Spatial Phenotypic Signatures:

Novel Biomarker Class for Predicting

Immunotherapy Outcomes
 Which cancer patients will respond to
PD-1 / PD-L1 checkpoint inhibitor therapy?

<50%
Fewer than half of patients identified by current FDA-
approved companion diagnostics achieve a positive result,
and not everyone who tests negative ends up doing poorly.
This uncertainty is keeping patients from receiving the most
efficacious treatment, adding to the cost of care, and slowing the
development of next-generation immunotherapies.

Published clinical trial data show that fewer than half of all cancer patients
recommended for immunotherapy treatment based on approved companion
diagnostics are classified as responders at the end of therapy.1

1. Per Keytruda, Tecentiq and Libtayo package inserts: https://www.accessdata.fda.gov/scripts/cder/daf/.

 Modality AUC
PD-L1 IHC 0.650
TMB (DNA) 0.688

Raising the bar for predictive value in

GEP (RNA) 0.650
mIHC/IF 0.790a

identifying patients most likely to respond

to immunotherapy.
A meta-analysis in JAMA Oncology looking at over eight
thousand cases and 10 different cancer types showed that the
tools used for spatial phenotyping outperformed the three most
widely used assays for predicting response to immunotherapy.2

In the MITRE study, Taube, et al. demonstrated the suitability of

standardizing an automated mIF-based spatial biology workflow
even when used across labs with multiple operators.3

A meta-analysis of diagnostic techniques currently used for predicting response

to PD-1/PD-L1 checkpoint inhibitor immunotherapy shows that multiplexed
mIF/mIHC methods have significantly higher predictive value than PD-L1
immunohistochemical (IHC) staining, tumor mutational burden (TMB), or gene
expression profiling (GEP).2
2. Lu S, Stein JE, Rimm DL, et al. Comparison of biomarker modalities for predicting response to PD-1/PD-L1
checkpoint blockade: a systematic review and meta-analysis. JAMA Oncol. 2019;5(8):1195-1204.
3. Taube JM, Roman K, Engle EL, et al. Multi-institutional TSA-amplified multiplexed immunofluorescence
reproducibility valuation (MITRE) study. J Immunother Cancer. 2021;9(7):e002197.
 IHC
Multiplex IF
Equivalence analyses show no loss of resolution between cells labelled
for CD8 using traditional immunohistochemistry (IHC) staining versus
advance multiplex immunofluorescence (mIF). TOP: IHC with single CD8
labeled biomarker. BOTTOM: CD8 as one of multiple biomarkers applied
simultaneously to the same tissue sample using mIF with no loss of detail.4
Multiplexed assays increase predictive
value through simultaneous analysis of
multiple markers.5,6
Each Spatial Phenotypic Signature starts with multiplex IF. By
expanding your analysis beyond one or two markers per slide, you
reveal correlations between cell types and expression patterns
not apparent with serial analyses. In one recent study, co-analysis
of tertiary lymphoid structures (TLS) with PD-L1 or CD8 density
increased the predictive value of the assay by 70-100% over the
PD-L1 or CD8 density value alone.7
4. Data on file. Akoya Biosciences. 2021.
5. Hofman P, Badoual C, Henderson F, et al. Multiplexed immunohistochemistry for molecular and immune
profiling in lung cancer—just about ready for prime time? Cancers. 2019;11(3):283.
6. Nguyen QH, Pervolarakis N, Blake K, et al. Profiling human breast epithelial cells using single-cell RNA
sequencing identifies cell diversity. Nat Commun. 2018;9(1):2028.
7. Vanhersecke L, Brunet M, Guegan JP, et al. Mature tertiary lymphoid structures predict immune checkpoint
inhibitor efficacy in solid tumors independently of PD-L1 expression. Nat Rev Cancer. 2021;2:794-802.

This analysis of the proximity of PD-1+ and PD-L1+ cells in a sample of breast
cancer tissue is shown to have higher value in predicting response to
immunotherapy than does traditional PD-L1+ expression using IHC. Taube, et
al, showed that this relatively complex analysis can be performed with high
reproducibility at multiple labs across the US, and by multiple operators within
each lab.8,9
Spatial phenotypic signatures advance
our understanding of tumor biology
through visualization of the tissue and
cellular phenotypes in context. 
Increasingly, oncologists and pathologists are recognizing
that predicting therapeutic response and understanding
tumor heterogeneity are best done in the context of a tumor
microenvironment (TME). Spatial phenotypic signatures provide
insight into the dynamic ecosystem of the TME where selective
pressures shape which cells are present, how those cells
organize, and the balance between immune stimulating and
inhibiting factors.10
8. Akturk G, Parra ER, Gjini E, et al. Multiplex tissue imaging harmonization: a multicenter experience from CIMAC-
CIDC immuno-oncology biomarkers network. Clin Cancer Res. 2021;27(18):5072-5083.
9. Taube JM, Akturk G, Angelo M, et al. The Society for Immunotherapy of Cancer statement on best practices for
multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation. J Immunother
Cancer. 2020;8(1):e000155.
10. Schurch CM, Bhate SS, Barlow GL, et al. Coordinated cellular neighborhoods orchestrate antitumoral immunity
at the colorectal invasive front. Cell. 2020;182(5):1341-1359.
 Spatial phenotyping methodologies
SITE 1 SITE 2 SITE 3 SITE 4 SITE 5 SITE 6
advance precision medicine with high
predictive value and strong inter-lab and
RUN 1

inter-operator concordance.
Immuno-oncologists and pathologists from Johns Hopkins, Yale
University, The Earle A. Chiles Research Institute, MD Anderson
SITE 1 SITE 2 SITE 3 SITE 4 SITE 5 SITE 6
Cancer Center, and Bristol Myers Squibb collaborated with Akoya
to conduct the MITRE study that demonstrated high inter-lab and
RUN 2

inter-operator concordance using standardized automated mIF-

based spatial phenotyping workflows, demonstrating its practical
potential for supporting clinical trials and future applications in
routine clinical testing.

Analyses of similar assays on similar tissue samples performed by multiple

operators at six geographically dispersed labs were performed by blinded third-
party reviewers and showed “strong intersite and intrasite concordance.” 11

11. Taube JM, Roman K, Engle EL, et al. Multi-institutional TSA-amplified multiplexed immunofluorescence
reproducibility valuation (MITRE) study. J Immunother Cancer. 2021;9(7):e002197.
 Spatial phenotyping: Automated and
adapted to complement your workflows
and research.
The Akoya Phenoptics™ Solution allows any lab to gain novel,
spatially informed insights into the structure, progress, and
potential response to therapy of different tumor types using
standard sampling techniques, reagents, and markers.

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The Phenoptics Solution allows any lab to perform spatial phenotyping

using standard sampling techniques, reagents, and markers as part of a
typical daily workflow.