Introduction: These guidelines represent systematically developed statements to assist in the provision of quality assured HER2 testing in breast and gastric/ gastro-oesophageal carcinoma. They are based on current published international guidelines. They were presented to the Faculty of Pathology Quality Assurance Working Group and to the Academy of Medical Laboratory Scientists for comment by their membership before adoption and recommendation for implementation. Each year the authors will consider, in conjunction with current literature update, whether the guidelines need to be revised Version 1 of these Guidelines were ratified by the Professional bodies and adopted in 2011. Version 2 of these Guidelines were drafted in response to published updated ASCO/CAP HER2 test Guideline Recommendations- Wolff AC et al. Recommendations for Human Epidermal Growth Factor Receptor Testing in Breast Cancer: American Society of Clinical Oncology/ College of American Pathologists Clinical Practice Guideline Update. Journal of Clinical Oncology: 2013; 31 (31): 3997-4013 Guideline Update history: American Society of Clinical Oncology (ASCO) Practice Guideline Committee approval: April 26, 2013 College of American Pathologists (CAP) approval: June 2013.
Proposal for HER2 National Testing Criteriarecommendations by the HER2 sub-group This group was initially convened in May 2010 from a larger group of pathologists and medical scientists involved in laboratory testing for HER2 across Irish pathology departments. All pathologists known to be involved in HER2 testing across Irish laboratories were invited to initial meetings to discuss the wider issues relating to HER2 testing and the need to refine testing recommendations and guidelines for the Irish laboratories. From this group, a sub-group was appointed. The aim was to have representation on the subgroup from pathologists and senior/chief medical scientists from the centres who were represented in the main group. The remit of the sub-group was to review current international recommendations for HER2 testing in the literature (1-10) and, from this, to make recommendations for Irish laboratories involved in IHC and ISH (FISH and CISH) HER2 testing for breast and gastric cancer. HER2 testing sub- group membership: Dr Mairead Griffin, St James s Hospital Mr John Harford, St Vincents University Hospital Dr Susan Kennedy, St Vincents University Hospital Ms Deirdre Mc Mahon, St Vincents University Hospital Mr Kieran Mc Allister, Mater Hospital Dr Fionnuala O Connell, Cork University Hospital Dr Tony O Grady, Royal College of Surgeons Ireland & Beaumont Hospital Dr Cecily Quinn, St Vincents University Hospital Dr Margaret Sheehan, Galway University Hospital (Chairperson) Ms Jan Walker, St James s Hospital Dr Michael Jeffers, AMNCH- Chairperson of the Advisory Board, in attendance at sub-group meetings and correspondence. Subsequent to publication of the revised guidelines by ASCO/ CAP in 2013 (11), the group re-convened to review the updated Guidelines and presented here as Version 2. Review Authors for Version 2, HER2 National Testing Criteria Dr Margaret Sheehan, BreastCheck/ Galway University Hospital Dr Cecily Quinn, BreastCheck/ St Vincent s University Hospital Dr Tara- Jane Browne, Cork University Hospital Dr Mairead Griffin, St James s Hospital, Dublin Version 2 circulated to wider group March 2014. Discussed at open forum 25/3/14. Feedback/ comments incorporated in the final document Version 2 finalised 31st March 2014 **indicates changes to guidelines in Version 2 update The HER2 subgroup meetings have been facilitated by Roche.
HER2 Testing Criteria- consensus from sub-group Specimen for testing: Core biopsy or excision specimen as per local practise No evidence in literature that either specimen is preferable Mandatory Criteria GENERAL CRITERIA IHC and ISH Formalin fixation (10% neutral buffered Formalin) Immediate fixation of core Slicing of excision specimens with fixation same day/next day Duration fixation 6-72 hours ** Sections for IHC <6 weeks old Positive and negative controls with each IHC run Participation in technical IHC NEQAS (or other recognised EQA scheme) Laboratory accredited/in process of accreditation Comment: Slicing of excision and fixation same day/next day may present a problem for specimens received late on a Friday- local steps should be taken to avoid this where possible INTERPRETATION CRITERIA Reporting categories IHC (0, 1+, 2+, 3+) Changes in categorisation criteria for IHC scores 0,1+,2+,3+ see Fig 1 ** **Key changes in IHC scoring: Critical threshold of cell membrane staining reduced from 30% tumour cells to 10% tumour cells for all reporting categories IHC score 0 now includes some weak/barely perceptible staining in 10% or less of tumour cells (previously scored 1+) IHC incomplete weak/ moderate membrane staining in >10% tumour cells now categorised as IHC score 2+ (previously scored 1+) ISH testing of all IHC 2+ cases IHC score only invasive tumour (not DCIS) EXCLUSION CRITERIA Core biopsies fixed <1 hour
Strong IHC membrane staining in internal normal ducts/lobules Controls not stained as expected Excessive crush/edge artefact in core Excess cytoplasmic staining obscuring membrane staining Very little tumour in core repeat on excision specimen Comment: individual cases need to be evaluated on criteria above on a case by case basis VALIDATION OF TEST CRITERIA Number of cases >50 >95% concordance for completion validation process Validation of IHC against ISH Any alteration to the technique requires revalidation of the technique ISH TESTING CRITERIA ISH testing all IHC 2+ cases Chromosome 17 probe included in ISH test Minimum 20 cells counted for a homogenous case (amplified or non amplified) Score only invasive carcinoma (not DCIS) IHC 2+ area used to guide ISH assessment Borderline category ratio 1.8-2.2 ** this category now eliminated **Categories of ISH reporting See Fig 2, 3. Heterogeneity CISH testing validated against FISH ISH negative- Average HER2 copy number <4.0 signals/cell and HER2:CEP17 ration<2.0 ISH equivocal- average HER2 copy number >=4.0 and <6.0 signals/cell ISH positive- Average HER2 copy number >=6.0 signals /cell and/or HER2:CEP 17 ratio >=2.0 Use of HER2 copy numbers and/or HER2:CEP 17 ratio to categorise using dualsignal (HER2 gene) assay (dual-probe ISH) Additional fields counted **heterogenous tumours- second contiguous population of cells with increased HER2 signals (>10% of tumour cells on slide), separate counting of at least 20 nonoverlapping cells in this area to be included in report REPORTING CRITERIA IHC reporting- clone used to be stated in report
ISH- probes used to be stated in report WORKLOAD CRITERIA HER2 testing (IHC and ISH) for both medical scientists and pathologists should be restricted to small numbers of individuals in accordance with the workload of the laboratory to ensure the individual has the optimum experience in this area.(*) (See comment) An individual pathologist must be reporting a reasonable number of cases (*) A laboratory performing a small number of cases has responsibility to reconsider it appropriate to meet with the testing guidelines and should consider referral to a larger laboratory for testing QUALITY ASSURANCE CRITERIA Regular internal audits of results (every 6-12 months depending on volume of workload being tested). Comparison of results with international anticipated overall rates for each category of IHC reporting and ISH amplification rates NEQAS (or other recognised EQA scheme) results to be reviewed by the pathologists and scientists REPEAT TESTING ** Repeat testing of recurrent / metastatic disease should be carried out where material is available Optional Criteria: GENERAL CRITERIA Positive control on each test case slide Use of TMAs for controls INTERPRETATION CRITERIA ISH testing of % of 0, 1+ and 3+ cases VALIDATION OF TEST CRITERIA Number of cases >100 REPORTING CRITERIA IHC double reporting of all cases ISH- number of cells counted stated in the report
QUALITY ASSURANCE CRITERIA Test reporting medical scientist and pathologist participation in scoring workshops as available REPEAT TESTING ** Repeat testing of HER2 suggested where certain morphological features of the tumour are regarded as challenging the molecular test result, or other equivocal test outcomes These include: initial HER2 negative result in Grade 3 carcinoma carcinoma on excision which is morphologically distinct from that seen on NCB biopsy initial IHC score 2+ and reflex ISH test equivocal (see above for definition)- consider an alternative ISH assay or repeat testing on new tissue Indeterminate for HER2 (see below for definition) Comment: Repeat testing is recommended in the ASCO/CAP 2013 updated guidelines in the above circumstances. Critical review of these updated guidelines by the Nottingham group (12) points out that supportive evidence for these Repeat Testing guidelines is not provided. For this reason we have included these, for now, under Optional Criteria in this guideline update, subject to revision pending further published data. INDETERMINATE FOR HER2 ** Category defined more specifically in the updated ASCO/CAP guidelines 2013 by the presence of technical issues interfering with test performance or assay. These include: inadequate specimen handling artefacts which limit interpretation test failure due to any other cause Repeat testing is recommended using another specimen. * Comment: Workload volumes for a testing laboratory The UK Group have made recommendations that a laboratory should carry out a minimum of 250 IHC tests per year and 100 FISH tests a year 5,9. The CAP guidelines do not specify a workload minimum 6.
This subgroup discussed this in relation to Irish laboratories where 8 centres of cancer care have been designated, each of which performs breast work. An occasional additional laboratory will also do such work affiliated with one of the 8 cancer centres. It was felt that the workload per laboratory guidelines did not address number of tests carried out per individual medical scientist or pathologist, which is felt to be as critical as overall workload volume per laboratory. With specialised breast reporting, individual pathologists in laboratories with differing HER2 testing workload may report a similar and adequate number of cases.
Gastric HER2 testing: There was agreement that this area of HER2 testing is still evolving with similarities and certain differences (interpretation/ scoring of IHC) from breast HER2 testing. The following overall guidelines were agreed at this stage, with anticipation that this area will be updated as literature and experience in this area expands. The following guidelines were agreed based on current literature: Criteria for specimen fixation, processing, test validation are as per breast HER2 testing Definition of negative, borderline, positive for gastric HER2 IHC is not the same as for breast and also differs for biopsy material and excision material. The interpretation and scoring criteria for IHC of Hofmann et al should be currently applied (8) QA for gastric HER2 testing should be as for breast and it is recommended that a laboratory involved in gastric HER2 testing should have QA HER2 expertise with breast (to include NEQAS or equivalent participation) HER2 is currently recognised in the treatment of metastatic or locally advanced gastric cancer. The sub-group would recommend testing all such patients, as identified by the MDM group locally. Testing of all gastric cancers (ie not just metastatic/locally advanced) would need to be decided locally as directed by local policy and laboratory facility to carry out same. **The current Updates ASCO/ CAP guidelines refer to HER2 testing in breast only and do not address gastric/ oesophago-gastric adenocarcinoma. Abbreviations: IHC- Immunohistochemical staining FISH- Fluorescent in situ hybridisation CISH- Chromogenic in situ hybridisation QA Quality assurance NEQAS- National external quality assessment scheme NCB- Needle core biopsy TMA- Tissue microarray
References: 1. Ellis IO et al. Recommendations for HER2 testing in the UK. J Clin Pathol 2000; 53:890-892 2. Bilous M et al. Current Perspectives on HER2 testing: A Review of National Testing Guidelines. Mod Pathol 2003; 16(2): 173-182 3. Ellis IO, Bartlett J, Dowsett M et al Updated recommendations for HER2 testing in the UK. J Clin Pathol 2004; 57; 233-237 4. P Fitzgibbon et al. Interlaboratory comparison of immunohistochemical testing for HER2.Results of CAP 2004 and 2005 tissue microaray study. Arch Pathol Lab Med. 130: 144.0-1445 5. M Dowsett et al. HER2 testing in the UK: Consensus from a national consultation. J Clin Pathol 2007; 60: 685-689 6. Wolff A et al. American Society of Clinical Oncology/ College of American Pathologists Guideline Recommendations for Human Epidermal Growth factor Receptor 2 Testing in Breast Cancer. Arch Pathol Lab Med 2007; 131: 18-43 7. Dowsett M et al. Standardisation of HER2 testing: results of an international proficiency- testing ring study. Mod Pathol 2007; 20: 584-591 8. Hofmann M, Stoss O, Buttner R, van de Vijver M et al. Assessment of a HER2 scoring system for gastric cancer: results from a valiation Study. Histopathology 2008; 52; 797-805. 9. Walker R, Bartlett J, Dowsett M et al. HER2 testing in the UK: further update to recommendations. J Clin Pathol 2008; 61; 818-824 10. T Garcia-Caballero et al. Determination of HER2 amplification in primary breast cancer using dual in situ hybridisation is comparable to fluorescence in situ hybridisation. Histopathology 2010, 56: 472-480 11. Wolff AC et al. Recommendations for Human Epidermal Growth Factor Receptor Testing in Breast Cancer: American Society of Clinical Oncology/ College of American Pathologists Clinical Practice Guideline Update. Journal of Clinical Oncology: 2013; 31 (31): 3997-4013 E-pub http://jco.ascopubs.org/cgi/doi/10.1200/jco.2013.50.9984 12. Rakha EA, Lee AHS, Ellis IO. The updated ASCO/CAP guideline recommendations for HER2 testing in the management of invasive breast cancer: critical review of their implications for routine practise. Histopathology 2013 (acepted Dec 25th) Accepted Article, doi: 10/1111/his.12357.
Appendix HER2 Testing Algorithinms Fig 1. Algorithm for evaluation of human epidermal growth factor receptor 2 (HER2) protein expression by immunohistochemistry (IHC) assay of the invasive component of a breast cancer specimen. Although categories of HER2 status by IHC can be created that are not covered by these definitions, in practice they are rare and if encountered should be considered IHC 2_ equivocal. ISH in situ hybridization. NOTE: the final reported results assume that there is no apparent histopathologic discordance observed by the pathologist. (*) Readily appreciated using a low-power objective and observed within a homogeneous and contiguous invasive cell population.
Fig 2. Algorithm for evaluation of human epidermal growth factor receptor 2 (HER2) gene amplification by in situ hybridization (ISH) assay of the invasive component of a breast cancer specimen using a single-signal (HER2 gene) assay (single-probe ISH). Amplification in a single-probe ISH assay is defined by examining the average HER2 copy number. If there is a second contiguous population of cells with increased HER2 signals per cell, and this cell population consists of more than 10%of tumor cells on the slide (defined by image analysis or visual estimation of the ISH or immunohistochemistry [IHC] slide), a separate counting of at least 20 nonoverlapping cells must also be performed within this cell population and also reported. Although categories of HER2 status by ISH can be created that are not covered by these definitions, in practice they are rare and if encountered should be considered ISH equivocal (see Data Supplement 2E). NOTE: the final reported results assume that there is no apparent histopathologic discordance observed by the pathologist. (*) Observed in a homogeneous and contiguous population.
Fig 3. Algorithm for evaluation of human epidermal growth factor receptor 2 (HER2) gene amplification by in situ hybridization (ISH) assay of the invasive component of a breast cancer specimen using a dual-signal (HER2 gene) assay (dual-probe ISH). Amplification in a dual-probe ISH assay is defined by examining first the HER2/CEP17 ratio followed by the average HER2 copy number (see Data Supplement 2E for more details). If there is a second contiguous population of cells with increased HER2 signals per cell, and this cell population consists of more than 10% of tumor cells on the slide (defined by image analysis or visual estimation of the ISH or immunohistochemistry [IHC] slide), a separate counting of at least 20 nonoverlapping cells must also be performed within this cell population and also reported. Although categories of HER2 status by ISH can be created that are not covered by these definitions, in practice they are rare and if encountered should be considered ISH equivocal (see Data Supplement 2E). NOTE. The final reported results assume that there is no apparent histopathologic discordance observed by the pathologist. (*) Observed in a homogeneous and contiguous population. ( ) See Data Supplement 2E for more information on these rare scenarios.