1. About these slides SPEC – Short Presentation in Emerging Concepts
Provided by the CAP as an aid to pathologists to facilitate discussion on the topic.
Content has been reviewed by experts at the CAP, but does not necessarily reflect the official opinion of the College of American Pathologists.
Non-CAP material with identified copyright source may only be copied or distributed under a license (permission) from the copyright holder, or under the doctrine of fair use.
Version 1.0, rev. 5/28/2014

2. HER2 Testing in Breast Cancer: 2013 ASCO/CAP HER2 Guideline Update
Short Presentation in Emerging Concepts (SPEC)

3. Why is accurate HER2 Testing Important?
Inaccurate HER2 testing by both IHC and ISH remains a clinical concern in routine practice given the significant demonstrated benefit from HER2-targeted therapy for HER2 positive breast tumors.
Prospective sub-studies from two adjuvant trials demonstrate that up to 20% of HER2 results may be inaccurate.
The ASCO/CAP HER2 Guideline provides standards for improving the accuracy and reliability of HER2 testing.
In 2007, a joint Expert Panel convened by ASCO and CAP met to develop guidelines for when and how to test for the human epidermal growth factor receptor 2 gene (HER2, also referred to as ERBB2), which is amplified and/or over-expressed in approximately 15-20% of primary breast cancers. This unprecedented collaboration was triggered by the substantial therapeutic benefit observed in most of the first generation randomized phase 3 clinical trials of trastuzumab in HER2-positive disease, and led to the approval in the adjuvant setting of this humanized monoclonal antibody directed against the extra-cellular domain of the HER2 protein. Trastuzumab had previously been shown to improve progression free and overall survival (PFS/OS) when combined with chemotherapy in the metastatic setting. Since 2005 several of the first generation adjuvant trials have been updated and confirmed the disease free and overall survival (DFS/OS) benefit offered by one year of trastuzumab administered with or after adjuvant chemotherapy.
Prospective sub-studies (secondary aims) from two of the adjuvant HER2 clinical trials demonstrated up to 20% of the HER2 testing being performed in the community at that time may be inaccurate. This finding lead the two organizations to come together and draft the HER2 testing guidelines of 2007, which has provided standards for improving the accuracy and reliability of HER2 testing. To help address ongoing testing needs and challenges in light of new published literature, the ASCO/CAP HER2 Guideline was updated in 2013.
To address ongoing testing needs and challenges in light of new published literature, the ASCO/CAP HER2 Guideline was updated in 2013.
Wolff AC, et al., Arch pathol lab med. 2007;131:18-43.

4. 2013 HER2 Testing in BC Guideline Update
What Changed?
Recommendations
2013 Updates
Perform HER2 testing on every primary invasive tumor and any subsequent reoccurrence including metastatic sites.
Optimal tissue specimen handling procedures
Maximum time in fixative: 72 hours
New algorithms for test interpretation and reporting
Language on repeat testing (reflex and new tests)
The recommendations that changed in the 2013 HER2 testing update are summarized on this slide. Many of these changes will be further elaborated in this presentation.
Need for enhanced communication between pathologists and oncologists
Guidance for communicating with patients
Revised test validation requirements to align with ER/PgR recommendations
Wolff AC et al. Arch pathol lab med. 2014;138: 4

5. 2013 HER2 Testing in BC Guideline Update What Remains the Same?
Recommendations
No Change from 2007
Optimal tissue specimen handling procedures
Tissue acquisition (i.e., minimize cold ischemic time) < 1 hour
Fixative: 10% neutral buffered formalin (NBF)
Minimum duration of fixation: 6 hours
Must document fixation time points in accession or report
The recommendations that have stayed the same between the 2007 guidelines and the 2013 HER2 testing update are summarized on this slide.
Laboratory quality assurance processes, including proficiency testing and lab accreditation
Wolff AC et al. Arch pathol lab med. 2014;138: 5

6. 2013 HER2 Testing in BC Guideline Update Tumor Specimens to be Tested
2007 Guidelines1
Resection specimens preferred sample for HER2 testing
More representative sample of the patient’s tumor, more tumor tissue for evaluation
2013 Guideline Update2
Increasing use of core for testing
Core biopsies can be used for initial test (likely better pre- analytics)
Repeat testing on the excision may be necessary if a HER2 result is negative on the core in certain circumstances
The 2007 guideline stated that the resection specimen was the preferred sample for HER2 testing. The thinking was that the re-sected tumor sample would provide more tumor tissue for analysis. Since that time, we have clearly seen an increasing use of the initial diagnostic needle core biopsy for breast predictive factor testing. One advantage of the needle core biopsy would be a negligible ischemic time for the tissue and rapid tissue fixation.
The panel felt that the core biopsy was an acceptable sample for the initial HER2 analysis, however repeat testing on the excision may be necessary if a HER2 result is negative on the core in certain circumstances:
Tumor is grade III
Amount of invasive tumor in the core biopsy is small
Resection specimen contains high grade carcinoma that is morphologically distinct from that in the core
There is doubt about specimen handling of the core biopsy:
Long ischemic time,
Short time in fixative,
Different/alternative fixative used, and/or
Test is suspected by the pathologist to be negative based on testing error
Some of the limitations of using core biopsies for HER2 testing are shown on the next slide.
1. Wolff AC, et al., Arch pathol lab med. 2007;131:18-43.
2. Wolff AC, et al. Arch pathol lab med. 2014;138:

7. HER2 IHC stain obtained by core needle biopsy
2013 HER2 Testing in BC Guideline Update Tumor Specimen Selection
Core samples may not be optimal in some situations
Crushing and surface artifacts in cores may hamper interpretation
Tumor on resection may show morphologic heterogeneity
Tumor on resection may show intratumoral heterogeneity
Tissue is not fixed for adequate length of time
HER2 IHC stain obtained by core needle biopsy
Heterogeneity
This slide explains the limitations of using needle core biopsies for HER2 testing in breast cancer.
Intratumoral Heterogeneity
(HER2 IHC)
Edge Artifact (HER2 IHC)
Crush(HER2 IHC)
If core results are questionable, test the excision specimen 7

8. 2013 HER2 Testing in BC Guideline Update Duration of Fixation
Time in fixative
6 – 72 hours
2007 Guidelines
Time in fixative
6 – 48 hours
Applies to both excision and core specimens
In 2013 the duration of fixation time was changed based on prospective data reported in the literature, so that both the ASCO/CAP HER2 and ER/PgR Testing Guidelines now share the same recommendation for the duration of fixation.
Both the ASCO/CAP HER2 and ER/PgR Testing Guidelines now share the same recommendation for the duration of fixation. 8

9. 2013 HER2 Testing in BC Guideline Update IHC HER2 Positive Interpretation Criteria, Redefined
2007 Guidelines
Positive for HER2 is 3+ (defined as uniform intense membrane staining of > 30% of invasive tumor cells).
Positive for HER2 is 3+ (defined as uniform intense membrane staining of >10% of invasive tumor cells*.
2013 Guideline Update
HER2 (3+) in 10% of tumor
(HER2 Positive)
HER2 (3+) in >90% of tumor
(HER2 Positive)
The 2013 guidelines placed the threshold for HER2 positive by IHC back to >10% of the invasive tumor cells showing intense, circumferential membrane staining. The 10% threshold was the eligibility requirement for enrollment in the first generation adjuvant clinical trials and is also the threshold provided in the package insert for the FDA approved IHC test.
The threshold was moved to 30% in 2007 with the idea that this would help improve the concordance between IHC and ISH results. Emerging clinical data supported moving the threshold back to 10%. Some of the data supporting this changes came from a retrospective review of the 9831 trial published by Dr. Perez where about 4% of the HER2+ cases enrolled on that trial had strong circumferential membrane staining seen in 10-30% of the invasive tumor cells. These patients appeared to receive the same benefit as those who had strong circumferential membrane staining in >30% of their tumor.
If the pathologist or oncologist observes an apparent histopathologic discordance after HER2 testing, the need for additional HER2 testing should be discussed.
*Readily appreciated at low power. 9

10. 2013 HER2 Testing in BC Guideline Update IHC HER2 Negative Interpretation Criteria, Redefined
2007 Guidelines
Negative result for HER2 IHC is 0 or 1+
IHC 0: no staining
IHC 1+: weak, incomplete membrane staining in any proportion of tumor cells or weak, complete staining in <10% of cells
Negative result for HER2 IHC is 0 or 1+
IHC 0: No staining* or incomplete membrane staining (faint/barely perceptible) and within ≤ 10% of tumor cells
IHC 1+: Incomplete membrane staining (faint/barely perceptible) and within > 10% of tumor cells
2013 Guideline Update
A comparison of the criterion for HER2 negative by IHC between 2007 and 2013 is shown here. These changes are minor, however the 2013 update asks the pathologist to think about the result and interpret a negative HER2 IHC result in the morphologic context of the patient’s tumor. A weak to moderate partial membrane staining pattern in a patient with a grade 1 tumor which is strongly ER/PR+ and has a low proliferative index is consistent with a negative result. The same staining pattern in a patient with a grade 3 tumor and a high proliferative index should raise concern about HER2+ disease and consideration should be given to additional, either reflex or repeat, testing to rule-out HER2-driven cancer.  Decisions about repeat testing are best made in discussion with the patient’s medical oncologist.
HER2 IHC
If the pathologist or oncologist observes an apparent histopathologic discordance after HER2 testing, the need for additional HER2 testing should be discussed. 10

11. 2013 HER2 Testing in BC Guideline Update ISH HER2 Positive Interpretation Criteria, Redefined
2007 Guidelines
Positive for HER2 is FISH amplified (ratio of HER2 to CEP17 of > 2.2 or average HER2 gene copy number > six signals/nucleus for those test systems without an internal control probe).
2013 Guideline update
Positive for HER2 is ISH amplified ratio of HER2/CEP17 of ≥ 2.0 (with average HER2 signals >4) or if average HER2 signals are ≥6 signals/cell (regardless of ratio) in population of >10% of tumor cells.
A comparison of HER2 positive criteria by ISH between the 2007 and 2013 guidelines is shown here.
Rarely, one can encounter a case with a HER2/CEP17 ratio that is >2 but with a HER2 copy number that is less than 4. While these cases are rare, it is important to point out that patients with a HER2/CEP17 ratio of >2 were eligible for the adjuvant trial, even if HER2 was < 4. No one would expect a breast cancer with <4 copies of the HER2 gene to behave as a HER2 positive breast cancer, and in the unlikely event that the ratio is >2 it is probably due to chromosome 17 monosomy. The challenge here is that these patients were eligible for enrollment in the adjuvant Herceptin trial. Several members of the ASCO/CAP guideline panel felt that if someone was eligible for the trial, they should be eligible for therapy in spite of the fact that there is no good clinical data to support benefit. In cases where this scenario is encountered, one needs to do a careful review of all of the data for the case (tumor grade, proliferative index, ER/PR) and have a discussion with the patient’s medical oncologist. The decision about therapy becomes a clinical decision at that point.
If the pathologist or oncologist observes an apparent histopathologic discordance after HER2 testing, the need for additional HER2 testing should be discussed.
CEP17
Chromosome 17
HER2

12. 2013 HER2 Testing in BC Guideline Update ISH HER2 Negative Interpretation Criteria, Redefined
2007 Guidelines
Negative for HER2 is FISH HER2/CEP17 ratio of < 1.8 or average HER2 gene copy number of < 4 signals/nucleus for test systems without an internal control probe.
2013 Guideline update
Negative for HER2 ISH is HER2/CEP17 ratio < 2 or HER2 signals/nucleus < 4, regardless of ratio.
A comparison of the criteria for a HER2 negative result by ISH between the 2007 and 2013 guidelines is shown here.
Patients with a ratio of >2 were eligible for the adjuvant trial, even if HER2 was < 4. Although these cases are rare, given the eligibility criterion for the adjuvant trial and the demonstrated benefit from HER2 targeted therapy for patients with a ratio of greater than or equal to 2, the 2013 update considers a patient eligible for treatment with a ratio of > 2, even if the HER2 copy number was less than 4. It is important to carefully review all the pathologic features (grade, proliferative index, ER/PR results and HER2 IHC) for such cases before making a final determination of the HER2 status.
Fluorescence in situ hybridization (FISH) assay for HER2 quantitatively measures the level of HER2 gene amplification in breast cancer tumor cell nuclei. This image shows the appearance of the dual-colored FISH assay (DAKO HER2 FISH pharmDXTM, x1000 original magnification). Invasive tumor cell nuclei are highlighted by the blue fluorescence of a DNA counter stain (4’,6-diamidino-2-phenylindole [DAPI]). This example shows a non-amplified breast cancer with roughly equal numbers of HER2 (red signals) and CEP17 (green signals) with a calculated HER2/CEP17 ratio of less than <2.
If the pathologist or oncologist observes an apparent histopathologic discordance after HER2 testing, the need for additional HER2 testing should be discussed.

13. 2013 HER2 Testing in BC Guideline Update HER2 Equivocal Results Require a Reflex or Repeat Test
2007 Guidelines
Equivocal for HER2 IHC is 2+
ISH: FISH HER2/CEP17 ratio of or average HER2 gene copy number 4-6 HER2 signals/nucleus for test systems without an internal control probe
2013 Guideline update
Must report HER2 test result as Equivocal (HER2 tumor status Unknown) and order reflex test using the alternative test if:
IHC: (2+) circumferential membrane staining, incomplete and/or weak/ moderate in >10% of the invasive tumor cells; or complete and circumferential membrane intense staining within ≤10% of the invasive tumor cells
ISH: Dual Probe HER2/CEP17 ratio <2.0 with an average HER2 copy number ≥4.0 and <6.0 signals/cell
A comparison of the criteria for a HER2 equivocal result between the 2007 and 2013 guidelines is shown here.
It is important to emphasize that an equivocal result by either IHC or ISH requires additional reflex or repeat HER2 testing. If a reflex test on a HER2 Equivocal result does not render a (+) or (-) HER2 result, one must review the clinical and pathologic features of the case and confer with the oncologist about further testing.
Note: Equivocal is an interpretation category requiring additional testing.
If a reflex test on a HER2 Equivocal result does not render a (+) or (-) HER2 result, must review clinical and pathologic features of case and should confer with the oncologist about additional testing.

14. 2013 HER2 Testing in BC Guideline Update Changes to Testing Algorithm Help Address Heterogeneity
Intratumoral heterogeneity for HER2 can be seen in breast cancer by IHC and ISH.
With heterogeneity, the fields selected for evaluation will determine whether or not the tumor is reported as ISH amplified.
Mean score/ratio depends on the number of amplified cells counted and gene copy # after dilution with non-amplified cells.
Can lead to discordant results for HER2 analysis
Between IHC and ISH, cores vs excision, between blocks
Easier to detect with IHC (can be used to target ISH analysis)
Clinical significance of heterogeneity remains unclear however:
Patients with HER2 IHC 3+ (10-30%) and ISH ratio (2-2.2) appear to benefit from treatment with HER2-targeted therapy.
It is important to carefully review all the pathologic features (grade, proliferative index, ER/PR &HER2 results) for such cases.
The majority of tumors that demonstrate HER2 gene amplification show a uniform increase in the number of HER2 gene signals relative to the number of copies of chromosome 17 appearing diffusely through all areas of the invasive tumor (Hicks and Tubbs 2005). Occasionally, one encounters heterogeneity of gene amplification with scattered individual cells or a cluster of cells with amplification amid cells with a normal HER2 gene status (Tubbs et al. 2007).
Many new papers have described heterogeneity as a more frequent occurrence than previously thought and that could identify a subset of patients that might benefit from HER2-targeted therapy (Hanna 2014). Genomic heterogeneity can occur in three separate patterns:
>Discrete clustered populations (clones) of amplified and non-amplified tumor cells
>Diffuse intermingling of amplified and non-amplified cells across the tumor
>Isolated amplified cells in a predominantly non-amplified tumor.
Experts contend that the only type of amplified population that is significant is the discrete aggregated or clustered amplified cells. Most commonly, these separate populations are randomly counted, which could result in inconsistent test results by ISH. As such, cases of HER2 genomic heterogeneity can lead to discordant HER2 results between IHC and ISH, cores versus excision, between different blocks from the primary tumor or primary versus nodal metastasis.
In terms of the clinical significance of HER2 intratumoral heterogeneity, there was a retrospective review of the 9831 trial published by Dr. Perez where about 4% of the HER2+ cases enrolled on that trial had strong circumferential membrane staining seen in 10-30% of the invasive tumor cells (which I would interpret as intratumoral heterogeneity). These patients, who where enrolled and treated on the trial, appeared to receive the same benefit as those who had strong circumferential membrane staining in >30% of their tumor.
Hanna, et al., Mod Pathol Jan;27(1):4-18

15. ASCO/CAP 2013 HER2 Guideline Definition, HER2 Positive: Heterogeneity
Immunohistochemistry:
>10% of tumor cells with strong circumferential membrane staining observed in a homogenous or clustered contiguous population of at least 10% of invasive tumor cells.
Strong membrane staining is readily appreciated at low power.
HER2/CEP17 = 1.5
HER2/CEP17 = 7.1
Intratumoral heterogeneity for HER2 is rare. It was seen in about 4% of the HER2+ cases that were enrolled on the 9831 clinical trial. Most of the time when there is heterogeneity, the positive tumor cells are seen as discrete clusters of cells that are intermixed with negative cells. This is easiest to recognize with IHC but can also be detected with a careful low power scan of the ISH slide.
The criterion of 10% of the tumor being positive was not intended in the literal sense (1 in 10 cells or 2 in 20 cells). For IHC it is acceptable that if >10% of the invasive tumor cells show strong circumferential membrane staining the case should be considered positive for treatment eligibility.
The same rule should apply for ISH. If >10% of the invasive tumor shows gene amplification then the case is considered positive. This requires a careful low power scan of the slide looking for pockets or homogeneous/contiguous clustered of amplified cells. If this constitutes at least 10% of the invasive tumor then the case should be reported as amplified. The amplified ratio and counts should be reported separately from the non-amplified component of the tumor. Do not combine the ratio between the amplified and non-amplified components of the tumor as this may dilute the average numbers for the amplified component of the tumor and become misleading. The important point here is that if there is intratumoral heterogeneity within an amplified component to the tumor that we recognize it and call the breast cancer HER2 positive if this population represents at least 10% of the invasive tumor cells.
The 2013 guideline update recommends a standardized method for ISH interpretation that includes scanning of the entire slide prior to counting and/or using an IHC HER2 test to define areas of potential amplification. Any aggregate population of amplified cells comprising >10% of the total tumor cell population on the slide must be counted separately. The number of CEP17 and HER2 signals should be counted in a minimum of 20 non-overlapping and contiguous invasive cancer cell nuclei in at least 2 tumor areas of each population of tumor cells (unamplified and amplified areas). The HER2/CEP17 ratio should then be calculated for each population of cells individually including the average HER2 signals/cell and ratio of HER2 signals/CEP17 signals, if available. Cases containing amplified and non-amplified areas (using this definition) should be reported as positive for HER2. The percentage of the total tumor population with amplification should also be reported.
In Situ Hybridization:
HER2/CEP17 ratio of >2 observed in a homogenous or clustered contiguous population of at least 10% of the invasive tumor cells.
HER2 copy# >6

16. 2013 HER2 Testing in BC Guideline Update Histopathologic Discordance and Repeat Testing
If the initial test result is HER2 NEGATIVE on Core:
Order a New Test on the Excision if:
DO NOT order a New Test if:
Tumor is Histologic Grade-3
Small amount of invasive tumor on core
Resection contains high-grade component not present on core
Core biopsy equivocal diagnosis by both IHC and ISH
Questionable specimen handling of core or result is suspect to be negative due to testing error
Tumor is histologic grade-1
Infiltrating ductal or lobular carcinoma that is strongly ER/PR positive
Tubular carcinoma (>90% pure)
Mucinous carcinoma (>90% pure)
Cribriform carcinoma (>90% pure)
Adenoid cystic carcinoma (typically these tumors are triple negative)
The 2013 guideline update asks the pathologist to interpret the pattern of HER2 staining in the morphologic context of the case.  Because high grade breast cancer are more likely to be HER2 positive, the guidelines states that reflex or repeat testing should be considered if the initial HER2 test is negative and the tumor is high grade to help insure that an opportunity to offer HER2-targeted therapy is not missed.
A weak to moderate membrane staining pattern in a patient with a grade 1 tumor which was strongly ER/PR+ and has a low proliferative index is entirely consistent with a negative HER2 result. The same staining pattern in a patient with a grade 3 tumor which has a high proliferative index should raise a concern about HER2-positive disease and consideration should be given for additional, either reflex or repeat, testing to make sure that we have ruled out a HER2-driven cancer.  Decisions about repeat testing are probably best made after discussion with the patient’s medical oncologist.
Wolff AC, et al. Arch pathol lab med. 2014;138:

17. Chromosomal Abnormalities involving CEP17 (Aneusomy)
Chromosome 17
Polysomy 17 = increased copy number of HER2 & CEP17 signals
Most frequently defined as average CEP17# >3 by ISH
HER2/CEP17 <2 (not amplified)
aCGH studies have shown true chromosome 17-polysomy is rare
CEP17 copy number >3.0 in ISH is frequently related to gain or amplification of the centromeric region
Typically high grade tumor and HER2 IHC is (2+) or (3+)
HER2/CEP17 ratio < 2 may be misleading in such cases
CEP17
HER2
HER2 FISH
Some breast cancers demonstrate an abnormal number of copies of chromosome 17 within the tumor cells. This is referred to as chromosome 17 polysomy in the literature. This is defined in ISH assays as an average of > 3 copies of chromosome 17 within tumor cells (CEP17 > 3); this exists in 5 – 10% of breast cancers (Tubbs et al. 2007, Downs-Kelly et al. 2005).
Using the average number of CEP17 to define polysomy assumes that the number of CEP17 signals directly reflects the number of copies of chromosome 17 in the tumor cells and those tumors with increased CEP17 signals display chromosome 17 polysomy. Each extra copy of chromosome 17 in such cases would be expected to have a copy of the HER2 gene, which results in an overall increase in the average number of copies of HER2 genes. The HER2/CEP17 ratio corrects the number of HER2 copies for the increased number of chromosome 17 and the resulting ratio may be greater than or less than 2. For such cases, a ratio of < 2 has been interpreted as non-amplified for HER2. It is interesting that patients with a HER2/CEP17 ratio > 2 appear to benefit from HER2 targeted therapy regardless of the chromosome 17 copy number (Perez et al. 2010).
Recent pangenomic studies have demonstrated that true polysomy 17 is rare (Tse et al. 2011, Yeh et al Mod Pathol, Moelans et al Breast Cancer Res Treat) and in some cases, CEP17 may undergo gene amplification events in the same way as HER2 or other gene loci. Breast tumors with a HER2/CEP17 ratio <2, where the average HER2 copy number is > 6 and the average CEP17 copy number is > 3 may, in fact, represent HER2 positive cancers with amplification of both HER2 and CEP17 (Tse et al. 2011). In support of this, cases with these findings frequently are high grade and show over-expression of HER2 protein (2+ or 3+) by IHC.
HER2 IHC
Hanna, et al., Mod Pathol Jan;27(1):4-18, Tse, et al., J Clin Oncol Nov 1;29(31):

18. 2013 HER2 Testing in BC Guideline Update Changes to Testing Algorithm Help Address Chromosomal Abnormalities involving CEP17 (Aneusomy)
Co-amplification of CEP17 region is observed in many ISH assays with increased HER2 and CEP17 copy #
May lead to a HER2/CEP17 ratio < 2.0 suggesting lack of HER2 amplification and discordant IHC/ISH results
If the HER2 copy number is >6, the HER2 test result must be reported as Positive regardless of the HER2/CEP17 ratio
HER2 amplification defined by ratio criterion (>2), HER2 copy# criterion(>6) or both
Some labs may choose to repeat HER2 testing in the same specimen using an alternative chromosome 17 reference probe (another gene on chromosome 17 not expected to co- amplified with HER2) to help demonstrate an amplified ratio (>2)
Although increases in CEP17 copy number have been interpreted as representing polysomy 17, recent pangenomic studies have demonstrated that true polysomy 17 is rare (Tse et al. 2011, Yeh et al Mod Pathol, Moelans et al Breast Cancer Res Treat) and in some cases, CEP17 may undergo gene amplification events in the same way as HER2 or other gene loci. Breast tumors with a HER2/CEP17 ratio <2, where the average HER2 copy number is > 6 and the average CEP17 copy number is > 3 may, in fact, represent HER2 positive cancers with amplification of both HER2 and CEP17 (Tse et al. 2011).
Based on this emerging literature, the ASCO/CAP panel concluded in the 2013 update that if the average HER2 copy number was > 6 then the HER2 test result must be reported as positive, regardless of the HER2/CEP17 ratio. Furthermore, in such cases, repeat ISH analysis for HER2 with an alternative chromosome 17 reference probe in such cases (such as RARA, or TP53) will frequently demonstrate a HER2-to-chromosome 17 ratio that is in the amplified range, supporting the idea that the HER2 gene and the centromere is co-amplified in these cases. It is important to carefully review all the pathologic features (grade, proliferative index, ER/PR results and HER2 IHC) for such cases before making a final determination of the HER2 status. Clearly, more investigation of the clinical outcomes for such patients is needed, particularly for patients whose HER2 status may be reclassified as amplified rather than non-amplified due to polysomy 17.

19. Key Messages Guidelines are living document which change
From user feedback
From new publications and data
Iteration of guidelines leads to greater clarity
Algorithm changes in the HER2 testing guideline update will provide better safety for patients
Positive patients will be found and treated
Equivocal patients will have further work done to better define their HER2 status
Negative patients will be spared unnecessary treatment
Scrutiny of cases by physicians will find patients with unusual situations and generate discussion between Pathologists and Medical Oncologists 19

20. Selected Resources
Wolff AC1, Hammond ME, Schwartz JN, 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.
Wolff AC1, Hammond ME, Hicks DG, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. Arch pathol lab med. 2014;138:
Hanna WM, Rüschoff J, Bilous M, et al. HER2 in situ hybridization in breast cancer: clinical implications of polysomy 17 and genetic heterogeneity. Mod Pathol Jan;27(1):4-18.
Tse CH, Hwang HC, Goldstein LC, et al. Determining true HER2 gene status in breast cancers with polysomy by using alternative chromosome 17 reference genes: implications for anti-HER2 targeted therapy. J Clin Oncol Nov 1;29(31):

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