Abstracts of the Scientific Posters, 2013 AACC Annual Meeting ...

Cancer/Tumor Markers
Tuesday, July 30, 9:30 am – 5:00 pm
CNE-2) have been widely studied over the past decade due to their different levels
of radiosensitivity. CNE-1 cells have high differentiation with a radiosensitive
phenotype, whereas CNE-2 cells have low differentiation with a radioresistant
phenotype.
Methods/Results: We found that CNE-1 and CNE-2 cells were infected with highrisk
HPV 18. To our knowledge, no other report links CNE-1 and CNE-2 to HPV
infection. Our studies showed that relative copy number in CNE-1 is lower than in
CNE-2 (0.448 vs. 0.831, respectively). Their copy numbers were higher than those
found in cervical cancer C-4 II cells infected with HPV 18 (0.199). Our studies
detected 2 HPV oncogenes, E6 and E7 mRNA, in the CNE-1 and CNE-2 cell lines.
Independent of cell lines, the E6 mRNA level was significantly higher than the E7
mRNA level. The relative E6/E7 mRNA in CNE-1 was significantly higher than in
CNE-2. Although no significant differences between E6 and E7 mRNA levels in CNE-
1 and C-4 II were found, the E6 and E7 mRNA levels in CNE-2 were significantly
lower than in C-4 II. Mitochondrial DNA plays a key role in intrinsic sensitivity to
radiation. We found that the relative mtDNA copy number (mtDNA/nDNA ratio) in
CNE-1 was significantly lower than in CNE-2 (1 vs. 2.92, respectively). A similar
trend in mtDNA mutation (4,977-bp common deletion) was also found; the relative
mitochondrial common deletion in CNE-1 was significantly lower than in CNE-2 (1
vs. 4.25, respectively). These results were confirmed by real-time polymerase chain
reaction (RT-PCR) and branched DNA methods (QuantiVirus® HPV Detection Kit,
DiaCarta, Hayward, CA).
Conclusion: HPV may be the etiologic factor in some Epstein-Barr virus-negative
NPC cases. Further studies are warranted. Moreover, our studies also showed that
mitochondrial DNA may be responsible for the difference in radiosensitivity between
CNE-1 and CNE-2.
A-29
Molecular characterization of FLT3 mutations in Acute Myeloid
Leukemia from Pakistan with different FAB subtypes
M. Faiz, M. Ishfaq, T. Bashir, A. Shahid. Institute of Nuclear Medicine and
Oncology, Lahore, Pakistan
Introduction: FLT3 mutations are common genetic changes reported to have
prognostic significance in acute myeloid leukemia (AML). Methods: Peripheral
blood samples of 94 AML Pakistani patients were collected to determine FLT3
internal tandem duplication (ITD) incidence by PCR in exons 14 and 15 of FLT3
gene and D835 activating mutation in the tyrosine kinase domain (TKD) on isolated
DNA stored at -20oC. Results: Among 94 AML patients, 60 were males and 34
were females with male to female ratio 2:1. The age ranged between 15 to 78 years
with a median age of 32 years. Among 81 patients whose FAB subtype was known,
AML-M2 was the predominant subtype (37%) followed by M4 (23.5%), M3(15%),
M1 (11%), M5 (11%), M6 (2.5%) . The incidence of FLT3/ITD and TKD was 22%
and 6.3% respectively. Majority of the FLT3/ITD mutation was most common in
AML-M4 (63%) patients while D835 mutation was found in FAB M1, M2. Presence
of mutation was not related to gender or age. However, presence of FLT3/ITD was
clearly associated with hyperleukocytosis. No significant relationship was found
between clinical features and FLT3/ITD positivity. Conclusion: FLT3/ITD mutation
was common genetic abnormality found in Pakistani AML patients and unfavorable
prognostic marker that should be included in molecular diagnostic testing of AML.
Moreover, effective therapy with FLT3 targeting agents may be considered to improve
the prognosis in patients.
A-30
Restoration of miR-638 induces SPC-A1 cells apoptosis via down
regulation of HGF
S. Pan, Y. Cao, J. Xu, B. Zhang, J. Ma, E. Xie, D. Chen, L. Gao, Y. Zhang.
The fi rst clinical medical college, Nanjing, China
Background: Aberrant expression of miRNAs has been correlated with various
human diseases including cancers, and this small non-coding RNA has been identified
which have oncogenic or tumor suppressor properties Emerging evidence showed that
miRNAs are important regulators in cancer cell proliferation, apoptosis, metastasis,
chemosensitivity and so on. In the previous study, we produced a monoclonal
antibody designed NJ001, which exhibited its anti-tumor activity both in vitro and in
vivo by inducing apoptosis. This study was aimed to investigate the role of miRNAs
in SPC-A1 cells undergoing apoptosis following treatment with NJ001 and find the
pro-apoptosis miRNA in tumor cells for further study on cancer therapy.
Methods: Affymetrix GeneChip® miRNA 2.0 Array was performed to acquire
dynamic miRNA expression profile of SPC-A1 after treatment with NJ001.
Quantitative real-time-PCR was carried out to validate the results of microarray
approach. After that, we used Cluster Analysis of up-regulated expression in SPC-A1
incubated with NJ001 to focus interesting miRNA. In the gain of function study,
Images of CY-3 labeled miRNA mimics and qRT-PCR was used to confirm augmented
expression. After successfully over-expression of miRNA, double staining with FITC-
Annexin V and PI was carried out to evaluate the apoptosis rate. Members in apoptosis
pathway were detected by western blot. We used five programs_miRanda, miRDB,
miRWalk, RNA22 and Targetscan_to predicte targets of miR-638. The wild-type and
mutation-type 3’-UTRs of these potential targets were cloned into the PGL4 plasmid
and dual-luciferase assays was carried out after co-transfection miRNAs and reporter
plasmids into SPC-A1 cells to evaluate the changes of luciferase activity. Changes
of mRNA levels and protein levels of target genes were confirmed by qRT-PCR and
western blot respectively.
Results: After treatment with NJ001, The high percentage of Annexin V + cells in
NJ001 groups was observed at 24 h, 48 h and 72h compared to cells in the control
groups (44.3%, 74.0% and 81.4% vs. control respectively, P < 0.05 for all time points).
The expression of miR-638 was the first to show a significant change and found to
be dynamically increased accompanied with the climbing apoptosis rate according
to the result of Cluster Analysis of microarray approach. In addition, we observed
that miR-638 is significantly suppressed in SPC-A1 when compared with human
embryonic lung fibroblast (HFL-1) and functional studies indicated over-expression
of miR-638 positively regulated the apoptosis of SPC-A1 through both extrinsic and
intrinsic pathway via activating caspase 8, caspase 9, caspase 3 and shifting the bax/
bcl-2 ratio. By transcriptomic analysis and computational algorithms, we identified
the anti-apoptotic protein hepatocyte growth factor (HGF) as a target gene of miR-
638. Dual-luciferase reporter assay confirmed that miR-638 negatively regulated HGF
by interaction between miR-638 and complementary sequences in the 3’ UTR of HGF.
MiR-638 repressed HGF at post-transcriptional levels as revealed by quantitative RT-
PCR and Western blot analysis.
Conclusion: In summary, our findings demonstrate that miR-638 has a critical role in
regulating apoptosis of SPC-A1 cells, implying the function of miR-638 as a putative
tumor suppressors miRNA and provide a basic rationale for the use of miR-638 in the
treatment of NSCLC.
A-31
NJ001 antibody specific antigen is the key molecule for outcome
evaluation of lung adenocarcinoma patients
P. Huang, Y. Han, F. Wang, R. Yang, L. Zhang, T. Xu, Q. Li, H. Wang, S.
Pan. The fi rst clinical medical college, Nanjing, China
Objective: To evaluate the relationship between NJ001 specific antigen and
clinicopathological features, so as to further explore its role in the prognosis of lung
adenocarcinoma.
Methods: The expression of NJ001 specific antigen was examined by means of the
envision system immunohistochemical staining with monoclonal antibody NJ001 in
110 lung adenocarcinoma and 46 benign lung disease, as well as a tissue microarray
(TMA) containing 75 lung adenocarcinoma and the adjacent normal tissue.
Immunohistochemistry results were reckoned by multiplication of the percentage and
staining intensity of positive tumor cells. Then we evaluated the associations of the
antigen expression with several clinicopathologic parameters. Overall survival rates
were determined by using the Kaplan-Meier method with log-rank test for comparison
among groups with different expression of the specific antigen.
Results: We observed that NJ001 specific antigen was predominantly located on the
cell membrane and in the cytoplasm of tumor cells. The positive rate was respectively
84.70% in lung adenocarcinoma, 8.22% in the adjacent normal tissue and 8.70% in
benign lung disease. The specific antigen expression in lung adenocarcinoma was
significantly associated with the poor and moderate differentiation grade of the tumor
(P= 0.017) and lymph node metastasis (P