What Breast Medical Oncologists Need From Pathologists 

Updated: Mar 20, 2019
Author: Oudai Hassan, MD; Chief Editor: Chandandeep Nagi, MD 

Overview

Breast medical oncologists use the information provided by pathologists to make decisions about systemic therapy. The image below provides an example of a schematic representation of a decision tree used by medical oncologists when deciding whether to administer systemic therapy and/or which systemic therapy to recommend to a patient.

The flow chart shows the idealized decision making The flow chart shows the idealized decision making process of the medical oncologist when deciding whether and what systemic therapy to give. Remember that the decision to give a specific systemic therapy to an individual patient is based on the expected benefit as compared with the possible risks in each individual patient. The blue text provides links to the text below explaining the role of the pathologist in providing the necessary information to make these decisions. * The cut off is 2 cm for pure mucinous carcinomas. **OncotypeDX can help decisions in patients with tumors between 1 and 2 cm.

Providing an easy-to-read summary or synoptic report, including the key prognostic and predictive factors, is particularly helpful to the medical oncologist. Although many of the pathology elements are continuous variables, clinical decisions are not. Being aware of the critical factors that influence decisions in a particular patient when evaluating a specimen is important for pathologists. Medical oncologists should be knowledgeable about sources of controversy and should look for potential pitfalls in the pathology report. Tumor size, grade, axillary lymph node status, steroid hormone receptor status, and human epidermal growth factor receptor 2 (HER2)/neu overexpression are the factors that most commonly influence treatment decisions and are discussed in detail below.

In 2007, the American Society of Clinical Oncology (ASCO) updated their recommendations for the use of tumor marker tests in the prevention, screening, treatment, and surveillance of breast cancer. An overview of these recommendations is presented in Table 1.[1]

Table 1. Overview of the 2007 ASCO Guidelines for the Use of Tumor Markers in Breast Cancer (Open Table in a new window)

Marker

Recommendation

Cancer antigen (CA) 15-3 and CA 27.29

CA 15-3 and CA 27.29 as markers for breast cancer, as screening, diagnostic, or staging tests

Present data are insufficient to recommend CA 15-3 or CA 27.29 for screening, diagnosis, and staging.

CA 15-3 and CA 27.29 to detect recurrence after primary breast cancer therapy

Present data do not support the use of CA 15-3 and CA 27.29 for monitoring patients for recurrence after primary breast cancer therapy.

CA 15-3 and CA 27.29 to contribute to decisions regarding therapy for metastatic breast cancer

For monitoring patients with metastatic disease during active therapy, CA 15-3 or CA 27.29 can be used in conjunction with diagnostic imaging, history, and physical examination. Present data are insufficient to recommend the use of CA 15-3 or CA 27.29 alone for monitoring response to treatment. However, in the absence of readily measurable disease, an increasing CA 15-3 or CA 27.29 level may be used to indicate treatment failure. Caution should be used when interpreting a rising CA 15-3 or CA 27.29 level during the first 4-6 weeks of a new therapy, as spurious early rises may occur.

Carcinoembryonic antigen (CEA)

CEA for screening, diagnosis, staging, or routine surveillance of breast cancer after primary therapy

CEA is not recommended for screening, diagnosis, staging, or routine surveillance of breast cancer after primary therapy.

CEA to contribute to decisions regarding therapy for metastatic breast cancer

For monitoring patients with metastatic disease during active therapy, CEA can be used in conjunction with diagnostic imaging, history, and physical examination. Present data are insufficient to recommend use of CEA alone for monitoring response to treatment. However, in the absence of readily measurable disease, an increasing CEA level may be used to indicate treatment failure. Caution should be used when interpreting a rising CEA level during the first 4-6 weeks of a new therapy, as spurious early rises may occur. There is no change from the guideline published in 2000.

 Estrogen receptors (ERs) and progesterone receptors (PgRs)

ERs and PgRs

ER and PgR should be measured in every primary invasive breast cancer and may be measured on metastatic lesions if the results would influence treatment planning. In both premenopausal and postmenopausal patients, steroid hormone receptor status should be used to identify patients most likely to benefit from endocrine forms of therapy in both early breast cancer and metastatic disease. In patients with DCIS who are candidates for hormonal therapy, data are insufficient to recommend routine measurement of ER and PgR for therapy recommendations.

DNA flow cytometry–based parameters

DNA flow cytometry–based parameters

Present data are insufficient to recommend use of DNA content, S phase, or other flow cytometry–based markers of proliferation to assign patients to prognostic groups.

Immunohistochemically based markers of proliferation

Immunohistochemically based markers of proliferation

Present data are insufficient to recommend measurement of Ki67, cyclin D, cyclin E, p27, p21, thymidine kinase, topoisomerase II, or other markers of proliferation to assign patients to prognostic groups.

HER2

HER2 evaluation in breast cancer

HER2 expression and/or amplification should be evaluated in every primary invasive breast cancer, either at the time of diagnosis or at the time of recurrence, principally to guide selection of trastuzumab in the adjuvant and/or metastatic setting. Other utilities for HER2 evaluation are also discussed separately below.

HER2 to define prognosis for early-stage breast cancer in the absence of systemic therapy

HER2 amplification, HER2 overexpression, and the presence of HER2 extracellular domain are generally associated with a poorer prognosis. However, the value of this information in clinical practice is questionable, and the use of HER2 for determining prognosis is not recommended.

HER2 to select patients for anti-HER2–based therapy

High levels of tissue HER2 expression or HER2 gene amplification should be used to identify patients for whom trastuzumab may be of benefit for the treatment of breast cancer in the adjuvant or metastatic disease settings.

The utility of HER2 for predicting response to specific chemotherapeutic agents

Level II evidence (prospective therapeutic trials in which marker utility is a secondary study objective) suggests that overexpression of HER2 (3+ by protein or >2.0 FISH ratio by gene amplification) identifies patients who would have a greater benefit from anthracycline-based adjuvant therapy. If a clinician is considering chemotherapy for a patient with HER2-positive breast cancer, it is recommended that an anthracycline be strongly considered in the absence of contraindications to anthracycline therapy. In the context of trastuzumab therapy, there is level I evidence (single, high-powered, prospective, randomized, controlled trials specifically designed to test the marker or a meta-analyses of well-designed studies) that a nonanthracycline regimen may produce similar outcomes. At present, the Update Committee does not recommend that HER2 be used to guide use of taxane chemotherapy in the adjuvant setting.

HER2 to determine sensitivity to endocrine therapy

HER2 should not be used to withhold endocrine therapy for a patient with hormone receptor–positive breast cancer, nor should it be used to select one specific type of endocrine therapy over another.

Utility of circulating extracellular domain of HER2

Measuring circulating extracellular domain of HER2 is not currently recommended for any clinical setting.

p53

p53 as a marker for breast cancer

Present data are insufficient to recommend p53 measurements for the management of patients with breast cancer.

Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1)

uPA and PAI-1 as a marker for breast cancer (Note: This topic is new to the guideline)

uPA/PAI-1 measured by ELISA on a minimum of 300 mg of fresh or frozen breast cancer tissue may be used to determine prognosis in patients with newly diagnosed node-negative breast cancer. IHC for these markers is not accurate, and the prognostic value of ELISA using smaller tissue specimens has not been validated. Low levels of both markers are associated with a sufficiently low risk of recurrence, especially in hormone receptor–positive women who will receive adjuvant endocrine therapy, so chemotherapy will only contribute minimal additional benefit. Furthermore, CMF-based adjuvant chemotherapy provides substantial benefit compared with observation alone in patients with a high risk of recurrence as determined by high levels of uPA and PAI-1.

Cathepsin D

Cathepsin D as a marker for breast cancer

Present data are insufficient to recommend the use of cathepsin D measurements for the management of patients with breast cancer.

Cyclin E

Cyclin E fragments as markers for breast cancer (Note: This topic is new to the guideline)

Present data are insufficient to recommend the use of whole-length or fragment measurements of cyclin E for the management of patients with breast cancer.

Proteomic analysis

Proteomic analysis for breast cancer (Note: This topic is new to the guideline)

Present data are insufficient to recommend the use of proteomic patterns for the management of patients with breast cancer.

Multiparameter gene expression analysis

Multiparameter gene expression analysis for breast cancer (Note: This topic is new to the guideline)

In newly diagnosed patients with node-negative, ER-positive breast cancer, the Oncotype DX assay can be used to predict the risk of recurrence in patients treated with tamoxifen. Oncotype DX may be used to identify patients who are predicted to obtain the most therapeutic benefit from adjuvant tamoxifen and may not require adjuvant chemotherapy. In addition, patients with high recurrence scores appear to achieve relatively more benefit from adjuvant chemotherapy (specifically (C)MF) than from tamoxifen. There are insufficient data at present to comment on whether these conclusions generalize to hormonal therapies other than tamoxifen or whether this assay applies to other chemotherapy regimens. The precise clinical utility and appropriate application for other multiparameter assays, such as the MammaPrint assay, the "Rotterdam Signature," and the Breast Cancer Gene Expression Ratio are under investigation.

Bone marrow micrometastases

Bone marrow micrometastases as markers for breast cancer (Note: This topic is new to the guideline)

Present data are insufficient to recommend the assessment of bone marrow micrometastases for the management of patients with breast cancer.

Circulating tumor cell (CTC) assays

CTC assays as markers for breast cancer (Note: This topic is new to the guideline)

The measurement of CTCs should not be used to diagnose breast cancer or to influence any treatment decisions in patients with breast cancer. Similarly, the use of the recently FDA-cleared test for CTC (CellSearch assay) in patients with metastatic breast cancer cannot be recommended until further validation confirms the clinical value of this test.

CMF = cyclophosphamide, methotrexate, and fluorouracil; DCIS = ductal carcinoma in situ; ELISA = enzyme-linked immunosorbent assay; FDA = US Food and Drug Administration; FISH = fluorescence in situ hybridization; IHC = immunohistochemistry.

 

Tumor Size

Definition

Tumor size is measured as the largest dimension of the invasive tumor only, in millimeters.

Practical considerations

When available, the pathologic tumor size takes precedence over the clinical tumor size. The tumor size reported in the pathology report should reflect the pathologist’s best assessment of the largest dimension of the invasive tumor only. This is usually the microscopic invasive tumor size; however, in many circumstances, correlation with the findings from gross examination, prior specimens, different imaging modalities, and clinical information is needed to most accurately estimate the tumor size. Benign and in situ mass-forming lesions, biopsy site changes, fixation, and processing can all affect tumor size measurements.

Tumor size should be measured with a precision of 1 mm. Tumor size cutoffs of 1 cm and of 2 cm are particularly important for treatment decisions. A statement about how the measurement was obtained or estimated is helpful (eg, "Microscopic, gross, largest dimension in prior core biopsy is…, estimated by…, at least…, likely not more than…, correlation with imaging and clinical findings is needed.").

Controversies

Multiple tumor foci are measured separately. A comment about their relative location and the intervening breast tissue is needed. The size of the largest focus is used for staging.[2]

In patients who have undergone preoperative chemotherapy, the presence of residual invasive and in situ disease in the tumor bed and lymph nodes provides important information. However, at this time, the only validated pathologic measure is pathologic complete response (ie, lack of invasive disease at surgery), in addition to the size of the residual tumor. Cellularity of the residual tumor is also an important prognostic indicator. Classification of the response either by Miller-Payne scoring or residual cancer burden (RCB) scoring appears to predict clinical outcome.[3]  Although the latter scoring systems require additional validation for widespread use in clinical practice, they may provide useful information for the oncologist.

After neoadjuvant chemotherapy, tumors with a partial response often present as scattered foci of the tumor with variable cellularity and with a residual tumor bed that can be identified as reactive changes, including fibrosis, histiocytes, and fat necrosis, but is sometimes microscopically indistinguishable from normal breast tissue. Tumor stage after neoadjuvant therapy should be decided based on the size of the largest residual foci with the M descriptor added if there is more than one residual foci within the tumor bed.[4] The Miller-Payne score divides the reduction in overall cellularity in five grades, with grade 1 as no significant reduction in overall cellularity and grade 5 as a complete pathologic response at the site of the primary tumor. RCB scoring[4] combines the size of the primary tumor bed in two dimensions, the cellularity of the invasive cancer, and the amount of residual disease in the lymph nodes (size of the tumor bed, tumor cellularity, and lymph node status are needed to calculate the score) into a single score.

 

Histologic Grade

Histologic grade reflects how far the tumor architecture and cytology deviate from normal, and how rapidly the tumor proliferates. The College of American Pathologists (CAP) recommends the use of the Nottingham combined histologic grade, also called the modified Scarff-Bloom-Richardson grading system (see Table 2).[5]

Table 2. The Nottingham Combined Histologic Grade (Modified Scarff-Bloom-Richardson Grade) (Open Table in a new window)

Feature

 

Score

Tubule formation

>75%

1

10-75%

2

< 10%

3

Nuclear pleomorphism

Small, regular uniform cells

1

Moderate increase in size and shape

2

Marked variation

3

Mitotic counts per 10 high-power field (HPF) (field diameter, 0.52 mm)

0-7

1

8-15

2

≥16

3

Scoring

3-5 points

Well differentiated

Grade 1

6-7 points

Moderately differentiated

Grade 2

8-9 points

Poorly differentiated

Grade 3

Practical considerations

The size of the high-power field (HPF) (40×) needs to be taken into account when assessing mitotic counts. Mitotic counts should be assessed at the periphery of the tumor, in the most mitotically active area. One should first look around at low power to look for mitotic figures. Only definite mitotic figures should be counted.

Controversies

Due to tumor heterogeneity, assessment of tubule formation and mitotic count on core biopsy may not be representative of the entire tumor. When more than the nuclear grade is reported on the core biopsy, a possible change in grade on the excision specimen should be expected.

 

Axillary Lymph Node Status

Definitions

See Table 3 below.[2]

Table 3. Definitions of Terms Used to Describe Axillary Lymph Node Metastases (Open Table in a new window)

Feature

Definition

Pathologic N Stage

Isolated tumor cells (ITCs) only

Tumor cell deposit ≤0.2 mm

pN0(i+)

Positive molecular findings Positive molecular findings by reverse-transcriptase polymerase chain reaction (RT-PCR); no ITCs detected pN0 (mol+)

Micrometastasis

Tumor cell deposit >0.2 mm and ≤2 mm

pN1mi

Macrometastasis

Tumor cell deposit >2 mm

pN1

Number of positive axillary lymph nodes (LNs)

If there is one tumor cell deposit >2 mm, then all LNs with micrometastases or macrometastases are counted as positive LNs. Cancerous nodules in the axillary fat without histologic evidence of residual LN tissue are classified as regional LN metastasis.

pN1a (1-3 positive axillary LNs)

pN2a (4-9 positive axillary LNs)

pN3a (≥10 positive axillary LNs)

Practical considerations

Although no consensus on how to process lymph nodes to maximize the detection of isolated tumor cells (ITCs) and micrometastases exists, pathologic evaluation of axillary lymph nodes should identify all macrometastases (>2 mm). This requires submitting grossly negative lymph nodes entirely sectioned at 2-mm intervals and examining at least one hematoxylin and eosin (H&E)–stained section of each block.

Because the number of positive axillary lymph nodes is taken into consideration for pN staging of breast carcinoma, counting the number of positive lymph nodes is necessary. Therefore, each separate lymph node must be identified in a separate cassette or by inking. A representative section of each grossly positive lymph node is sufficient. Cytokeratin immunostains and estrogen-receptor (ER) immunostains (for ER-positive tumors) can be helpful to confirm the presence of tumor cell deposits, especially for lobular carcinomas or carcinomas with a histiocytoid appearance. The presence of extracapsular extension should be mentioned in the pathology report, as this has both local and systemic recurrence implications.

Controversies

Protocols used to increase the likelihood of identifying micrometastases and ITCs vary widely from laboratory to laboratory. The techniques used include step sectioning at different intervals through the block and cytokeratin immunohistochemical stains. When interpreting immunohistochemical stains, correlation with H&E and morphologic features (particularly nuclear features) is needed. Only cytokeratin-positive tumor cells (morphologically identifiable as such by comparison with H&E, by cell size, and by nuclear morphology), not cytokeratin-positive cells, are reported as ITC. It is helpful to state how the lymph nodes were examined (eg, number of H&E levels, cytokeratin immunostain).

 

Tissue-Based Tumor Markers

Indications for testing

In 2007, the American Society of Clinical Oncologists (ASCO) updated their recommendations for the use of tumor marker tests in the prevention, screening, treatment, and surveillance of breast cancer.[1] An overview of these recommendations is presented in Table 1 (see the Overview section).

Practical considerations

For the results of any prognostic/predictive test to be clinically meaningful, rigorous quality-control measures must be applied and followed. Any new test should be compared with a reference test for which there has been clinical validation. Clinical validation requires a study demonstrating that the test predicts clinical outcome.

When formalin-fixed paraffin-embedded (FFPE) tissue is used for testing, the importance of appropriate fixation cannot be overemphasized. The standard fixative is 10% neutral buffered formalin. Fixing within 1 hour of removal from the patient is recommended. Specimens should be sliced into 0.5- to 1-cm sections prior to fixing and placed in sufficient volumes of formalin. The duration of fixation should be kept between 6 and 72 hours. Sections for processing should be 2-4 mm thick.

Controversies

Excellent concordance for human epidermal growth factor receptor 2 (HER2) and estrogen-receptor (ER) testing on core biopsy and excision specimens has been reported.[6] Adequate fixation appears to be a more important factor than biologic tumor heterogeneity. HER2 and ER testing is preferably performed on core biopsy specimens in the authors’ practice, because it is easier to standardize fixation times for core biopsy specimens and because test results are available at an earlier step in the management of the patient.

 

Estrogen Receptor and Progesterone Receptor Status

Indications for testing

The result of estrogen receptor (ER) and progesterone receptor (PgR) testing is used to identify patients who are most likely to benefit from endocrine therapy (tamoxifen, ovarian ablation, aromatase inhibitors, irreversible ER inhibitors). ER and PgR should be measured in every primary invasive breast cancer, and they may be measured on metastatic lesions if the results would influence treatment planning. In both premenopausal and postmenopausal patients, steroid hormone receptor status should be used to identify patients who are the most likely to benefit from endocrine forms of therapy in the settings of early breast cancer and metastatic disease. In patients with ductal carcinoma in situ (DCIS) who are candidates for hormonal therapy, the data are insufficient to recommend routine measurement of ER and PgR for therapeutic guidance.[1]

Practical considerations

Currently preferred method of testing

Immunohistochemistry (IHC) is now the method of choice in pathology laboratories. Three ER antibodies are frequently used (1D5, 6F11, and SP1).

Heat-induced epitope retrieval times/techniques and optimal dilutions must be determined by individual laboratories.

Interpretation and reporting

The percentage or proportion of cells expressing the antigen should be specified in the pathology report.[7] ER and PgR are nuclear stains. When clinicians decide to use hormonal therapy, any ER staining is considered relevant. At some low levels of expression, there is minimal or absent benefit of administration of endocrine therapy.

Scoring systems that incorporate both intensity of staining and the proportion of cells that stain, such as the Allred score, have been correlated with response to therapy.[8]  However, there is currently no generally accepted scoring system. The pathologist plays an important role in avoiding false-negative results (see below).

Quality concerns[6]

The most important concern in testing for ER is false-negative results. These can occur from fixation times that are too short (< 6 h)[9] or too long (weeks),[10] decalcification and mercury-containing fixatives (B5), storage of unstained sections for more than 1 week, and insufficient antigen retrieval.

Insufficient antigen retrieval is thought to be one of the most important factors leading to false-negative findings.[11] Table 4 in the next section ("HER2/neu Status") provides a number of practical tips to avoid false-negative results.

Some of the challenges that may lead to a false-positive result are entrapped normal cells, cytoplasmic positivity, and mistaking the control sample on the same slide with carcinoma.

Avoiding false-negative ER and PgR immunohistochemistry results

Considerations concerning preanalytic/analytic variables are as follows:

  • Appropriate fixation: The time from tissue acquisition to fixation should be as short as possible; specimens should be fixed in neutral buffered formalin for 6-72 hours, sliced at 5- to 10-mm intervals, and placed in a sufficient volume of neutral buffered formalin; the time to fixation and the duration of fixation should be recorded for each sample

  • Appropriate antigen retrieval (to be determined by individual laboratories; at least 25 minutes has been suggested)

  • Appropriate controls (ideally, controls with both high and low levels of ER should be used)

  • Other preanalytic factors that might lead to a false-negative results

Considerations during pathologist interpretation are as follows:

  • When possible, the test should be performed on a block with an internal control, because this internal control will be an indicator of fixation issues; normal benign ducts and lobules show patchy to diffuse ER immunopositivity.

  • ER positivity is expected in virtually all grade 1 tumors, in pure tubular carcinomas, in colloid carcinomas, and in classic lobular carcinoma.[12] Negative results in these tumors should be questioned with repeat testing, if indicated; ER-negative, PgR-positive tumors are very uncommon (< 5%); the test should be repeated, if indicated.

  • If the results on core biopsy are questioned, repeat testing on an excision is warranted.

  • Negative results on unstained sections stored for prolonged periods should be questioned; unstained sections can be stored in a freezer to slow down oxidation-mediated reduction of antigenicity.

  • Negative results in bone biopsies should be questioned because of the possible effects of decalcification or B5 fixative. (Fine-needle aspiration or touch preparation samples are a good alternative.)

Controversies

The role of quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) assays measuring the level of mRNA expression is not yet clear.

In newly diagnosed patients with node-negative, ER–positive breast cancer, the Oncotype DX assay can be used to predict the risk of recurrence in patients treated with tamoxifen. Oncotype DX may be used to identify patients who are predicted to obtain the most therapeutic benefit from adjuvant tamoxifen and may not require adjuvant chemotherapy. In addition, patients with high recurrence scores appear to achieve relatively more benefit from adjuvant chemotherapy (specifically (C)MF) than from tamoxifen. There are insufficient data at present to comment on whether these conclusions generalize to hormonal therapies other than tamoxifen, or whether this assay applies to other chemotherapy regimens.

 

HER2/neu Status

Indications for testing

Human epidermal growth factor receptor 2 (HER2) expression and/or amplification should be evaluated in every primary invasive breast cancer either at the time of diagnosis or at the time of recurrence, principally to guide selection of trastuzumab in the adjuvant and/or metastatic setting. Other uses for HER2 evaluation are also discussed separately in Table 1 in the Overview section.[1]

Practical considerations

There is no criterion standard assay for HER2 in breast cancer. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are both used. Available data do not clearly demonstrate the superiority of either IHC or in situ hybridization as a predictor of benefit from anti-HER2 therapy. In 2007, the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) published a joint guideline regarding HER2 testing in breast cancer.[13] A list of the US Food and Drug Administration (FDA) approved tests is provided in Table 4.

Table 4. FDA-Approved Tests for HER2* (Open Table in a new window)

Assay

Methodology

Link to Package Insert of Manufacturer

Immunohistochemistry (IHC)

HercepTest (DAKO Denmark A/S, Glostrup, Denmark)

A085 polyclonal antibody

https://www.agilent.com/cs/library/packageinsert/public/PD04086US_01.pdf 

(Revision 2019.01)

Pathway HER2 (4B5) rabbit monoclonal primary antibody (Ventana Medical Systems, Inc, Tucson, AZ)

CB11 monoclonal antibody

http://reagent-catalog.roche.com/product/98?type=93

Fluorescence in situ hybridization (FISH)

PathVysion HER2 DNA probe kit (Abbott Molecular, Inc, Des Plaines, IL, USA)

Hybridization of fluorescent DNA probes to the HER2 gene (orange) and chromosome 17 centromere (CEP17, green). CEP17 is a centromeric probe for chromosome 17 on which the HER2 gene resides used as an internal control probe.

https://www.molecular.abbott/us/en/products/oncology/pathvysion-her-2-dna-probe-kit

Dual ISH DNA Probe

INFORM HER2/neu Dual ISH DNA Probe Cocktail Assay (Ventana Medical Systems, Inc, Tucson, AZ)

Enumeration of the ratio of the HER2 gene to chromosome 17 with the use of standard light microscopy on one slide, via co-hybridization of HER2 probe (labeled with the hapten dinitrophenyl [DNP]) and a chromosome 17 probe (labeled with the hapten digoxigenin [DIG]). 

http://reagent-catalog.roche.com/product/1553?type=2013

 

http://productlibrary.ventana.com/ventana_portal/OpenOverlayServlet?launchIndex=1&objectId=780-44221003271US (6/2/2017; 1003271US Rev E)

*In addition to these commercially available tests, "home brew" IHC and FISH assays are accepted, if properly validated.

 

A previously available chromogenic in situ hybridization (CISH) assay (SPoT-Light HER2 CISH Kit, Invitrogen Corporation, Carlsbad, CA) was recalled in 2012, and subsequently discontinued by the manufacturer, owing to potential contamination of the diagnostic reagent with a fungal contaminant.[14]

Interpretation and reporting[13]

In 2018, ASCO/CAP updated their recommendations on how to report HER2 (see Table 5, below).[15]

Table 5. Interpretation and Reporting of HER2 Tests (Open Table in a new window)

Immunohistochemistry (IHC)

Positive

Negative

Equivocal

Only membranous staining is relevant.

Score 3+

Circumferential membrane staining that is complete and >10% of tumor cells

Score 0: No staining observed or

Membrane stating that is incomplete, is faint/barely perceptible, and within ≤10% of tumor cells

 

Score 1+: Incomplete membrane staining that is faint/barely perceptible and within >10% of tumor cells

Score 2+

Weak to moderate complete membrane staining in >10% of tumor cells or

 

Circumferential membrane staining that is intense but within ≤10% of tumor cells

Fluorescence in situ hybridization (FISH) Positive Negative  

At least 20 nonoverlapping cells in two separate areas of invasive carcinoma should be counted.

 

 

 

Tests systems with internal control probe (chromosome enumeration probe [CEP17]): The FISH ratio (ratio of HER2 gene signals to chromosome 17 signals) is reported.

Dual-probe assay

  • HER2/CEP17 ratio ≥2.0; < 4.0 HER2 signals/cell and concurrent IHC 3+
  • HER2/CEP17 ratio < 2.0; ≥6.0 HER2 signals/cell and concurrent IHC 2+ or 3+
  • HER2/CEP17 ratio < 2.0; ≥4.0 and < 6.0 HER2 signals/cell and concurrent IHC 3+
  • HER2/CEP17 ratio ≥2.0; ≥4.0 HER2 signals/cell 

Dual-probe assay

  • HER2/CEP17 ratio < 2.0; < 4.0 HER2 signals/cell
  • HER2/CEP17 ratio ≥2.0; < 4.0 HER2 signals/cell and concurrent IHC 0-1+ or 2+ 
  • HER2/CEP17 ratio < 2.0; ≥6.0 HER2 signals/cell and concurrent IHC 0-1+
  • HER2/CEP17 ratio < 2.0; ≥4.0 and < 6.0 HER2 signals/cell and concurrent IHC 0-1+ or 2+

 

Patients with positive results are considered eligible for trastuzumab therapy

Patients with negative results are not considered eligible for trastuzumab therapy

 

No currently available assay is perfectly accurate in identifying all patients expected to benefit from anti-HER2 therapy. Therefore, a combination of tests may be required on a particular specimen. The pathologist plays an important role in avoiding false-negative and false-positive results.

Quality concerns[13]

Both false-negative and false-positive results are an important concern in HER2 testing.[16, 17] HER2 testing should aim to identify all patients who would benefit from trastuzumab therapy. However, false-positive results are a significant concern because of the small risk of serious cardiac toxicity and the cost associated with trastuzumab therapy (>$70,000/year). Also, incorrect classification of cancer impedes efforts to evaluate the effectiveness of HER2-targeted therapy in clinical trials. False-negative results can occur from fixation times that are too short or too long, decalcification and mercury-containing fixatives (B5), storage of unstained sections for more than 1 week, insufficient antigen retrieval, and nonoptimal enzymatic digestion.

Excessive antigen retrieval and accidental assessment of in situ rather than invasive lesions can lead to false-positive results. Other causes of false-positive results includes edge artifact (lobular carcinoma can appear falsely positive on the edge), cytoplasmic positivity, and overinterpretation. ASCO and CAP developed a joint guideline to improve the accuracy of HER2 testing.[13]

Considerations concerning pre-analytic/analytic variables are as follows:

  • Appropriate fixation: Time from tissue acquisition to fixation should be as short as possible; specimens should be fixed in neutral buffered formalin for 6-72 hours; they should be sliced at 5- to 10-mm intervals and placed in sufficient volume of neutral buffered formalin; time to fixation and duration of fixation should be recorded for each sample

  • Strict adherence to test protocols is essential

  • Appropriate controls include positive controls and negative controls (eg, cell line controls and/or tissue-based references) and an internal control (normal breast epithelium)

Considerations during pathologist interpretation of immunohistochemistry are as follows:

  • Care should be taken to make sure only the invasive component is assessed and reported, as HER2 is often overexpressed and/or amplified in in situ breast lesions.

  • When possible, the test should be performed on a block with an internal control, because this internal control will be an indicator of excessive antigen retrieval; if normal ducts and lobules show strong membrane staining, excessive antigen retrieval may have occurred, and repeat testing or testing via FISH should be performed.

  • If cytoplasmic staining obscures membrane staining or if obscuring artifacts (edge, retraction, crush artifact) are present, the assay should be repeated or FISH should be performed.

  • HER2 overexpression correlates with high tumor grade and is less common in pure tubular carcinomas, colloid carcinomas, and classic lobular carcinoma[18, 19] ; results that are unexpected should be questioned with repeat testing or testing with an alternative method or a different sample, if indicated.

  • If the results on core biopsy are questioned, repeat testing on an excision is warranted.

  • Negative results on unstained sections stored for prolonged periods should be questioned; sections ideally should not be used if they were cut more than 6 weeks earlier.

  • Negative results in bone biopsies should be questioned because of the possible effects of decalcification or B5 fixative. (Fine-needle aspiration or touch preparation samples are a good alternative.)

  • Equivocal results (2+) must be subjected to FISH testing.

  • If samples were known to be fixed for periods shorter than 6 hours or longer than 72 hours, the report should qualify any negative result with a statement about fixation.

Regarding FISH, according to ASCO/CAP guidelines, counting can be performed by a trained technologist, but the pathologist must confirm that the result (count) is correct, that invasive tumor was counted, and that the sample is surveyed for genomic heterogeneity.[13] Other considerations are as follows:

  • Care should be taken to make sure only the invasive component is assessed; HER2 is often overexpressed and/or amplified in in situ breast lesions; corresponding hematoxylin and eosin (H&E) and/or IHC slides should be reviewed to localize the invasive carcinoma.

  • Control samples should be reviewed; if the findings are not as expected, the test should be repeated.

  • At least 20 nonoverlapping cells in two separate areas of invasive carcinoma should be counted; counting of additional cells and/or by a different observer may be appropriate.

  • Samples with nonuniform signals (weak signals in >25% of cells), high autofluorescence, poor nuclear resolution (indistinct nuclei), or high background-obscuring signal resolution (>10% of signals over cytoplasm) should be rejected.

  • HER2 overexpression correlates with high tumor grade and is less common in pure tubular carcinomas, colloid carcinomas, and classic lobular carcinoma[18, 19] ; results that are unexpected should be questioned with repeat testing or testing with an alternative method or a different sample, if indicated.

  • If the results on core biopsy are questioned, repeat testing on an excision is warranted.

  • Negative results on unstained sections stored for prolonged periods should be questioned; sections ideally should not be used if they were cut more than 6 weeks earlier.

  • Negative results in bone biopsies should be questioned because of the possible effects of decalcification or B5 fixative. (Fine-needle aspiration or touch preparation samples are a good alternative.)

  • Equivocal FISH results are to be confirmed by counting additional cells or repeating the FISH test; if the FISH result remains equivocal, IHC is recommended.

  • If samples were known to be fixed for periods shorter than 6 hours or longer than 72 hours, the report should qualify any negative result with a statement about fixation.

Controversies

Aneuploidy of chromosome 17

Polysomy 17 is seen in approximately 8% of all specimens, mostly among cases with 4-6 HER2 gene copies; these were previously labeled as equivocal. If polysomy 17 is defined as three or more CEP17 copies, most are not associated with mRNA overexpression. Most tumors with a HER2 gene copy number between 4 and 6 are also not associated with protein mRNA overexpression.

For tumors that are heterogeneous for HER2 amplification on in situ hybridization (1-2% of cases), the maximal HER2 values should be reported with a note that the tumor demonstrated heterogeneity for HER2 amplification.[20]

Discordant results

Discordant results (IHC 3+ with negative FISH result or IHC < 3+ with positive FISH result) are found in approximately 4% of patients. The available clinical data are insufficient to determine whether these represent distinct biologic subgroups with therapeutic implications or whether they are inaccurate test results.

HER2 overexpression does not equal benefit from anti-HER2 therapy. Some patients exist who truly overexpress HER2 but have upstream or downstream anomalies that render the interaction with trastuzumab ineffective.

Quantitative image analysis is encouraged by ASCO/CAP for cases with weak membrane staining 1-2+ to improve the consistency of interpretation.

 

Questions & Answers

Overview

How do medical oncologists use pathology findings in the selection of systemic therapy for breast cancer?

What are the ASCO guidelines for the use of tumor markers in the prevention, screening, treatment, and surveillance of breast cancer?

How is the tumor size defined in breast cancer pathology?

How is breast tumor size determined following preoperative (neoadjuvant) chemotherapy?

What is the Nottingham combined histologic grade (modified Scarff-Bloom-Richardson grade) system for breast tumors?

How are mitotic counts assessed in breast cancer tumor grading?

What are the limitations of core biopsy in the histologic grading of breast tumors?

What terms are used to describe axillary lymph node metastases in breast cancer?

What is included in the pathologic evaluation of axillary lymph nodes in breast cancer?

Which techniques are used to identify micrometastases and ITCs in axillary lymph nodes of patients with breast cancer?

Which quality control measures should be taken when performing prognostic breast tumor marker testing?

What is the role of fixation in FFPE tissue testing of breast tumors?

When should HER2 and ER testing of core biopsy and excision breast specimens be performed?

When is ER and PgR testing indicated in patients with breast tumors?

When is the preferred method for ER and PgR testing in breast tumors?

How are ER and PgR testing results interpreted in breast tumors?

What causes false-negative ER and PgR results in breast tumors and how can they be avoided?

What is the role of a quantitative RT-PCR assay in the pathologic assessment of breast tumors?

What is the role of the Oncotype DX assay in the pathologic assessment of breast tumors?

What is the role of HER2 testing in the pathologic assessment of breast tumors?

What are the FDA-approved HER2 tests of breast tumors?

How are HER2 testing results interpreted in breast tumors?

What is the accuracy of HER2 testing results in breast tumors?

How does aneuploidy of chromosome 17 affect the results of HER2 tests of breast tumors?

What causes discordant results in HER2 tests of breast tumors?