Elsevier

Pathology

Volume 49, Issue 6, October 2017, Pages 575-581
Pathology

Anatomical pathology
HER2 testing in advanced gastric and gastro-oesophageal cancer: analysis of an Australia-wide testing program

https://doi.org/10.1016/j.pathol.2017.05.009Get rights and content

Summary

This Australian human epidermal growth factor receptor 2 (HER2) testing program aimed to analyse >800 cases tested in a coordinated setting to further evaluate the criteria to establish HER2 status in advanced gastric and gastro-oesophageal junction (GOJ) cancer. Heterogeneity, and minimum number of biopsy fragments for reliable HER2 assessment were also examined in a subset of samples.

Five laboratories tested 891 samples referred to determine HER2 status for potential anti-HER2 treatment. Cancer site, specimen type (endoscopic biopsy/resection/metastases), immunohistochemistry (IHC) score, HER2 gene and CEP17 copy number (CN) and HER2:CEP17 ratios were recorded. Samples were derived from stomach (53.1%), GOJ (28.2%) or metastases (18.5%). IHC for HER2 and dual probe HER2:CEP17 in situ hybridisation (ISH) were performed in parallel. A stringent definition (SD) of HER2 positivity was used (IHC2+/3+ plus CN>6 and ratio>2) and compared with other published criteria.

HER2 positive rate was 13.9% (114/820) by SD, and 12.9–16.0% using other definitions. There was higher concordance between IHC and HER2 CN by ISH than with ratio. The HER2 positive rate was significantly higher in GOJ samples than others (p = 0.03) and in endoscopic biopsies than resections (p = 0.047). In a subset of 98 positive cases, 39 (39.8%) showed heterogeneity, and in 282 endoscopic biopsies positivity rate plateaued at five tumour fragments, suggesting this is the minimum number of biopsies that should be examined.

Introduction

Adenocarcinoma of the stomach is the fifth most common cancer globally and the third leading cause of cancer death.1, 2 Gene amplification and overexpression of human epidermal growth factor receptor 2 (HER2) is associated with more aggressive disease and a poor outcome in patients with gastric and gastro-oesophageal junction (GOJ) cancers.3, 4, 5

As with breast cancer, trastuzumab is now a standard-of-care for treatment of HER2 positive advanced gastric cancer. The ToGA (Trastuzumab for Gastric Cancer) trial demonstrated that trastuzumab, combined with chemotherapy, improved median overall survival in HER2 positive advanced gastric or GOJ cancer.6 An exploratory post-hoc analysis showed that patients with tumours with high levels of HER2 protein expression [immunohistochemistry (IHC) 3+ or IHC 2+ and in situ hybridisation (ISH) positive] demonstrated the greatest benefit.

Predicting which patients will most benefit from trastuzumab treatment requires accurate determination of HER2 status using a rigorous testing algorithm and robust definitions of gene amplification and protein overexpression. There has been no universally accepted definition of HER2 positive status: it has been defined by protein expression with an IHC 3+ result or a HER2:chromosome enumeration probe 17 (CEP17) ratio by ISH of ≥2 alone, or a combination of IHC, HER2:CEP17 ratio and HER2 copy number (CN).6, 7, 8, 9, 10

In addition to issues with cut-off, there are limited evidence-based scientific data on practical issues in testing, such as tissue fixation, selection of sample type (endoscopic biopsies, resections, metastatic sites including cytology samples), quantity and quality of tumour tissue samples and impact of heterogeneity on results.9, 11, 12, 13, 14, 15, 16 Implementation of biomarker protocols including HER2 testing in GOJ cancers is lagging, even in specialist centres.17

To address these shortfalls, a centralised program overseen by the GaTHER (Gastric HER2 Testing) group was initiated in Australia in 2010 to establish a robust testing algorithm for samples from patients being considered for trastuzumab therapy. The GaTHER group previously evaluated inter-laboratory agreement on HER2 scoring in advanced gastric and GOJ cancers in Australia,7 demonstrating good inter-laboratory agreement on IHC 3+ scoring (as determined by kappa values), and there was good/very good agreement between IHC positivity (defined as IHC 3+) and ISH when HER2 CN was used.7 Agreement was lower when HER2:CEP17 ratio was used suggesting that HER2 CN is the critical criterion for determining HER2 status in gastric and GOJ cancers.

The aims of this study were to analyse over 800 cases tested in a coordinated setting to (1) further evaluate criteria to establish HER2 status in advanced gastric and GOJ cancer, (2) assess the rate of heterogeneity, and (3) determine the minimum number of biopsy fragments required for reliable HER2 assessment.

Section snippets

Materials and methods

HER2 testing was performed in gastric and GOJ cancers between December 2010 and November 2013, overseen by the GaTHER group,7 in five laboratories around Australia: SydPath, St Vincent's Hospital Pathology Service, NSW; Peter MacCallum Cancer Centre, Vic; Princess Alexandra Hospital Pathology Service, Qld; PathWest, Queen Elizabeth II Medical Centre, WA; and SA Pathology, SA.

The basic principles of the testing program, including identification of laboratories, their training, certification, and

Results

A total of 891 patient samples were assessed, of which 820 had complete data for analysis. These comprised 457 primary endoscopic biopsies, 251 resections and 110 biopsies from metastatic sites. There were 114 (13.9%) tumours that were HER2 positive using the SD criteria, and 119 (14.5%) tumours that were HER2 positive when all IHC 3+ samples were included irrespective of ISH status. The number of HER2 positive patients ranged from 106 (12.9%) using the Gomez-Martin definition to 131 (16.0%)

Discussion

In the ToGA subgroup analysis, greatest survival benefit was demonstrated in those with a high level of protein expression (IHC 2+ or IHC 3+ and FISH positive, compared to the IHC 1+ and 0 and FISH positive group). Others have suggested that a cut-off of HER2:CEP17 ratio of 4.7 and a HER2 gene CN of 9.4 were optimal for discriminating between patients who were sensitive or refractory to trastuzumab therapy.8 They concluded that the level of HER2 gene amplification significantly predicts

Conclusion

The overall positivity rate in Australia is 13.9% and heterogeneous amplification was identified in approximately 40% of all positive cases. Analysis of a large number of samples from across Australia in a coordinated setting has allowed us to establish a standardised testing algorithm for HER2 testing in gastric/GOJ cancers. We recommend confirmation of IHC equivocal (IHC 2+) and positive (IHC 3+) cases by ISH with the use of HER2 CN to assess amplification. Endoscopic biopsies should contain

Acknowledgements

Dr Belinda Butcher and Dr Sophie Gibb, WriteSource Medical Pty Ltd, provided statistical and medical writing services (respectively) funded by Roche Products Pty Limited.

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