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

680s Tumor Biology
10598 General Poster Session (Board #49G), Mon, 1:15 PM-5:15 PM
The efficacy of targeted next-generation sequencing for detection of
clinically actionable mutations in cancer. Presenting Author: Yu-Ye Wen,
Baylor College of Medicine, Houston, TX
Background: The emergence of next-generation sequencing (NGS) technologies
has significantly accelerated the identification of cancer-causing
mutations and the development of personalized cancer care. However, the
clinical application of these technologies to detect cancer gene mutations
has been extremely limited due to the long turn around time, the high cost,
and large amount of input DNA required by existing NGS-based tests.
Methods: We have assessed the performance of a novel NGS technology that
merges multiplex PCR with ion semiconductor sequencing (AmpliSeq, Life
Technologies, Inc.) in our clinical diagnostic laboratory. The test interrogates
739 common mutations in 46 cancer genes including many clinically
actionable mutations concurrently. First, we studied 12 tumor samples
including 4 archived FFPE, 4 blood/bone marrow, and 4 cell line samples
with known mutations to evaluate the sensitivity and specificity of the test.
We then studied 34 de-identified, archived FFPE tumor samples of
unknown genotype to further evaluate the efficacy of the test. Results: We
successfully identified all known mutations previously detected by Pyposequencing
or Sanger sequencing technologies. Multiple serial dilution
studies showed that the test could detect mutations at frequencies as low
as 5% with 99% confidence. For the samples of unknown genotype, we
detected 23 COSMIC mutations in 16 samples including HRAS, BRAF,
MET, TP53 mutations in lung cancer, KRAS, PIK3CA, TP53, APC, BRAF,
ERBB2 mutations in colon cancer, TP53 and KRAS mutations in breast
cancer, and KIT and PDGFRA mutations in GIST. Analysis of the variant
call data showed that a minimum of 100X coverage is required in order to
detect mutations at 10% frequency or above; a minimum 300K final library
reads are necessary in order to minimize/eliminate amplicon dropout.
Conclusions: The targeted NGS test can effectively detect cancer gene
mutation with input DNA as low as a few nanograms, turn around time can
be as short as two days, and can significantly lower cost compared to
traditional Sanger sequencing. Our experience demonstrates that this
technology holds great potential for clinical use, including diagnostic and
therapeutic applications.
10600 General Poster Session (Board #50A), Mon, 1:15 PM-5:15 PM
A circulating epithelial cell type merging stem cell, EMT, and hypoxic
stress markers in early and advanced breast cancer. Presenting Author: Leo
Habets, Metares e.V, Aachen, Germany
Background: MAINTRAC as developed by our coworkers from Jena finds
more CETC. They found CETC in 90% of high risk patients. Circulating cells
in epithelial mesenchymal transition (EMT�) should be detectable in early
disease. Methods: 2 colours (7AAD, EP-FITC) detected living and dead
cells. Cells in EMT (EP-FITC, Vim-PE) were analysed in paralell. Complex
marker expression urged us to introduce a three colour technique. 1.
EPCAM-FITC, Vimentin-PE and DAPI , marking living or dead cells in EMT.
2. EP-FITC, Vim-PE and CD44 PB for stemness. ACA9 (hypoxia) and
PARP-1 expression (genotox) were used too. Results: In the 2 colour phase
we examined CEC in 110 patients with early disease and 44 patients with
metastasis. Cells were detectable in both disease situations in 50 and 60%
of patients, in a control population only in few patients low numbers were
found. In non cancer situations e.g liver disease high numbers of EMT�cells
were found.The results of 3 colour analysis are shown in the table. No clear
differences between the classical risk groups are found, other subgroups
are too small yet to be considered. Conclusions: Our findings show that in
both disease situations cells merging EMT, stemcells ( EP�,Vim�.CD44�)
and other markers are detectable. This cell type however is found also in
non cancer conditions.We hypothesize that hypoxia is the common driver.
Metastasis gives different patterns. Highly aggressive cancers show often
none or few cells. In less agressive disease high cell numbers are found. We
need to characterize the �benign� EMT phenomenon, as it could mingle
with �real� CTC. Definitely liver is the source of these benign cells. Multi
colour analysis will reveal the differences. Nevertheless monitoring of this
special cell type could give new insights in the metastatic process in early
and late disease.
Cell type EMT living EMT total EMT�CD44
CEC per ml �250 �250 �250 �250 �250 �250
ALL 56 44 21 79 25 75 n�60
N0 80 20 41 59 57 43 n�36
N� 64 26 51 49 34 66 n�24
Lum A 48 52 20 80 34 66 n�30
Lum B 48 52 31 69 23 77 n�29
Advanced 39 61 27 73 37 63 n�40
Controls 98 2 91 9 81 19 n�112
�Liver � 10 90 22 78 n�84
10599 General Poster Session (Board #49H), Mon, 1:15 PM-5:15 PM
Serum HER2 in the context of circulating tumor cells in patients with
metastatic breast cancer. Presenting Author: Volkmar Mueller, University
Medical Center Hamburg, Gynecology, Hamburg, Germany
Background: Circulating tumor cells (CTC) reflect an aggressive tumor
behavior by hematogenous tumor cell dissemination. Overexpression of
HER2 in breast cancer (BC) is associated with increased angiogenesis and
therefore potentially linked to increased hematogenous tumor cell spread.
The aim of the analysis was to investigate whether concentrations of serum
HER2 (sHER2) deliver prognostic information in the context of CTC
detection in metastatic BC patients. Methods: Blood was obtained in a
prospective multicenter setting from 254 patients with metastatic BC at
the time of disease progression. sHER2 was determined using a commercial
ELISA-kit (Wilex). CTC were detected with the CellSearch system
(Veridex). Patients received systemic treatment according to national and
international guidelines including HER2-targeted treatment. Results: Five
or more CTC were detected in 122 of 245 evaluable patients (49.8%).119
of 251 (47%) metastatic patients had serum sHER2 levels above 15ng/
mL. Median PFS was 9.2 months (95%-CI: 9.9 – 13.0 mths) with elevated
sHER2 versus 11.4 mths (9.9 – 13.0 mths) with non-elevated levels
(p�0.07). OS was 17.4 mths (14.6 – 20.3 mths) vs. 26.5 mths
(23.1-29.8 mths; p�0.01). In patients with 5 or more CTC, serum levels
were above the cut-off for sHER2 in 61% vs. 33% in those with less than 5
CTC (p� 0.01). Patients with elevated sHER and 5 or more CTC hat a PFS
of 9.1 mths (7.2 – 11.1 mths) and a OS of 14.5 mths (11.8 – 17.2 mths),
those with non-elevated sHER2 and less than 5 CTC a PFS of 12.1 (10.1 –
14.1 mths) and a OS of 29.5 month (25.4 – 33.6 mths) (p�0.15 for PFS
and p� 0.01 for OS). Including sHER2, CTC and established prognostic
factors in the multivariate analysis, the presence of CTC, line of therapy, ER
and HER2 status of the primary tumor remained independent predictors of
OS. Conclusions: Elevated serum levels of sHER2 are associated with the
presence of CTC and indicate poor clinical outcome. However, sHER2 has
no independent prognostic value when presence of CTC were taken into
account.
10601 General Poster Session (Board #50B), Mon, 1:15 PM-5:15 PM
Exploratory study of histopathological and biomolecular characteristics of
breast cancer in two different populations: African (Tanzania) and Caucasian
(Italy). Presenting Author: Patrizia Serra, Unit of Biostatistics and
Clinical Trials, IRST, Meldola, Italy
Background: There is little information available on the clinical and
molecular epidemiology of breast cancer in Sub-Saharan African women. In
Tanzania, breast cancer mortality is the second cause of death after cancer
of the uterine cervix, with the majority of tumors diagnosed at a very
advanced stage. We aimed to compare the distribution of histopathological
and biomolecular characteristics (ER, PgR, Ki67, HER2, grading) of breast
cancer in African (Tanzania) and Caucasian (Italy) patients. Methods: 142
women with invasive breast cancer, 71 from Tanzania and 71 from Italy,
were considered. Histopathological classification and biomolecular determinations
were performed at the Biosciences Laboratory of the Cancer
Institute of Romagna (I.R.S.T., Meldola, Italy) as part of a joint Cancer
Control Project between I.R.S.T and the Bugando Medical Center in
Mwanza, Tanzania. All cases were evaluable for histopatological characterization,
while biomolecular determination was possible in only 61 of the
African patients. Hormone receptor status and Ki67 were determined by
IHC; HER2 was evaluated by IHC for the African population and by FISH for
the Italian one. Results: Whilst histopathological patterns were similar in
the two populations, some biomolecular characteristics differed substantially.
Of note, 64% of African breast cancer patients had ER negative
tumors compared to only 17% of the Caucasian population. Important
differences were observed in high Ki67 (�15%) values: 76% in Tanzanian
women vs 58% in Italian breast cancer cases. HER2 positivity rates also
differed in the two populations (25% in Tanzanian and 17% in Italian
patients), as did the incidence of triple negative disease (13% and 7% in
African and Caucasian populations, respectively). Conclusions: This preliminary
study shows that breast cancer in African (Tanzanian) women is
characterized by a high frequency of HER2 positive, ER negative, highly
proliferating tumors. These biological patterns, together with very advanced
stage at diagnosis, could be considered the main prognostic factors related
to the high mortality rates for breast cancer in this African population.
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