HER2 Testing Guidelines in Breast Cancer

· Who should be tested:

· All newly diagnosed patients with breast cancer

· Every primary invasive breast cancer

· Patients who then develop metastatic disease must have a HER2 test performed in a metastatic site, if tissue sample is available

· Especially should be considered for a patient who previously tested HER2 negative in a primary tumor and presents with disease recurrence with clinical behavior suggestive of HER2-positive or triple-negative disease

· Recurrent invasive breast cancer

· What samples should be tested:

· Core biopsy routinely (CCO)

· Excisional specimen, if repeat testing is indicated:

· Equivocal test result:

· On the core, retest on the excision (CCO)

· On the excision, testing of additional blocks and/or specimen on equivocal cases may depend on pathologist judgement (CCO)

· If HER2 copy number is between 4 and 5, repeat testing is not recommended (CCO)

· If HER2 copy number is between 5 and 6, or if there are other clinical concerns, testing of one additional block may be recommended (CCO)

· If the disease is node positive, testing of the lymph node metastasis may be considered (CCO)

· Reflex testing, OR:

· on the same specimen (unless the pathologist has concerns about the specimen) using an alternative test

· New test on a new specimen, if available (same or alternative method)

· If equivocal result remains after reflex testing

· Oncologist should confer with the pathologist regarding need for HER2 testing on the same or another tumour specimen, if two separate HER2 tests

· Histopathologic discordance with HER2 result, then should be discussed

· HER2 positive AND:

· Grade 1, AND:

· IDC or lobular carcinoma, ER+ PR+

· Tubular carcinoma (>= 90% pure)

· Mucinous carcinoma (>= 90% pure)

· Cribriform carcinoma (>= 90% pure)

· Adenoid cystic carcinoma (>= 90% pure) (often triple neg)

· No need to repeat if HER2 negative with those mentioned histologies

· HER2 negative AND:

· Grade 3 (on biopsy, or morphologically distinct on resection)

· Small amount of invasive tumour in the biopsy

· Doubts about the specimen handling of the biopsy:

· Long ischemic time

· Short fixation time

· Different fixative

· Suspicion of testing error on the core

· Indeterminate test result (another specimen should be requested)

· Ex. specimen handling is not in accordance with guideline recommendations

· Controls are not as expected

· Observer cannot find and count at least two areas of invasive tumour

· > 25 % of signals are unscoreable due to weak signals

· > 10 % of signals occur over cytoplasm

· Nuclear resolution is poor

· Autofluorescence is strong

· After neo-adjuvant treatment

· Negative result, but there remains significant clinical concern about the result after consulation between the pathologist and the oncologist

· If tests on three different blocks (core biopsy plus two excision specimen blocks) are negative, no additional testing is recommended.

· ER- on core biopsy (HER2 may also be retested, if HER2 was negative or equivocal)

· Other circumstances in which the pathologist deems repeat testing to be appropriate based on their clinical judgement (CCO)

· These repeat tests are to be viewed as medically necessary, and should be supported by CCO reimbursement.

· Specimen handling:

· Time to fixation (cold ischemic time):

· Fixation process should be initiated quickly by the laboratory (as short as possible)

· Time to fixative within 1 hour

· This should be recorded and considered in defining the test result

· 5-10 mm thickness of slices after appropriate gross inspection and margins designation

· Fixative: 10% neutral buffered formalin

· Cytology specimens must be fixed in formalin

· Volume sufficient

· Fixation time: 6 to 72 hours

· This should be recorded and considered in defining the test result

· Processing according to standardized, analytically validated protocols

· > 6 weeks old tissue sections should ideally not be used for HER2 testing; this may vary with primary fixation or storage conditions

· Methods of testing:

· FDA approved assay is preferred

· LDT may be chosen by a CLIA-certified laboratory

· Single-probe or dual-probe ISH

· Bright-field ISH:

· Must be validated by comparing to an FDA-approved FISH assay

· If tumour cell pattern is neither normal nor clearly amplified, test should be submitted for expert opinion

· When IHC result is clearly negative, ISH should not be performed, but rather reported as HER2 negative based on the IHC result (CCO)

· mRNA expression assays: insufficient evidence to support this method

· DNA microarray assays: insufficient evidence to support this method

· IHC interpretation:

· Review controls

· If not as expected, test should be repeated

· Score only infiltrating ductal carcinoma (avoid scoring DCIS)

· Positive

· IHC 3+ and no apparent histopathologic discordance

· IHC 3+:

· > 10 % of tumour with intense, uniform, circumferential membrane staining (resembling chicken wire) (readily appreciated using a low-power objective)

· Equivocal

· IHC 2+ and no apparent histopathologic discordance

· IHC 2+:

· Circumferential membrane staining that is incomplete and/or weak/moderate in > 10 % of tumour cells, OR

· CCO clarifation: “circumferential” pattern, defined as having at least a cluster of cells with “rim-like” or “honeycomb” membranous staining that comprises > 10% of the tumour, is required.

· The presence of the circumferential pattern is REQUIRED

· “360-degree” staining throughout every cell present is not required, but it is recommended that enough staining be present to appreciate this pattern using 10x objective

· At least a cluster of cells with rim-like or “honeycomb” membrane staining, and clusters of cells with this pattern should comprise > 10 % of the tumour

· < 10 % of tumour cells with intense, complete circumferential membrane staining

· Includes “scattered heterogeneity” cases (CCO term) showing scattered single cells with “360-degree” strong staining comprising < 10 % of the overall population

· Note that some rare gland-forming and micropapillary carcinomas show intense but incomplete (basolateral or U shaped) HER2 staining that is strictly considered 1+, but these should be considered equivocal and request reflex testing using an alternative test

· Negative

· IHC 1+ or IHC 0, and no apparent histopathologic discordance

· IHC 1+:

· Incomplete, faint/barely perceptible membrane staining in > 10 % of tumour cells

· IHC 0:

· No staining, OR

· Incomplete, faint/barely perceptible membrane staining within <= 10 % of tumour cells

· Indeterminate

· Technical issues prevent interpretation of positive, equivocal or negative

· Inadequate specimen handling

· If cytoplasmic staining obscures membrane staining

· Controls (batch or on-slide) show inappropriate staining

· Normal ducts and lobules show obvious cytoplasmic staining (unless in areas of apocrine metaplasia) (reject sample)

· Strong membrane staining of normal breast ducts (internal control)

· Crush or edge artifact (reject sample)

· Artifacts involve most of the sample

· CCO recommends to add a statement to the core biopsy biomarker report if repeat testing is indicated on the excision specimen (see above)

· ISH interpretation:

· Review corresponding H&E and/or IHC slide to localize the invasive cancer and avoid carcinoma in situ

· Review controls; if not as expected, test should be repeated (indeterminate result)

· Review the entire ISH slide to determine whether there is more than one population of cells with variable numbers of signals / cell (must be done by a pathologist prior to counting the sample), OR ALTERNATIVELY, use areas of 3+ IHC staining for ISH

· Count at least 20 non-overlapping cells in 2 separate areas of invasive cancer (at least 10 cells per area)

· If ratio is between 1.8 and 2.2, have an additional person count an additional 20 non overlapping cells

· Heterogeneity:

· Regional Heterogeneity (CCO term):

· If two populations of tumour cells show different levels of amplification, and each comprise > 10 % of total tumour cells on the slide, each should be separately counted, with at least 20 non-overlapping cells in each population. (ASCO/CAP)

· The percentage of the entire tumour on the slide that shows HER2 gene amplification must be defined by IHC. (ASCO/CAP)

· Only amplified populations of 10% or more of the entire cell population on the tissue sample should be reported. (ASCO/CAP)

· Scattered Heterogeneity (CCO term):

· the ISH score should include areas where these cells are present (compare to IHC if present on IHC) (CCO)

· Monosomy:

· When cells with both 1 and 2 CEP17 signals are present in an ISH study, it is recommended to selectively count the cells with two CEP17 signals (CCO)

· Reject if:

· Variable staining intensity of signals (> 25 %)

· Autofluorescence is high

· Nuclear resolution is poor (FISH)

· Background obscures signal resolution

· Brightfield ISH:

· Compare signals of normal breast to tumour cells to assure that normal and amplified areas can be readily distinguished. If the pattern is not clearly either unamplified or amplified, slides should be submitted for expert opinion.

· Personnel:

· Counting can be done by a trained technologist

· Pathologist must confirm that result (count) is correct and that invasive tumour was counted

· Interpretation:

· Positive:

· HER2 signals per cell > 6.0 (regardless of ratio)

· Ratio >= 2.0 (regardless of HER2 copy number)

· Cases containing amplified and non-amplified areas (discrete populations of tumour cells, not intermixed or scattered isolated cells), and the amplified cell population consists of more than 10% of tumour cells on the slide (defined by image analysis or visual estimation of the ISH or IHC slide) should be reported as positive

· The percentage of the total tumoru population with amplification should also be reported

· Equivocal:

· Mean HER2 per cell >= 4.0 and < 6.0, AND ratio < 2.0 (if dual-probe method used)

· Assessment of additional nuclei (at least double the usual number of cells) or by a second observer is recommended (CCO)

· Laboratories may pursue one of two options if coamplification of CEP17 is suspected:

· Repeat on the same specimen using an alternative probe for CEP17, or for another gene on chromosome 17 that is not expected to coamplify with HER2

· Reflex test, or order a new HER2 test on another available specimen

· Note: Equivocal cases as defined here are not elible for trastuzumab in Ontario (CCO)

· Negative:

· Ratio < 2.0 and mean HER2 per cell < 4.0

· Indeterminate:

· Technical issues prevent interpretation of positive, equivocal or negative

· Crush or edge artifact (reject sample)

· Inadequate specimen handling

· Controls (batch or on-slide) are not as expected

· Note: for single probe ISH test:

· Positive: >= 6.0 signals / cell

· Equivocal: >= 4.0 and < 6.0 signals / cell

· Negative: < 4.0 signals / cell

· Reporting:

· Elements to include:

· Case and block number

· Specimen site and type

· IHC:

· Antibody clone/vendor

· ISH:

· Probe(s) identification

· Results for each discrete population of tumour cells (if present)

· Only amplified populations of 10% or more of the invasive tumour on the slide should be reported

· Number of invasive tumour cells counted

· Number of observers

· Method used (test/vendor and if FDA approved)

· If FDA-approved method has been modified, a statement should be included indicating what modifications were made

· If not FDA approved, include a statement that the lab follows requirements of CAP or other accreditor for LDT reporting

· Image analysis method (if used)

· Controls (positive, equivocal, negative, internal)

· Adequacy of sample for evaluation (adequate number of invasive tumour cells present)

· Results

· Mean HER2 signals per cell

· Mean CEP17 signals per cell (if dual probes used)

· Ratio of mean HER2 / mean CEP17 (if dual probes used)

· Percentage of invasive tumour cells exhibiting complete membrane staining

· Uniformity of staining: present/absent

· Homogeneous, dark circumferential pattern: present/absent

· Statement about whether specimen handling falls within guideline recommendations

· Disclaimer statement required if the specimen handling requirements are not met

· Clear comment in the pathology report should be made if specimen handling falls outside of recommended guidelines

· If additional testing is pending, this should be included in the comment section

· CCO recommends to add a statement to the core biopsy biomarker report if repeat testing is indicated on the excision specimen (see above)

· (EQUIVOCAL CASES) A comment stating that the tumour is not considered eligible for trastuzumab treatment may be included, to clarify the significance of “Equivocal” to clinicians. (CCO)

· Cases that remain difficult to resolve (i.e. intratumoural heterogeneity), should be considered on a case-by-case basis. (CCO)

· Heterogeneity:

· Regional Heterogeneity (CCO term):

· Reporting recommendation (ISH): Heterogeneous or “Focally amplified”, with the percentage and size of the surface area amplified. (CCO)

· These cases may be considered for targeted therapy under the Evidence Building Program at CCO (even if the amplified population is < 10 %)

· Scattered heterogeneity (CCO term):

· Negative/equivocal/positive based on the average ratio and HER2 copy number, according to the 2013 ASCO/CAP criteria. A comment describing the scattered calls can be provided. (CCO)

· Monosomy:

· For cases of monosomy classified as “HER2-positive”, clinical judgment must be applied to determine the most appropriate management. As it is acknowledged that monosomy remains controversial and that there is a lack of definitive evidence for decision-making in these cases, a disclaimer may be included in the interpretation and/or report. (CCO)

· Terminology equating “monosomy” with “HER2-negative” should be avoided (CCO)

· example: “HER2 Interpretation: Positive based on Ratio. (HER2 copy number > 4. See Comment.) Comment: Although this case is considered Positive by 2013 ASCO/CAP guidelines, the predictive significance of this finding is not entirely clear.” (CCO)

·

· Who should be treated:

· HER2 positive cancer: recommend HER2 targeted therapy, if there is no apparent histopathologic discordance with HER2 testing, and if clinically appropriate.

· HER2 equivocal cancer:

· delay treatment decision, confer with pathologist regarding need for additional HER2 testing on the same or another tumour specimen.

· If ultimately equivocal after reflex testing with an alternative assay, HER2 targeted therapy may be considered on an individualized basis.

· HER2 negative: treatment not recommended, if there is no apparent histopathologic discordance with HER2 test result

· Validation:

· 20 negative and 20 positive for FDA-approved assays

· 40 negative and 40 positive for LDTs

· Compare IHC to ISH, or a modified IHC assay, or other such comparisons

· Typically 95% concordance (overall concordance rate) should be strived for (but specific concordance requirements are not required)

· Due to variability in the estimated concordance on a finite number of cases, the observed concordance in a particular testing session may drop slightly below 95%; however, a laboratory should strive for a long term average concordance of 95% or better

· If not achieved then a rigorous clinical valdation must be performed including clinical outcome to establish that the new assay reliably predicts clinical benefit from anti-HER2 therapy

· Cases included should be representative of the types of cases that would be seen in routine practice

· Specimen acquisition and handling

· Representative range of strong and weak positive and negative results

· Preferably separate validations should be carried out for positive cases and negative cases

· Testing analytic validation requirements:

· Concordance study comparing to established HER2 test

· Include a broad representation of patients with breast cancer with ~15-20% HER2+ rate

· QC/QA:

· 15-20% of primary breast cancers are HER2 amplified and/or overexpressed

· Internal quality assurance:

· Review and document external and internal controls with each test and each batch of tests

· Initial and ongoing competency assessment:

· Laboratory professionals as required under CLIA 88

· Pathologists as required under CLIA 88

· Should perform ongoing competency assessment and document the actions taken as a part of the laboratory record

· Acceptable performance standard remains the same as in the original guideline

· Ongoing QC and equipment maintenance

· Use of standardized operating procedures including routine use of control materials

· Revalidation of procedure if changed

· Optimal laboratory accreditation:

· Onsite inspection every other year with annual requirement for self-inspection

· Review:

· Laboratory validation

· Procedures

· QA results

· Processes

· Results

· Reports

· Unsatisfactory performance results in suspension of laboratory testing for HER2 for that method

· External proficiency testing is manadatory for CAP-accredited laboratories

· At least two testing events (mailings) a year

· >= 90% correct responses on graded challenges for either test for satisfactory performance

· Unsatisfactory performance requires investigation and corrective action according to accreditation agency program requirements, before the lab can continue to offer HER2 testing

· Examples of International QA programs:

· UK NEQUAS

· CAP

· RCPA

· ESP

· Nordiqc

· Routine periodic performance monitoring:

References:

· Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer. Arch Pathol Lab Med. 2014;138:241–256.

· CCO - Summary Statement: 2013 ASCO/CAP HER2 Guidelines: Building a Consensus for Ontario (June 2016)