Breast Cancer

Dx
Screen
Monitor
PGx
Background
Lab Tests

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 gene amplification and protein overexpression
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 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
  - Immunohistochemistry used as initial screening test
    - If equivocal, followup with FISH is appropriate since gene amplification and not overexpression correlates with response to therapy
  - Conflicting data for use 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 oncotype Dx, MammaPrint genomic grade index, topoisomerase II-alpha, Rotterdam signature, microRNA
  - Genomic Health Oncotype DX (National Cancer Institute [NCI])
    - Refer to the web site of the National Cancer Institute for information on TAILORx (Trial for Assigning Individualized Options for Treatment [Rx]) – study of genes associated with recurrence risk for use in determining most effective treatment options
      - Help to predict effectiveness of tamoxifen in postmenopausal women with node-negative, ER+, early breast cancer
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
- 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
- Lymphoma
  - B-cell
  - T/NK-cell
- Sarcoma
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 therapy of patients with metastatic breast cancer in conjunction with diagnostic imaging, history, physical
  - Cannot be used alone for monitoring breast cancer patients
    - Serial measurements are most useful
  - American Society of Clinical Oncology (ASCO) 2007, European Society for Medical Oncology (ESMO) 2009, National Comprehensive Cancer Network (NCCN) 2010, and National Academy of Clinical Biochemistry (NACB) 2008 do not support use for monitoring recurrence after primary therapy
  - European Group on Tumor Markers (2010) does recommend use for monitoring
- Carcinogenic embryonic antigen – can be used in conjunction with imaging, history, physical for monitoring therapy in metastatic disease, particularly if CA 15-3 or CA 27.29 not elevated (ASCO, 2007)
- Circulating tumor cell count – use in metastatic tumors to monitor disease progression and response to therapy
- 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

Prevalence – in U.S., 207,090 new cases and 39,840 deaths in 2010 (SEER estimate)
Age – prevalence increases with age
Sex – most common cancer in females
- Rare in males (2,030/year in U.S.)

Risk Factors

10% linked to genetics – BRCA1, BRCA2, p53 mutations
Early menarche
Late menopause
First childbirth >30 years
Menopausal estrogen and progesterone therapy
Chest radiation <30 years
Moderate alcohol intake
Family history of breast cancer
See the web site of 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 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
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+, FISH 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+, FISH 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 0049218 Method: Fluorescence in situ Hybridization	Triage for trastuzumab therapy (predictive indicator, FDA approved) Confirm positive or equivocal immunohistochemical result	FDA approved for formalin-fixed tissue only
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%)
Circulating Tumor Cell Count 0093399 Method: Immunomagnetic Separation/Immunofluorescent Stain/Computer Assisted Analysis	Monitor metastatic tumors in conjunction with clinical data and imaging Serially monitor disease progression and response to therapy; assess prognosis when compared to baseline values Prognostic marker for recurrent disease and overall survival	Prognostic marker, do not use for diagnosis CTCs not detected for cells with No EpCAM expression (epithelial cell adhesion molecule) EpCAM expression with no CK 8, CK 18, CK 19 Results should be interpreted with caution if specimens drawn within 7 days of doxorubicin therapy
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 Mutations and Gene Duplication 0051232 Method: Polymerase Chain Reaction/Primer Extension	Detect CYP2D6 mutations affecting tamoxifen metabolism; determine risk of impaired benefit from adjuvant tamoxifen therapy >95% clinical sensitivity in Caucasians; sensitivity unknown in other ethnicities	Only the targeted CYP2D6 mutations will be detected Mutation detection is not a substitute for therapeutic drug monitoring
Cytochrome P450 2C19 (CYP2C19) 9 Mutations 0051104 Method: Polymerase Chain Reaction/Primer Extension	Detect CYP2C19 mutations affecting drug metabolism	Only the targeted CYP2C19 mutations will be detected Mutation detection is not a substitute for therapeutic drug monitoring
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 Number	Comments
ERBB2 (HER2) (4B5) by Immunohistochemistry 2007329 Method: Immunohistochemistry
ERBB2 (HER2) (HercepTest)™ by Immunohistochemistry 2007332 Method: Immunohistochemistry
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
ERBB2 (HER2/neu) (HercepTest) with Interpretation by Immunohistochemistry, Tissue 0049174 Method: Immunohistochemistry
Cytology, Fine Needle Aspirate 8209706 Method: Microscopy
IGH-BCL2 (BCL-2/JH) Translocation, t(14;18) by PCR 0055616 Method: Polymerase Chain Reaction	Prognostic marker in some studies; recently less utilized Offered only in conjunction with consultation
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