2014 San Antonio Breast Cancer Symposium

1508 Pages·2014·3.24 MB·English

2014 San Antonio Breast Cancer Symposium
Publication Number: S1-01
Title: TransCONFIRM: The correlative analysis of breast tumors from patients with advanced hormone receptor positive disease
identifies a genetic signature associated with decreased benefit from single agent fulvestrant
Rinath M Jeselsohn1, William T Barry1, Jin Zhao1, Gilles Buchwalter1, Cristina Guarducci2, Ilenia Migliaccio2, Chiara Biagioni2,3,
Martina Bonechi2, Naomi Laing4, Yuri Rukazenkov4, Eric P Winer1, Myles Brown1, Angelo Di Leo2,3 and Luca Malorni2,3.
1Dana-Farber Cancer Institute, Boston, MA; 2Hospital of Prato, Istituto Toscano Tumori, Prato, Italy; 3"Sandro Pitigliani" Medical
Oncology Unit, IstitutoToscano Tumori, Prato, Italy and 4AstraZeneca Pharmaceuticals, Macclesfield, United Kingdom.
Body: Introduction: Several multi-gene expression based assays have been developed to assess the prognosis and predict
response to endocrine treatments in early stage hormone receptor positive (HR+) breast cancer. Although a significant number of
patients with metastatic ER+ disease will not respond to endocrine treatments, molecular assays to predict response in this
setting are limited. In addition, tissue specimens of metastatic lesions for molecular studies are not always available. In this study
we sought to identify a molecular profile in the primary tumors of patients who developed disease recurrence that could predict
response to endocrine treatment in metastatic disease.
Methods: We used the primary breast tumor samples from a subgroup of patients participating in the randomized phase III
CONFIRM trial, which compared 500mg versus 250mg of fulvestrant in post-menopausal women with HR+ advanced breast
cancer. Formalin- fixed paraffin embedded tumors were collected from 130 of the participants and were centrally reviewed for ER,
PR, HER2 and Ki67. RNA was sufficient for gene expression profiling in 112 of the cases using the NuGEN Ovation FFPE WTA
System and Affymetrix HTA 2.0 GeneChip. The majority of the patients in this analysis developed metastatic disease during
adjuvant endocrine treatment (N=55) or had de-novo metastatic disease (N=39) versus relapse after adjuvant treatment (N=18).
The association between gene expression and progression free survival (PFS) was investigated using a multivariate Cox
proportional hazard model adjusting for statistically significant clinicopatholgical factors. In addition we performed pathway-level
analysis and evaluated the PAM50 subtype predictor and Risk of Relapse (ROR) score.
Results: The median PFS was 8 months in our cohort. HER2 level by immunohistochemistry above 1+, high PR level, defined as
Allred score of above 6, and Ki67 of above 50% were significantly associated with PFS and were included in the multivariate
model. Dose of fulvestrant was not associated with PFS in this cohort. We identified a signature of 25 genes that is inversely
associated with PFS on fulvestrant treatment (FDR 20%). When compared to other published datasets of breast cancer tumors,
these genes are enriched in tumors with poor outcome and triple negative cancers. Pathway analysis revealed an association
between activation of the EGFR pathway and decreased PFS (P=0.01). PAM50 subtypes varied with the luminal subtype being
the most common (65%) and were generally concordant with the clinical subtype. However, we did not detect a significant trend
between PAM50 subtype or ROR score and PFS or overall survival.
Conclusions: In this cohort of patients with early and de-novo metastatic disease we identified a gene signature in the primary
tumors that is associated with decreased response to fulvestrant treatment in metastatic disease. This signature warrants further
validation to determine it’s predictive value and potential to assist in treatment decision making for patients with HR+ metastatic
disease.
2014 San Antonio Breast Cancer Symposium
Publication Number: S1-02
Title: Prognostic effects of gene mutation in estrogen receptor positive breast cancer
Obi L Griffith1, Malachi Griffith1, Jingqin Luo1, Jasreet Hundall1, Christopher A Miller1, David E Larson1, Robert Fulton1, Richard K
Wilson1, Shuzhen Liu2, Samuel Leung2, Torsten O Nielsen2, Elaine R Mardis1 and Matthew J Ellis1. 1Washington University School
of Medicine, St Louis, MO and 2University of British Columbia, Vancouver, BC, Canada.
Body: Background: Relationships between recurrent somatic mutations and outcome in estrogen receptor positive (ER+) breast
cancer has not been extensively studied as the original discovery efforts were from either heterogeneously treated patients or
follow up was too brief. Targeted massively parallel sequencing (MPS) analysis was therefore conducted on DNA extracted from
archived formalin-fixed breast primaries from a cohort of over 600 patents from British Columbia treated with five years of
adjuvant tamoxifen monotherapy and followed for over 10 years (Nielsen et al CCR 16:5222, 2010).
Methods: Genes were selected for targeted sequencing by meta-analysis of five large-scale breast cancer sequencing studies
and manual review of breast cancer literature. In total 83 genes were identified and 3286 probes were designed to tile across all
known exons. Minimum starting input DNA was 50ng (mean=189.1ng). Illumina sequencing libraries were constructed, indexed,
pooled, and enriched for target sequences by hybrid-capture followed by paired-end 100bp reads. The Genome Modeling System
was used to perform single-tumor somatic variant prediction. Variant calls were filtered to include only targeted regions and
exclude variants with global mutant allele frequencies greater than 0.1% in 1000 genomes or NHLBI exome datasets.
Kaplan-Meier analysis and multivariable analysis (clinical features and intrinsic subtype by qPCR) was performed for
breast-cancer-specific and relapse free survival.
Results: A total of 638 samples met minimum quality controls of 80% targeted space covered at 20X or greater. On average each
sample had 332M of aligned bases and a mean coverage of 134.3X. In total 7,159 variants were identified including 3,696
missense, 494 nonsense, and 1,047 frameshift insertions or deletions. Preliminary results indicate significant associations
between mutation status and improved survival for PIK3CA, ARID1B, ERBB3, MAP3K1 and GATA3 or worse survival for PTEN,
DDR1, TP53 and JAK2. Five Y537N/C, two E380Q and 5 potentially novel ligand-binding-domain mutations were identified in
ESR1. Such mutations were recently reported to be associated with resistance to hormone therapy but were discovered here in
as much as 1.9% of pre-treatment samples. Analysis will be presented regarding the use of relapse events to differentiate
passenger from driver mutations.
Conclusion. Multiple recurrently mutated genes have both positive and negative associations with prognosis in tamoxifen
montherapy treated breast cancer populations. Associations with poor outcome suggest that PTEN, DDR1, and JAK2 are high
priorities for pharmacological interventions.
Table 1: Mutations and survival in ER+ breast cancer
Gene P-Value Hazard Ratio
PTEN 0.002 2.11
PIK3CA 0.007 0.69
DDR1 0.008 2.41
ARID1B 0.01 0.57
ERBB3 0.02 0.36
MAP3K1 0.01 0.5
TP53 0.04 1.48
GATA3 0.04 0.44
JAK2 0.05 2.1
2014 San Antonio Breast Cancer Symposium
Publication Number: S1-03
Title: Identification of base pair mutations and structural rearrangements acquired in breast cancer metastases including a novel
hyperactive ESR1-DAB2 fusion gene specifically in hormone-resistant recurrence
Ryan J Hartmaier1,2, Shannon L Puhalla1, Steffi Oesterreich1,2, Amir Bahreini1,3, Nancy E Davidson1,2, Adam M Brufsky1 and Adrian
V Lee1,2,3. 1Women's Cancer Research Center, University of Pittsburgh Cancer Institute & Magee Women's Research Center,
Pittsburgh, PA; 2University of Pittsburgh, Pittsburgh, PA and 3University of Pittsburgh, Pittsburgh, PA.
Body: DNA structural variations (SVs) are a major source of genetic instability in cancer, but they remain understudied.
Large-insert mate-pair sequencing (MPS) is a powerful method designed to detect SVs, even in highly repetitive regions. Using
MPS and other methods, we performed a comprehensive analysis of genomic alterations in breast cancer progression.
Matched primary/recurrent frozen tumor samples from 6 patients, including two patients from our rapid autopsy program with
multiple metastatic tissues (20 total samples; average 5.5 years to recurrence) were examined by multiple large-insert library (3-5,
5-8, 8-12kb) MPS to identify metastatic acquired SVs. This was supplemented with RNAseq (n=15), whole exome sequencing
(n=18; ~75x), whole genome sequencing (n=3; 40-65x), and SNP arrays.
A relatively small fraction (~10%) of somatic single nucleotide variants (SNVs) in the primary tumor were identified in matched
metastatic samples, and the majority of metastatic SNVs were not found in the matched primary tumor. This indicates that a rare
sub-clone colonizes the metastatic site and evolves extensively before becoming clinically evident. For example, in one patient
with an ER+ tumor who initially declined anti-estrogen therapy, the recently described ESR1 Y537S mutation was not present in
the primary tumor or in metastatic disease 5 years later. However, after extensive anti-estrogen treatment for metastatic disease,
the mutation was identified at rapid autopsy, indicating that this mutation can be acquired even after initial metastatic spread.
We observed extensive patient-to-patient variability in the number and types of SVs. In general, the overall pattern of SVs was
remarkably similar between matched primary and metastatic samples, however, we identified a number of metastatic specific SVs
that likely contribute to disease progression. Specifically, in one patient with an ER+ primary tumor treated with adjuvant
Tamoxifen, we identified a novel fusion gene between ESR1 (estrogen receptor-a, ERa) and DAB2 (disabled-2) only in a lymph
node recurrence. RT-PCR and western blot analysis confirmed that the fusion RNA/protein was expressed/translated only in the
recurrent disease. The fusion retains the DNA-binding domain (DBD) and hinge region of ERa while the ligand-binding domain
(LBD) is replaced with the majority of DAB2. We hypothesized that this is a functional genetic alteration conferring
ligand-independent ERa-mediated signaling and growth. Confirming this, in vitro ERE-Tk-luc reporter assays showed that the
ESR1-DAB2 fusion has ligand-independent activity that is 13-290x higher than wild-type ERa. Chromatin immunoprecipitation
assays in metastatic tissue from tumors with mutant ERa show strong enrichment for ERa at classical ERa target genes. We are
currently assessing the genome-wide binding of ESR1-DAB2 and the functional contribution of DAB2 to the fusion protein.
This study represents the most comprehensive analysis to date of genomic changes in breast cancer progression and indicates
extensive changes occur during metastatic spread. A number of acquired changes likely represent therapeutically targetable
metastatic dependencies.
2014 San Antonio Breast Cancer Symposium
Publication Number: S1-04
Title: Exome sequencing of post-menopausal ER+ breast cancer (BC) treated pre-surgically with aromatase inhibitors (AIs) in the
POETIC trial (CRUK/07/015)
Pascal Gellert1, Corrinne V Segal1, Qiong Gao1, Tiandao Li2, Christopher A Miller2, Elaine Mardis2, Lesley-Ann Martin1,
Christopher Holcombe3, Anthony Skene4, Judith Bliss1, John Robertson6, Ian Smith5, Mitch Dowsett5 and POETIC Trial
Management Group and Trialists7. 1Institute of Cancer Research, London, United Kingdom; 2Genome Institute at Washington
University, St Louis, MO; 3Royal Liverpool University Hospital, Liverpool, United Kingdom; 4Royal Bournemouth Hospital,
Bournemouth, United Kingdom; 5Royal Marsden Hospital, London, United Kingdom; 6University of Nottingham, Nottingham,
United Kingdom and 7POETIC Trial Management Group and Trialists.
Body: Aims
1. To determine the variability of mutational profiles and sub-clonality in core-cut biopsies from ER+ BC and the impact of
2-weeks’ AI therapy on these.
2. To identify mutations or patterns of mutations associated with poor anti-proliferative response to AI treatment.
Background
DNA alterations may lead to de novo and acquired resistance to medical therapies including AIs. Assessing this requires single
time-point or sequential sampling usually with core-cuts but there is little information on their ability to represent mutational
profiles or sub-clonal structure. We studied this in paired biopsies from ER+ BC primaries in 60 selected postmenopausal patients
from the Peri-Operative Endocrine Therapy for Individualising Care (POETIC) trial (CRUK/07/015) before and after 2-weeks’
non-steroidal AI or no AI (randomised 2:1).
Methods
DNA was extracted from RNAlater-preserved diagnostic and surgical 14-gauge core-cut samples and peripheral blood from 20 no
AI (Control) and 40 AI-treated patients (15 poor and 25 good Ki67-responders [PR and GR, respectively]). Patients with low ER+
BC or unsuppressed estradiol on treatment were not considered. Exome sequencing (Illumina HiSeq 2000) achieved >60%
coverage across the exome at 15x depth. Variants were validated by re-sequencing (median >100x) together with 79 genes of
interest curated from COSMIC and selected publications. Statistically significant genes (SMGs) were determined using MuSiC.
Sub-clonality was analysed by SciClone.
Results
Good quality exomes were obtained on 102 samples including 44 pairs (control n=14; PR n=10; GR n=20). There were 5684
mutations (including 3616 missense and 1322 silent) affecting 3261 genes. SMGs in this series were PIK3CA (35.3%), TP53
(27.5%), CDH1 (13.8%), HEATR7B2 (8.8%), GATA3 (5.9%), CENPF (5.9%), MAP3K1 (5.9%), MAP2K4 (4.9%), HTR1A (2.9%)
and C22orf23 (1%). PR had more mutations than GR (median 65 vs 36, p=0.04). More PR than GR were HER2+ (5/14 vs 1/24,
p=0.019) and/or TP53-mutated (5/10 vs 3/20, p=0.08) but similar proportions were PIK3CA-mutated. The correlation of diagnostic
vs surgical variant allele frequencies was strong for the control (r=0.75) and treated (r=0.89) groups (for treated GR r=0.83; for
treated PR r=0.65). In the treated group there were fewer mutations at surgery vs diagnosis (p<0.026). PIK3CA and TP53
mutation status was identical between the paired samples in 41/44 and 40/44 cases; less frequently mutated genes showed lower
concordance. SciClone plots to infer sub-clonality were possible in 37 pairs; for 8 pairs (22%) there was clear evidence of at least
one sub-clone being present in only one core-cut sample.
Conclusion
This is the largest reported study of exome reproducibility in ER+ BC for mutation profiles based on core-cut biopsy. Multiple
sub-clones are identifiable in ER+ primary BC. In c.20% tumours, a single core-cut does not allow inference of all sub-clonal
populations, probably due to spatial heterogeneity. TP53 mutations but not PIK3CA mutations are associated with PR. Large
numbers of BC will be needed to identify any associations of lower frequency mutations with resistance. A trend to fewer
mutations after just 2 weeks AI needs confirmation.
2014 San Antonio Breast Cancer Symposium
Publication Number: S1-05
Title: In-depth genomic analysis of ER+ breast cancers during development of endocrine resistance
J Michael Dixon1, Arran K Turnbull1, Chris Fan2, Joel S Parker2, Xiaping He2, Laura Arthur1, Carlos Martinez-Perez1, Lorna
Renshaw1 and Charles Perou2. 1University of Edinburgh, Edinburgh, United Kingdom and 2Comprehensive Cancer Centre,
Chapel Hill, NC.
Body: Background: Aromatase inhibitors (AIs) have an established role in the treatment of estrogen receptor alpha positive
(ER+) post-menopausal breast cancer. Response rates are only 50-70% in the neoadjuvant setting and up to 40-50% of all
adjuvantly treated patients will eventually relapse. Mutations in certain genes have been previously shown to confer resistance to
therapy, and molecular subtype has associations with poor outcome (i.e. LumB and HER2E). In order to improve the outcomes of
non-responders or patients who become resistant to endocrine treatment, the identification of key mutations, and their interaction
with subtype, is crucial. Dynamic profiling of the same tumour demonstrating de novo or developing resistance after responding to
one or more lines of endocrine treatment in the neoadjuvant setting provides a unique opportunity to identify such genomic
changes.
Methods: This series is unique in that it includes 17 post-menopausal women with ER+ breast cancer treated with neoadjuvant
letrozole. 13 of these patients progressed on treatment or initially responded to treatment and then developed acquired resistance
and 4 responded well. Dynamic clinical response was assessed for each patient using periodic 3D ultrasound measurements
performed during treatment. Fresh tissue was taken before treatment and when the tumor was resistant to treatment (4 patients
had 2 biopsies, 9 patients had 3 and 4 patients had 4 available biopsies taken). RNA and DNA were extracted from tumour and
normal DNA obtained from either matched blood or normal lymphatic tissue. In total, 51 tumour samples were available and have
completed RNA-Seq, with exome sequencing shortly to be completed.
Results: From the RNA-seq data, the intrinsic subtype distribution was 9 LumA, 7 LumB, and 1 HER2-enriched; when stratified
according to response, the "progressors" were 7 LumB, 5 LumA and 1 HER2-enriched, while "responders" were 3 LumA and 1
LumB. When examined in an unsupervised hierarchical clustering analysis along with >800 TCGA Breast tumor samples, 13/17
patients had all of their samples grouped immediately together, suggesting that the overall tumor phenotype was maintained.
Interestingly, the most dominant change in gene expression was the observation that there were 5 "progressor" patients where
the pre-treatment sample was LumB and all subsequent samples were LumA; we only observed one instance of a patient starting
as LumA and changing to a LumB, who was also labelled as a "progressor". Full exome sequencing is underway and these
results will be presented.
Conclusion: Genomic analysis of progression suggests that an apparent "subtype shift" appears in a number of patients where a
shift to LumA is seen; this apparent change may be reflective of decreased proliferation rates caused by therapy, or the
acquisition of a true LumA phenotype. We cannot differentiate between these two hypotheses at this time, but expect that the
exome sequencing will help to differentiate between these two hypotheses given the large number of mutations and copy number
alterations that can differentiate between LumA vs. LumB.
2014 San Antonio Breast Cancer Symposium
Publication Number: S1-06
Title: Stromal tumor-infiltrating lymphocytes(S-TILs): In the alliance N9831 trial S-TILs are associated with chemotherapy benefit
but not associated with trastuzumab benefit
Edith A Perez1, Karla V Ballman2, S Keith Anderson2, E Aubrey Thompson1, Sunil S Badve3, Helen Bailey4 and Frederick L
Baehner4,5. 1Mayo Clinic, Jacksonville, FL; 2Mayo Clinic, Rochester, MN; 3Indiana University, Indianapoils, IN; 4Genomic Health
Inc, Redwood City, CA and 5University of California, San Francisco, CA.
Body: Background: Tumor-infiltrating lymphocytes (TILs) at diagnosis are reported to be prognostic in triple-negative breast
cancer (BC). Analysis of a small subset of 209 HER2+ patients (pts) with 49 events concluded that higher levels of S-TILs are
associated with increased trastuzumab benefit (Loi, 2014). Here we report the largest study to date evaluating S-TILs and their
prognostic and predictive association with clinical outcome in N9831 pts treated with either chemotherapy or chemotherapy plus
trastuzumab.
Methods: Samples assessed were from primary tumors of pts on N9831 arm A (standard ACfiT chemotherapy) and arm C
(concurrent chemotherapy with trastuzumab) (Perez, 2011). S-TILs were evaluated on H&E whole tumor slides by a single
pathologist with ~10% of cases read by two pathologists in tandem. The percent of stromal lymphocytic infiltrates (S-TILs) was
quantitated in deciles; ‡60% S-TILs was used for the categorical cutoff (Denkert, 2010). The association between S-TILs,
treatment (tx) and recurrence-free survival (RFS) was studied and the interaction between S-TILs, trastuzumab benefit and RFS
was calculated.
Results: 489 pts from arm A (chemo) and 456 pts from arm C (chemo with trastuzumab) were assessed and were similar to pts in
the overall trial; all had RFS information and a median follow-up of 4.4yr. Tumors from 54% of pts in arms A and C were HR+;
14% were node-negative. Tumors with high S-TILs were more likely to be hormone receptor-negative (p< 0.0001). In
multivariable analyses including nodal status, hormone receptor status, tx arm, tumor size, tumor grade, and age, ‡60% S-TILs
was significantly associated with RFS (HR 0.20; 95%CI 0.064–0.65, p=0.007) in arm A but not in arm C (HR 1.1; 95%CI
0.42–2.8, p=0.87); the interaction term of arm and ‡60% S-TILs was significant (p=0.042). Semi-continuous deciles were
associated with RFS in arm A (p<0.0002) but not in C (p=0.37). Hormone receptor status was an independent prognostic factor in
arm A (HR 0.61; 95%CI 0.41-0.93, p=0.02) but not in C (HR 0.79; 95%CI 0.44-1.41, p=0.42). In arm A the 10yr Kaplan-Meier
estimates for RFS were 90.9% and 64.5% for high S-TILs and low S-TILs pts, respectively (HR 0.23; 95%CI 0.073–0.73,
p=0.013). In arm C the 10yr Kaplan-Meier estimates for RFS were 80.0% and 80.1% for high S-TILs and low S-TILs pts,
respectively (HR 1.26; 95%CI 0.5–3.2, p=0.63).
Arm A (N=489) Arm C (N=456)
Variable HR (95% CI) p-value HR (95% CI) p-value
Nodal status
Lymph node(-) 1.00 (ref) <0.0001 1.00 (ref) 0.013
1-3+ 0.58 (0.25, 1.35) 2.99 (0.40, 22.70)
4-9 1.46 (0.64, 3.35) 3.29 (0.42, 25.80)
10+ 2.42 (1.04, 5.64) 8.22 (1.06, 63.60)
HR status
negative 1.00 (ref) 0.0198 1.00 (ref) 0.42
positive 0.61 (0.41, 0.93) 0.79 (0.44, 1.41)
S-TILs status
<60% 1.00 (ref) 0.007 1.00 (ref) 0.87
‡60% 0.2 (0.06, 0.65) 1.08 (0.42, 2.79)
Tumor grade
Grade 1 or 2 1.00 (ref) 0.50 1.00 (ref) 0.51
Grade 3 1.18 (0.73, 1.91) 1.24 (0.65, 2.39)
Tumor size 1.08 (0.98, 1.19) 0.12 1.00 (0.93, 1.07) 0.92
Age 0.99 (0.97, 1.00) 0.13 1 (0.98, 1.03) 0.87
Conclusions: In exploratory analyses from this subset HER2+ population from N9831, S-TILs were associated with RFS in
patients treated with chemotherapy alone, and were not shown to be associated with RFS in patients treated with chemotherapy
plus trastuzumab.
2014 San Antonio Breast Cancer Symposium
Publication Number: S1-07
Title: HER2 T cell dependent bispecific antibody (HER2-TDB) for treatment of HER2 positive breast cancer
Teemu T Junttila1, Ji Li1, Jennifer Johnston1, Maria Hristopoulos1, Robyn Clark1, Diego Ellerman1, Bu-Er Wang1, Yijin Li1, Mary
Mathieu1, Guangmin Li1, Judy Young1, Elizabeth Luis1, Gail Lewis Phillips1, Eric Stefanich1, Cristoph Spiess1, Andrew Polson1,
Bryan Irving1, Justin M Scheer1, Melissa R Junttila1, Mark S Dennis1, Robert Kelley1, Klara Totpal1 and Allen Ebens1. 1Genentech,
San Francisco, CA.
Body: Based on recent clinical success of tumor immunotherapies that block immune suppressive mechanisms to restore T cell
function, there is a profound interest in the clinical development of T cell targeted therapies. We have produced a
trastuzumab-based HER2 T cell dependent bispecific antibody (HER2-TDB) that conditionally activates T cells resulting in lysis of
HER2 expressing cancer cells at low picomolar concentrations. Due to its unique mechanism of action, which is unrelated to
HER2 signaling or sensitivity to chemotherapeutic agents, HER2-TDB can eliminate cells refractory to currently approved HER2
therapies. The potent anti-tumor activity of HER2-TDB was demonstrated using four model systems including MMTV-huHER2
and huCD3 transgenic mice. We demonstrate inhibitory effect of PD-L1 expression on the activity of bispecific T cell recruiting
antibodies. This resistance mechanism is reversed by anti-PD-L1 treatment and combination of HER2-TDB with anti-PD-L1
immune therapy resulted in enhanced inhibition of tumor growth, increased response rates and durable responses.
Significance:
This report presents a new immunotherapy for HER2+ breast cancer with an alternative, extremely potent mechanism of action
that is effective in cells resistant to current HER2 targeted therapies. Several significant advances are provided to bispecific T cell
recruiting antibodies: we characterize a critical resistance mechanism, a potential diagnostic marker, a novel transgenic efficacy
model and significantly improve the drug-like properties by using technology based on full length antibodies with natural
architecture. Finally we demonstrate the benefit of combining two immune therapies: direct polyclonal recruitment of T cell activity
together with inhibiting the T cell suppressive PD-1/PD-L1 signaling results in enhanced and durable long term responses.
2014 San Antonio Breast Cancer Symposium
Publication Number: S1-08
Title: Reduced tumor lymphocytic infiltration in the residual disease (RD) of post-neoadjuvant chemotherapy (NAC)
triple-negative breast cancers (TNBC) is associated with Ras/MAPK activation and poorer survival
Justin M Balko6, Carsten Denkert2, Roberto Salgado3, Martin O'Hely4, Peter Savas1, Paul A Beavis1, Phil K Darcy1, Susan
Combs5, David L Rimm5, Jennifer M Giltnane6, Monica V Estrada6, Melinda E Sanders6, Rebecca S Cook6, Kai Wang7, Vincent A
Miller7, Phillip J Stephens7, Roman Yelensky7, Joseph A Pinto8, Franco Doimi8, Henry Gomez9, Carlos L Arteaga6 and Sherene
Loi1. 1Peter MacCallum Cancer Center, Sydney, Australia; 2Charité University, Berlin, Germany; 3Institute Jules Bordet, Brussels,
Belgium; 4WEHI University, Melbourne, Australia; 5Yale University, New Haven, CT; 6Vanderbilt University, Nashville, TN;
7Foundation Medicine, Cambridge, MA; 8Oncosalud, Lima, Peru and 9Instituto Nacional de Enfermedades Neoplásicas (INEN),
Lima, Peru.
Body: Backgound: Tumor-infiltrating lymphocytes (TILs) are associated with improved prognosis in TNBCs, with several
retrospective analyses demonstrating that TNBCs with high baseline TILs have higher rates of pathologic complete response
(pCR) to NAC. Moreover, the TIL burden in the RD of patients who do not achieve pCR to NAC is also correlated with prognosis.
However, insight into the molecular pathways in TNBC which modulate heterogeneity in host anti-tumor immune responses is
lacking. To address this gap in knowledge, we analyzed TILs retrospectively in a cohort of clinically and molecularly characterized
TNBCs with RD after NAC.
Methods: TILs were scored in H&E stained slides by expert pathologists in the post-treatment tumors of 92 NAC-treated TNBC
patients with RD at the time of resection and in 44 matched baseline diagnostic biopsies. Genomic alterations in the RD were
assayed using targeted next-generation sequencing (tNGS) while selected transcriptional signatures were evaluated by
NanoString as previously published (Balko et al, Cancer Discovery 2014). Differences in pre- and post-NAC TILs were compared
between tumors harboring alterations in cell cycle, PI3K/mTOR, growth factor receptors, Ras/MAPK and DNA repair pathways.
Associations of TILs with transcriptional signatures were also tested.
Results: A strong positive association of TILs in NAC-treated specimens was observed with RFS (coxPH p=0.0001, relative risk
reduction of 3.4% for each % of TILs) and OS (p=0.0016; relative risk reduction of 2.8% for each % of TILs). In multivariate
analysis with stage, age, node status and RD tumor cellularity, TILs in the post-NAC disease remained a significant predictor of
RFS and OS (p=0.0008 and p=0.007, respectively). TILs tended to decrease with NAC in paired samples, although this decrease
was not statistically significant (p=0.07).
Genetic alterations in the Ras/MAPK (amplifications in KRAS, BRAF, RAF1 and truncations in NF1) and cell cycle pathway
(alterations in CCND1-3, CDK4, CDK6, CCNE1, RB, AURKA and CDKN2A) were associated with lower TILs in RD (p=0.005 and
p=0.05, respectively). A significant inverse linear correlation was detected between a transcriptional signature of Ras/MAPK
activation (Pratilas et al, PNAS 2009) and TILs in the RD (Spearman’s r=-0.42; p=0.00028). Total number of alterations of likely
functional significance detected by tNGS showed no association with TILs, suggesting that the association of Ras/MAPK
deregulation and cell cycle alterations with TILs may be a pathway-specific effect.
In TNBC cell lines, chemical inhibition of MEK transcriptionally up-regulated MHC-I and MHC-II molecules, while simultaneously
down-regulating mRNA expression of the immune checkpoint inhibitor PD-L1 (MDA-231 p=0.00002, BT549 p=0.0003, and
SUM159PT p=0.009). In vivo experiments confirming these associations are underway.
Conclusions: Our data suggest a strong correlation of Ras/MAPK pathway activation with immune-evasion and outcome in TNBC.
With additional mechanistic understanding, rational design of clinical trials combining MEK inhibitors with PD-L1 antibodies in
TNBC may be warranted.
2014 San Antonio Breast Cancer Symposium
Publication Number: S1-09
Title: A phase Ib study of pembrolizumab (MK-3475) in patients with advanced triple-negative breast cancer
Rita Nanda1, Laura Q Chow2, E Claire Dees3, Raanan Berger4, Shilpa Gupta5, Ravit Geva6, Lajos Pusztai7, Marisa Dolled-Filhart8,
Kenneth Emancipator8, Edward J Gonzalez8, Jennifer Houp8, Kumudu Pathiraja8, Vassiliki Karantza8, Robert Iannone8, Christine
K Gause8, Johnathan D Cheng8 and Laurence Buisseret9. 1University of Chicago, Chicago, IL; 2University of Washington, Seattle,
WA; 3UNC Lineberger Comprehensive Cancer Center, Chapel Hill, NC; 4Sheba Medical Center, Tel Hashomer, Israel; 5H. Lee
Moffitt Cancer Center and Research Institute, Tampa, FL; 6Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; 7Yale School of
Medicine, New Haven, CT; 8Merck & Co., Inc, Whitehouse Station, NJ and 9Institut Jules Bordet, Université Libre de Bruxelles,
Bruxelles, Belgium.
Body: Introduction: The PD-1 receptor-ligand pathway can be used by tumors to evade immune surveillance, thereby allowing
neoplastic growth. Pembrolizumab is a highly selective, humanized IgG4/kappa isotype mAb designed to block PD-1 interaction
with its ligands PD-L1 and PD-L2, thereby reactivating the immune system to eradicate tumors.
Methods: This is a multi-center, non-randomized trial of single agent MK-3475 treatment given intravenously at 10 mg/kg every 2
weeks, in patients with recurrent/metastatic triple-negative (ER, PR, and HER2 negative) breast cancer (TNBC). PD-L1
expression in tumor or stroma was required for study entry. PD-L1 status was determined by immunohistochemical analysis of
patient’s tumor tissues using the Merck proprietary 22C3 antibody. Primary objectives of this study were to determine the safety,
tolerability, and anti-tumor activity of MK-3475 in patients with PD-L1 positive, advanced TNBC. Secondary objectives included
assessments of progression-free survival, overall survival, and response duration. Adverse events (AEs) reported in any patient
receiving at least 1 dose of study treatment were monitored and graded using NCI CTCAE v. 4.0. Radiographic imaging was
obtained every 8 weeks and evaluated by both investigator and an independent radiologist to assess clinical responses as
defined by RECIST 1.1. This study (Clinicaltrials.gov: NCT01848834) is being conducted in conformance with Good Clinical
Practices.
Results: A total of 32 female patients with a median age of 50.5 years (range 29 – 72 years) with PD-L1 positive,
recurrent/metastatic TNBC were enrolled in the study. Most of these patients had received and progressed on multiple lines of
therapy for advanced disease. A preliminary analysis of data collected as of 23May2014 indicates that 5 patients (15.6%)
experienced at least one drug-related serious adverse event (SAE); each of 4 patients experienced one of the following: Grade 3
anemia, headache, aseptic meningitis or pyrexia, and a fifth patient experienced disseminated intravascular coagulation (DIC)
with thrombocytopenia and decreased blood fibrinogen. The patient who experienced DIC died. Preliminary analysis of data
collected from investigators as of 23May2014 indicates that no patient had a complete response, 16.1% of patients had a partial
response, 9.7% had stable disease, and 64.5% had progressive disease. As of 23May2014, all but one of the responders, in
addition to three patients with stable disease, remain on treatment.
Conclusion: This is the first report of clinical activity of an immune checkpoint inhibitor in TNBC. The preliminary results from this
study suggest that single agent MK-3475 is a well-tolerated and effective treatment with significant therapeutic activity in a subset
of heavily pre-treated patients with recurrent/metastatic triple-negative breast cancer.