Journal Thoracic Oncology

Abstracts Journal of Thoracic Oncology • Volume 12 Issue S1 January 2017
by flow cytometry. Colony formation assay was performed to determine
the radiosensitivity. Sphere formation assay in serum-free medium was
performed to evaluate the self-renewal capacity. γH2AX foci were analyzed
by immunofluorescence. The monoclonal antibody of CACNA2D1 was applied
alone or combined with radiation to CACNA2D1+ cells and colony formation
and sphere formation assays were performed to determine its effect on
CACNA2D1+ cells. Results: CACNA2D1+ cells had higher sphere-forming
efficiency, and were resistant to radiation compared with CACNA2D1- cells.
In unsorted cells the CACNA2D1+ percentage was enhanced after radiation.
These data suggest CACNA2D1+ NSCLC cells are relatively radio-resistant.
Knockdown of CACNA2D1 in CACNA2D1-high A549 enhanced radiosensitivity,
while overexpression of CACNA2D1 in CACNA2D1-low PC9 and H1975 reduced
radiosensitivity, suggesting CACNA2D1 converts the radio-resistance. The
number of γH2AX foci increased after radiation, and decreased more rapidly in
CACNA2D1-overexpression cells than control group. Moreover, in CACNA2D1-
overexpression cells, the baseline phosphorylation level of CHK2 and ATM was
higher than control group, and were more activated after radiation, suggesting
CACNA2D1 overexpression resulted in an increase in the DNA damage repair
capacity. The monoclonal antibody of CACNA2D1 had a synergetic effect
with radiation to block self-renewal of CACNA2D1+ A549 cells. The antibody
also enhanced radiosensitivity in CACNA2D1+ cells in colony formation assay.
Conclusion: CACNA2D1 marks radio-resistant cancer stem-like cells in NSCLC.
CACNA2D1 converts resistance to radiation, partially by enhancing the DNA
damage repair efficiency. The monoclonal antibody of CANCA2D1 enhances the
radio-sensitivity of CACNA2D1+ cells, suggesting its potential to improve the
treatment outcome when combined with radiation on NSCLC.
Keywords: CACNA2D1, NSCLC, Cancer stem cell, radio-resistance
POSTER SESSION 2 – P2.05: RADIOTHERAPY
BIOLOGY –
TUESDAY, DECEMBER 6, 2016
P2.05-003 PIK3CA MUTATION IS ASSOCIATED WITH INCREASED
LOCAL FAILURE IN LUNG STEREOTACTIC BODY RADIATION
THERAPY (SBRT)
Natalie Lockney 1 , T. Jonathan Yang 1 , Kelly Panchoo 1 , Daphna Gelblum 1 , Ellen
Yorke 2 , Andreas Rimner 1 , Abraham Wu 1
1 Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York/NY/United
States of America, 2 Medical Physics, Memorial Sloan Kettering Cancer Center, New
York/NY/United States of America
Background: Hyperactivation of the phosphatidylinositol-3-kinase (PI3K)
pathway has been associated with radioresistance. It is unclear whether such
mutations also confer suboptimal local control for patients who receive lung
stereotactic body radiation therapy (SBRT). Our objective was to examine
whether mutations in the EGFR/AKT/PIK3CA signaling pathway are associated
with local failure (LF) after lung SBRT. Methods: We retrospectively reviewed
134 patients who underwent SBRT to primary or metastatic lung lesions from
2007-2015 for whom molecular testing data was available for EGFR, AKT,
and PIK3CA genes. For tumors of lung origin (n=122), molecular testing data
was included from the lung tumor. For metastatic tumors to the lung (n=12),
molecular testing data from either a primary or metastatic tumor site was used.
Association between clinical factors, including molecular mutation status,
and LF was evaluated with Cox regression analysis. The Kaplan-Meier method
was used to assess differences in LF rates based on PIK3CA mutation status.
Results: The most common histology was adenocarcinoma (90%) among all
tumors. Six patients (4%) had PIK3CA mutation, 31 patients (23%) had EGFR
mutation, and one patient (0.7%) had AKT mutation. Median lesion size was 2.0
cm (range, 0.6–5.6 cm), median dose was 48 Gy (range 30–70 Gy), and median
number of fractions was 4 (range, 3–10). Median follow-up was 20 months
(range, 0.2–70 months). LF was observed for 16 patients (12%). Median time to
local failure was 15 months (range, 7–31 months). On univariate analysis, PIK3CA
mutation presence was associated with LF (HR 5.3 [95% CI 1.1-25.0], p=0.03),
while tumor histology (adenocarcinoma vs. other), tumor size (≤2cm vs. >2cm),
primary tumor site (lung vs. other), and EGFR or AKT mutation presence were
not. By multivariate analysis, PIK3CA mutation trended toward association
with LF (HR 5.0 [95% CI 1.0-25.3], p=0.051). At one year, probability of LF in
lesions with PIK3CA mutations was 12.4% vs. 5.7% in lesions without mutations
(p=0.02). Lesions with PIK3CA mutations were associated with a decreased
time to LF (mean 17.9 months [95% CI 12.7–23.2 months]) compared to those
without PIK3CA mutation (mean 58.6 months [95% CI 52.6–64.7 months]).
Conclusion: We explored EGFR/AKT/PI3KCA pathway mutations and found that
patients with PIK3CA mutations are at higher risk for LF after lung SBRT. Due
to the limitation of small numbers, this data needs to be validated in a larger
patient cohort. Nonetheless, this is a novel finding and hypothesis-generating
for future studies.
Keywords: Radioresistance, local failure, PIK3CA, Stereotactic body radiation
therapy
POSTER SESSION 2 – P2.05: RADIOTHERAPY
BIOLOGY –
TUESDAY, DECEMBER 6, 2016
P2.05-004 ABT-737, A BH3 MIMETIC, ENHANCES THERAPEUTIC
EFFECT OF IONIZING RADIATION IN MURINE LUNG CANCER MODEL
Jung Mo Lee 1 , Yoon Soo Chang 1 , Eun Young Kim 2
1 Department of Internal Medicine, Yonsei University College of Medicine, Seoul/
Korea, Republic of, 2 Division of Pulmonology, Department of Internal Medicine,
Yonsei University College of Medicine, Seoul/Korea, Republic of
Background: Radiotherapy is one of the main treatment modalities of lung
cancer, but its effectiveness is often hampered because of dose dependent
radiation toxicity. Aberration of apoptotic pathway after irradiation is
another mechanism attenuating therapeutic effect of radiation. ABT-737, a
‘first-in-class’ of BH3 mimetics, disrupts the BCL-2/BAK complex and initiates
BAK-dependent intrinsic apoptotic pathway. In this study, we sought
that ABT-737 is able to maximize the therapeutic effects of radiation in
experimental animal models. Methods: Kras:p53 fl/fl double mutant mice were
obtained by genotyping of offspring from LSL Kras G12D and p53 fl/fl mouse.
Lung cancer was induced by inhalation of 5 X 10 7 PFU AdCre viral particles
at 8 weeks age. After 12 (± 2) weeks of inhalation, the mice were randomized
and treated with either vehicle or ABT-737 (50 mg/kg, i.p., daily) for 3 days.
Then mice underwent microCT and were irradiated in the left lung at a dose
of 20 Gy using X-rad 320. In 2 weeks, 2 nd round microCT was performed and
lungs were harvested for histological analysis. Results: When the changes
in the expression of pro-apoptotic and anti-apoptotic molecules after 20 Gy
of irradiation were evaluated by immunoblotting, the decrease of BCL2-like
11 (BCL2L11) was most prominent in the irradiated lung. The tumor area was
decreased in the irradiated lung of both vehicle and ABT-737 pretreated
mice and inhibitory effect was remarkable when the mice were pretreated
with ABT-737. Disputed tumor structure with apoptotic bodies were most
frequently observed in the irradiated lung of ABT-737 pretreated mice. To
quantify the apoptotic effect of this combination, immunohistochemical
analysis against activated caspase-3 was performed. Counts of activated
caspase-3 were significantly higher in the irradiated lung with ABT-737
pretreatment, suggesting ABT-737 possesses radiosensitizing property.
Conclusion: Decrease of BCL2L11 expression in the irradiated lung is one of
prominent findings, which might compromise therapeutic effect of radiation.
Pretreatment of ABT-737 enhanced anti-tumor effect of ionizing radiation
in Kras:p53 fl/fl lung cancer model, suggesting BH3 mimetics would be a good
candidate of radiosensitizer in lung cancer. Further studies are warranted for
identification of optimal dosing and schedule of this combination treatment.
Keywords: apoptosis, radiosensitizer, BH3 mimetics, ABT-737
POSTER SESSION 2 – P2.05: RADIOTHERAPY
BIOLOGY –
TUESDAY, DECEMBER 6, 2016
P2.05-005 MECHANISM OF RADIOTHERAPY IN REDUCTION/DELAY
OF T790M-MEDIATED EGFR TKI RESISTANCE
Yasi Xu 1 , Shirong Zhang 1 , Bing Xia 2 , Lucheng Zhu 3 , Shenglin Ma 4
1 Center for Translational Medicine, Hangzhou First People’s Hospital, Nanjing
Medical University, Hangzhou/China, 2 Department of Radiation Oncology,
Hangzhou Cancer Hospital, Hangzhou/China, 3 Department of Radiation Oncology,
Hangzhou First People’s Hospital, Nanjing Medical University, Hangzhou/China,
4 Department of Oncology, Hangzhou First People’s Hospital, Nanjing Medical
University, Hangzhou/China
Background: EGFR T790M mutation accounts for more than 50% of acquired
resistance to TKI. In pre-clinical, EGFR-TKI resistant cells with T790M
exhibited enhanced sensitivity to radiation, suggesting the potential of
radiotherapy in reduction and delay of T790M-mediated EGFR TKI resistance.
Methods: Under different radiotherapy dose and times, we use droplet
digital PCR to observe the emerging time of T790M and its proportion during
chronic exposure to gefitinib in TKI-sensitivity cell lines, and to evaluate the
anti-tumor effect of early radiation combined with gefitinib in xenograft
model with different proportion of T790M. Furthermore, we performed
miRNA microarray to screen miRNAs differentially expressed in the paired
NSCLC gefitinib-sensitivity cell lines and gefitinib resistant cell lines and find
potential molecular markers of T790M mutation. Results: Our data showed
radiation combined with gefitinib delayed the occurrence of EGFR T790M
mutation compared to gefitinib alone in T790M wildtype (TKI-sensitive) cell
line and it also reduced the T790M mutation abundance in de novo T790M
mutation (TKI-resistant) cell line. The phenomena was also confirm in mice
xenograft model. In addition, our results showed TKI-resistant (induced
T790M mutation) cells had higher radiosensitivity than TKI-sensitive cells.
miRNA array showed miR-1275 was the one of the most significantly elevated
miRNAs in TKI-resistant cells. Knockdown of miRNA-1275 significantly
decreased the radiosensitivity of TKI-resistant cells. Western blot showed
Copyright © 2016 by the International Association for the Study of Lung Cancer
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