Breast Cancer

Diagnosis

Indications for Testing

  • Malignant histology compatible with breast cancer

Histology

  • Therapeutic decisions are made using combination of tumor features
    • Histologic grade – includes nuclear probe and mitotic index
    • Estrogen receptor (ER) and progesterone receptor (PR) status
    • HER2 (ERBB2) status
      • Methods for initial HER2 determination – immunohistochemistry (IHC), in situ hybridization (ISH)
        • ISH includes fluorescence or Bright-field assays; most current ISH assays consist of two differentially labeled probes, one for HER2 and one for chromosome 17 centromere
        • Concordance between IHC and ISH may vary due to subjective interpretation
          • Either test may be used in the initial evaluation
        • Equivocal results should be resolved with reflex test (ASCO/CAP 2013 guidelines)
          • Equivocal (2+) IHC – ISH recommended follow-up test 
          • Equivocal ISH – follow-up testing options
            • Repeat ISH on same specimen using an alternative control probe
            • IHC for HER2 expression status using the same block or a different block from the same specimen, if not already performed
            • Repeat testing (ISH or IHC) on a different specimen from patient’s tumor
  • Other immunohistochemical staining
    • Immunoreactivity of normal breast epithelium
      • Luminal: CK 7, CK 8, CK 18, CK 19
      • Basal cells: CK 5/6, CK 14, CK 17
      • Myoepithelial cells: CK 5, CK 14, CK 17, SMA, calponin, p63
    • IHC staining may be used to differentiate ductal versus lobular carcinoma
      • E-Cadherin
        • Ductal (+)
        • Lobular (-)
      • Keratin 903 – more commonly used to differentiate usual ductal hyperplasia (UDH) from atypical ductal hyperplasias (ADH)/low-grade ductal carcinoma in situ (LG-DCIS)
        • Ductal (-)
        • Lobular (+)
      • CAM 5.2
        • Ductal (peripheral)
        • Lobular (perinuclear)

Prognosis

  • American Society of Clinical Oncology (ASCO)/National Academy of Clinical Biochemistry (NACB) recommend the following
    • ER/PR
      • ER positivity alone is a weak prognosis indicator; use in combination with progesterone
      • Two molecular subtypes – luminal A and luminal B
        • B has best prognosis
      • Positivity associated with improved prognosis when treated with anti-estrogen therapy (tamoxifen)
    • HER2/neu 
      • Positivity occurs in 15-20% of breast cancer patients
      • Graded scale of 0-3 for immunohistochemistry
        • 0 and 1 – negative
        • 2 – equivocal
        • 3 – positive
    • Triple negative tumors (ER-/PR-/HER2-
      • More common in
        • African Americans – significant number have BRCA1 mutations
        • Young age of onset
        • Premenopausal women
      • Predominantly high-grade tumors
        • 75% have basal-like gene expression (CK5/14+, EGFR+) – worst prognosis of all breast cancer subtypes
      • Worst prognosis for breast cancer
  • Other markers
    • p53 
      • Positivity may be associated with worse prognosis
      • Insufficient data to recommend use in disease management (ASCO, 2007)
    • Aneuploid and high S-phase tumors
      • Associated with worse prognosis in node-negative cancers
      • Low S-phase and diploid DNA content associated with better prognosis
      • Insufficient data to recommend use in disease management (ASCO, 2007)
    • Ki-67 (MIB-1) – cell proliferative-associated antigen
      • Elevation associated with aggressive tumor behavior
      • Insufficient data to recommend use in disease management (ASCO. 2007)
    • uPA/PAI-1 (urokinase-type plasminogen activator/plasminogen-like activator inhibitor)
      • If both markers are low in node-negative patients, risk of relapse is low
    • PIK3CA mutation
      • Associated with shorter breast cancer-specific and disease-free survival
    • Fibroblast growth factor receptor 2 (FGFR2)
      • ~ 1% of breast cancers show copy number gains in the FGFR2 gene
      • Reported in triple-negative breast cancers (negative for ER, PR, and HER2 expression)
      • Patients whose tumors demonstrate FGFR2 gene amplification may benefit from FGFR2-targeting antibodies or FGFR-specific tyrosine kinase inhibitors
  • Prognostic panels include Prosigna prognostic test, Oncotype Dx, MammaPrint genomic grade index, Rotterdam signature
    • Prosigna prognostic test
      • Use to assess risk of distant recurrence in post- or perimenopausal women with early stage (stage I or stage II), hormone receptor-positive (ER+ and/or PR+) breast cancer
      • Useful in both node-negative and node-positive (1-3 nodes) disease
      • Scores report risk as low, intermediate, or high based on
        • Molecular subtype of tumor
        • Proliferation status
        • Tumor size and node status
      • May help decrease overtreatment with chemotherapy
      • Does not provide information about which chemotherapy regimen should be given if at high risk for distant recurrence
    •  Genomic Health Oncotype DX (National Cancer Institute [NCI]) 
      • TAILORx (Trial for Assigning Individualized Options for Treatment [Rx]) – study of genes associated with recurrence risk for use in determining most effective treatment options 
        • Helps to predict effectiveness of tamoxifen alone in postmenopausal women with node-negative, ER+, early breast cancer
  • Minimal invasive disease detection helps determine risk or presence of metastatic disease and includes measures of tumor cells in
    • Bone marrow 
    • Axillary nodes/sentinel nodes
    • Circulation (circulating tumor cells – CTCs)
      • Most helpful in prognosis of advanced disease
      • Should not be used for diagnosis or treatment decisions

Screening

  • Screening (mammogram, clinical breast exam, breast self-exam)

    Society

    Mammogram

    Clinical Breast Exam (CBE)

    Breast Self-Exam (BSE)

    AAFP (2009)

    <50 yrs – should be evaluated on individual basis  

    50-74 yrs – every 2 yrs

    Insufficient evidence to assess harms or benefits

    Recommend against teaching BSE

    ACS (2013)

    Annual screening for all women beginning at 40 yrs until no longer in good health

    Annual screening for all women beginning at 40 yrs until no longer in good health

    Every 3 yrs for women in their 20s and 30s; every year for women ≥40 yrs 

    Women should know how their breasts normally look and feel and report any breast change promptly to their health care provider

    BSE is an option for women starting in their 20s

    ACOG (2011)

    Every 2 yrs starting at 40 yrs

    Annually starting at 50 yrs

    Annually at 40-45 yrs

    Recommend monthly BSE

    AGS (2010)

    Stop screening healthy women ≥85 yrs; ≥75 yrs consider life expectancy and quality of life when screening

    USPSTF (2009)

    Recommend against routine screening mammography in women 40-49 yrs

    Biennial screening mammography for women 50-74 yrs

    No recommendation

    Recommend against teaching BSE

    USPSTF = U.S. Preventive Services Task Force; ACS = American Cancer Society; AAFP = American Academy of Family Physicians; AGS = American Geriatrics Society; ACOG = American Congress of Obstetricians and Gynecologists

  • Breast cytology screening not yet recommended, but may be useful in high-risk patients (eg, ductal lavage)
  • No tumor markers recommended in screening (ASCO, 2007)
    • All markers have low sensitivity and specificity when used in a screening setting
  • BRCA testing should be considered in patients with multiple female relatives with breast cancer, or early onset breast cancer (eg, premenopausal)
    • If mutation is detected, screening should begin at 25-30 years

Monitoring

  • Annual mammography
    • Most organizations (American Cancer Society, National Cancer Institute, American Medical Association, American Geriatrics Society) recommend annual screening beginning at 50 yrs (see table in Screening)
  • Annual gynecological examination for patients receiving tamoxifen therapy due to increased risk of endometrial cancer
    • Endometrial ultrasound and biopsy indicated in patients with abnormal vaginal bleeding or atypical endometrial cells on a PAP smear
  • Cancer markers
    • CA 15-3 and CA 27.29
      • May be used to monitor advanced disease in conjunction with diagnostic imaging, history, physical (ESMO, NACB, NCCN)
      • Cannot be used singly for monitoring breast cancer patients
        • Serial measurements are most useful
    • Carcinogenic embryonic antigen – use in conjunction with imaging, history, physical for monitoring therapy in metastatic disease (NACB, NCCN)
    • Circulating tumor cell count – use to determine prognosis, assess treatment efficacy, and aid in treatment decisions for patients with metastatic breast cancer
    • HER2/neu (serum) – preliminary evidence suggests potential value in monitoring trastuzumab therapy in advanced disease

Pharmacogenetics and Therapeutic Drug Monitoring

  • Tamoxifen is an anti-estrogen drug used in treatment of ER+ breast cancer
    • Tamoxifen metabolites, particularly endoxifen and 4-hydroxy-tamoxifen bind the estrogen receptors and suppress breast cancer cell proliferation
  • Cytochrome P450 2D6 (CYP2D6) and tamoxifen
    • Metabolism of tamoxifen to endoxifen depends on a CYP2D6-mediated reaction
    • Decreased metabolite production (due to nonfunctional or poorly functional CYP2D6) could put patients at risk for recurrence of breast cancer
    • CYP2D6 genotype is associated with the following phenotypes
      • Poor metabolizer – little or no metabolism; alternate drug recommended
      • Intermediate metabolizer – possible impaired metabolism
      • Extensive metabolizer – no impairment
      • Ultrarapid metabolizer – faster than normal metabolism; implications for tamoxifen therapy not well-characterized
  • Cytochrome P450 2C19 (CYP2C19) and tamoxifen
    • Metabolism of tamoxifen to 4-hydroxy-tamoxifen can be accelerated by the presence of the CYP2C19*17 allele that confers an ultrarapid metabolizer phenotype
  • Tamoxifen metabolism is also affected by concomitant administration of SSRIs and other strong inhibitors of CYP2D6
    • Strong inhibitors of CYP2D6 should be avoided in patients taking tamoxifen

Clinical Background

Primary carcinoma of the breast, the most common type of breast malignancy, usually begins as a neoplastic proliferation of epithelial cells lining the ducts or lobules of the breast.

Epidemiology

  • Incidence – >200,000 new breast cancers diagnosed in U.S. per year (CDC, 2010)
  • Age – prevalence increases with age 
  • Sex – most common cancer in females
    • Rare in males (~2,000/year in U.S.) (CDC, 2010)

Risk Factors

  • Gene mutations – BRCA1, BRCA2, p53; other rarer syndromic mutations
    • ~10% of all breast cancers
  • Early menarche
  • Late menopause
  • First childbirth at >30 years
  • Menopausal estrogen and progesterone therapy
  • Chest radiation at <30 years
  • Moderate alcohol intake
  • Family history of breast cancer
  • See the National Cancer Institute for the Breast Cancer Risk Assessment Tool  (based on the GAIL model) to further estimate breast cancer risk

Pathophysiology

  • Tumors are usually epithelial cell in origin
  • Noninvasive forms may be present alone or in association with invasive carcinoma

Clinical Presentation

  • Breast mass
  • Nipple discharge
  • Breast asymmetry
  • Retraction of nipple, skin changes
  • Redness or tenderness – inflammatory breast cancer (rare)

Indications for Laboratory Testing

  • Tests generally appear in the order most useful for common clinical situations
  • Click on number for test-specific information in the ARUP Laboratory Test Directory
Test Name and Number Recommended Use Limitations Follow Up
Estrogen/Progesterone Receptor with Interpretation by Immunohistochemistry 0049210
Method: Immunohistochemistry

Aids in prediction of response to anti-estrogen therapy

Stained and resulted by ARUP

For paraffin-embedded, formalin-fixed tissue

 
ERBB2 (HER2/neu) (4B5) by Immunohistochemistry, Tissue with Reflex to Dual ISH if 2+ 2007782
Method: Immunohistochemistry

Aids in prediction of response to trastuzumab (Herceptin) therapy

Alternate test to confirm equivocal FISH result

Reflex – if  HER2+, ISH testing is added to confirm gene amplification

Results graded 0-3 in a semiquantitative manner

Stained and resulted by ARUP

FDA approved for formalin-fixed tissue only

 
ERBB2 (HER2/neu) (4B5) by Immunohistochemistry, Tissue with Reflex to FISH if 2+ 2002217
Method: Immunohistochemistry

Aids in prediction of response to trastuzumab (Herceptin) therapy

Alternate test to confirm equivocal FISH result

Reflex – if IHC 2+, FISH testing is added

Measures protein overexpression

Testing using tissue fixed in alcohol-based or non-formalin fixatives has not been validated using this method

Specimens placed in decal may have a false negative result

Repeat testing is recommended for discordant results

 
ERBB2 (HER2/neu) (4B5) with Interpretation by Immunohistochemistry, Tissue 2002218
Method: Immunohistochemistry

Aids in prediction of response to trastuzumab (Herceptin) therapy

Alternate test to confirm equivocal FISH result

   
ERBB2 (HER2) (HercepTest) by Immunohistochemistry 2007332
Method: Immunohistochemistry

Measure protein overexpression

   
ERBB2 (HER2/neu) (HercepTest) with Interpretation by Immunohistochemistry, Tissue 0049174
Method: Immunohistochemistry

Measures protein overexpression

   
ERBB2 (HER2/neu) Gene Amplification by FISH, Tissue 2008603
Method: Fluorescence in situ Hybridization

Measure gene amplification

Utilizes fluorescence, which limits visualization of tissue morphology

FDA approved for formalin-fixed tissue only

 
ERBB2 (HER2/neu) Gene Amplification by Dual in-situ Hybridization 2007410
Method: Dual in situ Hybridization

Measure gene amplification

Alternative to FISH; highly concordant with FISH

   
ERBB2 (HER2/neu) Gene Amplification by PCR 0049390
Method: Polymerase Chain Reaction

Detect amplification in HER2 gene for predicting response to trastuzumab (Herceptin) therapy

Confirm or validate FISH test results for HER2 gene amplification

Resolve discrepancies between HER2 FISH and IHC results (ie, IHC 3+/FISH negative)

Clinical sensitivity – 90.5%

Clinical specificity – 95%

Analytical sensitivity – limit of detection is 25% of tumor area

Analytical specificity – 100%

Testing using tissue fixed in alcohol-based or non-formalin fixatives has not been validated using this method

Not FDA approved for clinical use

Interpret test result within the clinical context; do not use alone for a diagnosis of malignancy

Not recommended for detection of minimal residual disease

 
ERBB2 (HER2/neu) (HercepTest) by Immunohistochemistry, Tissue with Reflex to Dual ISH if 2+ 2007785
Method: Immunohistochemistry

Aids in prediction of response to trastuzumab (Herceptin) therapy

Alternate test to confirm equivocal FISH result

Reflex – if HER2+, ISH testing is added to confirm gene amplification

Results graded 0-3 in a semiquantitative manner

Stained and resulted by ARUP

FDA approved for formalin-fixed tissue only

 
HER2/neu Quantitative by ELISA 2004672
Method: Quantitative Enzyme-Linked Immunosorbent Assay

Triage for trastuzumab therapy when solid tissue unavailable

Positive results are reliable; however, this serum assay has a 30% false-negative rate

 
PIK3CA Mutation Detection 2004510
Method: Polymerase Chain Reaction/Pyrosequencing

Use to prognosticate in breast cancer

   
p53 with Interpretation by Immunohistochemistry 0049250
Method: Immunohistochemistry

Histologic prognosis of breast cancer

Determine treatment method for node-negative breast cancer

Stained and resulted by ARUP

   
Ki-67 with Interpretation by Immunohistochemistry 2007182
Method: Immunohistochemistry

Histologic prognostic indicator for node-negative breast cancer

Stained and resulted by ARUP

   
DNA Content/Cell Cycle Analysis - Ploidy and S-Phase 0095155
Method: Quantitative Flow Cytometry

Prognostic indicator for node-negative breast cancer

Tumor-specific S-phase used when possible

Average histogram S-phase used

  • For diploid and some aneuploid tumors – where tumor and host S-phases cannot be separated
  • When percentage of aneuploid cells in histogram low – <25%
   
Cytology, Breast Nipple Secretion 8209700
Method: Microscopy

Cytomorphologic screening for breast cancer cells and precursor lesions

Potential risk-assessment tool

Known false negatives and false positives

 
Prosigna Breast Cancer Prognostic Gene Signature 2010248
Method: Hybridization/gene expression

Assess the risk of distant recurrence in post- or perimenopausal women with early stage (stage I or stage II), hormone receptor-positive (ER+ and/or PR+) breast cancer

Useful in both node-negative and node-positive (1-3 nodes) disease

May help decrease overtreatment with chemotherapy

Minimum 10% tumor required

Test is intended for women with hormone receptor-positive breast cancer only

Not intended to provide information about what chemotherapy regimen should be given if at high risk for distant recurrence

 
Circulating Tumor Cell Count 0093399
Method: Immunomagnetic Separation/Immunofluorescent Stain/Computer Assisted Analysis

Use to determine prognosis, assess treatment efficacy, and aid in treatment decisions for patients with metastatic breast cancer

Cutoffs vary by tumor cell type

CTC is not as accurate as imaging in assessing whether a patient has progressive disease

Doxorubicin therapy patients – allow at least 7 days following administration of dose before testing

Not detected – CTCs that do not express EpCAM; CTCs that express EpCAM but not cytokeratins 8, 18, and 19

 
Keratin 903 (K903) High Molecular Weight by Immunohistochemistry 2003978
Method: Immunohistochemistry

Histologic diagnosis of adenocarcinomas of ductal origin in breast

Stained and returned to client pathologist for interpretation; consultation available if required

   
E-Cadherin by Immunohistochemistry 2003869
Method: Immunohistochemistry

Histologic diagnosis of morphologic subtype of breast cancer

Stained and returned to client pathologist for interpretation; consultation available if required

   
Cytokeratin 8,18 Low Molecular Weight (CAM 5.2) by Immunohistochemistry 2003493
Method: Immunohistochemistry

Histologic diagnosis of breast cancer

Stained and returned to client pathologist for interpretation; consultation available if required

   
PAX8 by Immunohistochemistry 2010787
Method: Immunohistochemistry

Distinguish ovarian cancer from breast cancer

Stained and returned to client pathologist for interpretation; consultation available if required

   
Cytochrome P450 2D6 (CYP2D6) 14 Variants and Gene Duplication 0051232
Method: Polymerase Chain Reaction/Primer Extension

Pretherapeutic testing to identify individuals who should avoid or have different dosing of medications metabolized by CYP2D6 such as tamoxifen

Screening of individuals with personal or family history of adverse drug event or therapy failure when exposed to CYP2D6-metabolized drugs

Variants tested

  • Functional
    • *2 (2850C>T)
    • *2A (-1584C>G; 2850C>T)
  • Decreased function
    • *9 (2613-5delAGA)
    • *10 (100C>T)
    • *17 (1023C>T)
    • *29 (1659G>A)
    • *41 (2988G>A)
  • Nonfunctional
    • *3 (2549delA)
    • *4 (1846G>A)
    • *5 (gene deletion)
    • *6 (1707delT)
    • *7 (2935A>C)
    • *8 (1758G>T)
    • *12 (124G>A)
    • *14 (1758G>A)
  • Increased function
    • Duplicated functional alleles

Clinical sensitivity – >95% in Caucasians; unknown in other ethnicities

Only the targeted CYP2D6 mutations will be detected

Phase and copy number of detected CYP2D6 mutations may not be determined

Mutations in other genes associated with drug metabolism or drug response will not be detected

Drug metabolism may be affected by nongenetic factors

Mutation detection is not a substitute for therapeutic drug or clinical monitoring

Diagnostic errors can occur due to rare sequence variations

Genotype results should be interpreted in the context of the individual clinical situation

 
Cytochrome P450 2C19 (CYP2C19) 9 Variants 0051104
Method: Polymerase Chain Reaction/Primer Extension

Pretherapeutic testing to identify individuals who should avoid or have different dosing of medications metabolized by CYP2C19 such as tamoxifen

Screening of individuals with personal or family history of adverse drug event or therapy failure when exposed to CYP2C19-metabolized drugs

Variants tested

  • Decreased function
    • *9 (c.431G>A)
    • *10 (c.680C>T)
  • Nonfunctional
    • *2 (c.681G>A)
    • *3 (c.636G>A)
    • *4 (c.1A>G)
    • *6 (c.395G>A)
    • *7 (c.819+2T>A)
    • *8 (c.358T>C)
  • Increased function
    • *17 (c.-806C>T, increased gene transcription)

Clinical sensitivity – ~99%  in Asians; ~87% in Caucasians

Only the targeted CYP2C19 mutations will be detected

Mutations in other genes associated with drug metabolism or drug response will not be detected

Drug metabolism may be affected by nongenetic factors

Mutation detection is not a substitute for therapeutic drug or clinical monitoring

Diagnostic errors can occur due to rare sequence variations

Genotype results should be interpreted in the context of the individual clinical situation

 
FGFR2 Gene Amplification by FISH 2007099
Method: Fluorescence in situ Hybridization

Consider ordering for patient with triple negative (ER-, PR-, HER2-) status to determine eligibility for treatment with FGFR inhibitors

   
Additional Tests Available
 
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
ERBB2 (HER2) (4B5) by Immunohistochemistry 2007329
Method: Immunohistochemistry
ERBB2 (HER2/neu) (HercepTest) by Immunohistochemistry, Tissue with Reflex to FISH if 2+ 0049178
Method: Immunohistochemistry
ERBB2 (HER2/neu) (HercepTest) with Interpretation by Immunohistochemistry, Tissue 0049174
Method: Immunohistochemistry
Cytology, Fine Needle Aspirate 8209706
Method: Microscopy
Cancer Antigen-Breast (CA 15-3) 0080464
Method: Quantitative Electrochemiluminescent Immunoassay

May be used to monitor stage II-III breast cancer

Use in conjunction with other clinical methods

Cancer Antigen 27.29 0080392
Method: Quantitative Chemiluminescent Immunoassay

May be used to monitor patients treated previously for stage II-III breast cancer and who are clinically free of disease

Use in conjunction with other clinical methods for early detection of recurrent disease

May be used to monitor disease progress and response to therapy in patients with metastatic disease

Carcinoembryonic Antigen 0080080
Method: Quantitative Electrochemiluminescent Immunoassay

May be used to monitor stage II-III breast cancer

Serial testing should be used in conjunction with other clinical methods

Solid Tumor Mutation Panel by Next Generation Sequencing 2007991
Method: Massively Parallel Sequencing

Prognosis/treatment of individuals with solid tumor cancers at initial diagnosis or with refractory disease