1. HER2 Testing in Gastric/GEJ Adenocarcinoma
Short Presentation on Emerging Concepts
Version 2.0e rev. 7/12/2017

2. Why is HER2 Testing of Gastric/GEJ Adenocarcinomas Important?
In 2010, the results of the first (and only) randomized trial of trastuzumab in gastric/GEJ adenocarcinoma were published
Trastuzumab in Gastric Adenocarcinoma (ToGA)
Prolonged median survival and progression-free survival in patients treated with trastuzumab+chemotherapy compared to those treated with chemotherapy alone (13.8 vs 11.1 months, p=0.0046, and 6.7 vs 5.5 months, p=0.0002)
TOGA trial was an open label phase 3, randomized controlled trial based in Europe involving patients from Europe, Asia and South America (no North American sites) While uncommon in the United States (21,000 cases and 10,00 deaths), gastric cancer is a huge burden worldwide with almost one million diagnoses annual. It is the third most common cause of cancer death (~700,000 annually).
© 2018 College of American Pathologists. All rights reserved.

3. Who was eligible for ToGA?
Patients with inoperable, locally advanced, recurrent or metastatic HER2 positive adenocarcinoma of the stomach or GEJ
© 2018 College of American Pathologists. All rights reserved.

4. How is HER2 status determined?
There are several methods of determining HER2 status
There are two clinically validated methods
Immunohistochemistry (IHC) which assesses protein expression on the cell membrane
Fluorescence in situ hybridization (FISH) which assesses gene amplification
In the ToGA trial, patients were dual-tested (i.e. underwent both IHC and FISH testing)
Examples of methods include PCR (gene amplification), Northern blot (mRNA), Western blot (protein)
In breast cancer HER2 testing, IHC is usually the initial test; if results are equivocal FISH testing is performed. In breast cancer, IHC negative cases do not usually undergo FISH testing (this is different than what was done in ToGA)
© 2018 College of American Pathologists. All rights reserved.

5. What defines a sample as IHC positive?
Moderate-strong membranous staining in ≥10% of tumor cells
A cluster of ≥5 cells with otherwise positive features in a biopsy specimen
Staining need not be circumferential as in breast cancer; due to differences in biology, staining may be basolateral or lateral
In a pre-ToGA preparatory trial (Hofmann), cases with the features described above were shown to be amplified with FISH
Source: Hicks DG, Whitney-Miller C. HER2 testing in gastric and gastroesophageal junction cancers: a new therapeutic target and diagnostic challenge. Appl Immunohistochem Mol Morphol Dec;19(6): doi: /PAI.0b013e31822c3a0f.
© 2018 College of American Pathologists. All rights reserved.

6. What defines a sample as FISH positive?
Most FISH testing uses two probes
One against the gene of interest (HER2)
One not usually amplified as a reference (CEP17)
In ToGA, if HER2 copy number/CEP17 copy number was ≥2 the case was considered amplified
HER2
CEP17
Subsequently, experts have recommended that if the HER2 copy number is >6 these cases should be considered amplified as well (similar to breast)
Image courtesy of Christa L. Whitney-Miller, MD: University of Rochester Medical Center
© 2018 College of American Pathologists. All rights reserved.

7. Isn’t HER2 Testing in Gastric/GEJ Adenocarcinoma the same as Breast Cancer?
While similar, there are important differences in the interpretation of HER2 IHC in breast vs gastric cancer
Gastric/GEJ
Score/Result
Breast
<10% of cells stain
0/Negative
No staining observed
Faint staining in ≥10% of cells
1+/Negative
Weak or incomplete staining in any proportion of invasive tumor cells
Weak-moderate, complete, basolateral or lateral membranous staining in ≥10% of tumor cells
2+/Equivocal
Weak to moderate complete membrane staining that is observed in ≥ 10% of tumor cells
Moderate-strong basolateral or complete membranous staining in ≥10% of cells
3+/Positive
Uniform intense membrane staining observed in ≥ 10% of invasive tumor cells
Cohesive cluster of cells with moderate to strong basolateral or complete membranous staining in a biopsy
Uniform intense membrane staining observed in ≥10% of invasive tumor cells
For the Hofmann study, investigators took 168 samples from stomach, GEJ and esophagus, and applied commercially available IHC and FISH kits (HercepTest and pharmDx from Dako) and applied the breast cancer scoring criteria. Knowing that with proper handling and interpretation, there is very good correlation between IHC and FISH in breast cancer, the investigators looked for concordance. They found good concordance applying the breast criteria, but there were a few cases that were not concordant. Closer examination of these cases prompted the modifications to the breast criteria that they recommended. It is important to note that Hofmann used the IHC stained slides to guide which areas to count on the FISH. * New criteria for interpretation of 2+/equivocal per J Clin Onc. 2015; 33(11):
Hofmann M, Stoss O, Shi D, et al. Assessment of a HER2 scoring system for gastric cancer: results from a validation study. Histopathology. 2008;52: doi: /j x
Wolff AC, Hammond MEH, Hicks DG, et al. Reply to E.A. Rakha et al. J Clin Onc. 2015; 33(11):
Bartley AN, Washington MK, Ventura CB, et al. HER2 Testing and Clinical Decision Making in Gastroesophageal Adenocarcinoma: Guideline From the College of American Pathologists, American Society for Clinical Pathology, and the American Society of Clinical Oncology. Arch Pathol Lab Med. 2016;140(12):

8. Should we follow the same algorithm as is used in breast cancer?
Even though samples were dual-tested in TOGA, subsequent exploratory analysis suggested response to trastuzumab was correlated to IHC result
Herceptin [prescribing information]. Genentech, Inc., South San Francisco, CA; October 29, 2010.
Updated survival analysis sorted by IHC result
Source: Herceptin [prescribing information]. Genentech, Inc., South San Francisco, CA; October 29, 2010.
© 2018 College of American Pathologists. All rights reserved.

9. Should we follow the guidelines for HER2 testing in breast cancer when testing a gastric sample?
CAP/ASCO/ASCP published specific guidelines for HER2 testing in gastric and esophageal adenocarcinoma (GEA) in 2016
© 2018 College of American Pathologists. All rights reserved.

10. © 2017 College of American Pathologists. All rights reserved.
Source: Bartley AN, Washington MK, Ventura CB, et al. HER2 Testing and Clinical Decision Making in Gastroesophageal Adenocarcinoma: Guideline From the College of American Pathologists, American Society for Clinical Pathology, and American Society of Clinical Oncology. Arch Pathol Lab Med. 2016;140(12):

11. Highlights of the HER2 GEA Guidelines
It is acceptable to test a biopsy, resection or metastasis
Laboratories must validate their GEA HER2 test independent of their breast test
IHC should be performed first, followed by ISH in equivocal cases
HER2 testing can be performed by request; alternatively, with agreement between oncologist and pathologist, reflexively in appropriate patients
© 2018 College of American Pathologists. All rights reserved.

12. © 2017 College of American Pathologists. All rights reserved.
Source: Bartley AN, Washington MK, Ventura CB, et al. HER2 Testing and Clinical Decision Making in Gastroesophageal Adenocarcinoma: Guideline From the College of American Pathologists, American Society for Clinical Pathology, and American Society of Clinical Oncology. Arch Pathol Lab Med. 2016;140(12):

13. nonoverlapping nuclei nonamplified/negative
FISH
QUALITY CHECK
indeterminate
correlate with IHC
count at least 20
nonoverlapping nuclei
HER2/CEP17 > 2
CEP17>3
nonamplified/negative
image courtesy of Christa Whitney-Miller, MD and Lisa Testa; University of Rochester Medical Center
amplified/positive
final result
HER2>6
resolved
count another 20 cells
HER2<4
alternative
Image courtesy of Christa L. Whitney-Miller, MD, and Lisa Testa: University of Rochester Medical Center

14. Summary
A subset of patients with GEA over-express HER2 and may benefit from trastuzumab
HER2 status can be determined by IHC or FISH
IHC should be performed first and interpreted using the Hofmann/Ruschoff criteria
Equivocal cases should under go ISH testing
Hoffmann/Ruschoff criteria should be utilized in ISH testing
© 2018 College of American Pathologists. All rights reserved.

15. SPEC Author/Editor Information
Christa L. Whitney-Miller, MD – University of Rochester Medical Center
Editors:
Matthew W. Anderson, MD, PhD – BloodCenter of Wisconsin Inc
Pranil K. Chandra, DO – PathGroup
Allison M. Cushman-Vokoun, MD, PhD – The University of Nebraska Medical Center
Michael J. Misialek, MD – Newton-Wellesley Hospital
© 2018 College of American Pathologists. All rights reserved.

16. SPEC Author/Editor Information (continued)
Editors:
Damon Olson, MD – Baylor College of Medicine
Andrew E. Schade, MD, PhD – Eli Lilly and Company
Gail H. Vance, MD – Indiana University
Jessica Wood, MD – University of Vermont Medical Center
Mary M. Zutter, MD – Vanderbilt Medical Center
© 2018 College of American Pathologists. All rights reserved.

17. Selected References
Hofmann M, Stoss O, Shi D, et al. Assessment of a HER2 scoring system for gastric cancer: results from a validation study. Histopathology. 2008;52:
Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-esophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. The Lancet :
© 2018 College of American Pathologists. All rights reserved.

18. Selected References (continued)
Rüschoff J, Dietel M, Baretton G, et al. HER2 diagnostics in gastric cancer-guideline validation and development of standardized immunohistochemical testing. Virchows Arch. 2010;457(3):
Wolff AC, Hammond MEH, Hicks DG, et al. Reply to E.A. Rakha et al. J Clin Onc. 2015; 33(11):
Bartley AN, Washington MK, Ventura CB, et al. HER2 Testing and Clinical Decision Making in Gastroesophageal Adenocarcinoma: Guideline From the College of American Pathologists, American Society for Clinical Pathology, and the American Society of Clinical Oncology. Arch Pathol Lab Med. 2016;140(12):