Breast Cancer

Diagnosis

Indications for Testing

  • Based on a malignant histology result

Histology

  • Therapeutic decisions are made based on tumor features
    • Histologic and nuclear grade
    • ER and progesterone-receptor (PR) status
    • Mitotic index
    • HER2 testing
      • Methods for initial HER2 determination
      • Concordance between IHC and FISH may vary due to subjective interpretation
      • Gene amplification – HER2 copy number or HER2/CEP17 ratio by ISH/FISH (based on counting ≥20 cells)
      • IHC (protein overexpression)
      • Repeat testing with other test should be considered if results are discordant
      • Use PCR to resolve discrepancies between IHC and ISH/FISH
  • Immunohistochemistry
    • Keratin 903 (K903) – potentially useful for identifying adenocarcinomas of ductal origin in breast
    • 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 to differentiate ductal and lobular carcinoma
      • E-Cadherin
        • Ductal (+)
        • Lobular (-)
      • Keratin 903
        • Ductal (-)
        • Lobular (+)
      • CAM 5.2
        • Ductal (peripheral)
        • Lobular (nuclear)
    • Hormone receptor type
      • PR (+/-)
      • ER (+/-)

Prognosis

  • American Society of Clinical Oncology (ASCO)/National Academy of Clinical Biochemistry (NACB)
    • ER/PR
      • Positivity associated with improved prognosis with anti-estrogen therapy (tamoxifen)
      • Two molecular subtypes – luminal A and luminal B
        • B has best prognosis
      • Test gives low/high recurrence scores to help assess risk in ER+/PR+, node-negative patients treated with tamoxifen
        • Treatment management trials are occurring to evaluate if therapeutic decisions can be based on high/low recurrence scores
      • ER positivity alone is a weak prognosis indicator; use in combination with progesterone
    • HER-2/neu 
      • Positivity occurs in 15-20% of breast cancer patients
      • Positivity is associated with worse prognosis in node-positive patients; provides target for trastuzumab therapy 
        • Graded scale of 0-3
          • 0 and 1 – negative
          • 2 – equivocal
          • 3 – positive
      • Conflicting data for HER-2/neu testing in node-negative patients
      • Predicts positive response to anthracycline therapy (if HER-2/neu is 3+ by immunohistochemistry or ≥2.0 gene amplification ratio by FISH)
    • p53 
      • Positivity may be associated with worse prognosis
      • Insufficient data to recommend use in disease management (ASCO, 2007)
    • Aneuploid and high S-phase tumors
      • Positivity associated with worse prognosis in node-negative cancers
      • Low S-phase and diploid DNA content associated with better prognosis
    • High Ki-67 (MIB-1) – cell proliferative-associated antigen
      • Elevation associated with aggressive tumor behavior
      • Insufficient data to recommend use in management of breast cancer
    • Triple negative tumors (ER-/PR-/HER2-
      • Worst prognosis
      • More common
        • African Americans – significant number have BRCA1 mutations
        • Earlier age of onset
        • Predominance of premenopausal women
      • Predominantly high-grade tumors
        • 75% have basal-like gene expression (CK5/14+, EGFT+) – worst prognosis of all tumors
    • 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; patient may not need chemotherapy
    • PIK3CA mutation
      • Associated with shorter breast cancer-specific and disease-free survival
  • Emerging markers include Prosigna prognostic gene test, Oncotype Dx, MammaPrint genomic grade index, topoisomerase II-alpha, Rotterdam signature, microRNA
    •  Prosigna prognostic test
      • 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
      • Does not provide information about what chemotherapy regimen should be given if at high risk for distant recurrence
    • Genomic Health Oncotype DX (National Cancer Institute [NCI]) 
  • 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
  • Minimal invasive disease detection helps prognosticate and includes measures of
    • Tumor cells in bone marrow 
    • Tumor cells in axillary nodes/sentinel nodes
    • Tumor cells in circulation
      • Most helpful in prognosis of advanced disease
      • Should not be used in diagnosis or treatment decisions

Differential Diagnosis

  • Breast cyst
  • Other malignancy
  • Cellulitis, mastitis
  • Idiopathic granulomatous mastitis

Screening

  • Screening (mammogram, clinical breast exam, breast self-exam)
    • U.S. Preventive Services Task Force (USPSTF), 2009
      • Recommends against routine screening mammography in women 40-49 years
      • Biennial screening mammography for women 50-74 years
      • Recommends against teaching breast self-examination
    • American Cancer Society (ACS), 2011
      • Clinical breast exam (CBE) about every 3 years for women in their 20s and 30s and every year for women 40 and over
      • Women should know how their breasts normally look and feel and report any breast change promptly to their health care provider
      • Breast self-exam (BSE) is an option for women starting in their 20s
      • Annual screening (mammography and clinical breast examination) for all women beginning at 40 years until no longer in good health
    • American Geriatrics Society (AGS), 2010
      • Stop screening healthy women ≥85 years
      • ≥75 years consider life expectancy and quality of life when screening
  • Breast cytology screening is 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
    • If mutation is detected, screening mammography 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
    • U.S. Preventive Services Task Force does not recommend annual screening
  • Annual gynecological examination for patients receiving tamoxifen therapy
    • 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 alone for monitoring breast cancer patients
        • Serial measurements are most useful
    • Carcinogenic embryonic antigen – can be used 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
    • HER-2/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 – ~229,000 new breast cancers diagnosed in U.S. per year
  • Age – prevalence increases with age 
  • Sex – most common cancer in females
    • Rare in males (2,030/year in U.S.)

Risk Factors

Pathophysiology

  • Tumors are usually epithelial cell in origin and rarely sarcoma or lymphoma (B-cell and T/NK cell)
  • Noninvasive forms may be present alone or in association with invasive carcinoma
    • Ductal carcinoma in situ
    • Lobular carcinoma in situ

Clinical Presentation

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

Treatment

  • Tamoxifen is anti-estrogen treatment of choice for estrogen-receptor positive (ER+) breast cancer
    • Consider genotyping to predict patient response to tamoxifen prior to initiating therapy (more information in Pharmacogenetics section)
  • Human epidermal growth factor receptor 2 positive (HER2+) breast cancer
    • Trastuzumab (Herceptin) is the treatment of choice; blocks signals for growth to the cancer cells

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

Triage for anti-estrogen therapy

Stained and resulted by ARUP

For paraffin-embedded, formalin-fixed tissue

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

Triage for trastuzumab therapy (predictive indicator)

Results graded 0-3 in a semiquantitative manner

Stained and resulted by ARUP

If HER2+, ISH testing is added to confirm gene amplification

FDA approved for formalin-fixed tissue only

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

Histologic prognostic and predictive indicator

Results graded 0-3 in a semiquantitative manner

Stained and resulted by ARUP

If HER2+, ISH testing is added to confirm gene amplification

FDA approved for formalin-fixed tissue only

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

Triage for trastuzumab therapy in metastatic breast cancer when solid tissue unavailable

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

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

Measure gene amplification

Triage for trastuzumab therapy (predictive indicator, FDA approved)

Confirm positive or equivocal immunohistochemical result

FDA approved for formalin-fixed tissue only

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

Detect amplification in ERBB2 gene for predicting response to trastuzumab therapy

Confirm or validate FISH test results for ERBB2 gene amplification

Resolve discrepancies between FISH and IHC results

Acceptable specimens – only formalin-fixed, paraffin-embedded tissue; area of tumor must be invasive

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

 
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, Breast (Paraffin) 0095735
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%)
 
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

 
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

 
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 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

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

Pre therapeutic 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

>95% clinical sensitivity 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

Pre-therapeutic 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

~99% clinical sensitivity in Asians, ~87% for 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

 
PIK3CA Mutation Detection 2004510
Method: Polymerase Chain Reaction/Pyrosequencing
   
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) (HercepTest) by Immunohistochemistry 2007332
Method: Immunohistochemistry

Measure protein overexpression

ERBB2 (HER2/neu) (4B5) by Immunohistochemistry, Tissue with Reflex to FISH if 2+ 2002217
Method: Immunohistochemistry
ERBB2 (HER2/neu) (4B5) by Immunohistochemistry, Tissue with Reflex to FISH if 2+ or 3+ 2002216
Method: Immunohistochemistry
ERBB2 (HER2/neu) (HercepTest) by Immunohistochemistry, Tissue with Reflex to FISH if 2+ 0049178
Method: Immunohistochemistry
ERBB2 (HER2/neu) (HercepTest) by Immunohistochemistry, Tissue with Reflex to FISH if 2+ or 3+ 0049172
Method: Immunohistochemistry
ERBB2 (HER2/neu) (4B5) with Interpretation by Immunohistochemistry, Tissue 2002218
Method: Immunohistochemistry
ERBB2 (HER2/neu) Gene Amplification by Dual in-situ Hybridization 2007410
Method: Dual in situ Hybridization

Measure gene amplification

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