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Background
PD-L1 expression levels on carcinoma cell lines were
identified by flow cytometry. For analytical validation,
H727, BT474 H358, HCC78 and H820 cells were spiked into
whole blood into CEE-SureTM blood collection tubes.
Samples were prepared in replicate and on different days,
incubated overnight and then processed. The leukocyte
fraction was incubated with our pan-CTC antibody capture
cocktail, labeled with biotinylated secondary antibody,
followed by enrichment in our streptavidin coated
microfluidic channels. Enriched cells were stained for
DAPI, cytokeratin, CD45, PD-L1 (clone 28-8) and CEE-
Enhanced (pan-CTC stain). After automated fluorescence
scanning, spiked tumor cells within the microchannel were
identified and average PD-L1 intensities were quantified
for each cell. Cut-off criteria were determined.
Fig. 3: PD-L1 stain on microfluidic enriched lung cancer cells spiked
into blood. Antibody stains are presented in following order:
UL: combined; cytokeratin (green); CD45 (red); CEE-Enhanced (yellow);
LL: PDL1 (yellow) (number indicates average intensity); DAPI (blue);
synthetic combined
Results Results
Table 1: Analytical performance of PDL1 assay. Negative control cell
lines and cell lines expressing high, medium, and low levels of PD-L1 are
depicted
Analytical Validation
Conclusions
References
• The Biocept PD-L1 assay can accurately detect PD-L1
expressing cells spiked into whole blood samples.
• The Biocept platform is able to capture CTCs from
cancer patient blood samples and identify the cells
expressing PD-L1 expressing CTCs
• The ability to detect PD-L1 expressing CTCs in blood
affords a way to identify patients likely to benefit from
immune therapy, as well as monitor the efficacy of such
treatments.
Phillips T et al, Development of an automated PD-L1
immunohistochemistry (IHC) assay for non-small cell
lung cancer. Appl Imm Mol Morphol. 2015 PMID: 26317305
Methods
The human immune system recognizes and eliminates
certain types of tumor cells, whereas other malignancies
are capable of suppressing immune function. A number of
cancer cell types express programmed cell death ligand 1
(PD-L1), which binds to its receptor PD-1 on T cells to
prevent their activation. High levels of PD-L1 expression
are typically associated with poor patient prognosis.
Researchers have developed immunotherapies (e.g.,
inhibitors of the PD-1/PD-L1 pathway) to stimulate the
immune system, allowing the body's natural defenses to
combat the tumor. To determine which patients are
suitable candidates for receiving immunotherapy, levels of
PD-L1 expression are often determined from tumor
biopsies. However, tumor heterogeneity can confound
these results and obtaining tumor tissue is often not
feasible. To enable non-invasive detection and sequential
monitoring of tumor-associated PD-L1 expression we have
developed a highly sensitive method of detecting PD-L1
levels in circulating tumor cells (CTCs). This work
describes the analytical validation of the Biocept PD-L1
assay in CTCs.
Fig. 2: Identification of PD-L1 expressing lung cell lines by flow
cytometry. PD-L1 epitopes were detected with either the MIH1 or 28-8
clone and presented as A) percent positives or B) mean fluorescent
intensity (MFI)
H820
H358
HC
C78
H3122
H1975
H1573
H1650
A549
DM
S114
H2228
H23
H727
0
2 0 0 0 0
4 0 0 0 0
6 0 0 0 0
8 0 0 0 0
1 0 0 0 0 0
L u n g c a n c e r c e ll lin e s
MF
I (P
DL
1)
P D L 1 M IH 1 M F I
P D L 1 2 8 -8 M F I
H820
H358
HC
C78
H3122
H1650
H1573
H1975
A549
H2228
H23
DM
S114
H727
0
5 0
1 0 0
L u n g c a n c e r c e ll lin e s
Pe
rc
en
t p
os
itiv
e (
PD
L1
)
P D L 1 M IH 1 % P o s itiv e
P D L 1 2 8 -8 % P o s it iv e
A.
B.
Fig. 4: This scatter plot (mean with SD) shows the mean fluorescent
intensity (MFI) measurement of PD-L1 protein in various blood spiked
lung cancer cell lines enriched and quantified in the Biocept
microfluidic device. (Cut-off value at 40 MFI is depicted by the dotted
line.)
Cell line tested Total CTC # cells PDL+ CTC PDL1 Status Cell Line PDL1 status Concordance
H727 400 2 Not Detected Not Detected Concordant
H727 400 2 Not Detected Not Detected Concordant
H727 400 3 Not Detected Not Detected Concordant
H727 400 9 Not Detected Not Detected Concordant
H727 400 4 Not Detected Not Detected Concordant
H727 400 2 Not Detected Not Detected Concordant
H727 400 5 Not Detected Not Detected Concordant
BT474 400 3 Not Detected Not Detected Concordant
BT474 400 7 Not Detected Not Detected Concordant
BT474 400 2 Not Detected Not Detected Concordant
BT474 400 8 Not Detected Not Detected Concordant
BT474 400 7 Not Detected Not Detected Concordant
BT474 400 17 Not Detected Not Detected Concordant
BT474 400 42 Not Detected Not Detected Concordant
BT474 400 19 Not Detected Not Detected Concordant
BT474 400 10 Not Detected Not Detected Concordant
BT474 400 13 Not Detected Not Detected Concordant
HCC78 400 364 Detected Detected Concordant
HCC78 400 335 Detected Detected Concordant
HCC78 400 317 Detected Detected Concordant
HCC78 400 387 Detected Detected Concordant
HCC78 400 280 Detected Detected Concordant
HCC78 400 287 Detected Detected Concordant
HCC78 400 239 Detected Detected Concordant
HCC78 400 385 Detected Detected Concordant
H820 400 391 Detected Detected Concordant
H820 400 371 Detected Detected Concordant
H358 400 260 Detected Detected Concordant
H358 400 254 Detected Detected Concordant
H358 400 304 Detected Detected Concordant
H358 400 286 Detected Detected Concordant
H358 400 240 Detected Detected Concordant
H358 400 319 Detected Detected Concordant
H358 400 328 Detected Detected Concordant
H358 400 329 Detected Detected Concordant
H358 400 301 Detected Detected Concordant
H358 400 304 Detected Detected Concordant
PD-L1 Accuracy Study (BioView “40” orange value cut-off)
Validation of PD-L1 Expression on Circulating Tumor Cells in Lung CancerTony J Pircher1, David L. Rimm2, Joshua J. Freitas1, Lyle Arnold1, Cecile Rose T. Vibat1, and Veena M. Singh1
1Biocept Inc., San Diego CA; 2Yale University, New Haven, CT
PD-L1 Expression in Lung Cancer Cell Lines
Methods
Results
A cut-off value (average fluorescence intensity value) was
established to yield 100% concordance between the result
of the PD-L1 test and the identity of the introduced cell
lines. The negative cut-off of 3.9% is based on a BETA
inverse of the 95% confidence level. In our microfluidic PD-
L1 assay we demonstrate H727 and BT474 cells to be negative
for PD-L1, while H358 cells have low-medium and HCC78 and
H820 cells high PD-L1 expression.
Fig. 5: PD-L1 reproducibility. Replicate measurements of negative and
positive PD-L1 expressing cells, spiked into blood, and enriched in
our microfluidic device are presented as MFI.
ReproducibilityPD-L1 Expression in Lung Cancer Cell Lines
Fig. 1: Biocept platform for CTC capture and staining. CTCs are
captured in transparent microfluidic channels and can be viewed in situ by
fluorescent microscopy. CTCs can be analyzed via immunofluorescence
(IF)
Plasma
ctDNA
Buffy Coat
CTCs
Red Blood
CellsProtein Detection
by IF
PD-L1 Expression in Clinical Samples
Fig. 6: Clinical examples. Six cancer patient cases are shown where
the Biocept platform was used to capture CTCs and the PD-L1 assay
identified cells expressing PD-L1 protein. PD-L1 positive CTCs were
identified in patents A-C, while PD-L1 negative CTCs were identified
in patents D-F.
Patient E Patient FPatient D
Patient B Patient CPatient A
1-H
358
2-H
358
3-H
358
4-H
358
5-H
358
6-H
358
7-H
358
8-H
358
9-H
358
10-H
358
11-B
T474
12-B
T474
13-B
T474
14-B
T474
15-B
T474
16-B
T474
17-B
T474
18-B
T474
19-B
T474
20-B
T474
0
5 0
1 0 0
1 5 0
MF
I (P
DL
1)
40
H820
H820
H820
H820
HC
C78
HC
C78
HC
C78
H2228
H2228
A549
A549
H1573
H1573
H727
H727
H727
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
MF
I (P
DL
1)
40