Annual Meeting Proceedings Part 1 - American Society of Clinical ...

10536 General Poster Session (Board #42A), Mon, 1:15 PM-5:15 PM
In vivo isolation of circulating tumor cells in non-small cell lung cancer
(NSCLC) patients by a medical device. Presenting Author: Lukasz Gasiorowski,
Medical University Poznan, Poznan, Poland
Background: Currently, circulating tumor cells (CTCs) are isolated in vitro
from small limited volumes of blood samples. Furthermore, CTC results for
NSCLC as a prognostic and stratification biomarker are scarce. The aim of
the study was to assess a functionalized and structured medical wire
(FSMW) for in vivo isolation of CTCs directly from the blood of NSCLC
patients, and to compare it to the Cell Search method. Methods: The device
was inserted in a cubital vein through a standard cannula for thirty minutes.
The interaction of target CTCs with the FSMW was mediated by an antibody
directed against the epithelial cell adhesion molecule (EpCAM). To confirm
the CTCs binding to the wire, the immunohistochemical staining against
EpCAM as well as CD45 for negative cell selection was performed. There
were 72 applications of the wire in 48 stage I-IIIB NSCLC patients (12
double applications) and 12 non cancer patients. Enumeration data was
available for 34 NSCLC patients and 8 non cancer patients. For 34
patients, samples were also tested in the Cell Search system. Results: The
device was well tolerated in all 72 applications without side effects. We
obtained in vivo isolation of CTCs in 32 of 34 NSCLC patients (94.1%) with
a median (range) of 13 (0-300) CTCs. The sensitivity was similar for early
and late stage NSCLC patients. In the non-cancer patients, no CTCs were
detected (100 % specificity). In 2 of 34 samples (5.8%), CTCs were
detected by the Cell Search method. In all matched pairs, the FSMW
detected the same number or more CTCs compared to the Cell Search
method. Conclusions: Whilst well tolerated without side effects, the CTC
detection rate of the FSMW in NSCLC patients was �90%. In contrast,
�10% detection was obtained using the Cell Search analysis. No CTCs
were detected in non-cancer patients. This proof of concept study may have
important clinical implications, as the implementation of the device into
clinical practice may improve early detection, prognosis and therapy
monitoring of NSCLC patients. The method may also allow the molecular
analysis of the CTCs, with the possibility of establishing more personalized
treatment regiments.
10538 General Poster Session (Board #42C), Mon, 1:15 PM-5:15 PM
Prognostic and diagnostic value of Ewing family of tumors (EFTs)associated
fusion transcripts detected in peripheral blood specimens.
Presenting Author: Joanna Przybyl, Department of Molecular Biology,
Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology,
Warsaw, Poland
Background: EFTs are characterized by chromosomal translocations leading
to formation of oncogenic EWSR1-FLI1 fusion gene in 85-90% of cases.
The aim of the study was to detect circulating tumor cells (CTCs) carrying
EWSR1-FLI1 fusion transcript in peripheral blood and assess their added
value to standard diagnostic procedures and utility as a prognostic marker
in EFTs. Methods: 10mL of whole blood was collected from 35 untreated
adult EFTs patients at the diagnosis (period: 2008-2011, median age 27
years) and 13 healthy controls. 13 patients presented metastatic disease
(M1) at the diagnosis. Nested RT-PCR was applied in triplicate for the
detection of EWSR1-FLI1 transcript. Blood specimen was regarded CTCpositive
when at least 2 out of 3 nested RT-PCR assays were positive and
results were confirmed by sequencing. FISH assay for EWSR1 rearrangement
was performed on FFPE tumor tissue in 28 available cases. Median
follow-up was 16 months. Results: EWSR1-FLI1 transcript was detected in
peripheral blood of 71.4% (n�25) of patients. FISH assay was positive for
EWRS1 rearrangement in 58% of cases. In 10 patients, where FISH assay
could not be performed due to insufficient quality or lack of material,
nested RT-PCR provided confirmation of immunopathological diagnosis.
Specificity of RT-PCR blood test in healthy control was 91.4% (12/13
negative; p�0.0001). Median overall survival (OS) was 18 months.
CTC-positive patients showed the trend for longer OS than CTC-negative
patients (1-year OS: 89% vs. 58%; p�0.07), but longer follow-up is
needed. Conclusions: Our results show that the fusion transcript detection
in peripheral blood specimens may be a useful additional test to the
standard clinicopathological diagnosis of EFTs. High detection rate of
EWSR1-FLI1 transcript in peripheral blood of EFTs patients at the
diagnosis may imply EFTs as a systemic disease with clinically evident
metastases or micrometastases at presentation. Prognostic significance of
CTCs in EFTs warrants further studies.
Tumor Biology
665s
10537 General Poster Session (Board #42B), Mon, 1:15 PM-5:15 PM
A new medical device for in vivo capturing of circulating tumor cells in
breast cancer (BC) patients. Presenting Author: Dawid Murawa, Wielkopolska
Cancer Centre, Poznan, Poland
Background: In BC, the number of circulating tumor cells (CTCs) is
discussed as a prognostic and stratification biomarker, and could also
reflect treatment efficacy. Currently, CTCs are isolated ex vivo from a small
volume of blood. Results from a larger volume of blood are scarce. The aim
of the study was to assess a functionalized and structured medical wire
(FSMW) for in vivo capturing of CTCs directly from the blood stream of BC
patients. Methods: The device was inserted in a cubital vein through a
standard cannula for thirty minutes. The interaction of target CTCs with the
FSMW was mediated by antibodies directed against the epithelial cell
adhesion molecule (EpCAM). To confirm binding of CTCs to the wire, the
immunohistochemical positive staining against EpCAM as well as negative
staining for CD45 was performed. There were 54 applications of the wire in
42 stage I-IV BC patients (12 double applications). Enumeration data from
37 BC patients with 49 applications (5 failed subsequent analyses) were
assessed. CTC counts on 23 devices were directly compared to counts by
CellSearch. Results: The device was well tolerated in all 54 applications
without side effects. We obtained in vivo isolation of CTCs in 44 of 49
applications to BC patients (89.7 %). The sensitivity was similar for early
and late stage BC patients. The median (range) of isolated EpCAM-positive
CTCs was 5 (0-515). The CellSearch method reached a sensitivity of
18.5%. In all paired samples the number of CTCs detected with the FSMW
was higher or equal to CellSearch, regardless of the disease stage. Linear
regression of the data of the double application of the FSMW showed a very
good concordance (r2 � 0.97, p�0.0001). Conclusions: Whilst well
tolerated without side effects, the CTC detection rate of the FSMW in BC
patients was nearly 90 %. CTC detection was obtained in 18.5% by the
CellSearch. Double application of FSMWs in the same patient indicates
ample precision. This proof of concept study may have important clinical
implications, as the device may improve early detection, prognosis and
therapy monitoring of BC patients. The molecular analysis of the captured
CTCs could become a breakthrough in personalized medicine.
10539 General Poster Session (Board #42D), Mon, 1:15 PM-5:15 PM
Circulating melanoma cells isolated from clinical blood samples and
characterized by full-length mRNA sequencing at single-cell level. Presenting
Author: Yu-Chieh Wang, Department of Chemical Physiology, The
Scripps Research Institute, La Jolla, CA
Background: Melanoma is the most aggressive type of skin cancer. Latestage
melanoma is highly metastatic and currently lacks effective treatment.
This discouraging clinical observation highlights the need for a better
understanding of the molecular mechanisms underlying melanoma initiation
and progression and for developing new therapeutic approaches based
on novel targets. Although genome-wide transcriptome analyses have been
frequently used to study molecular alterations in clinical samples, it has
been technically challenging to obtain the transcriptomic profiles at
single-cell level. Methods: Using antibody-mediated magnetic activated
cell separation (MACS), we isolated and individualized putative circulating
melanoma cells (CMCs) from the blood samples of the melanoma patients
at advance stages. The transcriptomic analysis based on a novel and robust
mRNA-Seq protocol (Smart-Seq) was established and applied to the
putative CMCs for single-cell profiling. Results: We have discovered distinct
gene expression patterns, including new putative markers for CMCs.
Meanwhile, the gene expression profiles derived of the CMC candidates
isolated from the patient’s blood samples are closely-related to the
expression profiles of other cells originated from human melanocytes,
including normal melanocytes in primary culture and melanoma cell lines.
Compared with existing methods, Smart-Seq has improved read coverage
across transcripts, which provides advantage for better analyzing transcript
isoforms and SNPs. Conclusions: Our results suggest that the techniques
developed in this research for cell isolation and transcriptomic analyses can
potentially be used for addressing many biological and clinical questions
requiring genomewide transcriptome profiling in rare cells.
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