Testing for her 2 in breast cancer: current pathology challenges faced in Canada

Review Article

Testing for her 2 in breast cancer: current pathology challenges faced in Canada


W. Hanna , MD , * , P. Barnes , BMedSc MD , R. Berendt , MD § , M. Chang , MD PhD * || , A. Magliocco , MD # , A.M. Mulligan , MD * ** , H. Rees , MD †† , N. Miller , MD * ‡‡ , L. Elavathil , MD §§ , B. Gilks , MD |||| , N. Pettigrew , MBChB ## , D. Pilavdzic , MD *** , S. SenGupta , MD †††

* University of Toronto, Toronto, ON.
Sunnybrook and Women’s College Health Science Centre, University of Toronto, ON.
QEII Health Sciences Centre, Dalhousie University, Halifax, NS.
§ Cross Cancer Institute, Edmonton, AB.
|| Mount Sinai Hospital, Toronto, ON.
# H. Lee Moffitt Cancer Center, Tampa, FL, U.S.A.
** Toronto General Hospital, University Health Network, Toronto, ON.
†† Saskatoon City Hospital, Saskatoon, SK.
‡‡ University Health Network, Toronto, ON.
§§ Juravinski Hospital and Cancer Centre, Mc-Master University, Hamilton, ON.
|||| Vancouver General Hospital and University of British Columbia, Vancouver, BC.
## University of Manitoba, Winnipeg, MB.
*** McGill University, Montreal, QC.
††† Queen’s University, Kingston, ON.

ABSTRACT

This review is designed to highlight several key challenges in the diagnosis of human epidermal growth factor receptor 2 ( her 2)–positive breast cancer currently faced by pathologists in Canada:

  • Pre-analysis issues affecting the accuracy of her 2 testing in non-excision sample types: core-needle biopsies, effusion samples, fine-needle aspirates, and bone metastases

  • her 2 testing of core-needle biopsies compared with surgical specimens

  • Criteria for retesting her 2 status upon disease recurrence

Literature searches for each topic were carried out using the medline , Embase, International Pharmaceutical Abstracts, and biosis databases. In addition, the congress databases of the American Society of Clinical Oncology (2005–2011) and the San Antonio Breast Cancer Symposium (2007–2011) were searched for relevant abstracts.

All authors are expert breast pathologists with extensive experience of her 2 testing, and several participated in the development of Canadian her 2 testing guidelines. For each topic, the authors present an evaluation of the current data available for the guidance of pathology practice, with recommendations for the optimization or improvement of her 2 testing practice.

KEYWORDS: her 2 testing, breast cancer , pathology

1.  INTRODUCTION

The human epidermal growth factor receptor 2 ( her 2) promotes cell proliferation and angiogenesis and inhibits apoptosis via the Ras/ mapk and pi 3 k /Akt pathways. The receptor is amplified or overexpressed (or both) in approximately 18%–20% of breast cancers1.

In breast cancer, her 2-positive status is a negative prognostic factor2. Before the introduction of her 2-directed therapies such as trastuzumab, the life expectancy of patients with her 2-positive disease was shorter than that of patients with her 2-negative tumours3. For patients with her 2-positive early breast cancer, 1 year of adjuvant therapy with trastuzumab has resulted in significant improvements in overall survival and disease-free survival4. Furthermore, trastuzumab-based therapy for early breast cancer is associated with reduced rates of disease relapse and reduced occurrences of metastatic disease5.

Trastuzumab-based therapy is the standard of care for her 2-positive metastatic breast cancer, and evidence is emerging that her 2-directed therapy remains a valid strategy even after disease progression6,7. Consequently, to accurately identify patients for whom her 2-directed therapy is appropriate, it is important that the tests and the testing procedures used to determine her 2 status both be reliable.

The American Society of Clinical Oncology ( asco ) and the College of American Pathologists ( cap ) have published guideline recommendations to improve the accuracy of her 2 testing in patients with invasive breast cancer1. The guidelines promote good general her 2 testing practice, but the information provided is insufficient in several key areas. In June 2010, an expert panel of practicing Canadian pathologists met to discuss current challenges in her 2 testing in patients with breast cancer. The aim of the discussions was to identify gaps in current her 2 testing guidelines and to provide recommendations for best practice. The outcomes of discussions relating to three key issues identified by the panel are presented here.

2.  PRE-ANALYSIS ISSUES IN NON-EXCISION SAMPLE TYPES AND BONE METASTASES

2.1  Background

Surgical excisions have long been the “gold standard” sample-type for the assessment of her 2 status8. When it is not possible or practical to obtain a surgical excision for her 2 testing (for example, in patients receiving neoadjuvant therapy or in patients with inoperable metastases), it may be desirable to assess her 2 status using a non-excisional sample such as a core-needle biopsy ( cnb ), fine-needle aspirate ( fna ), or effusion specimen.

Methods currently used to test her 2 status were developed and optimized for use with formalin-fixed, paraffin-embedded breast tissue specimens derived from surgical excisions9. However, tissue handling and fixation methods used with non-excisional samples may vary from those used with larger tissue samples. For example, biopsies taken from bone metastases usually require decalcification before her 2 status can be assessed. Consequently, it is important to establish whether any of these non-excision sample types can reliably be used for her 2 testing, and to determine whether sample handling and fixation protocols may be modified to improve the reliability of her 2 testing.

2.2  Evidence

2.2.1  Tissue Collection and Fixative

The asco / cap guidelines recommend that sample ischemia time (time from sample acquisition to fixation) should be as short as possible, because delaying fixation by more than 1 hour has been shown to negatively affect the detection of breast biomarkers1,10. That recommendation is underlined by the results of a recent study that compared immunohistochemistry ( ihc ) her 2 scores obtained for refrigerated and non-refrigerated excision samples with ihc her 2 scores obtained for corresponding cnb s (with negligible cold ischemia time). Significant reductions in ihc staining for her 2 were noted after only 2 hours of cold ischemia time for non-refrigerated samples and after 4 hours for refrigerated samples11.

The guidelines also specify that samples should be fixed in neutral buffered formalin and that use of any alternative fixative should be validated technically against results obtained from the same samples fixed in buffered formalin1. Current asco / cap guidelines do not provide guidance regarding sample handling or fixation techniques for use with cnb s, effusions, fna s, or bone metastases. Neutral buffered formalin is the only fixative recommended by manufacturers of her 2 testing kits such as HercepTest12, but fna s and pleural effusions are often fixed in ethanol, and concerns have been expressed that that approach may have a negative impact on the reliability of her 2 testing; nonspecific (false-positive) ihc staining for her 2 protein overexpression has been observed in some studies involving ethanol-fixed samples1315. Conversely, formalin fixation of fna s appears to improve the reliability of subsequent her 2 testing; concordance rates of up to 100% have been reported between formalin-fixed fna s or serous effusions and matched formalin-fixed tissue sections9.

In laboratories in which samples arrive already fixed in ethanol, in situ hybridization ( ish ) appears to be the most appropriate initial her 2-testing methodology. We identified several small studies in which liquid-based cytology samples were fixed in ethanolor methanol-based media and tested for her 2 status using ihc and ish (Table i). In the first of those studies, ethanol-fixed cytology samples and tissue blocks from 58 patients with invasive breast cancer were tested for her 2 status using ihc and ish . The ihc -based her 2 testing resulted in a concordance rate of only 75.9% compared with the corresponding formalin-fixed, paraffin-embedded excision samples, but chromogenic ish testing of cytology samples re-fixed in 4% paraformaldehyde resulted in an accuracy rate of 86.2% (50 of 58)16. A separate prospective study of 103 breast carcinoma samples found significant correlation between the chromogenic ish –determined her 2 status of cytology samples post-fixed in absolute ethanol and the corresponding histology samples17. As with ihc , ish -based her 2 testing of cytology samples appears most reliable when samples are fixed in formalin. A prospective study of 53 fna cytology samples fixed in ethanol and post-fixed in formalin detected a concordance rate of 100% (compared with matched formalin-fixed tissue blocks) when her 2 status was determined using cish 18. More recently, Almeida and Correia19 reported a chromogenic ish her 2 concordance rate of 94% (34 of 36) between formalin-fixed fna cytology samples and excision sample blocks.

TABLE I Accuracy of her 2 testing with chromogenic in situ hybridization using liquid-based cytology samples taken from breast tumours

 

2.2.2  Fixation Time

Although the asco / cap guidelines recommend a fixation time of 6–48 hours for excision samples, no practical guidance is provided regarding the optimum fixation time for cnb s or other non-excision sample types1. Although the permeation of formalin into cnb s is generally swifter than it is into excision specimens, there is general agreement that a fixation time of at least 6 hours is required to prevent under-fixation of cnb s. That period is supported by results of a study conducted by Dintzis and Allison20, who used formalin-fixed cell pellets, in which fixation times shorter than 6 hours resulted in a decrease in ihc her 2 staining intensity and a smaller proportion of cells with membrane staining. However, because fixation entails a chemical reaction process (known as the formaldehyde clock reaction) as well as tissue permeation, there is some suggestion that a fixation time of 24 hours is ideal to complete the fixation process. Pathologists should bear in mind that short or incomplete formalin fixation results in the specimen being sequentially fixed by ethanol during standard sample processing.

Although it has been suggested that prolonged fixation may lead to false-negative her 2 test results1, fixation time has been extended beyond 72 hours (median: approximately 79 hours)21 and even up to 96 hours22 without reducing ihc her 2 assay sensitivity.

2.2.3  Handling of Samples from Bone Metastases

The effect of decalcification on the reliability of her 2 testing with ihc was assessed in a prospective study of 10 randomly selected breast-tissue excision samples23. The authors found that membranous ihc her 2 staining was less intense and more heterogeneous in decalcified samples than in control samples. Compared with control samples, the decalcified samples showed a mean her 2 score that was reduced by 1.0 using the ihc 0 to 3+ scoring system23. Furthermore, fluorescence ish ( fish ) her 2 testing was unsuccessful in 2 of 2 cases. Thus, determination of her 2 status using fish may not be possible after sample decalcification. However, controlled decalcification using edta and daily radiography can result in a high concordance rate between ihc and ish . Zustin and colleagues24 used 10% edta to construct a tissue microarray using 149 breast-cancer bone metastases and decalcified samples, taking daily radiographs to assess the decalcification process. The success rate for her 2 testing by fish was 85.0% and a comparison of her 2 testing results obtained using ihc and fish revealed a concordance rate of 76.9% for ihc 3+ samples24. In some centres, current practice is to isolate soft-tissue cores from a bone metastasis sample for use in her 2 testing, because decalcification of such samples is not required.

2.3  Consensus

If the appropriate precautions are observed, her 2 testing of cytology samples and bone metastases is possible.

Non-excision samples should be immediately immersed in 10% phosphate-buffered formalin and fixed for at least 6 hours. Cytology specimens should always be fixed in formalin, not ethanol.

Immunohistochemistry is not suitable for determining the her 2 status of ethanol-fixed samples. In such cases, ish -based testing should be used.

Where soft-tissue cores can be isolated from bone metastases, those cores are preferred for determining her 2 status, because decalcification is not required. However, if such samples are not available, then use of a highly standardized decalcification protocol incorporating 10% edta and daily radiography is recommended, followed by her 2 testing using ish . If edta -based decalcification is not possible or practical, then decalcification by other methods can be considered, although the methods must be appropriately validated. Subsequent testing of her 2 status in such cases should be performed using ish . In the event that none of the foregoing approaches are either possible or successful, then we recommend referring to the her 2 status of the primary tumour, because high levels of concordance have been demonstrated between the her 2 status of primary tumour samples and metastases (see Section 4, “Criteria for Retesting her 2 Status on Disease Recurrence”).

In accordance with published guidelines, each laboratory should validate its her 2 testing methods wherever any one or a combination of the specimen type, handling, or fixation varies from that of the usual tissue specimen fixed in formalin.

3.  HER2 TESTING IN CNBs COMPARED WITH SURGICAL SPECIMENS

3.1  Background

As already outlined, the assumption is that, when an excision specimen is available, that specimen will be the primary source of material for her 2 testing. However, in circumstances in which obtaining an excision specimen is not possible or practical, or in which concerns arise about the quality of the excision specimen, it may be necessary to consider her 2 testing using a cnb . In cnb samples, her 2 testing raises several specific analysis considerations in addition to the pre-analysis considerations discussed earlier. Those considerations include

  • nonspecific staining caused by artifacts of compression or of edge and retraction; and

  • the small size of cnb specimens compared with surgical specimens, resulting in methodologic inconsistency and potential for sampling errors resulting from morphologic heterogeneity.

The first of those issues is addressed by the asco / cap and Canadian guidelines, which advise against her 2 testing using ihc in cnb s in which the entire core displays edge or retraction artifacts, or where crush artifacts are visible1,25. However, that problem is not common to all testing laboratories; some report that artifacts are not a significant feature in their analyses26. In practice, when such artifacts are present, the decision regarding whether and how to process the sample is left to the judgment of the individual pathologist.

The second issue, restricted sample size, also has potential implications for the reliability of her 2 testing, because equivocal ihc her 2 test results—and any consequent demand for confirmatory ish testing— are more likely to occur with small samples than with larger specimens (unpublished data). Morphologic heterogeneity can be addressed by testing additional tissue blocks chosen to represent the varied morphology of a heterogeneous tumour. However, heterogeneity poses particular challenges in cases in which only a small volume of tissue is available for technical and morphologic assessment. For instance, her 2-positivity occurs frequently in samples of ductal carcinoma in situ 27. Consequently, when small samples contain a mixture of ductal carcinoma in situ and invasive tumour, it may be difficult to identify invasive tumour so as to accurately gauge her 2 status. Finally, antigen retrieval and staining protocols may vary with the specimen size or type, resulting in methodologic inconsistencies compared with processing methods used for larger specimens.

Sample size is addressed by current Canadian guidelines (which specify that ish is the preferred initial testing method for small samples and cnb s in the neoadjuvant setting), but the asco / cap guidelines do not provide any specific discussion of the issue1,25. Consequently, there may be a perception that her 2 testing performed using cnb samples may be intrinsically less reliable than that using surgical excisions. However, if used appropriately, reliable her 2 testing is possible using cnb s8,28.

3.2  Evidence

When ihc or fish is used to determine her 2 status, concordance between the results obtained in cnb samples and in surgical specimens is generally excellent (about 85%–99%, Table ii). However, some studies found that results obtained using ihc staining were more consistent than those obtained using fish (99% and 94% respectively)30.

TABLE II Concordance between her 2 status of core-needle biopsy ( cnb ) and corresponding surgical ( ex ) samples in recent studies

 

The ihc her 2 score concordance between cnb samples and surgical excisions appears to vary according to the initial ihc score obtained for the cnb . For example, D’Alfonso et al. 29 reported 100% concordance between the her 2 status of cnb s scored as ihc 3+ and corresponding surgical samples tested using fish . Concordance rates were slightly lower for cnb s scored as ihc 1+ (90.6%) and ihc 0 (85.7%). In addition, the number of cores tested can have a bearing on the concordance rate between cnb and excision samples, although testing numerous cores may not be possible in all laboratories because of the cost implications. Some studies reported concordance rates of 100% when more than 3 cores were analyzed8,28. Accuracy may also be improved by obtaining larger cnb samples28,34 and by having samples evaluated by 2 or more observers34.

Tumour heterogeneity appears not to be a significant confounding factor in studies in which the her 2 status of cnb samples was determined using ish -based methods29. However, to ensure that patients whose cnb s were graded her 2-negative receive appropriate treatment, it has been suggested that ish retesting of cnb s scored as her 2 ihc 0/1+, or ihc retesting of a corresponding surgical specimen, may be advisable35.

The foregoing studies are published examples of her 2 testing in practice, and they reflect issues important to the accuracy and reliability of her 2 testing in cnb s. However, it should be acknowledged that it is not possible to evaluate whether the excision specimens and cnb s described in those studies were handled in a standardized manner. Certainly the pre-analysis conditions for processing cnb samples vary between studies. In some cases, fixation time was shorter than recommended by current guidelines8. Similarly, the antibodies or her 2 testing system used were not always specified. When defining her 2 status, half the studies did not use the current asco / cap cut-off limits for her 2-positivity by ihc 26,28,31,35, instead using older cut-off limits or failing to specify the cut-off used. Current asco / cap guidelines for ihc testing of her 2 status specify that, for a sample to be considered her 2-positive, more than 30% of invasive tumour cells should show intense uniform membrane staining1. Previously, the specified cut-off limit was 10%. Thus, as well as addressing concordance between her 2 testing in excisions and cnb s, the studies indirectly highlight the need for standardized pre-analysis and analysis measures and for adherence to current asco / cap guidelines for defining her 2 status.

3.3  Consensus

Concordance between her 2 testing results in cnb s and surgical specimens is generally good, but further improvements are possible. Strict adherence to pre-analysis and analysis measures is needed, and each laboratory should evaluate her 2 status concordance rates between ihc and ish in cnb samples before substituting one methodology for another. We recommend that reflex testing with ish should be carried out in cases in which ihc testing does not provide a clear positive ( ihc 3+) or negative ( ihc 0) result.

In the neoadjuvant setting, several cnb samples of non-necrotic tumour tissue should be tested to minimize the impact of tumour heterogeneity on result interpretation and to allow sufficient tissue for retesting by ihc or ish .

If the patient is not receiving neoadjuvant therapy, the excision specimen (where available) should be the primary specimen tested, and precedence should be given to the her 2 status of that sample unless concerns have arisen regarding its handling or preservation. If the excision specimen is of poor quality or does not contain an adequate volume of tumour, then retesting may be performed using cnb samples.

4.  CRITERIA FOR RETESTING HER2 STATUS UPON DISEASE RECURRENCE

4.1  Background

Whether her 2 status can change during disease progression is a matter of controversy. Furthermore, it is unclear whether anticancer therapy, particularly her 2-directed therapy, has any effect on her 2 status36. A proportion of the reported discordance in her 2 status is likely to stem from variations or improvements in her 2 testing methodology rather than from a true change in her 2 status between the primary tumour and metastasis37.

Retesting of her 2 status upon disease recurrence is not covered by current Canadian or asco / cap guidelines, although a recent update to the National Comprehensive Cancer Network guidelines recommends that the her 2 status of metastases should be tested if the her 2 status of the primary tumour is unknown or was originally negative, or if her 2 is not overexpressed38. We reviewed published studies addressing her 2 status in patients with metastases, including patients who received her 2-directed therapy for primary breast cancer.

4.2  Evidence

4.2.1  Retesting of Primary Tumour Tissue and Metastases in Treatment-Naïve Patients

The published data that explore the stability of her 2 status during disease progression are limited. Concordance between the her 2 status of primary tumour tissue and metastases has been investigated in several small studies. In retrospective studies in which her 2 status was compared between primary and metastatic tumour samples, discordance rates between 3.0% and 13.6% have been reported, although these examples are not exhaustive37,3945. Changes in her 2 status from the primary to the metastatic setting—both from her 2-positive to her 2-negative and from her 2-negative to her 2-positive—have been reported. The discordance rates reported in prospective studies have been similar. In one small prospective study, a discordance rate of 8% was calculated based on a review of 40 patients46. However, only 29 pairs of samples were evaluable for her 2 status, and discordant her 2 status from the primary to the metastatic setting was detected in only 2 patients. A more recent prospective study conducted in 60 patients with primary breast cancer and metastatic lymph nodes revealed concordance rates of 95% for her 2-negative primary tumours and 83.3% for her 2-positive primary tumours47.

4.2.2  Retesting Metastases in Patients Pretreated with Chemotherapy, With or Without Trastuzumab

There is little published evidence with which to evaluate any potential treatment effect on the her 2 status of patients who receive her 2-directed therapy for primary breast cancer and who subsequently develop metastases. A retrospective analysis by Xiao and colleagues36 demonstrated no significant difference in the her 2 status of primary and paired metastatic tumours in trastuzumab-treated patients compared with trastuzumab-naïve patients (86.6% and 82.1% respectively, p = 0.858). In patients whose her 2 status was discordant between the primary tumour and metastases, no apparent link could be drawn between changes to her 2 status and chemotherapy, endocrine therapy, metastasis site, or time to relapse36. However, variations in testing methodologies—and equivocal her 2 testing scores—were common in tumour pairs in which the her 2 status was discordant. A separate study of 137 primary breast tumours and metachronous metastases revealed discordant her 2 status in only 10% of cases ( n = 14). Given that some of the patients had received prior trastuzumab, it is interesting to note that a pairing of her 2-negative primary tumour with her 2-positive metastasis was significantly more common than was her 2-positive primary tumour with her 2-negative metastasis (12 cases vs. 2 cases, p = 0.04)48. However, no dedicated analysis was performed to assess the stability of her 2 status according to trastuzumab pretreatment.

The global phase ii shersig study (MO22004/NCT00885755) will assess changes in molecular marker expression in patients receiving her 2-directed therapy. The study will attempt to identify biomarkers associated with treatment efficacy and tumour biology from serial biopsy samples. It is expected to report during 2014.

4.3  Consensus

In accordance with the National Comprehensive Cancer Network guidelines, the her 2 status of metastases should be retested in patients whose primary tumours were her 2-negative or in whom the her 2 status of the primary tumour is unknown.

The mechanism by which her 2 status appears to change in some patients is not yet fully understood, and it is important that her 2 testing methods be standardized to ensure that the stability of her 2 status is accurately evaluated.

5.  CONCLUSIONS

5.1  Pre-analysis Issues in Non-Excision Sample Types and Bone Metastases

Excision specimens and cnb samples should be transferred to the laboratory for fixation without delay (ischemia time < 1 hour) and fixed in 10% phosphate-buffered formalin for at least 6 hours. Cytology specimens should always be fixed in formalin, not ethanol. If ethanol- or methanol-fixed samples are received for her 2 testing, then ish rather than ihc should be used as the initial testing methodology.

Soft-tissue cores are preferred for determining the her 2 status of bone metastases, because decalcification is not required. If soft-tissue cores are not available, then the following options may be considered (in order of preference):

  • Decalcification using a highly standardized decalcification protocol, incorporating 10% edta and daily radiography, followed by her 2 testing using ish

  • Decalcification using other methods (which must be appropriately validated), followed by her 2 testing using ish

If above approaches are not possible or successful, then refer to the her 2 status of the primary tumour.

In accordance with published guidelines, each laboratory should validate testing methods whenever any one or a combination of the specimen type, handling, or fixation varies from that of the usual tissue specimen fixed in formalin.

5.2  HER2 Testing in CNBs

Strict adherence to pre-analysis and analysis measures is needed when performing confirmatory her 2 testing using cnb s. In cnb samples, her 2 status concordance rates between ihc and ish should be evaluated before one methodology is substituted for another. Reflex testing with ish should be carried out if ihc testing does not provide a clear positive ( ihc 3+) or negative ( ihc 0) result.

In the neoadjuvant setting, tumour samples scored as ihc 2+ or 1+ should be retested using ish to confirm her 2 status. If the patient is not receiving neoadjuvant therapy, the excision specimen (when available) should be the primary specimen tested, and precedence should be given to the her 2 status of that sample unless there are concerns regarding its handling or preservation. If the excision specimen is of poor quality or does not contain an adequate volume of tumour, then retesting may be performed using cnb samples.

5.3  Criteria for Retesting HER2 Status on Disease Recurrence

Retesting of metastases is recommended if the her 2 status of the primary tumour is unknown or her 2-negative. The standardization of her 2 testing methods is desirable to ensure that the stability of her 2 status is accurately evaluated.

6.   ACKNOWLEDGEMENTS

Support for third-party writing assistance for this manuscript, furnished by Claire Cridland, was provided by F. Hoffmann–La Roche Ltd., Canada.

7.  CONFLICT OF INTEREST DISCLOSURES

Funding for the National her 2 Pathology Advisory Board meeting and support for third-party writing assistance were provided by F. Hoffmann–La Roche Ltd., Canada. All advisory Board members received an honorarium to compensate them for the time taken to attend the meeting.

WH has attended other advisory board meetings at the invitation of F. Hoffmann–La Roche Ltd. and has received associated honoraria. DP has participated in other advisory board meetings and an educational program for F. Hoffmann–La Roche Ltd. and has received associated honoraria. MC has received research funding from F. Hoffmann–La Roche Ltd.

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Correspondence to: Wedad Hanna, Sunnybrook Health Sciences Centre, 2075 Bayview Avenue, Room E4 32, Toronto, Ontario M4N 3M5., E-mail: wedad.hanna@sunnybrook.ca

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Current Oncology , VOLUME 19 , NUMBER 6 , 2012








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