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Volume 20 No.1Winter 20171-888-92-PHENO www.phenopath.com

Brandon Seaton, HTL (ASCP)

Intro

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Brandon Seaton is the clinical IHC supervisor at PhenoPath. He supervises the clinical IHCand Histology departments. These departments perform all clinical immunohistochemistry,histology, immunofluorescence, and chromogenic in-situ hybridization. As supervisor, Brandon plays an administrative role in ensuring the department has appropriate staffing, supplies,and training to perform IHC at the highest level of quality. He is also involved in both testand instrument validation, and fills in on the IHC bench when needed.

Before joining PhenoPath, Brandon worked as a histology trainer/histotechnologist at LabCorp in Seattle where he was responsible for all IHC and special stains in a fast paced, high volume labora-tory.  He also worked as a clinical histotechnologist at Seattle Cancer Care Alliance where he per-formed all tasks in the histology and IHC workflow in a lab uniquely designed to serve bone marrow transplant patients.

Brandon obtained a Biotechnology Laboratory Specialist certificate from Shoreline Community College.  He has a bachelor’s degree in Zoology from University of Washington. Brandon is ASCP certified as a histotechnologist (HTL).

Plasma Based “Liquid Biopsy” Testing for EGFR MutationsPhenoPath Offers

In a prior issue of Phenomena (Volume 19 No. 1), we introduced the Roche cobas® EGFR Mutation Test (v2), an FDA-approved companion diag-nostic assay designed to identify appropriate first and second line therapies for patients with non-small cell lung carcinoma (NSCLC). This assay was recently FDA-approved for use with “cell-free” DNA isolated from plasma specimens. Plasma based EGFR testing provides a non-invasive substitute for tissue biopsies in patients who have progressed on first line therapy and can identify the T790M mutation, indicating eligibility for treatment with Tagrisso® (osimertinib).

“Cell free” DNA refers to DNA extracted from the non-cellular components of peripheral blood (i.e. plasma). Multiple terms have emerged in the medical literature to describe “cell-free” DNA (cfDNA) in the setting of solid tumor molecular testing, such as “circulating tumor DNA” and “liquid biopsy.” Cell-free DNA is normally present in plasma as a by-product of normal cell turn-over. Increased cfDNA can be seen in a variety of clinical settings, including tissue damage (e.g. myocardial infarction), immunologic responses, and organ rejection. Sources of circulating tumor DNA include necrosis/apoptosis of tumor cells, secretion of DNA by tumors, and lysis of circulating tumor cells.

Molecular testing of cfDNA presents unique challenges to the molecular laboratory. Normal white blood cells present in the peripheral blood begin to lyse soon after blood is drawn, releasing normal genomic DNA into plasma. Increased normal genomic DNA dilutes the tumor derived cell-free DNA, making it more difficult to detect mutations. Therefore, sample processing is critical to optimize the chances of mutation detection. To minimize normal cellular degradation, plasma should be isolated from peripheral blood samples and frozen within four hours of collection. In addition, the amount of circulating tumor DNA varies from patient to patient, depending on the size, vascularity and location of the tumor. The proportion of circulating tumor DNA is also affected by physiologic clearance and filtering in the blood and lymphatic systems. For these reasons, EGFR mutation testing of circulating tumor DNA in plasma is inherently more likely to produce a false negative result than tissue-based testing.

When handled optimally, plasma based testing provides a non-invasive means of detecting EGFR mutations in NSCLC patients. Published stud-ies have shown concordance between plasma based and tissue based EGFR mutation detection; and the studies include reports of T790M mutation detection (indicative of resistance) before disease progression was clinically evident. The presence of a detectable plasma EGFR mutation has also been associated with a worse prognosis in some studies.

Investigation of PD-L1 Biomarker Testing Methods for PD-1 Axis Inhibition in Non-squamous Non-small Cell Lung CancerSheffield BS, Fulton R, Kalloger SE, Milne K, Geller G, Jones M, Jacquemont C, Zachara S, Zhao E, Pleasance E, Laskin J, Jones SJ, Marra MA, Yip S, Nelson BH, Gown AM, Ho C, Ionescu DN. J Histochem Cytochem 64(10):587-600, 2016Inhibitors of the programmed cell death 1 (PD-1) signaling axis have recently demonstrated efficacy and are rap-idly being incorporated into the treatment of non-small cell lung cancers (NSCLCs). Despite clear benefits to cer-tain patients, the association of these responses with a predictive biomarker remains uncertain. Several different biomarkers have been proposed, with differing results and conclusions. This study compares multiple methods of biomarker testing for treatment of NSCLCs with PD-1 axis inhibitors. Tissue microarrays of matched primary and metastatic NSCLCs were used to compare four different PD-1 ligand (PD-L1) IHC techniques, as well as RNA in situ hybridization (ISH). Eighty cases were included in the IHC study. Multiple IHC methodologies showed a high rate of agreement (Kappa = 0.67). When calibrated to RNA expression, agreement improved significantly (Kappa = 0.90, p=0.0049). PD-L1 status of primary and metastatic tumors was discordant in 17 (22%) cases. This study suggests that different IHC methodologies for PD-L1 assessment provide slightly different results.

Comparative Sensitivities and Specificities of Antibodies to Breast Markers GCDFP-15, Mammaglobin A, and Different Clones of Antibodies to GATA-3: A Study of 338 Tumors Using Whole SectionsKandalaft PL, Simon RA, Isacson C, Gown AM. Appl Immunohistochem Mol Morphol 24(9):609-614, 2016GATA-3 is a transcription factor that has recently been identified by immunohistochemistry to be highly expressed in urothelial and breast car-cinomas (CAs). We sought to determine the potential utility of GATA-3 in identifying metastatic breast CA, and to compare its utility with the standard breast markers, GCDFP-15, and mammaglobin A. We identified an archival series of 338 formalin-fixed paraffin-embedded whole-tissue sections of various CAs. Using standard immunohistochemical (IHC) techniques we used mouse monoclonal antibodies to GATA-3 (clones L50-823, HG3-31), GCDFP-15 (23A3), and mammaglobin A (31A5). Both clones of GATA-3 showed positivity in 96% of non-triple-negative breast carcinomas (TNBCs), L50-823 and HG3-31, demonstrating expression in 87% and 63% of TNBCs, respectively; GCDFP-15 and mammaglobin A were expressed in 69% and 61% of non-TNBCs, respectively, and 10% and 17%, of TNBCs, respectively. The L50-823 clone manifested a lower specificity in identifying breast CAs (84%) than did the HG3-31 clone (97%). Both monoclonal antibodies to GATA-3 are very sensitive reagents for the identification of breast CA, surpassing antibodies to GCDFP-15 and mammaglobin A, and offer a significant improvement in identifying TNBCs. However, the L50-823 clone showed a lower level of specificity, which may qualify its utility in the setting of CAs of unknown primary.

PhenoPath is on the forefront of biomarker testing, offering all FDA-approved PD-L1 clones, and having tested and analyzed over 3,000 specimens since the launch of PD-L1 testing in June of 2015. Please see the biomarker poster on the following page with up-to-date FDA-approval status to assist oncologists and pathologists navigate the various PD-L1 options, all of which PhenoPath offers. Refer to www.phenopath.com for the most up-to-date version upon additional FDA approvals.

PhenoPath is providing PD-L1 testing for both patient management in the clinical setting as well as for R&D and clinical trials, crossing over vari-ous tumor types (lung, breast, H&N, GU, GI, melanoma, etc.) and platforms (Dako and Ventana), in addition to comparison studies among the various PD-L1 clones. PhenoPath is currently supporting over 80 R&D and clinical trials, many of which include PD-L1 testing of various clones.

Comparative Sensitivities and Specificities of Antibodies toBreast Markers GCDFP-15, Mammaglobin A, and DifferentClones of Antibodies to GATA-3: A Study of 338 Tumors

Using Whole Sections

Patricia L. Kandalaft, MD,*w Rochelle A. Simon, MD,wChristina Isacson, MD,z and Allen M. Gown, MD*

Abstract:GATA-3 is a transcription factor that has recently been

identified by immunohistochemistry to be highly expressed in

urothelial and breast carcinomas (CAs). We sought to determine

the potential utility of GATA-3 in identifying metastatic breast

CA, and to compare its utility with the standard breast markers,

GCDFP-15, and mammaglobin A. We identified an archival

series of 338 formalin-fixed paraffin-embedded whole-tissue sec-

tions of various CAs. Using standard immunohistochemical

(IHC) techniques we used mouse monoclonal antibodies to

GATA-3 (clones L50-823, HG3-31), GCDFP-15 (23A3), and

mammaglobin A (31A5). Both clones of GATA-3 showed pos-

itivity in 96% of non–triple-negative breast carcinomas

(TNBCs), L50-823 and HG3-31, demonstrating expression in

87% and 63% of TNBCs, respectively; GCDFP-15 and mam-

maglobin A were expressed in 69% and 61% of non-TNBCs,

respectively, and 10% and 17%, of TNBCs, respectively. The

L50-823 clone manifested a lower specificity in identifying breast

CAs (84%) than did the HG3-31 clone (97%). Both monoclonal

antibodies to GATA-3 are very sensitive reagents for the iden-

tification of breast CA, surpassing antibodies to GCDFP-15 and

mammaglobin A, and offer a significant improvement in iden-

tifying TNBCs. However, the L50-823 clone showed a lower level

of specificity, which may qualify its utility in the setting of CAs

of unknown primary.

Key Words: GATA-3, mammaglobin A, GCDFP-15, breast

carcinoma

(Appl Immunohistochem Mol Morphol 2016;24:609–614)

GATA-3 is a transcription factor that was initiallyidentified by cDNA microarray analysis to be highly

expressed in urothelial and breast carcinomas (CAs),1,2

and is 1 of the 6 members of the GATA family of tran-scription factors crucial to the differentiation of a numberof different tissues including breast, hair follicles, T-lym-phocytes, trophoblasts, kidney, urothelium, parathyroid,autonomic nervous tissue, and endothelial cells.3–5 Sub-sequent to identification of GATA-3 by cDNA microarrayanalysis, a limited number of relatively small, predom-inantly tissue microarray (TMA)-based studies evaluatedIHC expression of GATA-3 utilizing a mouse monoclonalantibody (clone HG3-31). These studies showed GATA-3expression in 67% of bladder urothelial CAs and 96% ofovarian Brenner tumors. Low-level expression of GATA-3was associated with a poor outcome and estrogen receptornegativity in breast cancer patients.1,2,6 However, pub-lication of a study incorporating IHC evaluation of GATA-3 as a potential organ-specific marker was not undertakenuntil 2012. In a TMA-based evaluation of greater than 1100sections of CAs by Liu et al,5 utilizing the mouse mono-clonal clone HG3-31, 86% of transitional cell carcinomas(TCCs), and 94% of breast CAs showed expression ofGATA-3, with the remainder of the evaluated CAs neg-ative, with the exception of 2% of endometrial endome-trioid adenocarcinomas (ECs).

Given this high reported rate of GATA-3 expression inbreast CAs, we sought to determine the potential utility ofGATA-3 in identifying breast CA, comparing these resultswith the standard breast markers, GCDFP-15 and mamma-globin A, incorporating whole-tissue sections and 2 differentcommercially available clones of antibodies to GATA-3.

MATERIALS AND METHODSThe tissues used in this study were obtained from

the clinical case files of CellNetix Pathology, Seattle, WA,and PhenoPath Laboratories, Seattle, WA, consisting ofdeidentified cases, previously diagnosed using establishedmorphologic, IHC, and clinical criteria, and consisted of338 formalin-fixed, paraffin-embedded whole-tissue sec-tions of a variety of CAs, with tumor type and number ofcases indicated in Tables 2–5.

After epitope retrieval, IHC studies were performedon deparaffinized 4 mm sections of each case using anti-bodies to: GATA-3 (clone L50-823, BioCare, and clone

Received for publication May 1, 2015; accepted May 11, 2015.From *The PhenoPath Laboratories PLLC; wPacific Pathology Part-

ners; and zCellNetix Pathology and Laboratories, Seattle, WA.The authors declare no conflict of interest.Reprints: Patricia L. Kandalaft, MD, Pacific Pathology Partners, 550

17th Avenue, Suite 300, Seattle WA 98122 (e-mail: pkandalaft@pacificpathologypartners.com).

Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved.

RESEARCH ARTICLE

Appl Immunohistochem Mol Morphol � Volume 24, Number 9, October 2016 www.appliedimmunohist.com | 609

Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved.

Journal of Histochemistry & Cytochemistry 2016, Vol. 64(10) 587 –600© 2016 The Histochemical SocietyReprints and permissions: sagepub.com/journalsPermissions.navDOI: 10.1369/0022155416665338jhc.sagepub.com

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Article

Introduction

Immune checkpoint inhibitors have recently demon-strated marked efficacy in advanced non–small cell lung carcinoma (NSCLC)1–3 and a number of other advanced malignancies.4 These drugs, specifically pro-grammed cell death 1 (PD-1) axis inhibitors, will have an increasing role in the treatment of advanced lung cancer. With the 2015 approvals of both nivolumab and pembrolizumab by the US Food and Drug Administration

for previously treated NSCLC, and the development of other agents in this class, this therapeutic focus

665338 JHCXXX10.1369/0022155416665338PD-L1 testing in lung cancerSheffield et al.research-article2016

Received for publication April 12, 2016; accepted July 26, 2016.

Corresponding Author:Brandon S. Sheffield, Department of Pathology and Laboratory Medicine, University of British Columbia, 910 W 10th Ave., Vancouver, British Columbia, Canada V5Z 1M9. E-mail: Brandon.S.Sheffield@gmail.com

Investigation of PD-L1 Biomarker Testing Methods for PD-1 Axis Inhibition in Non-squamous Non–small Cell Lung Cancer

Brandon S. Sheffield, Regan Fulton, Steve E. Kalloger, Katy Milne, Georgia Geller, Martin Jones, Celine Jacquemont, Susanna Zachara, Eric Zhao, Erin Pleasance, Janessa Laskin, Steven J.M. Jones, Marco A. Marra, Stephen Yip, Brad H. Nelson, Allen M. Gown, Cheryl Ho, and Diana N. IonescuDepartment of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada (BSS, SEK, SZ, SY); PhenoPath Laboratories, Seattle, Washington (RF, CJ, AMG); Trev and Joyce Deeley Research Centre (KM, BHN), Division of Medical Oncology (GG, JL, CH), Canada’s Michael Smith Genome Sciences Centre (MJ, EZ, EP, SJMJ, MAM), and Department of Laboratory Medicine and Pathology (DNI), BC Cancer Agency, Vancouver, British Columbia, Canada; and Department of Pathology, Abbotsford Regional Hospital and Cancer Centre, British Columbia, Canada (BSS)

SummaryInhibitors of the programmed cell death 1 (PD-1) signaling axis have recently demonstrated efficacy and are rapidly being incorporated into the treatment of non–small cell lung cancers (NSCLCs). Despite clear benefits to certain patients, the association of these responses with a predictive biomarker remains uncertain. Several different biomarkers have been proposed, with differing results and conclusions. This study compares multiple methods of biomarker testing for treatment of NSCLCs with PD1-axis inhibitors. Tissue microarrays of matched primary and metastatic NSCLCs were used to compare four different PD-1 ligand (PD-L1) IHC techniques, as well as RNA ISH. Additional cases with whole genome and transcriptome data were assessed for molecular correlates of PD-L1 overexpression. Eighty cases were included in the IHC study. Multiple IHC methodologies showed a high rate of agreement (Kappa = 0.67). When calibrated to RNA expression, agreement improved significantly (Kappa = 0.90, p=0.0049). PD-L1 status of primary and metastatic tumors was discordant in 17 (22%) cases. This study suggests that different IHC methodologies for PD-L1 assessment provide slightly different results. There is significant discordance between the PD-L1 status of primary tumors and lymph node metastases. RNA ISH may be a useful adjunct to complement PD-L1 IHC testing. (J Histochem Cytochem 64:587–600, 2016)

Keywordsbiomarker, immunotherapy, lung cancer, non–small cell lung carcinoma, NSCLC, PD-1, PD-L1

PD-L1 Testing Update (see attached poster)

New Publications from PhenoPath Pathologists/Scientists

The IHC Laboratory at PhenoPath is proud to announce that, after an extensive validation, we have switched to the 4B5 HER2 antibody clone on our new Ventana Ultra platform for our assessment of HER2 expression by im-munohistochemistry. One of the motivating factors be-hind this switch is the extremely high scores given to the 4B5 antibody on the Ventana platform in the assessments by the NordiQC https://goo.gl/lzQLaI

HER

2 C

lone

4B5

H3K27me3 is a protein which, when trimethylated, is tightly associated with inactive gene promoters. Antibodies to H3K27me3 can be employed for the identification of malig-nant peripheral nerve sheath tu-mors (MPNST), approximately 50% of which show complete loss of expression of this nuclear protein; a smaller fraction dis-plays partial loss of this pro-tein. Interestingly, loss of expression of H3K27me3 is not a feature of epithelioid MPNSTs. This loss of expression in sarcomatoid MPNST is highly specific to this tumor, and it can help distinguish it from other sarcomas and spindle cell melano-mas. H3K27me3 offers superior specificity and sensitivity for MPNST over mark-ers such as S100 and SOX10. References: Schaefer IM, Mod Pathol 29:4-13, 2016; Preito-Granada CN et al., Am J Surg Pathol 40:479-89, 2016

H3K27me3

Breast cancer showing 3+ HER2 (4B5) Partial loss of expression of H3K27me3 in MPNST

Head & NeckSquamous Cell CA

Hodgkin Lymphoma

Melanoma

NSCLC 1st line

NSCLC 2nd line

Renal Cell CA

Urothelial CA(bladder)

KEYTRUDA®(pembrolizumab)anti-PD-1

PD-L1 22C3(tumor cells)

OPDIVO®(nivolumab)anti-PD-1

PD-L1 28-8(tumor cells)

TECENTRIQ®(atezolizumab)anti-PD-L1

PD-L1 SP142(immune cells / tumor cells)

PhenoPathDiagnoses you can count on®

Biomarker Testing for Checkpoint Inhibitors

Δ = FDA approved companion diagnostic (required); † = FDA approved complementary diagnostic (optional)

TPS = Tumor proportion score; TC = tumor cells; IC = immune cells

FDA approved; no testing required

FDA approved with

22C3Δ≥50% TPS

FDA approved; no testing required

FDA approved;no testing required

PD-L1 testing available NOWat PhenoPath

22C3 28-8 SP142 E1L3NDako Link 48 Dako Link 48 Ventana Ultra Generic

FDA approved; no testing required

Specimen requirements: FFPE block or 4 to 5 unstained slides cut at 4µmTo submit a specimen: Request supplies or use any requisition form at www.phenopath.com; indicate PD-L1 clone desired; ship FedEx standard overnight in secure shipping container (provided by PhenoPath upon request)Turnaround time: 24-48 hours from receipt of specimen

References: keytruda.com, opdivo.com, tecentriq.com, dako.com, ventana.com, fda.gov, drugs.com

Disclaimer: The content of this poster should not be relied upon as the sole source of information to guide specimen testing or patient treatment.

www.phenopath.com • 888-927-4366

FDA approved with

22C3Δ≥1% TPS

FDA approved with

28-8†

≥1% TPS

FDA approved with

28-8†

≥1% TPS

FDA approved with

SP142†

≥50% TC / ≥10% IC

FDA approved with

SP142†

≥5% IC

v. 14FEB2017

FDA approved; no testing required

FDA approved; no testing required

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PhenoPath at the USCAP, Mar 4-10, 2017, San Antonio, TX (Booth #411)Monday, March 6, 2017, 1:00PM-4:30PM, Exhibit Hall 1 / Poster Session II #288 “Robotic and Semi-Automated Microtomy Can Decrease Variability in HER2 Staining Intensity” (Abstract #2026), Studies performed in conjunction with Premier Lab, Longmont, CO; Array Science, Sausalito, CA; Sakura Finetek, Torrance, CA; Horizon Discovery, Cambridge, UK; Boulder Statistics, Boulder, CO; PhenoPath, Seattle, WA

Tuesday, March 7, 2017, 9:30AM-12:00PM, Exhibit Hall 1 / Poster Session III #135 “Concordance Study of 4 Anti-PD-L1 Antibodies in Primary and Metastatic Bladder Cancer” (Abstract #966), Studies performed in conjunction with Univ of Washington, Seattle, WA; Univ of Chicago, IL; PhenoPath, Seattle, WA

Tuesday, March 7, 2017, 1:00PM-4:30PM, Exhibit Hall 1 / Poster Session IV #120 “Comparison of 4 PD-L1 Antibodies in 560 Kidney, Bladder and Prostate Cancers” (Abstract #1062), Studies performed in conjunction with Univ of Washington, Seattle, WA; PhenoPath, Seattle, WA; Northwestern Univ, Chicago, IL

Wednesday, March 8, 2017, 9:30AM-12:00PM, Exhibit Hall 1 / Poster Session V #143 “Role of SATB2 in Distinguishing the Site of Origin in Glandular Lesions of the Bladder/Urethra: An Immunohistochemical Study” (Abstract #913), Studies performed in conjunction with Vanderbilt, Nashville, TN; PhenoPath, Seattle, WA; John Hopkins, Baltimore, MD

Wednesday, March 8, 2017, 12:00PM-1:00PM, RC Conf Room 1-4 / “Hot Topics in Pathology 03-Immunohistochemistry in Evaluating Tumors of Undetermined Origin”, Moderated by Jason L. Hornick, MD, PhD, Brigham and Women’s Hospital, Boston, MA in conjunction with Andrew M. Bellizzi, MD, Univ of Iowa Carver College of Medicine, Iowa City, IA; Allen M. Gown, MD, PhenoPath, Seattle, WA

PhenoPath at the NCCN, Mar 23-25, 2017, Orlando, FLVisit us at the following poster session:

Thursday, March 23, 2017 & Friday, March 24, 2017 / General Poster Sessions “PD-L1 Testing in Non-Small Cell Lung Cancer”, Allen M. Gown, MD and Regan Fulton, MD, PhD, PhenoPath, Seattle, WA