P1

SFX-01 targets STAT3 signalling to reverse endocrine resistance in ER+ breast cancer

Bruno M Simões1, Chiara Chiodo1, Tiago Moreira1, Daniel Conole2, Scott Lovell2, Denis Alferez1, Rachel

Eyre1, Katherine Spence1, Angélica Santiago‐Gómez1, Aida Sarmiento‐Castro1, Andy Sims3, Elisabetta

Marangoni4, Edward Tate2, Sacha J Howell1, Robert B Clarke1 1University of Manchester, Manchester, United Kingdom. 2Imperial College London, London, United

Kingdom. 3University of Edinburgh, Edinburgh, United Kingdom. 4Institut Curie, Paris, France

Introduction: Estrogen receptor (ER) positive breast cancer (BC) is frequently sensitive to endocrine

therapy. However, multiple mechanisms of endocrine therapy resistance have been identified,

including induction of cancer stem-like cell (CSC) activity (Simões et al, 2015). SFX‐01, a stabilised

formulation of synthetic sulforaphane, targets CSCs in preclinical models and has recently been shown

to reverse endocrine resistance clinically (STEM trial; ESMO 2019 abstract #5696).

Aim: To define the effects of SFX-01 on ER+ breast CSCs and elucidate its mechanism of action.

Methods: We investigated SFX-01 effects on breast CSC activity using mammosphere formation

efficiency (MFE) and aldehyde dehydrogenase (ALDH) activity in patient samples and patient-derived

xenograft (PDX) tumours. Early (HBCx34) and metastatic (BB3RC31) ER+ PDX tumours were treated

with SFX-01 (300mg/Kg/day) alone or in combination with tamoxifen (TAM, 10 mg/kg/day) or

fulvestrant (FULV, 200 mg/kg/week). Gene and protein expression of treated samples was assessed.

Results: SFX‐01 reduced MFE of both ER+ primary, n=12, (0.19% vs control 0.52%: p<0.001) and

metastatic, n=15, patient samples (0.43 vs control 0.93%: p<0.001). Analysis of ALDH+ cells from

endocrine-resistant patient samples revealed activation of STAT3 signalling.

Both TAM and FULV increased MFE and ALDH activity of PDXs, which was reversed by combination

with SFX‐01. SFX-01 significantly reduced tumour initiating cell frequency in secondary transplants at

limiting dilution and reduced the formation of lung micrometastases in PDX in NSG mice.

Mechanistically, both TAM and FULV induced STAT3 phosphorylation. SFX-01 suppressed this

phospho‐STAT3 induction by anti‐estrogen treatment. Expression of STAT3 target genes; MUC1,

OSMR and CCND1 predicted poor prognosis in patients with ER+ tumours and was inhibited by SFX-01

in patient samples.

Conclusion: Our data demonstrate the potential of SFX-01 for clinically meaningful improvements to

endocrine therapy in ER+ BC by inhibiting STAT3 signaling and reversing CSC-mediated resistance.

P2

Transcriptomic landscape of metastatic, unreactive and reactive cancer-free lymph nodes from

breast cancer patients

Thomas Hardiman1, Jelmar Quist1, Natalie Woodman1, Julie Owen1, Anargyros Megalios1, Anthony

Cheung1, Robert Harris1, Elinor Sawyer1, Sophia Karagiannis1, Todd Auman2, Joel Parker2, Jo Spencer1,

Cheryl Gillett1, Arnie Purushotham1, Anita Grigoriadis1 1King's College London, London, United Kingdom. 2The University of North Carolina at Chapel Hill,

Chapel Hill, USA

Introduction: Lymph node (LN) involvement is a major prognostic factor in breast cancer (BC). Changes

in the LN can precede metastasis. We recently reported on morphological effacements, in both

cancer-free and metastatic LNs, to be informative for the risk prediction of developing distant

metastasis in LN-positive BC patients.

Aims: By exploring the molecular and histological landscape of LNs, we seek to decipher how these

immune organs prepare the niche for metastatic spread.

Methods: Clinico-pathological data, including extensive immune and stroma characteristics

from cancer-free and metastatic LNs, and primary tumours were available for 143 LN-positive BC

patients. Using a previously established immune-stroma IHC score, patients were considered to be

either at high or low risk for developing distant metastasis. As a pilot, RNA-sequencing (150 million

paired-end reads) was performed for 5/143 patients, for whom (i) a cancer-free ‘unreactive’ (i.e: no

or few germinal centres (GCs)) LN, (ii) a cancer-free ‘reactive’ (i.e: several enlarged GCs) LN and a

partially metastatic LN were selected. The latter was macro-dissected into the (iii) cancer-free and (iv)

metastatic portions.

Results: In accordance with morphological changes, the transcriptomic landscape across these four

histologically distinct LNs varied. The uninvolved ‘reactive’ LNs in high and low risk patients were highly

divergent, with 343 genes differentially expressed (Q value < 0.05), including pathways involved in GC

formation and regulation. Manually curated B-cell activation and differentiation, along with IgG and

IgE class switching gene sets, revealed differing expression patterns across the histologically distinct

LNs between high and low risk patients.

Conclusions: The transcriptomic landscape of histologically distinct LNs reflect a diverse range of

immune cells present during the invasion of cancerous cells. Further planned RNA-sequencing in

combination with multiplexed immune cell characterisation are warranted and will define key turning

points for BC progression.

P3

Preventing the progression of breast cancer bone metastasis by CDK inhibition

Lubaid Saleh1, Penelope D Ottewell1, Janet E Brown1, Steven L Wood1, Nicola J Brown1, Caroline

Wilson2, Catherine Park1, Ingunn Holen1 1University of Sheffield, Sheffield, United Kingdom. 2Weston Park Hospital, Sheffield, United Kingdom

Background: CDK 4/6 inhibitors have demonstrated significant improved survival for patients with

oestrogen receptor positive breast cancer. However, the benefits of these promising agents in patients

with bone metastasis from triple negative BC (TNBC) remain to be established. We investigated the

effects of the CDK 4/6 inhibitor palbociclib using in vivo models of breast cancer bone metastasis,

alone and in combination with the anti-resorptive agent zoledronic acid (Zol).

Methods: MDA-MB-231 TNBC cell line sensitivity (IC50) to palbociclib was confirmed in vitro. Analysis

of the retinoblastoma protein was conducted by western blot. Modelling bone metastasis in vivo,

5x104 MDA-MB-231 luc2+ve cells were injected (intra-cardiac) into 6-week old female BALB/c nude

mice. Daily vehicle or Palbociclib (100mg/kg/p.o) treatment for 28 days +/- 100mg/kg/week of Zol

(i.p.) commenced on day 3 and tumour growth detected by IVIS imaging.

Results: We determined the IC50 of palbociclib to be 850nM in MDA-MB-231 cells, which caused

significant inhibition of Retinoblastoma protein phosphorylation in vitro. In vivo, vehicle treated

animals exhibited significantly higher numbers of skeletal tumours than palbociclib treated animals

(4.7±0.7 vs 0.375±0.26/mouse, p=0.0004). This was reflected by a higher proportion of tumour-

positive animals in the vehicle group (8/10) compared to palbociclib group (3/8). However, tumours

emerged in palbociclib treated animals within 7 days of treatment cessation; this was not inhibited by

administration of Zol or by a second cycle of palbociclib treatment, suggesting the emergence of

resistant tumours growing independently of bone-derived factors.

Discussion: Daily administration of palbociclib was required to prevent outgrowth of disseminated

TNBC cells into overt bone metastasis in vivo. Current palbociclib treatment strategies are based on a

3 week-on, 1 week-off cycle, our data suggest that this break may contribute to acquired resistance

as demonstrated by continuous tumour growth during the second palbociclib cycle. Funded by BCN.

P4

'A putative role for bone morphogenetic protein antagonist Gremlin in HER2 positive breast cancers'

Catherine Zabkiewicz, Rachel Hargest, Lin Ye

Cardiff University, Cardiff, United Kingdom

Introduction: Human epidermal growth factor receptor 2(HER2) over-expression denotes poor

prognosis in breast cancers and despite treatments targeting HER2, a significant number of patients

have disease relapse and death. Epidermal growth factor(EGF) and bone morphogenetic protein(BMP)

signalling pathways are thought to co-regulate breast cancer proliferation and metastasis, and in lung

cancer, active BMP signalling denoted resistance to treatment with an EGFR inhibitor.BMP signalling

may therefore be of importance in those malignancies that are treated by modulating or blocking

EGF/HER2.As an endogenous antagonist and regulator of BMP signalling, Gremlin is of interest in HER2

positive breast cancers, and as yet completely unexplored in this context.

Methods: The Gene Expression Omnibus (GEO) public gene expression microarray database was

examined for correlations of expression between GREM1 and HER2 in breast cancers and related to

clinicopathological parameters and outcomes in HER2 positive breast cancers. The largest cohort of

327 breast cancers (GSE 20685) was examined in more detail. For in vitro experiments GREM1

overexpression plasmid and pEF control plasmid were transduced into BT474 HER2 positive breast

cancer cells. After selection with blasticidin, overexpression was confirmed with PCR, qPCR and

western blotting. In vitro cell function tests for growth, transwell migration, and transwell invasion

were performed in triplicate. Quantitative real time PCR (qPCR) was used to examine expression of

markers of epithelial to mesenchymal transition (EMT) in control and GREM1 Overexpression BT474

cells. BT474 cells were treated with a small molecule HER2 inhibitor (CP724714) and the effect on

GREM1 expression examined with qPCR and PCR.A lentiviral vector HER2 knockdown and scramble

control were also transduced into BT474 cells, and after confirmation of knockdown, the effect on

GREM1 expression examined with qPCR and PCR.

Results: GREM1 expression significantly positively correlates with HER2 expression in breast tumours

(Pearson correlation co-efficient 0.12, p = 0.03) and is higher in HER2 positive patients with metastasis

compared to those without (p = 0.04). HER2 positive patients with higher than median primary tumour

GREM1 expression, have poor relapse free and distant metastasis free survival in comparison to those

with lower than median GREM1 expression(p = 0.03 and p < 0.0001 respectively)(see table ) .

Survival Outcome

Low GREM1

Expression

Median

Survival

(Months)

Low GREM1

Expression

patient number

(n)

High GREM1

Expression

Median Survival

(Months)

High GREM1

Expression

patient number

(n)

P value

Relapse Free

Survival(RFS) 23 120 15 131 0.03

Survival Outcome

Low GREM1

Expression

Median

Survival

(Months)

Low GREM1

Expression

patient number

(n)

High GREM1

Expression

Median Survival

(Months)

High GREM1

Expression

patient number

(n)

P value

Distant

Metastasis Free

Survival (DMFS)

98 56 15 63 <0.0001

In vitro, GREM1 overexpression in BT474 HER2 positive cells resulted in significantly increased cell

growth and transwell migration(p <0.05 and p <0.0001 respectively)but did not significantly alter

transwell invasion(p = 0.8) compared to control.GREM1 overexpression in these cells downregulated

E-Cadherin and up-regulated markers of EMT such as SNAI1 and SNAI2 compared to

control. Knockdown of HER2, or treatment with a small molecule HER2 specific inhibitor resulted in

down regulation of GREM1 expression compared to control.

Conclusions: GREM1 expression correlates with HER2 expression in breast cancer. A high GREM1

expression in primary tumours indicates a worse clinical outcome for HER2 positive breast cancer

patients. In vitro results support this, as high GREM1 expression promotes cellular growth and

migration in HER2 positive cancer cells, as reflected in the downregulation of E Cadherin and

upregulation of gene expression associated with EMT. Interestingly as GREM1 expression appears to

reduce when HER2 is inhibited, there is a suggestion of a regulatory mechanism between GREM1 and

EGF/HER2 cell signalling that could be of importance in clinical response to HER2 blockade treatments

and targeted therapy that would be worthy of further investigation.

P5

Inhibition of breast cancer bone metastasis through Interleukin-1B regulated tumour-associated

innate immune response

Claudia Tulotta, Charlotte K Moore, Diane V Lefley, Ana E Amariutei, Khawla Ahmed, Amy R Spicer-

Hadlington, Penelope D Ottewell

University of Sheffield, Sheffield, United Kingdom

Background: Interleukin-1B (IL-1B) is a pro-inflammatory cytokine and a biomarker for breast cancer

metastasis to secondary organs, including bone. We have previously shown that production of IL-1B

by breast cancer cells drives bone metastasis. Here, we consider the role of IL-1B from the

microenvironment. We hypothesise that the interplay between tumour- and microenvironment-

derived IL-1B affects primary tumour growth and breast cancer bone metastasis though regulating

anti-tumour immune responses.

Methods: In a humanised model of breast cancer metastasis to bone, IL-1B signalling was inhibited

using an anti-IL-1B antibody (10 mg/kg/14 days) or the IL1R1 antagonist Anakinra (1 mg/kg/day). In

syngeneic models, Anakinra (1 mg/kg/day) was administered in combination with the anthracycline

doxorubicin (2mg/kg/week) and zoledronic acid (100ug/kg/week). Effect on breast cancer metastasis

and immune response were analysed by Nanostring. IL-1B/IL1R1 knock out (KO) mouse models were

used to identify the role of IL-1B in breast cancer development at the primary site and formation of

bone metastasis.

Results: Anthacyclines are documented as inducing anti-tumour immunity. Combining Anakinra with

doxorubicin and zoledronic acid reduced primary tumour growth (P=0.0009 Anakinra vs combination

treatment), and inhibited the development of bone metastases by 71.41%. Microenvironment-

derived IL-1B displayed dual and opposing roles in primary breast cancer growth (2.3-fold increase in

IL-1B KO mice) and formation of bone metastases (95% reduction in IL-1B KO mice). These findings

recapitulated the effect observed upon treatment with anti-IL-1B antibody and Anakinra

demonstrating IL-1B driven increases in tumour growth at the primary site and prevention of tumour

growth in bone. Microenvironment-derived IL-1B promoted anti-tumour immunity at the primary site,

while supporting the development of overt metastases in bone, by regulating myeloid cell infiltration.

Conclusions: Combining drugs that stimulate anti-tumour immune response with IL-1B targeting may

provide a useful therapeutic approach to inhibit breast cancer growth and bone metastasis.

P6

Improved breast cancer radio-response with novel hypoxia activated prodrugs

Radhika Aiyappa1, Klaus Pors2, Stewart G Martin1 1Nottingham Breast Cancer Research Centre, School of Medicine, University of Nottingham,

Nottingham, United Kingdom. 2Institute of Cancer Therapeutics, Faculty of Life Sciences, University of

Bradford, Bradford, United Kingdom

Introduction: Radiotherapy is often used in breast cancer treatment, both in the neoadjuvant and

adjuvant setting. Unfortunately recurrences occur indicating treatment failure. Hypoxia is often found

in breast cancer and is associated with radiotherapy resistance, metastasis and poor survival. A

strategy to improve radioresponse in breast cancer is to target hypoxic tumour cells using hypoxia

activated pro-drugs (HAPs). This study investigates, AQ4N (control and comparator) and a novel AQ4N

analogue, KP167, as potential radiosensitisers in breast cancer.

Methods: 2D monolayer cultures and 3D spheroid models (>200 μM diameter) of triple negative

(MDAMB-231, MDAMB-468) and luminal (MCF-7 and T47D) breast cancer, were used to assess in vitro

cytotoxicity of AQ4N and KP167. Radiosensitisation was assessed by treating spheroids with single

agent IC50 doses of AQ4N or KP167 for 96 hours then irradiation with single doses of 195kV X-rays,

ranging 1-8Gy with subsequent disaggregation and seeding for assessment of clonogenic survival.

Results: In comparison to 2D normoxic treatment, AQ4N and KP167 exhibit potent single agent anti-

cancer activity in 3D spheroids with AQ4N IC50 values of 20-25 μM in triple negative and 5-7.5 μM in

luminal cells. KP167 IC50 values were 20 and 0.25 μM in MDAMB-231 and MDAMB-468 and 5 and 2

μM in MCF-7 and T47D spheroids respectively. Breast cancer cells responded as expected to radiation

with D0 values ranging from (0.8-0.9) Gy and α/β values in the range of 11-17. Significant

radiosensitisation was achieved by both AQ4N and KP167 in 3D spheroids compared to 2D monolayer

cultures. In comparison to AQ4N (SERs ranging 1.2-1.6), the novel analogue KP167 substantially

sensitised breast cancer cells with SER’s of 2.7 in MDAMB-231, 2.3 in MDAMB-468, 1.5 in MCF-7 and

1.8 in T47D cells.

Conclusions: Data indicate that KP167 is a highly potent and effective breast cancer radiosensitiser,

especially in triple-negative breast cancer models.

P7

Epigenetic regulation of breast cancer treatment response by a chromatin remodelling complex

protein AT-Rich Interaction Domain 1A, ARID1A

Sankari Nagarajan1, Shalini V Rao1,2, Igor Chernukhin1, Joseph Sutton1, Danya Cheeseman1, Shanade

Dunn3,4, Evangelia K Papachristou1, Jose-Enrique Gonzalez prada1, Dominique-Laurent Couturier1,

Sanjeev Srinivas Kumar1, Kamal Kishore1, Chandra Sekhar Reddy Chilamakhuri1, Silvia Elena Glont1,

Emily Archer Goode1, Cara Brodie1, Naomi Guppy5, Rachael Natrajan5, Alejandra Bruna1, Carlos

Caldas1, Alasdair Russell1, Rasmus Siersbaek1, Kosuke Yusa4,6, Jason S Carroll1 1Cancer Research UK Cambridge Institute, Cambridge, United Kingdom. 2Dept of Clinical and Molecular

Medicine, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology,

Trondheim, Norway. 3Astrazeneca, Cambridge, United Kingdom. 4Wellcome Trust Sanger Institute,

Hinxton, United Kingdom. 5The Breast Cancer Now Toby Robins Research Centre, The Institute of

Cancer Research, London, United Kingdom. 6Institute for Frontier Life and Medical Sciences, Kyoto

University, Kyoto, Japan

Introduction: Breast cancer patients possess better survival in past few years but the problem of drug

resistance still continues to affect the patient outcome. Studying the drug resistance mechanisms can

help us in improving their outcome.

Objectives and methods: We attempt to study the drug resistance mechanisms presented in ER+

breast cancer cell lines during Tamoxifen and Fulvestrant treatment using functional genome-wide

CRISPR screenings and transcription factor occupancy analysis.

Results: Using genome-wide CRISPR screenings in oestrogen receptor (ER)-positive breast cancer cells,

we discovered ATP-ase dependent chromatin remodelling complex proteins including ARID1A as the

most critical factors required for response to two classes of ER antagonists. ARID1A perturbation led

to endocrine resistance. Unexpectedly, ARID1A was also the top candidate for response to the BET

inhibitor JQ1, but in the opposite direction, where loss of ARID1A sensitized breast cancer cells to BET

inhibition. We show that ARID1A is a transcriptional co-repressor which binds chromatin at ER cis-

regulatory elements and can physically associate with ER. However, ARID1A elicits repressive activity

in an enhancer-specific, but ER-independent manner. The ER pioneer factor, FOXA1 promotes the

binding of ARID1A on key regulatory elements. Deletion of ARID1A resulted in loss of Histone

Deacetylase 1 (HDAC1) binding, increased histone 4 lysine acetylation and subsequent BRD4-driven

transcription and growth.

Conclusion: ARID1A mutations are more frequent in treatment-resistant disease and our findings

provide mechanistic insight into this process whilst revealing rational treatment strategies for these

patients using BET inhibitors.

P8

Reprogramming of amino acid transporters induces Aspartate and Glutamate dependency and

drives endocrine resistance in breast cancer

Marina Bacci1, Nicla Lorito1, Luigi Ippolito1, Matteo Ramazzotti1, Matteo Parri1, Bruno M Simões2,

Lesley-Ann Martin3, Elisabetta Marangoni4, Massimiliano Mazzone5, Paola Chiarugi1, Andrea Morandi1 1University of Florence, Florence, Italy. 2Manchester Cancer Research Centre, University of

Manchester, Manchester, United Kingdom. 3The Breast Cancer Now Toby Robins Research Centre, The

Institute of Cancer Research, London, United Kingdom. 4Institut Curie, Paris, France. 5VIB Center for

Cancer Biology, University of Leuven, Leuven, Belgium

Introduction: Endocrine therapy (ET) is the standard of care for oestrogen receptor (ER)-positive

breast cancers. Unfortunately, 30-40% of women relapse with ET-resistant (ETR) disease.

Aims: Understanding the metabolic behaviour during response and resistance to ET may offer a series

of predictive biomarkers and potential targetable pathways to be exploited to combat or delay ETR in

ER positive breast cancers.

Methods: Isogenic ER+ cell lines mimicking resistance to different endocrine agents (i.e., tamoxifen,

fulvestrant and aromatase inhibitors) were subjected to metabolic/metabolomic characterisation. In

vitro data were confirmed using patient-derived xenograft (PDX) material (sensitive and resistant to

ET), mouse models and retrospective clinical data.

Results: Gene expression analysis performed in ETR cells compared to parental cells revealed a

downregulation of the neutral and basic amino acid transporter SLC6A14 governed by enhanced miR-

23b-3p expression, resulting in impaired amino acids uptake. Biochemical and biological assays

showed that this deregulation of amino acid metabolism is supported by autophagy activation and an

increased import of acidic amino acids (i.e., aspartate and glutamate) mediated by the cognate SLC1A2

transporter in ETR cells. We then analysed aspartate and glutamate destiny by 13C-U-glutamate and 13C-U-aspartate flux analysis and detection of isotopologues using LC-MS. Notably, enhanced TCA cycle

intermediates were paralleled by a significant labelling of uridine-5'-triphosphate (DNA synthesis) and

glutamine (protein synthesis), indicating that both amino acids fuel the TCA and interrelated

anaplerotic pathways. Additionally, Seahorse analysis showed that the deprivation of aspartate and

glutamate in ETR cells significantly impaired oxygen consumption rate and subsequent oxidative

potential. The clinical significance of these findings is validated in large cohorts of ET-treated patients

and in PDX.

Conclusion: Targeting amino acid transporters reprogramming resensitises ETR cells to therapy and

impairs ETR cell aggressive features (e.g., proliferation, invasion, clonogenic capacity), including their

metastatic potential in in vivo experiments.

P9

Neratinib Reduces HER2+/Luminal B Breast Cancer Stem Cell Activity and Inhibits Resistance by

Blocking Efflux Mechanisms

Nathan J Hull1, Robert B Clarke2, Nigel J Bundred1,3 1University of Manchester, Manchester, United Kingdom. 2Breast Biology Group, University of

Manchester, Manchester, United Kingdom. 3University Hospital of South Manchester, Manchester,

United Kingdom

Introduction: Breast cancer stem cells (BCSCs) drive drug resistance and recurrence of disease

following treatment. BCSCs resist chemotherapy by various mechanisms including increases in

expression of the ATPase drug efflux pumps ABCB1 and ABCG2, reducing intracellular Paclitaxel and

Mitoxantrone drug concentration, respectively. Neratinib a pan-HER tyrosine kinase inhibitor is

reported to block both ABCB1 and ABCG2 pump activity. We aimed to determine the effect of

Neratinib on BCSC activity (primary and self-renewal) and determine if the mechanism includes pump

inhibition.

Methods: Effects of neratinib alone and in combination with tamoxifen, paclitaxel or mitoxantrone on

anoikis resistance culture were evaluated in multiple breast cancer cell lines. The mammosphere

forming efficiency (MFE) and generation of secondary mammospheres (self-renewal) were calculated.

Blockage of the ABCG2 by neratinib was assessed using flow cytometry analysis of side population.

Statistical significance was determined using unpaired t-test and one-way ANOVA.

Results

Cell line CSC activity Neratinib Paclitaxel Neratinib +

Paclitaxel

BT474 MFE 0.43 (P<0001) 0.30 (P<0001) 0.05 (P<0001)

MDA-MB-231 MFE 0.87 (ns) 0.26 (P<0001) 0.15 (P<0001)

SKBR3 MFE 0.96 (ns) 0.84 (ns) 0.69 (P<0.05)

MCF7 MFE 1.15 (ns) 0.85 (ns) 0.74 (P<0.05)

MCF7 Self-renewal 0.98 (ns) 0.22 (P<0.01) 0.07 (P<0.001)

Table 1. The MFE/self-renewal of various breast cancer cell lines following treatment normalised to

the control. ns, not significant

We found that combining neratinib with mitoxantrone further decreased BCSC activity than either

treatment alone in MCF7, MCF7HER2, SKBR3 and MDA-MB-453 cells. Neratinib also reduced the side

population in BT474 and SKBR3 cells in a dose response manner, demonstrating inhibition of ABCG2

activity.

Conclusions

These findings suggest that neratinib has the ability to overcome drug resistance caused by the ABCB1

and ABCG2 pumps. They also provide insight into a role for neratinib to enhance paclitaxel

chemotherapy in triple negative BCSCs.

P10

Tumour Cell and Immune Profiling of Lymph Nodes from Breast Cancer Patients

Inga Hansine Rye1, Kanutte Huse2, Oslo Breast Cancer Research Consortium (OSBREAC)3, Ellen

Schlichting4, Øystein Garred5, June Myklebust2, Hege Russnes1 1Department of Cancer Genetics, Institute for Cancer Research, Oslo University Hospital, OSLO,

Norway. 2Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital,

OSLO, Norway. 3*, *, Sweden. 4Department of Oncology, Oslo University Hospital, OSLO, Norway. 5Department of Pathology, Oslo University Hospital, OSLO, Norway

Introduction: A sentinel lymph node (SN) is the primary node draining the tumour and is assumed to

be affected early in the metastatic process. Detection of metastases in SN is a standard procedure in

breast cancer diagnostics based on microscopic evaluation (morphology and immunohistochemistry),

determining the need for removal of all axillary glands for inspection which again is crucial for tailoring

adjuvant therapy. The identification by microscopy is time-consuming and has a risk for false negative

results. We hypothesize that the immune profile of SN changes with the presence of tumour cells,

even at very low frequencies (micrometastases). By using a multi marker approach to characterize

thousands of cells from sentinel lymph nodes with and without metastases we aimed at identifying

both tumour cells but also characterize tumour specific immune responses. This dual approach might

provide an opportunity for a more sensitive test for SN diagnostics.

Aims:

1. Detect the presence of tumour cells by mass cytometry

2. Identify the effect of metastatic breast cancer cells on lymph node immune profiles

Material and Methods: We established a mass cytometry assay containing 38 markers (antibodies) to

combine immune profiling with identification of breast cancer cells. Cell suspensions from 18

metastatic axillary lymph nodes (ALNmet), 15 metastatic sentinel lymph nodes (SNmet) and 16 non-

metastatic sentinel lymph nodes (SN) from breast cancer patients from the clinical observational trial

Oslo2 (early, operable breast cancer patients representing all subtypes) were successfully analyzed by

the multimarker panel at a single cell resolution.

Results: Tumour cells gated as PanKer+CD45- were identified in 24 lymph nodes (14/18 ALNmet, 8/15

SNmet and 2/16 SN). In total, detection of tumour cell by mass cytometry has a 73% concordance with

pathology. We identified 23 immune subpopulations by manual gating. Comparison of immune

subpopulation abundance between the three lymph nodes groups revealed a significant skewing in

the CD4/CD8 population toward a memory phenotype in the ALNmet samples. The presence of T-

regulatory cells (Treg) and antigen presenting cells (APC) cells were found increased and associated

with the presence of tumour cells, and the activation markers CD38, TIGIT and HLA-DR were found

increased in Treg, CD8RO and CD4RO in the ALNmet samples compared to the SNneg and SNmet

samples.

Conclusion: We identified a significant difference in immune cell composition in lymph nodes with

and without metastases (ALNmet compared to SN samples), and we identified increased expression

of activation markers in T cells from ALNmet samples. We were also able to detect and identify

micrometastases in most lymph nodes where morphological examination had identified them, but in

addition found tumour cells in two samples scored as negative. The results will be validated in a larger

sample series.