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

660s Tumor Biology
10516 Poster Discussion Session (Board #9), Tue, 8:00 AM-12:00 PM and
11:30 AM-12:30 PM
Mutational analysis of circulating tumor cells in breast cancer patients by
targeted clonal sequencing. Presenting Author: Xunhai Xu, New York
Presbyterian-Columbia, New York, NY
Background: The molecular signature of circulating tumor cells (CTCs) may
serve as a surrogate marker for accurate description of the metastatic tumor
of interest, especially in the setting of treatment response and selection.
We present a method for mutational analysis of CTCs in metastatic breast
cancer (MBC) patients by using an emulsion-formulated, semiconductorbased,
targeted clonal sequencing platform. Methods: CTCs in MBC
patients were enriched by a microfluidic OncoCEE device using antibodies
against both epithelial and mesenchymal markers. Genomic DNA was
extracted from enriched CTC samples. Emulsion-based multiplex-PCR
targeted for various cancer genes was performed, after which semiconductorbased
deep sequencing was completed. The read error rates were analyzed
based on quality score and context of sequence including homopolymers.
Statistical significance for each mutational analysis was assessed using a
method based on beta-binomial distribution. Results: Of the 17 patients
samples obtained, we were able to enrich CTC samples in 9 of them (CTC
range 1-1063, median�12). Multiplex targeted sequencing was performed
on DNA from the enriched CTC patient samples (purity range
0.3-6%). Greater than 3000-fold coverage was accomplished. Missense
mutations at E545D on PIK3CA (p� 2.0e-07), F354L on STK11 (p�2.0e-
04), and Q61R on NRAS (p�2.0e-07) were detected. Novel mutations of
L540F and Q1033K within the hot spot regions of PIK3CA were observed
(p�1.3e-04). Genomic DNA from WBCs from a healthy female was
analyzed concurrently as a negative control, in which none of the mutations
were observed. Conclusions: Mutational analysis of CTCs in MBC patients
can be accomplished by deep sequencing. We developed a de novo protocol
for clonal mutation analysis on CTCs in MBC and detected various
significant and novel mutations. We anticipate reporting sequencing
results on CTCs and matched WBCs, as negative controls, from 40 MBC
patients. This will provide the foundation for the future studies in which we
will compare the mutational profile between CTCs and primary/metastatic
tumors. We intend to validate clonal mutational analysis of CTCs as a
predictive blood-based biomarker in subsequent trials.
10518 Poster Discussion Session (Board #11), Tue, 8:00 AM-12:00 PM and
11:30 AM-12:30 PM
Genetic variation in CYP19A1 and response to exemestane: Survival in early
breast cancer in the Dutch TEAM trial. Presenting Author: Daniel Houtsma,
Department of Clinical Oncology, Leiden University Medical Center, Leiden,
Netherlands
Background: In patients with endocrine-sensitive breast cancer treated with
adjuvant aromatase inhibitors (AI) it is unclear which patients will develop a
recurrence and who will benefit from AI’s. Variations in the aromatase gene
(CYP19A1) are associated with altered estrogen levels and altered aromatase
activity. The aim of this study was to examine the effect of SNPs in the CYP19A1
gene on survival in a prospective cohort of breast cancer patients treated with
adjuvant exemestane. Methods: Patients of whom tissue was available and who
were treated with five years of exemestane were selected from the Tamoxifen
Exemestane Adjuvant Multinational (TEAM) trial. DNA was isolated from tumor
samples and 30 SNPs were identified using a tagging SNP approach, aiming for
80% coverage of CYP19A1. Genotypes were determined with taqman assays.
Primary endpoint of the study was relapse-free survival (RFS) and secondary
endpoint was overall survival (OS). A Kaplan-Meier analysis was performed and
Cox proportional hazards models assessed survival differences. Analyses were
adjusted for age at diagnosis, tumor size, nodal status, histological grade,
surgery, adjuvant radiotherapy and chemotherapy. Results: 807 patients were
included in the analyses and genotypes were obtained in 722 cases. A significant
association with worse RFS was found with two SNPs: rs7176005 and
rs16964211, showing hazard ratios (HR) of 3.48 and 5.42 for the homozygeous
variant types respectively. These SNPs, as well as a third SNP, rs6493497, were
also significantly associated with OS (HR 5.87, 5.3 and 3.36 respectively).
Conclusions: Germline variations in the CYP19A1 gene are related to a worse
outcome in early breast cancer patients treated with exemestane. These findings
may contribute to the individualization of hormonal therapy in breast cancer. The
relation between RFS and SNP’s in CYP19A1.
Number of
genotype
Univariate HR
(95% CI) P value
Multivariable HR
(95% CI) P value
rs7176005 0.015 0.033
CC 578 1 (ref) 1 (ref)
CT 130 1.83 (1.15-2.91) 1.75 (1.05-2.92)
TT 8 2.78 (0.68-11.4) 3.48 (0.83-14.6)
rs16964211 0.021 0.019
GG 629 1 (ref) 1 (ref)
AG 67 1.04 (0.52-2.06) 0.52 (0.23-1.17)
AA 4 5.82 (1.43-23.7) 5.42 (1.25-23.4)
10517 Poster Discussion Session (Board #10), Tue, 8:00 AM-12:00 PM and
11:30 AM-12:30 PM
Intraoperative detection of lymph node metastasis using one-step nucleic
acid amplification (OSNA) in breast cancer patients: Effect on second
surgery rate and delay for adjuvant therapy. Presenting Author: Sophie
Klingler, Centre Alexis Vautrin, Vandoeuvre-lès-Nancy, France
Background: Sentinel lymph node (SLN) analysis is conventionally analysed
using HES and CK19 immunohistochemistry. In case of SLN involvement,
a second surgery is required for axillary lymph node (ALN) resection thus
delaying the initiation of adjuvant therapies. Methods: 381 pts with invasive
breast cancer were considered in this retrospective study. SLN were
detected using combined radio-isotope and color detection. SLN involvement
was analysed using OSNA for CK19 mRNA, in 100 pts (group 1) and
compared to conventional histopathology in 281 pts (reference population,
group 2). In all cases, control cytology was performed. Results: No
significant difference was found between group 1 and 2 regarding patients
characteristics, tumor localization, size, grade, steroid receptors and HER2
expression and the mean number of SLN analysed per pt, 2.4 (range 1-7)
and 2.5 (range 1-8), respectively. Considering positive SLN as “��”
(CK19 mRNA copy number�5000), “�” (250 � CK19 mRNA copy
number � 5000) and positive by inhibition in group 1 and macro-,
micrometastases and isolated tumor cells in group 2, no difference in
lymph node involvement rate was found between the two groups with 29.0
and 29.9% of positive SLN, respectively. Only one discordance case was
observed with negative OSNA analysis and positive cytology with isolated
tumor cells. Using OSNA intraoperatively, the mean time to process SLN
was 42 min (range 10-104) allowing immediate ALN resection. Significant
(P�.01) reduction of re-intervention rate was observed when OSNA was
used (9 vs 39%), including margins insufficiency (5 vs 13%), ALN
resection (3 vs 15%) or both (1 vs 11%). Delay before adjuvant therapy was
also significantly (P �.01) reduced when OSNA was used with 43 (range
20-82) vs 59 (range 14-212) days. Conclusions: Results achieved with
OSNA are fully consistent with those achieved using conventional immunohistochemistry
analysis. SLN analysis using OSNA avoids a second operation
in most patients for ALN resection and shortens the delay for adjuvant
therapy.
10519 Poster Discussion Session (Board #12), Tue, 8:00 AM-12:00 PM and
11:30 AM-12:30 PM
64Cu-DOTA-trastuzumab-PET imaging in patients with HER2-positive breast
cancer. Presenting Author: Kenji Tamura, Department of Breast and
Medical Oncology, National Cancer Center Hospital, Tokyo, Japan
Background: Targeting of HER2 with trastuzumab (T) is a well-established
strategy in the metastatic and adjuvant setting in HER2 positive breast
cancer (HER2-BC). Although HER2 status is routinely determined using
immunohistochemistry or fluorescence in situ hybridization, technical
problem can arise when lesions are poorly accessible. HER2 expression can
vary during the course of the disease, and there can be discordance in
HER2 expression across tumor lesion even in the same patients. Noninvasive
HER2 imaging is crucial needed to solve these problems. Previous
imaging using 111In or 89Zn- trastuzumab produced high radiation exposure
to patients by their long half life (�67.9and 78.4h) and low resolution
image. Half life of 64Cu is 12.7h. We performed a feasibility study of the
64Cu-1, 4, 7, 10- tetraazacylododecane-N,N’,N�,N�’-tetraacetic acid
(DOTA)-T to perform PET imaging in patients with HER2-BC. Methods:
Patients with HER2-BC received 150 MBq of 64Cu-DOTA-T and underwent
PET scan 1, 24 and 48h after the injection. Six patients were evaluated
internal dosimetry by collecting radioactivity data of blood and normal
tissue in each time point from PET study, and radiation exposure by
collecting clothes, linen and urine to test feasibility for outpatients. Results:
Fifteen patients who received T therapy were enrolled in the “first-inhuman”
trial. All patients had no severe toxicity. Radiation excretion
evaluating clothes, linen were under background level. Radiation exposure
of 64Cu-DOTA-T was equivalent to that of conventional 18F-FDG-PET. Distribution of liver, kidney, spleen, and blood vessel was 2-8 SUV, and
uptake in other normal tissue was low. At visual examination, in two
patients brain metastases were clearly visualized by 64Cu-DOTA-T-PET, suggesting that there are disruptions of the blood-brain barrier at the site of
the brain metastases. The sternum bone metastasis was well visualized and
quantitatively monitored according to the response by T. Primary breast
cancers, lymph node metastases and lung metastases could also be
visualized at the lesion indentified by CT. Conclusions: 64Cu-DOTA-T-PET was feasible test even for outpatient, and provides specific and high
resolution image in HER2 positive lesion.
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