Poster # 3188
Contact: Joseph Krueger, PhD
E-mail: jkrueger@Invicro.com

Examining “Her2 Low” patient populations for prediction to Her2-targeting antibody-drug conjugate response
in Her2 negative patients using a novel high sensitivity immunohistochemistry assay
Joseph Krueger1, Kenneth Bloom1, George Abe1, Hisatake Okada2, Hiroyuki Yokota2
1 Invicro, a Konica Minolta Company, Boston, MA 2 Konica Minolta Precision Medicine, JapanMethods
A breast tumor tissue FFPE microarray with 104 cases/208 cores (US Biomax, BR20810) was stained using anti-Her2/neu (clone4B5) immunohistochemistry (Ventana i-VIEW detection system) to recapitulate the staining expected with existing Her2/neu
companion diagnostic tests. The same microarray (serial section) was stained with Quanticell, Konica-Minolta’s Phosphor
Integrated Dot (PID) fluorescent nanoparticle technology as the detection system instead of DAB, while using the same
primary assay conditions. The DAB stained TMA was scored using the ASCO/CAP guidelines for Her2 scoring to stratify patients
into the standard 0+/1+/2+/3+ classes. The Quanticell stained TMA was scored using a novel quantitative approach, and thescoring methods compared.

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Quanticell
Fluorescent
Nanoparticle
TechnologyQuanticell Immunohistochemistry
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PID signal

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0+1+2+3+PID score (mean)

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141437Mann-Whitney
U testN/A0+ / 1+ difference
p=2.03*10-e8

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p=1.47*10-e7

Her2 0+ (L16)2+3+C8G51+H50
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Figure1: The comparison approach. A standard DAB assay used in the companion diagnostic setting (Ventana 4B5) to predict responseto anti-Her2 therapy was converted into a Quanticell-based assay using a novel fluorescent immunohistochemistry technology fromKonica-Minolta. In comparison to DAB, the Quanticell approach has a much higher sensitivity and larger dynamic range, allowing
clearer detection and precise quantification of Her2 across all patients regardless of the range of Her2 expression.

Her2 Score

Figure 2: The Quanticell-based assay using a novel fluorescent immunohistochemistry technology from Konica-Minolta. The Quanticell
approach shows much higher sensitivity compared to DAB, allowing clearer detection of “Her2 Low” examples. The assay also has avery large dynamic range, allowing the simultaneous detection of Her2 overexpressing (3+) examples as well as low Her2 expressing (1+)
examples. Importantly, the Quanticell assay is still able to detect and quantify very low Her2 expression even in samples which would beclassified as having no Her2 expression (0+).

C8Quanticell

Standard DAB Immunohistochemistry

PID signal

PID signal

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The positive results for trastuzumab deruxtecan (DS-8201) in Her2+ breast cancer from the pivotal phase II DESTINY-Breast01 trial
announced in May 2019 represents a pivotal change in the approach to anti-Her2 therapy. Specifically, this antibody-drug
conjugate (ADC) utilizes an exatecan derivative (Dxd) which targets Topoisomerase I and is thought to have a key benefit inmechanism of action over its comparator anti-Her2 ADC, Kadcyla (T-DM1, or ado-trastuzumab emtansine; a microtubule
inhibitor). Data from the phase 1 trial for DS-2801 has demonstrated tumor shrinkage even in patients with low Her2 expression(Her2 IHC 2+/1+) by Her2 immunohistochemistry using the Ventana HER2 (4B5) assay (Lancet Oncol. 2017 Nov;18(11):15121522). This is thought to be due to a strong bystander effect of deruxtecan (Int J Cancer. 2019 May 14. doi: 10.1002/ijc.32408)
and a resulting enhancement of anti-tumor immunity (Mol Cancer Ther. 2018 Jul;17(7):1494-1503). Thus, prediction of patient
response to trastuzumab deruxtecan goes well beyond the past approaches of evaluating only Her2 over-expression oramplification, and potentially involves complex tumor and immune interactions, which drive a potent response even inpatients who do not have high Her2 expression.
This represents an emerging “Her2 low” strategy in drug development which seeks to use antibodies against Her2 to direct
cytotoxic or immune-stimulating payloads to tumor cells which do not overexpress Her2, but still retain some Her2 expression,even if considered diagnostically “Her2 negative” by the existing Her2 companion diagnostic strategies. Currently, there is nomarketed approach to stratifying “Her2 low” patients for these therapies, as such an approach would require an improved
method of Her2 detection or IHC interpretation strategy that allows better segmentation of patients with low Her2 expression.
This demand is unlikely to be met through changing Her2 IHC interpretation, as the existing Her2 companion diagnostic strategy
still remains challenging in clinical practice after nearly 15 years of improvements in scoring interpretations led by ASCO/CAP
for improved testing performance.
This study demonstrates how we can meet this challenge using a novel approach to Her2 protein detection in human formalinfixed, paraffin embedded (FFPE) tissues, called Quanticell ™. Quanticell relies on Konica Minolta’s Phosphor-Integrated Dot(PID) technology for ultra-sensitive, in situ detection of Her2 protein in FFPE tissues. This approach is capable of detecting Her2expression in clinical tissues that are characterized as “Her2 negative” by current IHC approaches, facilitating thedetermination of a new approach to a diagnostic cutpoint to stratify patients between “her2 negative” and “Her2 low”; which
is distinct from the current “Her2 high” paradigm.

Her2 Classification by Assay Type

Comparison of Quanticell to DAB for Her2 Scoring

DABAbstract

Figure 3: Comparison of DAB staining to Quanticell staining. The Quanticell approach creates the potential to develop a new patient
stratification approach for newer anti-Her2 Antibody-drug Conjugate (ADC) therapies which have been shown to be efficacious inpatients normally considered diagnostically negative by the existing Her2 companion diagnostics.

=0+ by DAB IHC but considered 1+ by Quanticell=0+ by DAB IHC and Quanticell

Discussion

Figure 4: Comparison of DAB scoring to Quanticell scoring. The
current Her2 ASCO/CAP scoring approach using traditional DAB
staining techniques require the pathologist to report the 0+/1+/2+/3+classification based on the percent of cells which have staining of acertain type. The most critical determination is in classifying patients
as either 0+/1+ or 2+/3+ for ultimate determination of “Her2 positive”
vs “Her2 negative” for classic anti-Her2 therapy. Here, even patientsclassified as 2+ may still receive therapy if they are determined to bepositive by Her2 FISH testing.
However, modern anti-Her2 directed ADCs have been shown tohave efficacy in patients traditionally classified as “Her2 negative”(0+/1+). While examination of the clinical reason for this continues, aleading hypothesis is that a more sensitive or calibrated Her2 test
which has the ability to classify “Her2 Low” patients could direct thesepatients to Her2 ADC therapy optimally. Quanticell can meet thisdemand as it does not require the use of the typical 0/1/2/3 Her2score classifications, but rather provides a quantitative, linear scorethat can be used to determine the precise amount of Her2expression. The Quanticell approach can be use to create newpatient response classifications in the way that accurately reflects
response to anti-Her2 ADCs. The graph showing the relationshipbetween the Her2 Quanticell score and the DAB Her2 classifications
are shown to illustrate this potential.
Figure 5: Comparison of DAB classifications to presumptive
Quanticell classifications in a confusion matrix. The Quanticell
scores were assigned into novel 0+/1+/2+/3+ classifications basedon a statistical approach to reflect how they may be used fortherapy determination:•For traditional anti-Her2 therapy, only the patients assigned to theQuanticell 3+ category would be considered “Her2 positive”. Thisapproach matches to the traditional DAB-based 3+ classification
well.
•For traditional anti-Her2 therapy, the patients assigned to theQuanticell 2+ category would be reflex tested using Her2 FISH.
There is some disagreement between the traditional DAB-based
2+/1+ classifications and the Quanticell 2+/ 1+ classifications thathighlights the potential ability to define a differential Quanticell
classification for anti-Her2 ADCs here.
•For traditional anti-Her2 therapy, the patients assigned to 1+ or 0+
classes are considered “Her2 negative”. However, there issignificant discrepancy between the 1+ and 0+ classifications
between the two methods. Patients classified as 0+ by DAB canbe bifurcated into two novel Quanticell 0+ and 1+ categories
which may represent a key bifurcation of “Her2 Low” and “Her2negative” classes appropriate for anti-Her2 ADC therapy using theQuanticell method.

Based on clinical data being presented from novel anti-Her2 therapies, such as DS-8201, whichshow efficacy in “Her2 negative” patients, it may be necessary to redefine patient classes fromthe classical “Her2 positive” and “Her2 negative” assignments and include a novel “Her2 Low”
classification. Based on the available clinical studies, the existing DAB IHC approach is not able
to correctly identify a “Her2 Low” classification. Here, we introduce the Quanticell IHC method
as an approach which may enable ideal patient stratification for the novel anti-Her2 ADC
therapies by creating new classes of “Her2 high”, “Her2 low”, and “Her2 negative” which canbe used to predict patient response for these therapies.