HER2



HER2










This is a typical HER2-positive invasive ductal carcinoma with high nuclear grade and intense circumferential membrane immunoreactivity (“chicken wire” pattern), which is uniform throughout the tumor.






An increased number of HER2 genes (red signals image), compared to 1 or 2 copies of chromosome 17 centromere sequences (green signals image), would be classified as marked HER2 gene amplification.


TERMINOLOGY


Abbreviations



  • HER2-positive breast cancer (HER2+BC)


  • Human epidermal growth factor receptor-2 (HER2)


  • Chromosome 17 centromere enumeration probe (CEP17)


Synonyms



  • HER2, HER2/neu, NEU, NGL, TKR1, c-erb, CD340, ERBB2


Definitions



  • HER2 is a proto-oncogene located on chromosome 17q12



    • Expressed normally in a number of glandular epithelia including breast


  • Translated into a 185 kDa transmembrane growth factor receptor protein



    • HER2 transmits signals regulating normal cell growth, development, and survival


  • In 15-20% of breast cancers, HER2 protein is overexpressed



    • In > 95% of HER2+BC, the mechanism of overexpression is due to increased numbers of HER2 gene (gene amplification)



      • Amplification of the gene drives mRNA production and protein expression


      • Clinical assays evaluate DNA, mRNA, or protein


    • In ˜ 5% of HER2+BC, there may be other mechanisms resulting in protein overexpression



      • These mechanisms have not been well studied


  • HER2 is a member of HER-family of growth factor receptors



    • This family also includes HER1 (EGFR), HER3, and HER4



      • Regulate intracellular signaling through MAP-kinase and PI3-kinase pathways


      • Regulate normal cell proliferation and cell survival


      • Overexpression results in increased HER2 receptors on the surface of tumor cells


ETIOLOGY/PATHOGENESIS


Histogenesis



  • HER2 alteration is thought to be early event in pathogenesis of HER2+BC



    • May play important role in carcinogenesis and tumor development


    • Higher proportions of DCIS overexpress HER2 compared to invasive carcinoma


  • HER2 alteration is stable genetic change in tumor cells



    • HER2 overexpression is seen in primary tumor as well as metastases from HER2+BC


    • However, in some cases HER2 overexpression is lost in residual carcinoma after treatment or in metastases



      • Most likely due to initial tumor heterogeneity and the selective effects of targeted therapy


Molecular Pathology



  • Binding of high affinity ligands to HER-receptors leads to conformational change in molecule



    • Conformational change promotes receptor activation through dimerization



      • HER-family members form homo-dimers and hetero-dimers


    • Receptor dimerization leads to activation of intracellular tyrosine kinase portion of molecule



      • Tyrosine kinase activation initiates receptor signaling through phosphorylation


  • Overexpression of HER2 increases likelihood of receptor activation and signaling



    • Active HER2 pathway signaling



      • Promotes angiogenesis


      • Drives proliferation


      • Protects cells against apoptosis


      • Increases tumor cell motility and invasion



      • Contributes to more aggressive biologic behavior and malignant phenotype


CLINICAL IMPLICATIONS


Prognostic Implications



  • HER2 gene amplification/overexpression plays a pivotal role in driving tumor biology



    • Contributes to more aggressive clinical course for HER2+BC



      • Significantly decreased disease-free and overall survival


      • Higher incidence of local recurrence compared to luminal A cancers (ER positive and HER2 negative)


      • Increased incidence of lymph node metastases


      • More frequent metastases to brain, liver, and lung compared to luminal A cancers


      • 40-50% of patients with brain metastases have HER2+BC


  • HER2 overexpression significantly correlates with a number of unfavorable tumor characteristics



    • Higher proliferative index


    • Larger tumor size


    • Higher tumor grade


  • HER2 overexpression may predict response to certain adjuvant chemotherapy regimens and endocrine therapy


Treatment Implications



  • HER2 overexpression in breast cancer represents ideal target for therapy



    • Receptor located on surface of cell and is accessible


    • Receptor plays a pivotal role in driving clinical course of disease


    • Targeted therapy utilizes either a HER2 specific antibody or a HER2 small molecule inhibitor


  • Trastuzumab (Herceptin)



    • Humanized monoclonal antibody that targets the HER2 receptor


    • Combines mouse recognition sequence of monoclonal antibody (4D5) with human IgG1



      • Trastuzumab binds to extracellular epitope of HER2 receptor


  • Trastuzumab demonstrates high affinity and specificity for HER2 receptor



    • In preclinical studies, this drug inhibits growth of HER2 overexpressing breast cancer cells


    • Trastuzumab binding blocks receptor signaling



      • May stimulate immune-mediated tumor cell cytotoxicity


      • May act synergistically with chemotherapy to induce tumor cell apoptosis


  • In clinical trials, trastuzumab plus chemotherapy demonstrated remarkable efficacy against HER2+BC



    • Efficacy has been demonstrated for metastatic, adjuvant, and neoadjuvant therapy


    • Adjuvant trastuzumab plus chemotherapy can reduce relative risk of recurrence by 50% in early stage HER2+BC



      • Data suggest that only HER2+BC are likely to benefit from this therapy


    • Safety considerations



      • Cardiac dysfunction seen in 2-4% of patients treated with trastuzumab plus anthracycline-based chemotherapy


    • Data highlight the importance of accurate HER2 testing



      • Important to identify only those patients who will be most suitable candidates for treatment


      • Benefit from targeted therapy is not related to the degree of gene amplification


      • Cancers with low levels of gene amplification respond as well as cancers with large numbers of genes


  • Lapatinib (Tykerb)



    • Dual tyrosine kinase inhibitor that interrupts the kinase activity of both HER2 and EGFR (HER1)



      • Oral agent


    • Used for initial therapy in combination with chemotherapy or after carcinomas have progressed after treatment with trastuzumab


Clinical Assays for HER2 Status



  • Immunohistochemistry (IHC) detects HER2 protein overexpression


  • In situ hybridization (FISH or CISH) detects HER2 gene amplification



    • Both IHC and FISH have been clinically validated to help predict response to HER2-targeted therapy


  • Oncotype DX (Genomic Health; Redwood City, CA) assay detects HER2 mRNA overexpression using quantitative RT-PCR



    • Not currently used to make treatment decisions


    • Requires tissue microdissection if DCIS shows stronger HER2 positivity or if little carcinoma in relation to stroma is present


    • Will not detect heterogeneity of overexpression


  • HER2 testing has become an essential part of clinical evaluation for breast cancer patients



    • Treatment guidelines from ASCO and the NCCN recommend HER2 testing for all newly diagnosed breast cancer patients


  • Decisions about HER2-targeted therapy include concerns about cost and potential toxicities



    • HER2-targeted therapy should only be used in patients whose tumors have been evaluated by a validated HER2 assay


Specimen Handling



  • Breast specimens should be sectioned and placed in adequate volume of fixative within 1 hour from removal


  • If gross tumor is identified during initial specimen evaluation



    • Sample of tumor and fibrous normal tissue can be placed together into same cassette



      • Tissue is placed immediately into formalin; fixation start time should be recorded


      • Helpful to initiate good and rapid fixation


      • Helps ensure normal breast tissue is available as internal control for breast marker testing



Breast Tissue Fixation



  • Breast tissue samples must be fixed in 10% neutral buffered formalin



    • Formalin fixation is part of FDA approval for test kits that evaluate HER2 by IHC and FISH


  • Fixation time recommended to be no less than 6-8 hours and no more than 48 hours before processing



    • Underfixation can lead to technical problems with IHC assay


    • Only very long fixation times (weeks) have been demonstrated to alter results


    • Small biopsy samples require same amount of fixation time as larger resection samples


  • Acid decalcification can interfere with evaluation of specimens by FISH due to DNA degradation



    • Specimens treated with very careful EDTA decalcification and daily monitoring by specimen radiography can be used for FISH and IHC


    • Negative results using other methods of decalcification should be interpreted with caution


HER2 Assay Methodologies: IHC



  • Different antibodies have been used for evaluation of HER2 protein expression in formalin-fixed, paraffin-embedded samples



    • Antibody clones have varying sensitivities and specificities



      • 3B5 (mouse, monoclonal), predominantly used in older studies: C-terminus, preferentially recognizes unphosphorylated form


      • AO85 (rabbit, polyclonal), part of FDA-approved kit: Cytoplasmic portion


      • CB11 (mouse, monoclonal), part of FDA-approved kit: Internal portion of receptor


      • 4B5 (rabbit, monoclonal), part of FDA-approved kit


      • SP3 (rabbit, monoclonal): Extracellular domain


HER2 IHC Interpretation



  • Scoring of HER2 results by IHC needs to be semiquantitatively evaluated to be clinically relevant



    • Only areas of invasive carcinoma are scored


  • HER2+BC (IHC scored as 3+) ˜10-15% of cancers



    • Diffuse intense circumferential membrane “chicken wire” staining pattern in > 30% of invasive cancer



      • Score as HER2 IHC positive (3+)


      • In most cancers, the majority of the carcinoma is positive


      • If only focal positivity is present (e.g., strong positivity in 20% of the cancer), this should be described; these cases may correlate with genetic heterogeneity


    • Carcinomas with this staining pattern typically show good concordance with gene amplification by FISH (> 95%)


    • Patients with HER2 3+ carcinomas are candidates for treatment with HER2-targeted therapy


    • In rare cases, the associated DCIS overexpresses HER2 but the invasive carcinoma is HER2 negative



      • This finding should be documented to ensure this is taken into account during the evaluation of other assays


  • HER2-BC (IHC scored as 0 or 1+) ˜ 70-75% of cancers



    • Absent or weak incomplete membrane staining in invasive cancer



      • Score as HER2 IHC negative (0/1+)


    • Cancers with this staining pattern show a good concordance with absence of amplification by FISH (> 95%)


    • Breast cancer patients with HER2 IHC 0/1+ tumors are unlikely to benefit from targeted therapy


  • HER2 equivocal breast cancer (IHC scored as 2+) ˜ 15% of cancers



    • Weak, circumferential membrane staining &/or heterogeneity in staining distribution < 30% of invasive tumor



      • Score as HER2 IHC equivocal (2+)


    • In correlative studies, approximately 1/5-1/4 of 2+ cancers show HER2 gene amplification


    • Breast cancers with equivocal HER2 IHC result should be analyzed by FISH to assess for HER2 gene amplification



      • These cases are more likely to have low numbers of HER2 genes


    • If the studies are performed on a core needle biopsy, it is helpful to repeat on a larger area of carcinoma in the excision


    • Some cancers will have “equivocal” HER2 results by both IHC and FISH, reflecting that HER2 expression is continuous and not bimodal


  • HER2 IHC inadequate for interpretation (rejection); in some cases scoring is not possible



    • Needle core biopsies with crush artifact are inadequate for interpretation



      • Should not be overinterpreted as positive


    • Staining of adjacent normal breast tissue suggests that the assay is too sensitive



      • Results for the assay should be considered inadequate for interpretation


      • May lead to false-positive interpretation


    • Prolonged period of ischemia prior to initiation of formalin fixation



      • Time from tissue collection to fixation > than 1-2 hours


      • HER2 assay result may not be accurate


      • May lead to false-negative interpretation


    • Samples fixed for < 6-8 hours in neutral buffered formalin


    • Samples fixed for > 48 hours in neutral buffered formalin


    • Samples fixed in fixatives other than formalin



      • Alternative fixatives must be rigorously validated by laboratory


    • Samples with no residual invasive carcinoma on deeper levels


    • Samples on unstained slides stored for > 6 weeks prior to testing


  • Many laboratories utilize a HER2 testing algorithm in which tumor samples are initially screened by IHC



    • FISH testing is reflexively performed only on equivocal cases


    • Testing algorithm assumes that IHC assays are highly standardized




      • Laboratories must exercise rigorous quality control and follow published guidelines


      • Assay must be validated to show high degree of concordance with FISH results


HER2 Assay Methodologies: In Situ Hybridization (ISH)



  • Morphology-based assay to evaluate gene copy number



    • Single probe methods evaluate the number of HER2 genes


    • Dual probe methods evaluate the number of HER2 genes and chromosome 17 centromere sequences (CEP17)



      • Results are reported as the ratio HER2:CEP17


  • Multiple methods for ISH are available



    • FISH utilizes fluorescent labeled probes and fluorescence microscopy


    • Chromogenic/bright-field in situ hybridization (CISH, Duo-CISH) and silver-enhanced in situ hybridization (SISH) use light microscopy



      • Chromogen dye or silver deposition replaces fluorescent label for detection of gene copy number


      • Light microscopy facilitates correlation with histologic appearance


      • These methods correlate well with FISH


  • With FISH, quantitative interpretation of results more straightforward than with IHC



    • Concordance rates between observers are higher with FISH than with IHC in some studies


    • However, heterogeneity of expression is easier to detect with IHC


  • ISH and IHC assays are best viewed as complementary methodologies



    • Each assay examines a different aspect of HER2 biology


    • ISH assays can be used in conjunction with IHC or as primary methodology for HER2 testing


  • Chromosome 17 centromere sequences (CEP17)



    • Probe to the centromeric region of chromosome 17 is utilized in dual probe methods to determine the number of chromosomes present


    • 10-50% of HER2+BC are reported to have increased CEP17 sequences



      • “Polysomy” is usually defined as ≥ 3 CEP17 signals per nucleus


      • However, true polysomy (duplication of the entire chromosome) is only present in 1-2% of cancers


      • In the majority of cancers, increased CEP17 is due to duplication of a segment of centromeric DNA


    • Carcinomas with 3-5 copies of the HER2 gene and increased CEP17 generally do not show increased protein expression


    • Carcinomas with > 6 gene copies are usually associated with HER2 overexpression



      • > 90% of these cases will also have HER2:CEP17 ratios > 2.2, and the carcinoma is classified as HER2 amplified


      • In rare cases, the ratio is < 2.2 due to the increased CEP17 numbers, and the 2 methods for determining HER2 amplification have different interpretations


      • If not previously performed, an IHC assay should be performed


      • Carcinomas with 3+ scores by IHC are classified as HER2+BC


      • It is not yet clear if carcinomas with 2+ scores by IHC, > 6 gene copies, but ratios < 2.2 will benefit from targeted therapy


    • Monosomy for chromosome 17 occurs in < 5% of cancers



      • It is not clear if ratios > 2.2 in the presence of monosomy will predict benefit from targeted therapy


HER2 FISH Interpretation

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Jul 6, 2016 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on HER2

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