Tissue Evaluation for HER2 Tumor Marker

Etiology and Epidemiology

HER2 amplification or overexpression occurs in approximately 20% to 30% of human breast cancers and some ovarian and gastric cancers. When aberrant, HER2 generates worse biological behavior and clinical aggressiveness in breast cancer.[3] Overexpression of HER2 is primarily attributed to HER2 gene amplification and prompts activation of the HER2 downstream signaling pathway.[4] Breast cancers can possess up to 25 to 50 copies of the HER2 gene and up to a 40- to 100-fold increase in HER2 protein, resulting in 2 million receptors expressed at the tumor cell surface.[5]

HER2 amplification is a seminal event in human breast tumorigenesis, occurring in almost 50% of in situ carcinomas.[6] The fact that only 20% of invasive breast cancers show HER2 amplification suggests that many HER2-amplified in situ cancers never progress to the invasive stage. HER2-amplified breast cancers have unique biological and clinical characteristics such as increased proliferation rates, high histologic and nuclear grade, low estrogen and progesterone receptor expression levels, more aneuploidy, tendency to metastasize to central nervous system and viscera, relative resistance to endocrine therapy, and increased sensitivity to doxorubicin.[7]

Pathophysiology

HER2 is a member of the epidermal growth factor family of tyrosine kinase receptors. This family includes HER1 (ErbB1), HER3 ( ErbB3), and HER4 (ErbB4). HER receptors are essential for cell proliferation, differentiation, and survival.[8]  All HER proteins share the common structural features of the extracellular ligand-binding, transmembrane, and intracellular protein tyrosine kinase domains.[4] While HER1, HER3, and HER4 have 11 extracellular ligands, HER2 makes an exception as it does not bind to any ligand.[9]

Activation of HER2-mediated signaling pathways occurs by heterodimerization with ligand-activated epidermal growth factor receptor family (EGFR) or HER3 or by homodimerization when it is present in high concentrations, such as in cancer. These dimerizations would lead to the phosphorylation of tyrosine residues, which initiates downstream signaling cascades such as phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling cascade, rat sarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinases (Ras/MEK/ERK), and Janus kinase/signal transducer and activator of transcription (JAK/STAT), that regulate cell survival, proliferation, differentiation, motility, apoptosis, survival, invasion, migration, adhesion, and angiogenesis.[4]

Observation shows that HER3 plays an essential role in HER2-mediated oncogenic signaling.[10] HER3 lacks tyrosine kinase activity but relies on other receptor tyrosine kinases, most notably ERBB, to phosphorylate its tyrosine residue and activate the downstream signaling cascades. HER2-containing heterodimers have the highest mitogenic potential among all HER complexes. Between all HER2 pairs, HER2/HER3 heterodimer is the most potent activator of the PI3K/AKT signaling cascade via binding of the p85 subunit of PI3K to HER3.[11] Knocking down HER3 or targeting HER3 via neutralizing antibodies should theoretically reduce HER2-amplified human breast cancer growth.[10] Inhibition of HER2 dimerization prevents the activation of several intracellular signaling cascades, including the PI3K and mitogen-activated protein kinase (MAPK) pathways, which can cause carcinogenesis.[12]

Specimen Requirements and Procedure

Patients who are newly diagnosed with breast cancer or with metastatic breast cancer are strongly recommended to be tested for HER2 expression, as patients with a high level of HER2 expression are likely to benefit from agents targeting HER2.[13] Tests for HER2 are usually performed on tumor biopsy samples gathered via fine-needle aspiration, core needle biopsy, or vacuum-assisted breast biopsy via surgical excision. The sample attains a score based on the cell membrane staining pattern.[14] Alternately, the extracellular domain of HER2 may be shed from the surface of tumor cells and enter circulation. Using enzyme-linked immunosorbent assay (ELISA) to measure serum HER2 would provide a less invasive method of determining HER2 status than a biopsy.[15]

Diagnostic Tests

A tumor biopsy sample should be promptly prepared for pathology evaluation to ensure efficiency; minimizing the time from tissue acquisition to fixation is crucial. Samples for HER2 testing are fixed in 10% neutral buffered formalin for 6 to 72 hours. After appropriate gross inspection and margins delineated, samples should be sliced at 5 to 10 mm intervals and placed in a sufficient volume of neutral buffered formalin. Any exceptions to this process must be included in the report. If samples were cut more than 6 weeks prior, sections should not be used for HER2 testing. However, this may vary with primary fixation or storage conditions.[16]

Testing Procedures

Although different assays can be used to measure HER2 oncogene activity, there is still controversy over which is best.

The assays used clinically are: 

• HER2 gene amplification: Via in situ hybridization, fluorescence in situ hybridization, chromogenic in situ hybridization, and polymerase chain reaction [17]
• HER2 overexpression: Western blotting, enzyme-linked immunosorbent assay, immunohistochemistry [18]

Interfering Factors

The in situ hybridization test may be rejected and require repeating under specific criteria, including instances where the controls do not meet the expected standards or the observer cannot locate and quantify at least 2 areas of the invasive tumor. Additionally, the test may be rejected if more than 25% of signals cannot be scored due to weak signals or if over 10% occur within the cytoplasm. Poor nuclear resolution and strong autofluorescence are also factors that could lead to the rejection of the test.[19]

The immunohistochemistry test may be rejected and necessitate repetition or an alternative testing method, such as fluorescence in situ hybridization, under specific criteria; this includes situations where the controls do not meet the defined standards. Additionally, suppose artifacts significantly affect most of the sample, or there is intense membrane staining of normal breast ducts (serving as internal controls) in the sample. In that case, the test may be rejected; therefore, considering alternative testing methods for accurate and reliable results is imperative.[20]

Results, Reporting, and Critical Findings

The American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines for HER2 testing reporting and critical findings were updated in 2023. The update reaffirms the 2018 "HER2 Breast Testing Guideline Focused Update" and was prompted by the results of the 2022 DESTINY-Breast 04 trial, which led to the expansion of the approval of the HER2 antibody-drug conjugate, trastuzumab deruxtecan, by the United States Food and Drug Administration (US FDA) to include metastatic patients with HER2 immunohistochemistry (IHC) 1+ or 2+/in situ hybridization (ISH) negative results. However, the guideline update does not support the use of a HER2-Low interpretive category due to the lack of evidence to support that IHC 1+ or 2+/ISH negative results are predictive of trastuzumab deruxtecan treatment response when compared to IHC 0 results.[21][22] 

The guidelines recommend the following best practices for HER2 testing:

• Repeat testing is recommended if the initial test was HER2-positive in a histologic grade 1 carcinoma of infiltrating ductal or lobular carcinoma, hormone receptor-positive, and the HER2/chromosome 17 centromere (CEP17) ratio is <2 and an average HER2 copy number is between ≥4 and <6 signals/cell.
• All patients with at least 90% pure mucinous, cribriform, tubular, or adenoid cystic histologies should be tested for HER2. If the HER2/CEP17 ratio is greater than or equal to 2 and an average HER2 copy number is less than 4 signals/cell, the patient should receive HER2-directed therapy.
• A definitive diagnosis will be rendered based on additional workup if a case has a HER2/CEP17 ratio of ≥2, but the average HER2 signals/cell is <4. If the count remains an average of <4.0 HER2 signals/cell and HER2/CEP17 ratio ≥2, the diagnosis is HER2 negative with a comment. If a case has an average of ≥6 HER2 signals/cell with a HER2/CEP17 ratio of <2, formerly diagnosed as ISH positive for HER2, a definitive diagnosis will be rendered based on additional workup. If the HER2/CEP17 ratio remains <2 with ≥6 HER2 signals/cell, the diagnosis is HER2 positive. If the case has an average HER2 signal/tumor cell of ≥4 and <6 and the HER2/CEP17 ratio is <2, formerly diagnosed as ISH equivocal for HER2, a definitive diagnosis will be rendered based on additional workup. If the count remains an average of ≥4 and <6 and the HER2/CEP17 ratio is <2, the diagnosis is HER2 negative with a comment.

Clinical Significance

Several HER2-targeted therapies have US FDA approval for breast cancer, including trastuzumab, pertuzumab, ado-trastuzumab emtansine, lapatinib, trastuzumab-deruxtecan, and tucatinib. Additional therapies are still under clinical trials. The presently used agents include:

• Trastuzumab: This is a monoclonal antibody that binds to the extracellular segment of the HER2 receptors. The mechanism of action is still not fully understood, but this therapy has the most significant effect on tumors with increased HER2 homodimers. Although trastuzumab does not block the autophosphorylation of HER2, it does inhibit HER2 downstream signaling.[23] Additionally, this may disrupt the HER2/Src (a non-receptor tyrosine kinase) interaction and enhance antibody-mediated cytotoxicity, inducing the immune-mediated response that causes internalization and downregulation of HER2.
• Pertuzumab: This is also a monoclonal antibody that binds to the extracellular dimerization domain of HER2 and prevents binding to itself or other members of the EGFR family. Dosing in combination with trastuzumab is used rather than as a single agent.[24]
• Ado-trastuzumab emtansine: This is an antibody-drug conjugate composed of trastuzumab and emtansine, the antimicrotubule agent.[25]
• Lapatinib: This is a tyrosine kinase inhibitor against EGFR1 and HER2, inhibiting the signaling pathways down from HER2 level.[26]
• Trastuzumab-deruxtecan: This is an antibody-drug conjugate composed of trastuzumab and deruxtecan, a topoisomerase I inhibitor.[27]

Cases of HER2-positive metastatic breast cancer are usually managed with a combination of chemotherapy and HER2-directed agents. Patients with hormone-receptor-positive and HER2-positive metastatic breast cancer may receive a combination of endocrine therapy and HER2-directed therapy. No ideal treatment strategy for hormone-receptor-negative HER2-positive metastatic breast cancer exists. The preference for these patients who have not received therapy in the past is a combination of trastuzumab, pertuzumab, and a taxane, as data showed this to have improved clinical outcomes.[28] For patients previously treated with trastuzumab and with metastasis following a treatment-free interval of 6 months or longer, the preferred treatment is ado-trastuzumab emtansine, as this improves clinical outcomes.[29] However, for patients who relapse within 6 months of completing adjuvant trastuzumab therapy, intravenous trastuzumab is effective as a single agent. In combination with chemotherapy, this therapy significantly improves the median time to disease progression and survival time in patients with metastatic breast cancer overexpressing the HER2 receptor compared with chemotherapy alone.[30]

Quality Control and Lab Safety

Every laboratory must implement a quality management system for all procedures conducted within its scope to guarantee high-quality performance. A necessary quality management system includes essential components such as quality assurance, ongoing quality improvement, and quality control.[31] Quality assurance for HER2 testing is imperative for maintaining the highest standards of accuracy and reliability in breast cancer diagnosis and treatment. Thoroughly review and document external and internal controls with each test and every batch of tests conducted.[32] This meticulous approach ensures the accuracy and reliability of the results, promoting patient safety and trust in the laboratory's services.

Ongoing quality control and equipment maintenance must be prioritized to ensure that instruments and procedures remain in optimal working condition. Furthermore, continuous training and competency assessment of laboratory personnel are paramount, as they are the backbone of accurate testing.[33] Standardized operating procedures, including the routine use of control materials, are a foundation for consistent and high-quality testing. In the event of any changes to testing procedures, revalidation becomes necessary to maintain the integrity of the process.[34] Regular competency assessments of laboratory personnel are a vital component, and all actions taken in this regard must be meticulously documented as an essential part of the laboratory's records, ensuring accountability and transparency throughout the testing process.[33]

Method validation is utilized to confirm a test procedure for an analyte yields accurate and precise results. The laboratory regulations require performance for any new method to be "verified" before reporting the patient's test results.[35] The laboratory should ensure that initial validation conforms to the published 2010 ASCO-CAP recommendations for immunohistochemistry testing of estrogen and progesterone receptor guideline validation requirements, with 20 negative and 20 positives for FDA-approved assays and 40 negative and 40 positives for laboratory-developed tests. Equivocal samples are unnecessary for validation studies.[16] The characteristics of the cases used for validation should be similar to those seen in the laboratory's patient population (ie, core biopsies vs open biopsy material, or primary vs metastatic tumor, etc).

Lab accreditation holds immense significance in ensuring the quality and reliability of testing and measurement services. Further, accreditation formally recognizes the laboratory meets specific, internationally recognized standards of competence and compliance.[36] Optimal laboratory accreditation includes onsite inspection every other year with an annual requirement for self-inspection; reviews of laboratory validation, procedures, quality assurance results, processes, results, and reports; and unsatisfactory performance results in suspension of laboratory testing for HER2 for that method.[16]

Participation in and completion of an external proficiency testing program is a cornerstone of maintaining high-quality laboratory standards.[37] This program entails engaging in at least 2 testing events annually, where the laboratory's performance is evaluated through rigorous assessments. The laboratory must achieve a minimum of 90% correct responses on the graded challenges for either test to achieve the label of "satisfactory." This benchmark ensures the laboratory consistently delivers accurate and reliable results, meeting the rigorous demands of proficiency testing.[38] If an unacceptable proficiency testing result is identified, the method must be investigated for possible causes, and corrective action must be taken.[39]

Ensuring lab safety is paramount in clinical laboratories, where accurate and dependable results are crucial for patient diagnosis and influence treatment regimes. Maintaining a secure environment requires strict adherence to safety protocols.[40] Personal protective equipment (PPE) should be utilized appropriately in the designated laboratories; this ensures that no blood, potentially infectious materials, or hazardous chemicals come into contact with the skin, eyes, mouth, or other mucous membranes, work clothes (such as scrubs), street clothes, or undergarments under typical usage conditions. All PPE must be promptly removed before leaving the work area or as soon as feasible and placed in a designated location or container within the laboratory for storage, washing, decontamination, or disposal.[41]

The lab staff must exercise caution when handling and transporting specimens. Do not carry a specimen container without a lid if there is a fixative. If laboratory staff accidentally drops the container or spills any fixative on the floor, promptly employ spill absorbent and paper towels for cleaning. Laboratory personnel should avoid carrying a dripping specimen in their hand and instead utilize a paper towel, a lid, or a surgical towel.[42] Sharps containers shall be located where needles and sharps are used and shall be secured so they will not be knocked over and their contents spilled. Used needles must not be cut, bent, broken, or recapped by hand before disposal in a proper sharps container due to the increased chance of injury when needles are manipulated.[43] 

Proper operation of the microtome requires the utilization of its integrated safety features. The blade must be covered with the knife guard when not cutting sections, and the hand-wheel brake must be engaged to avoid any risk of the laboratory staff's fingers getting caught. New staff unfamiliar with operating and using a microtome must be carefully trained and monitored until they become proficient with the instrument and its safety features.[44]

Histopathology labs use many chemicals that are identified as hazardous, including flammables, irritants, corrosives, sensitizers, and toxic chemicals. Disastrous results can occur when incompatible chemicals come into contact with each other. Understanding chemical compatibilities in labs and ensuring that incompatible chemicals are properly segregated to prevent accidental contact is essential. Equally important is rinsing out waste bottles thoroughly before adding chemical waste.[45]

Formaldehyde is a chemical compound commonly utilized in histopathology laboratories for its preserving and fixative properties. Due to the effects of formaldehyde exposure, maintaining a formalin vapor monitoring system in all laboratories with formalin standard operating procedures.[46] According to the Occupational Safety and Health Administration, the legal airborne permissible exposure limit for formaldehyde in the workplace is 0.75 parts formaldehyde per million parts of air (0.75 ppm) measured as an 8-hour time-weighted average; this means that no employee should be exposed to an airborne concentration of formaldehyde that exceeds 0.75 ppm during an 8-hour working day.[47]

Ready-to-use formalin should be properly labeled and stored in a cool, dry, well-ventilated area. Used formaldehyde (from change of specimen containers or spillage) must be stored in a properly labeled hazardous waste container and sent for recycling or neutralization.[48] Prioritizing lab safety in the histopathology lab is not just a responsibility but a vital commitment to ensure laboratory personnel's well-being and the accuracy of diagnostic results.

Enhancing Healthcare Team Outcomes

HER2 expression is a key element of many breast cancers. Tissue evaluation for HER2 tumor marker status necessitates a comprehensive and collaborative approach among healthcare professionals to optimize patient outcomes. Physicians and advanced practitioners bring essential skills in interpreting diagnostic tests and integrating HER2 status into treatment plans. Nurses play a pivotal role in educating patients about HER2 testing and treatment protocols, while pharmacists offer critical knowledge on HER2-targeted therapies and medication management. An effective strategy involves multidisciplinary tumor boards to discuss HER2 status, treatment options, and tailored patient care plans based on evidence-based practices.

Interprofessional communication among team members ensures clear dissemination of HER2 status results, treatment plans, and timely updates to patients and families. Care coordination, guided by patient-centered approaches, involves continual follow-up, monitoring patient responses to therapies, and facilitating referrals to support services when needed. This collaborative effort harnesses the diverse expertise within the healthcare team to ensure informed decision-making, patient safety, and optimized outcomes in HER2-positive cases.