Real-time non-invasive chemoresistance profiling of circulating tumor associated cells in breast cancers to determine resistance towards mitotic inhibitors

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10.0%, p = 0.014) in the primary tumor. Among the cases with positive PD-
L1, 36.7% of VEGF positive cases had low TILs in the primary tumor, while
none of negative VEGF-A cases had low TILs in the primary tumor.
Conclusion: The present study demonstrated that VEGF-A expression in
breast cancer may be reflective of the expression of PD-L1 in the tumor.
VEGF-A may act as negative regulator of TILs in the PD-L1 positive BC. In
light of our results, VEGF-A may be predictive of immunological features and
be a useful biomarker for immuno-targeting therapy among patients with
breast cancer.
No conflict of interest.
301 Poster
Gene expression profiles in premenopausal women with HR+ HER2−
early breast cancer
H. Ni
1
, A. Kurt
1
, J. Kumbrink
2
, A. Seiler
1
, D. Mayr
2
, T. Degenhardt
1
,
F. Hagemann
1
,R.Wu
̈rstlein
1
, N. Harbeck
1
, T. Eggersmann
1
.
1
Ludwig
Maximilians University of Munich, Breast Center, Department of Gynecology
and Obstetrics and Comprehensive Cancer Center, Munich, Germany;
2
Ludwig Maximilians University of Munich, Department of Pathology,
Munich, Germany
Background: Early breast cancer (EBC) is not a single disease but consists
of several clinically relevant molecular subtypes. Within hormone receptor
positive (HR+), HER2 negative (HER2−) disease, different luminal subtypes
(A vs. B) impact on outcome and response to endocrine therapy. Gene
expression signatures predicting risk of recurrence are already part of clinical
management. Gene profiles correlated with important tumor pathways such
as metastasis and progression, immune response or proliferation also
correlate with clinical outcome and therapy response. Premenopausal
patients often have poorer prognosis compared to postmenopausal patients.
Even though the principle for treating premenopausal patients is consistent
with that for postmenopausal patients, the molecular properties of breast
cancer in young patients demand special attention in planning the
therapeutic strategy. Nevertheless, most studies on gene expression, in
particular for risk estimation, focussed on postmenopausal patients. The
purpose of our project is to determine gene expression profiles of tumor
samples from premenopausal patients with HR+, HER2−EBC. The gene
expression profiles will then be correlated to response to therapy response
and patient outcome.
Material and Methods: We comprised a collective of 162 premenopausal
EBC patients (77 with and 85 without relapse) treated at the LMU breast
center over a ten-year follow-up period. Diagnostic, therapeutic, and recent
follow-up data were documented and prepared for statistical analysis. Tissue
specimens were prepared for laboratory analysis which include a gene
expression profiling using a custom-made pan-cancer code set (n = 745
genes) and the Nanostring nCounter
®
analysis. Gene expression data will be
compared with conventional immunohistochemistry subtyping as well as
histopathological factors that can be used as surrogates forcertain pathways
(pan cancer pathways, pathways for tumor progression and tumor
immunology, etc.).
Results: Median patient age was 43.98 years of age (range 29–50). The
two patient groups (with/without relapse within 10 years) differed with regard
to clinical parameters: grade (2.06 ± 0.07/2.29 ± 0.06, p= 0.024), tumor
diameter (26.62 mm ± 2.11/21.89 mm ± 2.67, p= 0.033), percentage of
lymphnode metastasis [0.18 (range 0–1)/0.078 (range 0–0.92), p= 0.001]
(Table 1).
Table 1 Patients’clinical parameters
With relapse (n = 77)
Without relapse
(n = 85) p-value
Age at diagnosis 43.64 ± 0.58 (30–50) 44.29 ± 0.49 (29–50) 0.510
Grade 2.06 ± 0.07 (1–3) 2.29 ± 0.06 (1–3) 0.024
Tumor size (mm) 26.62 ± 2.11 (1–130) 21.89 ± 2.67 (2–110) 0.033
Nodal status 0.1844 (0.00–1.00) 0.0783 (0.00–0.92) 0.001
Conclusion: The project is ongoing. Updated results will be presented at
the conference.
Funding: The first author is funded by China Scholarship Council.
No conflict of interest.
302 Poster
Circulating tumor associated cells in breast cancers are resistance
educated towards prior anthracycline treatments
A. Srinivasan
1
, D. Akolkar
1
, D. Patil
1
, S. Limaye
2
, R. Page
3
, A. Ranade
4
,
R. Patil
1
, V. Datta
1
, S. Patil
1
, V. Mhase
1
, S. Apurwa
1
,S.Pawar
1
, R. Datar
1
.
1
Datar Cancer Genetics Limited, Research and Innovation, Nasik, India;
2
Kokilaben Dhirubai Ambani Hospital, Medical Oncology, Mumbai, India;
3
Worcester Polytechnic Institute, Bioengineering, Worcester, USA;
4
Avinash
Cancer Clinic, Medical Oncology, Pune, India
Background: Doxorubicin and Epirubicin are two anthracycline agents
commonly used in treatment of breast cancers. However, chemoresistance
towards these agents and subsequent treatment failures are commonly
reported. There are presently no means for real-time monitoring of innate and
acquired chemoresistance. Repetitive invasive biopsies to obtain tumor
tissue for in-vitro chemoresistance profiling (CRP) or viable tumor are not
feasible. We describe a non-invasive approach for CRP using peripheral
blood Circulating Tumor Associated Cells (C-TACs).
Materials and Methods: We obtained 15 mL peripheral blood from 1034
known cases of breast cancers, among whom 353 were therapy naïve and
681 were pretreated. Viable C-TACs were enriched and harvested from
PBMCs using an epigenetically active media that selectively kills normal cells
and simultaneously confers survival benefit on apoptosis-resistant cells of
tumorigenic origin. Surviving cells (C-TACs) confirmed by immunostaining
(EPCAM+, CK+, CD45±, GCDFP+). Viable C-TACs were seeded into multi-
well plates and treated with Doxorubicin or Epirubicin and surviving C-TAC
fraction was measured to determine % cell-death and chemoresistance.
Results: Among therapy naïve patients (n = 353), innate resistance
towards Doxorubicin and Epirubicin was observed in 44% and 46% of
samples respectively (overall innate resistance = 45%). Among pretreated
patients (n = 681), acquired resistance towards Doxorubicin and Epirubicin
was observed in 81% of samples.
Conclusion: Our study demonstrates the feasibility of CRR profiling of C-
TACs in therapy naïve and pretreated patients. Adoption of C-TAC –CRR
profiling can non-invasively provide real time oversight towards treatment
selection, monitoring of drug resistance and timely therapeutic course
correction.
Conflict of interest:
Ownership: Rajan Datar is the Founder of Datar Cancer Genetics Limited.
Other Substantive Relationships: Ajay Srinivasan, Dadasaheb Akolkar,
Darshana Patil, Revati Patil, Sanket Patil, Vishakha Mhase, Vineet Datta,
Sachin Apurwa and Sushant Pawar are full time employees of Datar Cancer
Genetics Limited.
303 Poster
Real-time non-invasive chemoresistance profiling of circulating tumor
associated cells in breast cancers to determine resistance towards
mitotic inhibitors
A. Srinivasan
1
, D. Akolkar
1
, D. Patil
1
, S. Limaye
2
, R. Page
3
, A. Ranade
4
,
R. Patil
1
, S. Patil
1
, V. Mhase
1
, V. Datta
1
, S. Apurwa
1
,S.Pawar
1
, R. Datar
1
.
1
Datar Cancer Genetics Limited, Research and Innovations, Nasik, India;
2
Kokilaben Dhirubai Ambani Hospital, Medical Oncology, Mumbai, India;
3
Worcester Polytechnic Institute, Bioengineering, Worcester, USA;
4
Avinash
Cancer Clinic, Medical Oncology, Pune, India
Background: Paclitaxel, Docetaxel and Vinorelbine exert anti-tumor activity
by interfering with microtubule dynamics, leading to mitotic arrest. Though
these agents are commonly used in treatment of breast cancers, therapy
failures are noted due to innate and acquired chemoresistance. Real-time
monitoring of chemoresistance towards such treatment agents is an unmet
clinical need since conventional methods for chemoresistance profiling
(CRP) necessitate invasive biopsies to obtain viable tumor tissue. We
evaluated the utility of peripheral blood Circulating Tumor Associated Cells
(C-TACs) for real-time non-invasive CRP in breast cancers.
Materials and Methods: We obtained 15 mL peripheral blood from 1034
known cases of breast cancers, among whom 353 were therapy naïve and
681 were pretreated. Viable C-TACs were enriched and harvested from
PBMCs using an epigenetically active media that selectively kills normal cells
and simultaneously confers survival benefit on apoptosis-resistant cells of
tumorigenic origin. Surviving cells (C-TACs) confirmed by immunostain-
ing (EPCAM+, CK+, CD45±, GCDFP+). Viable C-TACs were seeded into
multi-well plates and treated with Paclitaxel, Docetaxel or Vinorelbine.
Surviving C-TAC fraction was measured to determine % cell-death and
chemoresistance.
Results: Innate resistance towards Docetaxel, Paclitaxel and Vinorelbine
was observed in 42%, 59% and 56% of samples respectively in therapy
European Journal of Cancer 138, Suppl. 1 (2020) S18–S124 S76
Abstracts, EBCC 12 Posters A

naïve patients’samples. Acquired resistance towards Docetaxel, Paclitaxel
and Vinorelbine was observed in 78%, 72% and 66% of pretreated patients’
samples.
Conclusion: Our study demonstrates the feasibility of CRR profiling of C-
TACs in therapy naïve and pretreated patients. Adoption of C-TAC –CRR
profiling can non-invasively provide real time oversight towards treatment
selection, monitoring of drug resistance and timely therapeutic course
correction.
Conflict of interest:
Ownership: Rajan Datar is the Founder of Datar Cancer Genetics Limited.
Other Substantive Relationships: Ajay Srinivasan, Dadasaheb Akolkar,
Darshana Patil, Revati Patil, Sanket Patil, Vishakha Mhase, Vineet Datta,
Sachin Apurwa and Sushant Pawar are full time employees of Datar Cancer
Genetics Limited.
304 Poster
BRCA variant classification of ClinVar submitter content from ENIGMA,
ARUP laboratories and German cancer consortium compared to MH
BRCA
®
and correlation with response to PARP inhibition in MH GUIDE
®
K. Stecker
1
, U. Schmidt-Edelkraut
2
, J. El Hokayem
1
, M. Hartenfeller
3
,
F. Diella
2
, M. Stein
3
, J. Hermanns
4
, T. Holtrup
5
, S. Hettich
5
, T. Soldatos
6
,
D. Jackson
6
, S. Brock
3
, C. Meisel
3
.
1
Molecular Health GmbH, Scientific
Field Support, Heidelberg, Germany;
2
Molecular Health GmbH, Biomedical
Curation, Heidelberg, Germany;
3
Molecular Health GmbH, Research,
Heidelberg, Germany;
4
Molecular Health GmbH, Project Office, Heidelberg,
Germany;
5
Molecular Health GmbH, Sales and Business Development,
Heidelberg, Germany;
6
Molecular Health GmbH, Innovation, Heidelberg,
Germany
Background: Germline mutations in BRCA1 and BRCA2 genes confer a
high risk for the hereditary breast and/or ovarian cancer (HBOC) syndrome,
whereas both germline and somatic mutations are predictive biomarkers for
PARP inhibition. MH BRCA
®
classifies variants based on ACMG guidelines.
Clinical interpretation of NGS results by MH GUIDE
®
provides clinicians with
treatment recommendations to cancer based on expert curated biomarker
knowledge.
Material and Methods: MH BRCA
®
was compared to ClinVar submitter
content from ENIGMA, the international consortium of investigators on
clinical significance of BRCA1/2 variants, the ARUP laboratories, a clinical
testing lab of the university of UTAH, and the German Cancer Consortium
(combined total of 7840 BRCA1/2 variants). In each validation dataset the
concordance-rate was calculated, and discordant variant interpretations
were analyzed. Finally, based on functional evidence for DNA damage
response, we assessed the ACMG classification with the predicted response
to PARP inhibition by MH GUIDE
®
.
Results: In three independent validation datasets, MH BRCA
®
demon-
strated a concordance-rate between 74 and 99%. Subset analysis of the
pathogenic/likely pathogenic variants showed almost 100% concordance of
MH BRCA
®
with clinically assessed pathogenicity (4975 out of 4976
variants). Moreover, in the ARUP laboratories dataset, a re-classification of
variants of uncertain significance (VUS) was found in 32 out of 342 variants
(9%). The analysis of the ENIGMA dataset revealed that 9 variants are either
re-classified from VUS or change their classification from likely benign to
likely pathogenic based on functional evidence provided by the proprietary
variant annotation database of Molecular Health. Last, we assessed the
accordance of MH BRCA
®
variant classifications with treatment-decisions in
MH GUIDE
®
regarding PARP inhibition. The comparison demonstrated a
complete coverage of pathogenic classified variants with predicted response
to treatment. Interestingly, low-efficacy of PARP inhibition due to moderately
impaired homologous recombination repair activity was predicted in a subset
of variants classified as pathogenic due to hypomorphic BRCA1/2 mutations.
Conclusion: We showedthat MH BRCA
®
provides a standardized ACMG-
guided process for assessment of pathogenicity by concordant classification
of pathogenic BRCA1/2 variants with ClinVar submitter content of ENIGMA,
ARUP laboratories and the German Cancer Consortium. Moreover, we
identified clinically relevant likely pathogenic BRCA1/2 variants due to re-
classification in accordance with the ACMG-guided assessment using in-
house expert curated variant annotations. In addition, we showed that the
contextualization of BRCA1/2 variants classified as pathogenic with MH
GUIDE
®
supports the treatment strategy of PARP inhibition.
Conflict of interest:
Other Substantive Relationships: All authors/co-authors are employees of
Molecular Health GmbH.
305 Poster
Interaction of PAFAH and beta-catenin in BRCA1 mutant breast cancer
Y. Liao
1
,D.Mayr
2
, E. Schmoeckel
2
, A. Hester
1
, T. Kolben
1
, C. Zeder-Göß
1
,
R. Würstlein
1
, N. Harbeck
1
, S. Mahner
1
, U. Jeschke
1,3
, F. Trillsch
1
,
B. Czogalla
1
.
1
Department of Obstetrics and Gynecology, Breast Center,
University Hospital, LMU Munich, Munich, Germany;
2
Institute of Pathology,
Faculty of Medicine, LMU Munich, Munich, Germany;
3
Department of
Obstetrics and Gynecology, University Hospital Augsburg, Augsburg,
Germany
Background: Aberrant Wnt/beta-catenin signaling is a well-established
characteristic of breast cancer implicated in tumorigenesis and metastasis,
whereas platelet-activating factor acetylhydrolase (PLA2G7/PAFAH) is not
well understood in breast carcinogenesis so far. This study analyzes the
functional relationship between PAFAH and beta-catenin in breast cancer
tissue.
Material and Methods: PLA2G7/PAFAH expression was determined in
five breast cancer cell lines on mRNA- and protein-level. Beta-catenin
staining was performed in MDA-MB-231 (TNBC, BRCA1 wildtype) and
HCC1937 (TNBC, BRCA1 mutant) cell lines before and after siRNA
knockdown of PAFAH. Cell viability and proliferation were analysed via
MTT and BrdU assay after PAFAH knockdown. PAFAH and beta-catenin
expression was analyzed in BRCA1 wildtype and BRCA1 mutant breast
cancer tissue by immunohistochemistry using the IR-scoring System.
Results: mRNA and protein expression of PLA2G7/PAFAH were
significantly higher in HCC1937 cell line than in BRCA1 wildtype cell lines.
HCC1937 cells showed a distinct membranous beta-catenin staining
compared to the BRCA1 wildtype cell line MDA-MB-231 that showed
cytoplasmic/nuclear staining. After siRNA knockdown of PAFAH, HCC1937
cells lost this specific staining pattern. Viability and proliferation of HCC1937
cells were significantly improved after PAFAH knockdown. In breast cancer
tissue, nuclear PAFAH and membranous beta-catenin staining showed a
strong correlation (cc = 0.794, p < 0.001) with higher expression of both in
BRCA1 mutant cases (both p < 0.001).
Conclusions: Our data suggest a functional relationship between PAFAH
and the Wnt/beta-catenin pathway in BRCA1 mutant breast cancer and may
help to improve the understanding of breast cancer carcinogenesis.
No conflict of interest.
306 Poster
A Breast 3D model as a possible tool for non-invasive tumour
localization in breast surgery
P. Gouveia
1,2
, S. Bessa
3
, H. Oliveira
3
, E. Batista
1
, M. Aleluia
1
,J.Ip
1
,
J. Costa
1
,L.Nuno
4
, D. Pinto
1
, C. Mavioso
1
, J. Anacleto
1
, N. Abreu
1
,
P. Morgado
5
, M. Martinho
1
, J. Teixeira
3
, P. Carvalho
3
, J. Cardoso
3
,
C. Alves
1
, F. Cardoso
1
,M.Joa
̃o Cardoso
1,3,4
.
1
Champalimaud Clinical
Center, Breast Unit, Lisbon, Portugal;
2
University of Lisbon, Faculty of
Medicine, Lisbon, Portugal;
3
University of Porto, Inesc Tec, Oporto, Portugal;
4
Universidade NOVA de Lisboa, Nova Medical School, Lisbon, Portugal;
5
AI4MEDIMAGING, Research and development, Braga, Portugal
Background: For tumour location and surgical planning physicians need to
convert 2-D information into a 3D approach. Wire guided surgery is the most
common method for preoperative localization of breast cancer lesions. Other
localization methods can also be used, such as radioactive seeds, carbon
tattooing or ultrasound guided clips, but they are all invasive procedures. In
this work, we aimed at building an individualized 3D digital breast model
integrating the breast torso and tumor location through MRI/3D fusion
algorithms to help in pre-operative tumour locationwithou t the use of invasive
procedures.
Material and Methods: Patients with Tis/T1-T3 breast cancer proposed
for breast conservative treatment at the Champalimaud Clinical Centre
between April 2017 and January 2019 were assessed for inclusion in the
current study. Selected patients were proposed for image capturing with 3D
torso surface scans in the standing position with hands on hips; breast MRI
with gadolinium contrast was performed –patient in prone position with arms
up.
An image acquisition protocol was designed and applied to a validation
group to acquire breast Magnetic Resonance Imaging (MRI) and 3D surface
scan data using surface markers on the patient’s breasts and torso. Breast
MRI/3D surface scan fusion algorithms (a second-stage free form
deformation versus biomechanical model (BM)) with pose transformation
were applied to simulate patient-specific 3D digital breast models with
quantitative analysis of breast morphology and algorithm validation.
Results: A total of sixteen patients were selected and included in the
current study. Of these, seven patients were included in the validation group.
European Journal of Cancer 138, Suppl. 1 (2020) S18–S124 S77
Abstracts, EBCC 12Posters A