Annals of Oncology

abstracts
(Idylla, Biocartis) and MALDI-TOF (UltraSEEK, Agena Bioscience) accurately
identified 96% (all 100 copies & 23/25 50 copies input) and 93% (23/25 100 copies & 23/
25 50 copies input) of samples, respectively. Conversely, the digital PCR assay (KRAS
PrimePCR ddPCR,Bio-RadLaboratories,Inc.)wasnon-specific,identifyingthe
wrong mutation in 8 different mutation/allele frequency combinations. Turnaround time
from clinical sample to result ranged from ∼2 hours (Idylla CE-IVD) to 1 day (cobas®
CE-IVD) to >1 week for most NGS assays, while the level of required laboratory expertise
ranged from minimal (Idylla CE-IVD) to high (NGS platforms).
Conclusions: This comprehensive parallel assessment used high molecular weight
cell-line DNA as a model to address key questions for a clinical laboratory when
implementing routine KRAS testing. As most of the technologies are available for other
molecular biomarkers, results may be informative for other diagnostic functions.
Legal entity responsible for the study: AstraZeneca
Funding: AstraZeneca
Disclosure: J.L. Sherwood, H. Brown, A. Kohlman: Employee of and shareholder in
AstraZeneca London. A. Schreieck: institution received financial support for molecular
analysis from AstraZeneca. B. Claes: Reports a patent pending for isolation of nucleic
acids. B. Agrawal: Employee of Vela Diagnostics.A.O.H. Nygren: Employee of and
shareholder in Agena Bioscience. All other authors have declared no conflicts of interest.
92P
Impact of Kras mutant subtypes on PD-L1 expression in lung
adenocarcinoma
A.T. Falk 1 , N. Yazbeck 1 , L. Thon 1 , N. Guibert 2 , V. Hofman 3 , K. Zahaf 3 , V. Lespinet 3 ,
O. Bordone 4 ,V.Tanga 4 , K. Washetine 4 , C. Cohen 5 , N. Venissac 5 , J. Mouroux 5 ,
C-H. Marquette 6 , P. Brest 1 , M. Ilié 7 , P. Hofman 7
1 INSERM U1081/UMR CNRS 7284, Team 3, Institute for Research on Cancer and
Ageing, Nice, France, 2 Thoracic Oncology, CHU Toulouse, Hôpital de Larrey,
Toulouse, France, 3 Laboratory of Clinical and Experimental Pathology / Liquid
Biopsy Laboratory, Pasteur Hospital, Nice, France, 4 Hospital-Integrated Biobank
(BB-0033-00025), Pasteur Hospital, Nice, France, 5 Department of Thoracic
Surgery, Pasteur Hospital, Nice, France, 6 Department of Pneumology, Pasteur
Hospital, Nice, France, 7 INSERM U1081/UMR CNRS 7284, Team 3, Institute for
Research on Cancer and Ageing, Nice, France
Background: Clinical responses to immune checkpoint blockade by anti-PD-1/PD-L1
monoclonal antibodies in non-small-cell lung cancer (NSCLC) may be associated with
PD-L1 expression. This study was undertaken to determine the expression profile of
PD-L1 in patients with Kras-mutant lung adenocarcinoma (LUAD) and to investigate
the activation of Kras codon subtypes as a mechanism of PD-L1 regulation.
Methods: PD-L1 expression was evaluated by IHC (SP142 clone, Ventana) on 117
LUAD (Kras WT , n = 51; Kras mut , n = 66). Stable cell lines were generated by
transfection of Kras-G12D, G12V, G12C and WT plasmids into Beas2B bronchial cells.
Results: IHC analysis showed higher expression of PD-L1 in both tumor and immune
cells in Kras-mutant LUAD compared with Kras WT tumors (37% vs. 18%; P = 0.005).
Kras-mutant PD-L1 + tumors had increased CD66b + neutrophil infiltrates and lower CD8 +
T-cell content than PD-L1 − tumors. In vitro, mutant Kras led to significantly higher
cell-surface PD-L1 expression and PD-L1 transcripts, notably in Kras G12C and Kras G12V
cells, suggesting PD-L1 transcriptional regulation. There was differential activation of
NF-kB, ERK and Pi3k/Akt pathways between Kras-mutant subtypes. In addition, PD-L1
was upregulated 3-fold by stimulation with IFNγ, independently of the Kras codon
subtypes. Instead, hypoxia significantly increased PD-L1 expression in Kras G12C and
Kras G12D cells. Co-culture experiments with human PBMCs from healthy patients were
performed to determine the functional effect of altered PD-L1 expression. Increased PD-L1
expression by tumor cells induced by Kras mutations led to decreased PBMCs proliferation
and increased apoptosis. An anti-PD-L1 checkpoint inhibitor is currently being tested as
single agent or in combination with ERK or PI3K inhibitors in our Kras cell models.
Conclusions: PD-L1 is expressed in 37% of Kras mutant LUAD, suggesting PD-L1 as a
therapeutic target in this subset. According to the Kras mutation subtype, potential
drugs targeting the NF-kB, ERK or Pi3k/Akt pathways may additionally increase the
antitumor adaptive immune responses.
Legal entity responsible for the study: N/A
Funding: Pasteur Hospital, Nice
Disclosure: All authors have declared no conflicts of interest.
93P
KRAS status and HER2 targeted treatment in advanced gastric
cancer
E. Shinozaki, H. Osumi, K. Chin, D. Takahari, M. Ogura, T. Ichimura,
T. Matsushima, T. Wakatsuki, I. Nakayama, K. Yamaguchi
Department of GI Oncology, Cancer Institute Hospital of JFCR, Tokyo, Japan
Background: Trastuzumab targeted on HER2 has been shown to confer overall
survival benefit adding to fluoropyrimidine (Fp) plus CDDP in HER2-positive
advanced gastric cancer (AGC). HER2 is known to make the formation of heterodimer
with EGFR(HER1), HER3 and HER4. HER2 containing heterodimer activates the
common downstream of HER family such as MAPK pathway via RAS. RAS and BRAF
status has been established as predictive biomarkers for anti EGFR treatment in
metastatic colorectal cancer. However it remains unclear that the implications for these
status and HER2 targeted treatment in AGC. In this study we attempted to assess the
relationship with the efficacy of trastuzumab including treatment and mutational status
of HER family signaling pathway.
Methods: Out of 100 patients received Fp plus CDDP with trastuzumab as 1st-line
between March 2011 and November 2015, total 77 patients with sufficient specimen for
DNA extraction were enrolled in this analysis. Multiplex genotyping of HER family
common downstream was performed on archival samples using Luminex Assay
(MEBGEN and GENOSEARCH Mu-PACK, MBL) for KRAS and NRAS including
exon 2, 3 and 4, PIK3CA and BRAF. Tumor response was re-assessed by the
investigator retrospectively by RECIST1.1.
Results: KRAS mutation of exon2 was detected in only 6 patients of 77 patients (7.8
%). No mutations were found in NRAS, PIK3CA and BRAF in this HER2 positive
AGC series. An overall RR and the disease control rate (DCR) in KRAS wild type (WT)
vs. mutant type (MT) were following, RR; 66.2% vs. 16.7%, DCR; 87.3% vs. 66.7%,
respectively (CR2/0, PR 45/1, SD 15/3, PD 9/2). The median PFS and OS in KRAS WT
vs. MT were as followed, 8.9 months (m) vs. 3.6 m and 20.8 m vs. 10.3 m, respectively.
KRAS MT showed extremely shorter PFS and OS compared with KRAS WT
(P = 0.00008 and 0.0008).
Conclusions: Our data suggested HER2-positive AGC harbored KRAS mutation at the
low frequency. KRAS mutation might predict poor prognosis as receiving HER2
targeted treatment. Further investigation was warranted to confirm the predictive value
of KRAS status in HER2-positive AGC treated with trastuzumab to fluoropyrimidine
plus CDDP. We will present additional analysis of KRAS amplification in this cohort at
the convention.
Legal entity responsible for the study: N/A
Funding: Japanese Foundation for Cancer Research
Disclosure: All authors have declared no conflicts of interest.
94P An extended KRAS mutation test for the detection of 28
common mutations in FFPET and plasma specimens
J. Li 1 , C. Hoeppner 2 , S. Gan 1 , A. Blair 1 , K. Min 1 , A. Sims 2 , A. Tietz 2 , M. Vinas 2 ,
T.T. Rehage 2 , K. Malhotra 2 , H. Halait 2 , V. Brophy 1
1 Genomics and Oncology, Roche Molecular Systems, Pleasanton, CA, USA,
2 Assay Development, Roche Molecular Systems, Pleasanton, CA, USA
Background: The KRAS oncogene is frequently mutated in human cancers. In
addition to KRAS codons 12, 13, 61 hotspot mutations, recent clinical trial data
revealed that mutations in KRAS codons 59, 117, 146 also predict poor response to
anti-epidermal growth factor receptor (EGFR) therapy in patients with metastatic
colorectal cancer (mCRC). Detection of an extended set of KRAS mutations in
colorectal cancer tissues is now accepted clinical practice.
Methods: The KRAS Mutation Test v2 (Life Science Research, LSR) is a real-time PCR
assay, which detects 28 mutations in codons 12, 13, 59, 61, 117 and 146 of the KRAS
gene in DNA derived from formalin-fixed, paraffin-embedded tissue (FFPET) as well
as plasma specimens. Mutations are detected by allele-specific amplification in three
multiplex PCR reactions on the cobas z 480 analyzer. LOD studies were performed
using contrived tissue and plasma samples. For method correlation, 301 FFPET
specimens and 636 plasma specimens were tested with the KRAS Mutation Test v2
(LSR) and MiSeq (Illumina) sequencing as the reference method.
Results: Using preliminary analysis parameters, the KRAS test has shown it can detect
at least 1% mutant sequence in a background of wild-type DNA for tissue, and at least
100 mutant sequence copies per mL for plasma in the LOD studies. Preliminary
method correlation results revealed >98% overall concordance for tumor FFPET and
>90% overall concordance for cell-free DNA samples with MiSeq reference data. Final
results will be presented.
Conclusions: The KRAS Mutation Test v2 (LSR) demonstrated strong initial analytical
performance for the detection of 28 KRAS mutations. It is a sensitive, robust and reliable
assay with a quick turnaround time which can be used on both tissue and liquid biopsies.
Legal entity responsible for the study: Roche Molecular Systems
Funding: Roche Molecular Systems
Disclosure: All authors have declared no conflicts of interest.
95P
Annals of Oncology
Plasma YKL-40 as a biomarker for poor prognosis in patients
with metastatic colorectal cancer treated with 3. line
cetuximab and irinotecan
B.V. Jensen 1 , K-L.G. Spindler 2 , I.J. Christensen 3 , J.V. Schou 4 , D.L. Nielsen 1 ,
A. Jakobsen 5 , E. Høgdall 3 , P. Pfeiffer 6 , M.K. Yilmaz 7 , J. Johansen 1
1 Department of Oncology, Herlev and Gentofte Hospital, Herlev, Denmark,
2 Oncology, Aarhus University Hospital, Aarhus, Denmark, 3 Department of
Pathology, Herlev and Gentofte Hospital, Herlev, Denmark, 4 Department of
Oncology, Herlev and Gentofte Hospital, Copenhagen, Denmark, 5 Oncology, Vejle
Hospital Sygehus Lillebaelt, Vejle Sygehus, Vejle, Denmark, 6 Department of
Oncology, Odense University Hospital, Odense, Denmark, 7 Department of
Oncology, Aalborg University Hospital, Aalborg, Denmark
Background: YKL-40 (CHI3L1) plays a major role in inflammation and angiogenesis.
In patients with metastatic colorectal cancer (mCRC) we wanted to test if high
pretreatment levels of plasma YKL-40 and change of YKL-40 during therapy with
vi28 | abstracts Volume 27 | Supplement 6 | 2016