement conventional imaging for post-treatment monitoring ofSC

patients with LA-NSCLC.

M
ANU

ABSTRACT
Background:

Assays identifying circulating tumor cells (CTCs) may allow for noninvasive andsequential monitoring of lung cancer. We investigated whether serial CTC analysis
could complement conventional imaging for detecting recurrences after treatment in

Methods:TED

patients with locally advanced non-small cell lung cancer (LA-NSCLC).

EP

Patients with LA-NSCLC (stage II-III) who definitively received concurrent
chemoradiation were prospectively enrolled, with CTCs from peripheral blood samples

ACC

identified using an adenoviral probe that detects elevated telomerase activity present innearly all lung cancer cells. A "detectable" CTC level was defined as 1.3 GFP-positive
cells/mL of collected blood. Samples were obtained before, during (weeks 2,4,6), andafter treatment (post-radiation therapy (RT); months 1,3,6,12,18,24).
Results:
48 patients were enrolled. At a median follow-up of 10.9 months, 22 (46%) patientsrecurred at a median time of 7.6 months post-RT (range 1.3-32.0). Of the 20 of 22

pg. 2

ACCEPTED MANUSCRIPT

patients for whom post-RT samples were obtained, 15 (75%) had a rise in CTC counts
post-RT. In 10 of these 15 patients, CTCs were undetectable on initial post-RT draw but
were then detected again prior to radiographic detection of recurrence, with a median

RI
PT

lead time of 6.2 months and mean lead time of 6.1 months (range 0.1-12.0) between
CTC rise and radiographic evidence of recurrence. One patient with an early recurrence(4.7 months) had persistently elevated detectable CTC levels during and afterSC

treatment.
Conclusions:

M
AN
U

These results indicate that longitudinal CTC monitoring in patients with LA-NSCLC
treated with chemoradiation is feasible, and that detectable CTC levels in many patientsTED

meaningfully precede radiologic evidence of disease recurrence.

AC
C

EP

Keywords: radiation oncology, CTC, LA-NSCLC, prospective trial

pg. 3

ACCEPTED MANUSCRIPTINTRODUCTION
Lung cancer remains the second most commonly diagnosed cancer in both men
and women, with an estimated 228,150 new cases of lung cancer expected in 2019,

RI
PT

and accounts for about 13% of all cancer diagnoses. It is responsible for more deaths
than any other type of cancer in both men and women and is the leading cause ofcancer death in men and women above 40 years of age.1 Non-small cell lung cancer

SC(NSCLC) accounts for over 85% of these cases and the 5 year survival rate for locallyadvanced NSCLC (LA-NSCLC) ranges falls precipitously from 53% for stage IIB

M
ANU

disease, to 36% for stage IIIA disease, to 26% for stage IIIB disease.2,3
New developments in radiation therapy (RT), imaging techniques, chemotherapy,
surgery, and biological agents offer renewed hope for prolonging survival and improving
outcomes. Optimizing monitoring of disease status to guide treatment throughout eachTED

patient’s clinical course and after treatment completion, including with novel biomarkers,
could allow for improved efficacy of the combined modality therapies and improvedEPoutcomes for salvage therapies.4–6

Currently, the standard of care for patients having completed chemoradiation

ACC

treatment for LA-NSCLC usually includes serial history and physical examinations,along with follow-up imaging, such as positron emission tomography (PET) or chest
computed tomography (CT) scans. However, post-radiation changes and fibrosis often
render detection of recurrent disease difficult to identify on imaging. In such situations
where conventional imaging is unclear, circulating tumor cell (CTCs) assays could beilluminating. There is a relative paucity of information regarding CTC trends in patientswith localized, non-metastatic lung cancer, including monitoring for treatment response

pg. 4

ACCEPTED MANUSCRIPT

or assessing for tumor recurrence following definitive chemoradiation. We therefore
performed a prospective clinical trial with a primary endpoint to assess CTCs as apotential biomarker for patients with LA-NSCLC definitively treated with chemoradiation.

RIPT

A secondary goal was to assess whether CTC trends may precede conventional

AC
C

EP

TE
D

M
AN
U

SC

imaging in detecting recurrences.

pg. 5

ACCEPTED MANUSCRIPT

MATERIALS AND METHODSPatient Eligibility
All consecutive patients with LA-NSCLC (group stage II-III, as determined by 7th

RIPT

edition of the AJCC staging system) prospectively enrolled in this IRB-approved clinical
trial were included in the analysis. Enrollment criteria included patients age 18 years orolder who were planned to receive concurrent chemoradiation. Patients with a priorSC

active malignancy in the last 5 years were ineligible. All patients involved werediscussed in Thoracic Malignancy Multi-disciplinary Clinic and evaluated for surgery, but

MANU

were deemed unresectable by consensus typically due to tumor location, extent, or
excessive predicted peri-operative risks, generally due to pre-existing cardiopulmonary
comorbidities and/or limited performance status.
Circulating Tumor Cell AssayTED

The CTC assay utilized in this study has been previously described for patientswith NSCLC, glioma, melanoma, bladder and other types of solid tumors.7–10 It employs
a replication-competent adenovirus that detects elevated telomerase expressionEPmanifested by almost all tumor cells (but which is not elevated in the majority of normalACC

cells). The replication of the vector is regulated by the human telomerase reverse
transcriptase (hTERT) promoter element, ultimately driving GFP expression that can beimaged and quantified by fluorescence microscopy. The pre-clinical validation process
utilized testing of the probe in peripheral blood samples from healthy volunteers withand without spiking them with NSCLC cells.7 A logistic regression-based classifier wasapplied to these data to define the threshold for “CTC positivity” at 1.3 GFP-expressing
cells per mL of collected blood. Key aspects of the validation process and assay involvepg. 6

ACCEPTED MANUSCRIPT

depletion of erythrocytes, lymphocytes, granulocytes and mononuclear cells via
Oncoquick gradient centrifugation8 gating via cell size to further exclude the smaller
normal cells (such as monocytes),9 and utilizing a low titer of virus. These steps are(Supplemental Figure and data not shown).
Collection of Blood Samples from Patients

RI
PT

essential to minimize false positives and are far superior to simple RBC lysis.

SC

Peripheral blood samples (1 tube, typically between 7-10mL) were obtained fromM
ANU

patients to assess for CTCs using the above-mentioned assay. Patients were assayed
before chemoradiation (pre-RT), at 3 time points during concurrent chemoradiation(weeks 2, 4, and 6), and after chemoradiation completion (post-RT) (months 1, 3, 6, 12,18, and 24). All care providers and laboratory personnel were blinded to quantitative

results of these assays.

TED

and qualitative CTC analysis, and no treatment decisions were made based on theTreatment and Follow-Up

EP

Patients were treated to 6660 cGy in 37 fractions of external beam radiation
therapy (180 cGy/fraction). In all cases, the radiation therapy was delivered to theACCprimary tumor and to sites of nodal metastatic disease based on size criteria, PET
avidity, or pathologic confirmation of nodal metastasis. In all cases, patients received
concurrent chemotherapy that was a platinum based doublet, either cisplatin andetoposide or carboplatin and paclitaxel. Patients then underwent serial radiographic
surveillance with either PET/CT or CT scans obtained just prior to each of their follow up
appointments (every 3 months in years 1-2, every 4-6 months thereafter), or withpg. 7

ACCEPTED MANUSCRIPT

additional imaging at the discretion of the patient’s physicians, all of whom are blinded
to the results of the CTC analyses. The time to local failure was defined as at least a
20% increase in the diameter of the treated lesion or histologic confirmation of disease

RI
PT

and was marked from the date of histologic confirmation or empiric treatment ofdisease, while local control was defined as absence of local failure. Nodal and distantcontrol were defined as absence of disease in the lymph nodes or absence ofSC

extrathoracic metastases, respectively. Date of death was as recorded in the deathMANU

certificate or as noted in the electronic medical records of each patient.

Statistical Analysis

Overall survival was defined as the duration from the start of radiation treatment
to the date of death or last follow-up. Progression free survival was measured from theTEDstart of radiation treatment to the date of any disease event or last follow-up. Survival
curves were calculated via Kaplan-Meier methods. Statistical analysis was performed
using SAS/STAT software, version 9.4 of the SAS System (SAS Institute Inc., Cary, NC,

AC
C

EP

USA).

pg. 8

ACCEPTED MANUSCRIPTRESULTS
Patient Characteristics
Forty-eight patients were sequentially enrolled. Patient composition included the

RIPTfollowing: male (54%), Caucasian (69%) or African American (21%), and former (77%)
or current (21%) smokers. The median age of the patients was 66 years old (range, 3184). Adenocarcinoma (46%) or squamous cell carcinoma (48%) comprised most of theSChistologies of the tumors. The primary tumor median size was 3.7 cm (range, 1.5-

10.0). All patients were established to have histological confirmation of NSCLC andM
ANUclinically have LA-NSCLC, defined as clinical group stage II or III. Patients
predominantly were comprised of stage IIIA (54%) or IIIB (33%) disease, and they had
cN2 (60%) or cN3 (23%) nodal disease (Table 1). The median overall survival for thispatient population was 28.4 months (Fig. 1a), a duration comparable to the best arm

TEDsurvival rate seen in the RTOG-0617 clinical trial.11 Median progression free survival
was 7.1 months, within the range of 6-12 months often described for LA-NSCLC
patients (Fig. 1b).11–14 The number of patients at each stage and the correspondingEPblood draws are delineated in the Consort Diagram in the Supplemental Figure section.
CTC analysis provides lead-time notice of clinical recurrence of disease

ACC

Thirty-five of the 48 patients (73%) had detectable levels of CTCs in theirpretreatment samples, and all of these patients noted decreases in CTC counts after
completion of treatment. At a median follow-up of 10.9 months, 22 of 48 patients (46%)
had disease recurrence at a median time of 7.6 months post-RT (range 1.3-32.0). There
was a near even distribution between adenocarcinoma and squamous histology inthese patients and no obvious difference in outcomes between the two histologies was

pg. 9ACCEPTED MANUSCRIPT

observed. Post-RT blood samples for CTC analysis were obtained in 20 of 22 recurrent
patients (two patients were not able to return for CTC analysis). Of these 20 patients, 15(75%) had a detectable rise in CTC counts post-RT. In 10 of these 15 patients (67%),

RIPT

CTC counts were undetectable on initial post-RT draw and rose to detectable levels
prior to radiographic detection of recurrence, with a median lead-time of 6.2 months andmean lead-time of 6.1 months (range 0.1-12.0) between the CTC rise and radiographicSCevidence of recurrence. In these 10 patients, an average of 182 days (~6 months)

elapsed between when CTC increases were detected and when disease recurrenceMANU

was clinically noted. Specific details about the ten patients in whom CTC rises
preceded disease recurrence are included in Table 2.

Of the five patients in whom the number of CTCs increased into the detectable
range after radiographic detection of recurrence, one patient with an early recurrenceTED(4.7 months) had persistently detectable CTC levels during and after treatment. The
CTC counts in the four remaining patients decreased to a nadir, but then had
radiographic evidence of disease progression or recurrence prior to the rise in CTCs. It

EPshould be noted, however, that several of these patients missed clinic appointments or
had follow-up care conducted at outside facilities and so, were unable to undergo blood

ACCdraw and CTC analysis at protocol-specified intervals. While these missed serial CTC
assays were not classified as protocol violations, it is plausible that at least some ofthese would have also resulted in lead-time notice preceding radiographic evidence ofdisease progression or recurrence.
We also assessed whether CTC status prior to the start of treatment may havebearing on recurrence. As mentioned previously, 13 patients did not have detectablepg. 10ACCEPTED MANUSCRIPT

CTCs prior to treatment; of these 5 (or 38%) have been found to have radiographic
evidence of recurrence. In contrast, 17 of the 35 (or 49%) patients with CTCs detectableprior to treatment have recurred. The difference did not appear to be statistically

RI
PT

significant. Furthermore, as CTCs tend to be rare, additional pre-treatment blood draws
would likely increase the probability of detection. Other potential reasons for the lack of
CTC detectability are addressed in the discussion.

SC

Illustrative Clinical Vignette

A 70-year-old male presented with stage IIIB squamous cell lung cancer

M
ANU(T3N2M0). On pretreatment PET/CT, the patient was noted to have a 1.5cm mass with
SUV 13.9 in the left lung, with FDG avidity in the prevascular and left hilar nodes, anddetectable CTCs pre-treatment. At the end of chemoradiation, CTCs were no longer
detectable, and they continued to be undetectable during the first two post-treatment

TE
D

CTC evaluations, with a post-treatment CTC nadir of 0. At the patient’s 6-month posttreatment follow up visit though, CTCs were again detectable (1.4 CTCs/mL). However,
the official interpretation of the CT scan obtained for this visit was: “Evolution ofEPradiation changes and increase in prominence of mediastinal lymphoid tissue may bereactive.” Seven months later, the patient was unambiguously deemed to haveAC
C

recurrent disease with CT scans now demonstrating widespread progression in the left
lung and lymphadenopathy. One month later, the patient developed brain metastases
and passed away two months after that. In this patient’s case, 236 days passed
between when a positive CTC count was first noted and when the patient was clinicallydeemed based on imaging to have recurred. A graphical representation of this case,with imaging scans, is shown in Fig. 2.

pg. 11ACCEPTED MANUSCRIPTDISCUSSION
This pilot trial is, to our knowledge, the largest prospective study assessing CTCs
in patients with LA-NSCLC undergoing chemoradiation and the first compelling report

RI
PT

indicating that CTC monitoring in such patients is feasible and clinically useful. CTC
elevations into the detectable range in many patients meaningfully precede radiologic
evidence of disease recurrence. The six-month lead-time notice of recurrence affordedSC

by CTC analyses in this study (and in a population of patients with median progressionfree survival of only about seven months) potentially may allow the initiation of re-MANUtreatment or early salvage strategies at a time when tumor burden remains lower15.
The earliest possible detection of recurrences aided by CTC assays would allow
the greatest number of patients to be eligible for effective salvage treatment. For
example, an increasing body of prospective data indicate that reirradiation for local andTEDregional non-small cell lung cancer recurrences allows for long-term survival, aided byrecent advances in radiation therapy techniques and technology.16 Such treatment is,however, contingent on smaller treatment volumes to minimize the risk of complications.

EP

Prompt institution of effective salvage therapy may, therefore, lead to better outcomes
and better preservation of quality of life.14 The development and introduction ofACC

immunotherapy and other novel effective treatments such as immune checkpoint
inhibitors offer further opportunities for extending survival with early detection ofrecurrences. At the time of analysis, no subject in this trial had received immunotherapy.
The demonstrated efficacy of durvalumab, a PD-L1 inhibitor, in the recently reported
PACIFIC trial suggests the kind of additional salvage treatment options that may berapidly implemented with early detection of recurrence afforded by CTC analyses.17

pg. 12

ACCEPTED MANUSCRIPT

A number of factors may have accounted for the lack of detectable CTCs prior totreatment in some of the patients. The concentration of CTCs is likely to be very low
with estimates of 1 CTC per every 1 billion nucleated cells and thus a degree ofRIPTsampling error may be possible, especially when the volume of blood collected is low.18
In support of this possibility, wide variations in the rate of CTC release have been

observed with real-time continuous bloodstream monitoring in preclinical studies.19 DueSC

to discontinuous shedding of CTCs from the primary tumor, it may be ideal in futurestudies to include additional pretreatment draws for each patient in order to achieve

M
ANU

maximal sensitivity.20 We speculate that there may also be alterations in physiology or
biology (e.g. poorly perfused tumors, such that tumor cells have difficulty migrating into
the systemic circulation), and such factors may also account for why rises in CTC
counts were not observed in a subset (5 of 20 [25%]) of patients with clear evidence ofTED

disease recurrence.

It should also be noted that there were often logistical limitations in the time
points utilized to collect follow-up CTC samples. The protocol was designed for serialEP

CTC assays during a year after completion of treatment (at 1, 3, 6, 12 months).

AC
C

However, after completion of treatment at our quaternary care center, many patients
elect to have most or all of the follow-up care performed closer to home due to expense,
convenience, and other logistical factors. Consequently, for three of the four patients forwhom a rise in CTC counts was detected after conventional imaging, CTC collection
time points were missed due to such logistical reasons such as follow-up visits ordiagnostic imaging scheduled outside the home institution during which sample
collection for CTC analysis was not possible. In future studies, fewer time points may be

pg. 13

ACCEPTED MANUSCRIPT

missed if CTC collection could be performed in community settings. This would require
methods of shipping and handling that allow for the stable survival of cells, such as
through temperature-controlled shipping containers (different versions of which are

RIPT

commercially available right now). We are actively exploring such options.

Although these CTC assay results are promising, other circulating tumor

materials are also being investigated for diagnostic or prognostic monitoring of cancer,SCsuch as cfDNA/ctDNA (cell free DNA/circulating tumor DNA) and exosomes.21–24 FutureMANU

studies may indicate strategies through which CTC assays may complement assays ofthese other circulating tumor material. Furthermore, the standard of care for thesepatients now would be to receive one year of consolidation therapy with the PDL1
checkpoint inhibitor, durvalumab, which could impact the time to first rise in CTCs or thelead time in detecting recurrences, and further investigation in immunotherapy-treatedTED

patients is indicated. Ultimately, further analytical validation and testing are warranted
for any of these assays to guide treatment and management decisions to reduce lungcancer-associated morbidity or mortality. Based on these compelling findings, an NRG

EP

Oncology NCI-funded cooperative group study “NRG-LU004: Phase I Trial ofAC
C

Accelerated or Conventionally Fractionated Radiotherapy Combined With MEDI4736(durvalumab) in PD-L1 High Locally Advanced Non-Small Cell Lung Cancer (NSCLC)(ARCHON-1)” has just been activated that will incorporate CTC analyses in all patients
which aims to provide multi-center validation of our findings and test the assay inimmunotherapy-treated patients.

pg. 14

ACCEPTED MANUSCRIPT

Clinical Practice Points•

Analyses of circulating tumor material, including circulating tumor cells (CTCs),

RI
PT

have attracted much recent interest. These assays are based on peripheral blood
samples and are thus noninvasive and can be performed sequentially throughout
a patient’s treatment course. The clinical usefulness of CTC analysis for locally•SCadvanced non-small cell lung cancer (LA-NSCLC), however, is not yet defined.
We investigated a CTC assay that detects the elevated telomerase activity inMANU

almost all cancer cells and which helps confer lack of senescence andimmortality. In contrast, telomerase activity is not elevated in almost all normal
cells so normal cells undergo senescence.
•

Consecutive patients treated with chemoradiation (standard fractionation andfields with concurrent platinum-based chemotherapy) for stage II-III LA-NSCLC

TE
D

were prospectively assayed before, during, and at months 1, 3, 6, 12, 18, and 24
after radiation therapy completion (post-RT). Patients underwent serialEPradiographic imaging with either PET/CT or CT scans before treatment and ateach of their follow-up appointments.
22 of 48 (or 46%) patients recurred at a median time of 7.6 months post-RT. 15

AC
C

•

of 20 (75%) patients for whom post-RT samples were obtained had a rise in CTC
counts. In 10 of these 15 patients, CTCs were undetectable on initial post-RT
draw but were then detected again prior to radiographic detection of recurrence,with a median lead time of 6.2 months and mean lead time of 6.1 months
between CTC rise and radiographic evidence of progression.

pg. 15

ACCEPTED MANUSCRIPT•

CTC assay may enable implementation of salvage treatment at the earliest time

AC
C

EP

TE
D

M
AN
U

SC

RI
PT

of tumor recurrence or progression.

pg. 16

ACCEPTED MANUSCRIPTDISCLOSURE OF POTENTIAL CONFLICTS OF INTERESTThe University of Pennsylvania has filed patent applications based on associatedresearch and is ultimately the license owner. Drs. Hahn, Dorsey, and Kao are

RIPT

cofounders of Liquid Biotech USA through the University of Pennsylvania UPSTART
Program.
ACKNOWLEDGMENTSSC

We acknowledge Yasunari Kashihara and Yasuo Urata of Oncolys Biopharma,
Inc. for graciously supplying the adenoviral vector utilized in this research. We areMANUgrateful to Kaysee Baker and Susan Mazzoni of the University of Pennsylvania
Department of Radiation Oncology and the other clinical research coordinators, foressential assistance with the clinical protocol on which the patients reported here wereenrolled.

TE
D

GRANT SUPPORTThis work was supported by the following grants from National Institutes of
Health: grant R01 CA201071 from the National Cancer Institute and grant K08

EP

NS076548-01 from the National Institute of Neurological Disorders and Stroke. J.F.D.
was also supported by a Burroughs Welcome Career Award for Medical Scientists

ACC(1006792).

AUTHOR INFORMATION
G.D. Kao and C B. Simone are co-corresponding authors (the latter is presently

the Chief Medical Officer of the New York Proton Center, while GD Kao continues tolead the work at the University of Pennsylvania (“Penn”)). C. Chinniah contributed topg. 17

ACCEPTED MANUSCRIPT

this work as a clinical research fellow at Penn and is now a medical student at Albany

AC
C

EP

TE
D

M
AN
U

SC

RI
PT

Medical College.

pg. 18

ACCEPTED MANUSCRIPTREFERENCES
1. Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2019. CA. Cancer J. Clin. 69,
7–34 (2019).

RI
PT

2. Traynor, A. M. & Schiller, J. H. Systemic treatment of advanced non-small cell lungcancer. Drugs Today 40, 697 (2004).

3. Non-Small Cell Lung Cancer Survival Rates, by Stage. Available at:SC

https://www.cancer.org/cancer/non-small-cell-lung-cancer/detection-diagnosisstaging/survival-rates.html. (Accessed: 16th January 2019)

M
ANU

4. Vachani, A., Sequist, L. V. & Spira, A. AJRCCM: 100-Year Anniversary. The ShiftingLandscape for Lung Cancer: Past, Present, and Future. Am. J. Respir. Crit. CareMed. 195, 1150–1160 (2017).

5. Lim, C. et al. Improving molecular testing and personalized medicine in non-small-cellTEDlung cancer in Ontario. Curr. Oncol. 24, 103–110 (2017).

6. Kong, F.-M. (Spring), Hirsch, F. R. & Machtay, M. Potential future consideration for
imaging and blood-based biomarkers for precision medicine in lung cancer. Transl.

EP

Lung Cancer Res. 6, 713–715 (2017).

7. Dorsey, J. F. et al. Tracking Viable Circulating Tumor Cells (CTCs) in the Peripheral

AC
C

Blood of Non-Small Cell Lung Cancer Patients Undergoing Definitive Radiation
Therapy: Pilot Study Results. Cancer 121, 139–149 (2015).
8. Clawson, G. A. et al. Circulating tumor cells in melanoma patients. PloS One 7,
e41052 (2012).

pg. 19

ACCEPTED MANUSCRIPT

9. Yabusaki, M. et al. Detection and preliminary evaluation of circulating tumor cells in
the peripheral blood of patients with eight types of cancer using a telomerase-specificadenovirus. Oncol. Rep. 32, 1772–1778 (2014).

RI
PT

10. Xu, M. J. et al. Circulating Tumor Cells, DNA, and mRNA: Potential for Clinical Utility
in Patients With Melanoma. The Oncologist 21, 84–94 (2016).

11. Bradley, J. D. et al. Standard-dose versus high-dose conformal radiotherapy withSC

concurrent and consolidation carboplatin plus paclitaxel with or without cetuximab forpatients with stage IIIA or IIIB non-small-cell lung cancer (RTOG 0617): aMANUrandomised, two-by-two factorial phase 3 study. Lancet Oncol. 16, 187–199 (2015).
12. Blumenthal, G. M. et al. Overall Response Rate, Progression-Free Survival, andOverall Survival With Targeted and Standard Therapies in Advanced Non–Small-Cell
Lung Cancer: US Food and Drug Administration Trial-Level and Patient-Level

TE
D

Analyses. J. Clin. Oncol. 33, 1008–1014 (2015).

13. Provencio, M., Isla, D., Sánchez, A. & Cantos, B. Inoperable stage III non-small celllung cancer: Current treatment and role of vinorelbine. J. Thorac. Dis. 3, 197-204–EP

204 (2011).

14. Chao, H.-H. et al. Multi-Institutional Prospective Study of Reirradiation with Proton

AC
C

Beam Radiotherapy for Locoregionally Recurrent Non-Small Cell Lung Cancer. J.
Thorac. Oncol. Off. Publ. Int. Assoc. Study Lung Cancer 12, 281–292 (2017).
15. Lee, P. et al. Metabolic tumor burden predicts for disease progression and death inlung cancer. Int. J. Radiat. Oncol. Biol. Phys. 69, 328–333 (2007).
16. Vyfhuis, M. A. L. et al. Reirradiation for locoregionally recurrent non-small cell lungcancer. J. Thorac. Dis. 10, S2522–S2536 (2018).

pg. 20ACCEPTED MANUSCRIPT

17. Antonia, S. J. et al. Durvalumab after Chemoradiotherapy in Stage III Non–SmallCell Lung Cancer. N. Engl. J. Med. 377, 1919–1929 (2017).
18. West, H. (Jack) & Jin, J. O. Circulating Tumor Cells. JAMA Oncol. 1, 394–394

RI
PT(2015).

19. Juratli, M. A. et al. Dynamic Fluctuation of Circulating Tumor Cells during Cancer
Progression. Cancers 6, 128–142 (2014).

SC

20. Mocellin, S., Keilholz, U., Rossi, C. R. & Nitti, D. Circulating tumor cells: the‘leukemic phase’ of solid cancers. Trends Mol. Med. 12, 130–139 (2006).

M
AN
U

21. Calabuig-Fariñas, S., Jantus-Lewintre, E., Herreros-Pomares, A. & Camps, C.
Circulating tumor cells versus circulating tumor DNA in lung cancer—which one will
win? Transl. Lung Cancer Res. 5, 466-482–482 (2016).

22. Gold, B., Cankovic, M., Furtado, L. V., Meier, F. & Gocke, C. D. Do circulating tumorTED

cells, exosomes, and circulating tumor nucleic acids have clinical utility? A report of
the association for molecular pathology. J. Mol. Diagn. JMD 17, 209–224 (2015).

EP

23. Sandfeld-Paulsen, B. et al. Exosomal proteins as prognostic biomarkers in non‐small cell lung cancer. Mol. Oncol. 10, 1595–1602 (2016).

ACC

24. Ilie, M. et al. Current challenges for detection of circulating tumor cells and cell-freecirculating nucleic acids, and their characterization in non-small cell lung carcinoma
patients. What is the best blood substrate for personalized medicine? Ann. Transl.
Med. 2, 107 (2014).

pg. 21ACCEPTED MANUSCRIPTFIGURES AND TABLES

Tumor Size(cm)
Tumor SUVmax
Histology (%)
Stage (%)

T-Stage (%)

ACC

N-Stage (%)

pg. 22SC

Smoking
History (%)

MANUAge (yrs)

TE
D

Ethnicity (%)

Male: 26 (54)Female: 22 (46)White: 33 (69)
African American: 10 (21)Other: 5 (10)Median: 66
Mean: 66
Range: 31-84
Former: 37 (77)Current: 10 (21)Never: 1 (2)Median: 3.7
Mean: 3.98
Range: 1.5-10
Median: 13
Mean: 13.9
Range: 1.6-30
Adeno: 22 (46)Squamous: 23 (48)
Other: 3 (6)IIA: 1(2)IIB: 5 (11)
IIIA: 26 (54)
IIIB: 16 (33)
T1: 13 (27)
T2: 14 (29)
T3: 14 (29)
T4: 7 (15)N0: 6(13)N1: 2 (4)N2: 29 (60)
N3: 11 (23)EP

Sex (%)RI
PT

Table 1: Patient Characteristics

ACCEPTED MANUSCRIPT

AC
C

EP

TE
D

M
AN
U

SC

RI
PT

Table 2: CTCs Predating Recurrence

pg. 23

ACCEPTED MANUSCRIPT

Figure 1A and B: Overall and Progression Free Survival

M
AN
U

SC

RI
PT

A

AC
C

EP

B

TE
D

Median OS = 28.4 mo

pg. 24

Median PFS = 7.1 moACCEPTED MANUSCRIPT

AC
C

EP

TE
D

M
AN
U

SC

RI
PT

Figure 2: Sample Casepg. 25

AC
C

EP

TE
D

M
AN
U

SC

RI
PT

ACCEPTED MANUSCRIPT

pg. 26

AC
C

EP

TE
D

M
AN
U

SC

RI
PT

ACCEPTED MANUSCRIPT

pg. 27

ACCEPTED MANUSCRIPT

Figure and Table Legends
Table 1: Characteristics of the 48 patients that were sequentially enrolled

RI
PT

Table 2: Details of the ten patients in whom CTC rises preceded radiographic
evidence of disease recurrence.
Figure 1A and 1B: Overall and Progression Free Survival. The median overall
survival for this patient population was 28.4 months (Fig. 1A), while the medianprogression free survival was 7.1 months (Fig. 1B)

M
AN
U

SC

Figure 2: Temporal graphic illustrating the disease course of a representative patient(clinical vignette), with serial CTC assays and CT imaging scans showing the status of
the tumor. Periods when the CTC counts were elevated (“CTCs + “, in red) or not(“CTCs – “, in blue) are as indicated, along with when disease was detectedradiographically (red arrows). CTCs became detectable again on 6/14/2015, preceding
CT scans showing disease recurrence in 1/25/2016

Supplemental Figure: Testing of TelomeScan virus with blood from healthy
volunteers to establish ideal assay conditions associated with minimal “false positive”
background

AC
C

EP

TE
D

Consort diagram: Systematic delineation of the patients sequentially enrolled in the
study and the number active as the study progressedpg. 28

ACCEPTED MANUSCRIPT

Smoking
History (%)
Tumor Size(cm)
Tumor SUV
max

Histology (%)
Stage (%)

EP

T-Stage (%)

ACC

N-Stage (%)

SC

Age (yrs)

M
ANU

Ethnicity (%)

Male: 26 (54)Female: 22 (46)White: 33 (69)
African American: 10 (21)Other: 5 (10)Median: 66
Mean: 66
Range: 31-84
Former: 37 (77)Current: 10 (21)Never: 1 (2)Median: 3.7
Mean: 3.98
Range: 1.5-10
Median: 13
Mean: 13.9
Range: 1.6-30
Adeno: 22 (46)Squamous: 23 (48)
Other: 3 (6)IIA: 1(2)IIB: 5 (11)
IIIA: 26 (54)
IIIB: 16 (33)
T1: 13 (27)
T2: 14 (29)
T3: 14 (29)
T4: 7 (15)N0: 6(13)N1: 2 (4)N2: 29 (60)
N3: 11 (23)TE
D

Sex (%)RI
PT

Table 1: Patient Characteristics

ACCEPTED MANUSCRIPT

PostDate
Date RT RT
CTC
End
CTC
Rise
Nadir

CTC
Days
count Date ofelapsed: Vital
at
Recurrence rise tostatusrise
recur

6/17/13 0.3

1/19/142.5

1/19/143

12/3/13 0

1/13/15

1.5

1/20/157

RIPT

3/30/15 0.7

6/25/15

1.7

11/1/15

129

Alive

Adeno

9/16/15 0.1

12/2/15

1.5

5/18/16

168

Alive

Adeno

8/27/15 0.1

12/17/15 2

6/6/16

172

Alive

Adeno

6/22/15 0.7

1/12/16

2.3

8/1/16

202

Alive

Adeno

12/26/13 0.4

1/4/1636.5

8/24/16

233

Alive

Squamous

12/29/14 0

6/24/15

1/2/150

6/24/15

12/26/14 0

6/25/15TE
D

Table 2: CTCs Predating Recurrence

Deceased Squamous

M
AN
U

SC

Deceased Squamous

1.4

2/15/16236

Deceased Squamous

5.9

4/29/16

310

Alive

1.7

6/23/16364

Deceased Squamous

EP
AC
C

Histology

Adeno