Invasive breast cancer is the most frequently diagnosed cancer in women in the United States and is second to lung cancer as a cause of cancer-related death.¹ Improved detection with screening mammography has led to an increased reported incidence of both forms of noninvasive breast cancer (ie, ductal carcinoma in situ [DCIS] and lobular carcinoma in situ [LCIS]). Although invasive breast cancer in men is rare, its incidence is also increasing. Table 1 presents the predicted incidence and number of deaths from breast cancer for 2012.

The type of breast cancer must be determined to assess prognosis and select therapy. Though often curable, invasive breast cancer has a heterogeneous clinical course as reflected by variable 5-year survival rates: 98% for local, 83% for regional, and 26% for metastatic disease.¹ Noninvasive breast cancers, which are confined to the ducts (DCIS) or lobules (LCIS), are associated with increased risk of progression to invasive breast cancer relative to women in the general population: 2.0- to 8.6-fold for DCIS, and 3.0- to 4.2-fold for LCIS.²

This Clinical Focus discusses the role of laboratory testing in the diagnosis and management of breast cancer in women, with emphasis on biologic characteristics of the tumor. For an overview of the diagnosis and management of breast cancer in men, the reader is referred to a review by Giordano.³

• Women being evaluated for possible breast cancer

• Women with diagnosed breast cancer

Laboratory tests that can be used for diagnosis, determining prognosis, selecting and monitoring treatment, and predicting and detecting recurrence of breast cancer are listed in Table 2.

Diagnosis
Suspicion of breast cancer usually arises from an abnormal screening mammogram or the presence of a breast lump. Symptoms, if present, may include a generalized swelling of part of the breast, skin irritation or dimpling, nipple pain or retraction, redness or scaliness of the nipple or breast skin, or a discharge other than milk. Follow-up includes a detailed physical examination and medical history along with diagnostic mammography, ultrasound, and/or a biopsy. Pathologic review of a biopsy or surgical sample determines whether cancer is present or not.

The pathology review can distinguish between benign, atypical hyperplasia, DCIS, LCIS, and malignant disease. However, both DCIS and LCIS may be difficult to diagnose because their histological features frequently resemble those of atypical hyperplasia or carcinoma with early invasion.⁴ A second microscopic review may be necessary to confirm the diagnosis. Recommendations for follow-up of each of these diagnoses are detailed on the National Comprehensive Cancer Network (NCCN) Web site.⁴

As shown in Table 2, Quest Diagnostics offers histopathology testing including gross/microscopic tissue examination, and microscopic examination of breast fluid, fine needle aspirations, paraffin blocks, and slides. Special stains and/or specific immunohistochemical (IHC) procedures may be ordered at additional charge by the histopathologist for interpretation purposes.

Noninvasive Breast Cancer

Determining Prognosis

LCIS has classically been considered a nonsurgical disease with an excellent prognosis. Because LCIS is associated with an increased risk of invasive cancer, the NCCN recommends increased surveillance and consideration of risk-reduction therapy (see the NCCN Web site).⁴ 

DCIS, though noninvasive, may progress to invasive breast cancer. Histopathologic characteristics of DCIS tumors such as nuclear grade, surgical margin status, growth pattern, and histologic type are associated with variable risk of DCIS recurrence and development of invasive breast cancer. Among 1,036 patients with DCIS, nuclear grade was strongly associated with disease progression (Table 3). Relative to those with low-nuclear-grade tumors, patients with high- and intermediate-nuclear-grade lesions have a higher risk of invasive cancer or DCIS recurrence.⁵ In addition, patients with positive resection margins (ie, ink on the tumor) are more likely to have disease progression than those with disease-free margins ≥10 mm (Table 3). Accordingly, margins ≥10 mm are considered adequate and margins <1 mm are inadequate; no consensus exists for margins in between.⁴

Additional DCIS histopathologic variables were found to be associated with the appearance of local breast cancer in the European Organisation for Research and Treatment of Cancer (EORTC) trial (Table 4). After a median follow-up of 10.5 years, increased risk was associated with solid/comedo or cribriform vs clinging/micropapillary architecture and with intermediately or poorly differentiated vs well-differentiated DCIS.⁶

Selecting Therapy

For patients with DCIS, the NCCN recommends lumpectomy, with or without radiation therapy, or mastectomy. If lumpectomy is performed, the NCCN further recommends consideration of tamoxifen therapy, especially for patients with estrogen receptor (ER)-positive DCIS.⁴ In the National Surgical Adjuvant Breast and Bowel Project (NSABP) trial, patients with DCIS were treated with lumpectomy and radiation with or without tamoxifen. The recurrence rate at 5 years was 8.2% for patients treated with tamoxifen vs 13.4% with no further treatment (P <0.001).⁷ Increased levels of ER expression predicted tamoxifen benefit in reducing risk of both ipsilateral and contralateral breast cancer following surgery. The benefit of tamoxifen in women with ER-negative DCIS is uncertain.

Invasive Breast Cancer

Determining Prognosis

Primary prognostic markers useful for breast cancer include TNM staging information such as tumor size, nodal status, histologic type and grade and the status of hormone receptors and human epidermal growth factor receptor 2 (HER-2/neu) expression. These and additional markers that predict disease recurrence and patient survival are discussed in detail below.

Histopathologic Tumor Staging
Tumor staging is the foundation of a breast cancer prognostic assessment. After surgical removal, breast tumors undergo pathologic staging based on invasiveness and tumor size (T), number of lymph nodes (N) involved, and whether the cancer has spread (metastasis [M]) to other tissues. In January 2003, a revised TNM staging system was implemented for breast cancer.⁸ When the new system was compared to the previous one, stage-specific overall survival improved by as much as 15%.⁹

Estrogen/Progesterone Receptors
In a large study assessing the clinical utility of ER and PR analysis in early breast cancer, patients with ER+/PR+ tumors had better outcomes than those with ER–/PR– tumors, irrespective of endocrine adjuvant therapy (Table 5).⁸

HER-2/neu, Tumor Tissue

The HER-2/neu gene encodes 1 of 4 transmembrane receptor protein-tyrosine kinases that mediate cell growth, differentiation, and survival. HER-2/neu gene overexpression or amplification is found in ~20% of breast tumors and is associated with aggressive disease and a higher rate of disease recurrence and mortality.⁹ Because of its use in assessing prognosis and predicting response to therapy, tumor HER-2/neu testing is recommended for all patients with invasive breast cancer by an expert American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) Panel.⁹

Methods for tumor HER-2/neu assessment include fluorescence in situ hybridization (FISH), which assesses gene amplification, and IHC, which assesses protein expression. Overall method concordance rates of 82% to 92% have been reported and ASCO/CAP expressed no preference for one method over the other.⁹ Quest Diagnostics offers both methods and follows the ASCO/CAP guidelines.⁹

The FISH method uses fluorescent DNA probes for HER-2/neu and chromosome enumeration probe (CEP) 17. The ratio of HER-2/neu to CEP 17 is positive for gene amplification when >2.2 and is negative (no amplification) when <1.8. Ratios of 1.8 to 2.2 are reported as equivocal; equivocal results are only reported after recount by a second laboratorian.

IHC test results for HER-2/neu are based on cell membrane stain intensity and percent of tumor cells stained. IHC results that are negative (0 and 1+) or strongly positive (3+) are associated with negative and positive protein overexpression, respectively. Quest Diagnostics reflexes 2+ (equivocal) IHC results to the FISH method as recommended by the ASCO/CAP guidelines.⁹

Multivariate analyses of nearly 25,000 patients with breast cancer have typically demonstrated the association of HER-2/neu+ tumors with shortened disease-free survival (DFS) and overall survival (OS) independent of other prognostic indicators.¹⁰ These studies included both node-negative and
-positive disease.

HER-2, Serum

Similar to other transmembrane receptors, HER-2/neu protein is shed into the circulation and is elevated in ~43% and ~19% of patients with metastatic or primary breast cancer, respectively.¹¹ Increased levels of serum HER-2 are associated with a poor prognosis and are consistently associated with shorter time to progression (TTP) and/or OS.¹¹ In one study, patients with metastatic breast cancer (n = 566) and pretreatment serum HER-2 levels >15 ng/mL had a less favorable prognosis than those with lower levels; both TTP (HR = 1.87, P <0.001) and OS (HR = 1.84, P <0.001) were shorter.¹² Furthermore, patients with elevated serum levels of both HER-2 and CA 15-3 (>38.5 U/mL) had a poorer prognosis than those with elevated CA 15-3 alone.

CellSearch^®

CellSearch is a blood test that measures circulating tumor cells (CTCs). CTCs are rarely present in healthy individuals and patients with nonmalignant diseases but can be detected in patients with metastatic cancer. In patients with metastatic breast cancer, prospective clinical studies have found that the number of CTCs predicts progression-free survival (PFS) and OS before treatment and at first follow-up after initiation of new therapy (Table 6).^13,14

DNA Cell Cycle Analysis and Ki-67

The proliferative activity of breast carcinomas also has prognostic significance. Two approaches to assessing cell proliferation are DNA cell cycle analysis, which measures the number of cells actively synthesizing DNA (S-phase fraction [SPF]), and IHC measurement of Ki-67. Ki-67 is a nuclear non-histone protein that is present at low levels in quiescent cells but is increased in proliferating cells, especially in the G2, M, and latter half of the S phase. The Ki-67 assay has the advantage of requiring less tumor tissue for analysis.

DNA cell cycle analysis results are classified as low (<6%), intermediate (6%–10%), and high (>10%). High SPF is associated with worse DFS and OS: multivariate analysis on >28,000 patients with breast cancer found a relative risk of death of 1.29 (95% confidence interval [CI] 1.22–1.36, P <0.001) using an 11.0% cutpoint.¹⁵ In addition, the DNA cell cycle analysis includes ploidy results; aneuploidy (an abnormal complement of chromosomes) is a poor prognostic indicator.¹⁵

Data from a review of 18 studies suggest the prognostic value of Ki-67 is more useful in node-negative than in node-positive breast cancer.¹⁶ Nevertheless, supportive data are available for both node-negative and node-positive disease.¹⁷ IHC staining of <10% of tumor cells is associated with low proliferative activity, lower probability of recurrence, and longer survival and is interpreted as negative.

Micrometastasis Detection

Micrometastases are defined as metastatic lesions between 0.2 mm and 2.0 mm in diameter.¹⁸ One method of detecting such lesions is with IHC using monoclonal antibodies specific for low and high molecular weight cytokeratins that are expressed by epithelial cells. IHC detection of cytokeratins in bone marrow or lymph nodes is suggestive of an epithelial-derived metastatic tumor (eg, breast cancer) since cytokeratins are not normally expressed in these tissues.

Although axillary lymph node status is one of the most valuable prognostic indicators, the clinical relevance of lymph node micrometastases is marginal. In an analysis of data from 1724 patients with micrometastases, 12-year survival was reduced by 5.0% with solitary and 3.6% with multiple lymph node involvement when compared with node-negative patients.¹⁸ Furthermore, when comparing the clinical utility of micrometastases in lymph nodes vs bone marrow, lymph node micrometastases had no prognostic value.¹⁹ In the same study, bone marrow micrometastases conferred a moderate survival disadvantage.¹⁹

P53 Gene Mutation

The tumor suppressor gene P53 plays a key role in cell cycle control and apoptosis. In patients with breast cancer, mutation in tumor P53 was associated with increased disease-specific death independent of tumor size, node status, and hormone receptor status; the relative risk of death at 10 years was 2.27 relative to patients without a P53 mutation (n = 1,794, P <0.001).²⁰ Based on findings that plasma is enriched with tumor-specific nucleic acids in patients with breast cancer,^21,22 Quest Diagnostics offers a plasma-based test for P53 mutations. However, the clinical utility of this assay is not yet established. Overexpression of p53 protein detected by IHC is a surrogate marker of P53 mutation and is associated with a poor prognosis.

Selecting Therapy: Early-Stage Breast Cancer

The management of early-stage invasive breast cancer includes localized treatment of the tumor with surgery, radiation therapy, or both and treatment with endocrine therapy, chemotherapy, targeted biologic therapy, or combinations of these. In early-stage breast cancer, determining which course of action would be the most successful begins with the classification of risk.

At the 2007 St Gallen consensus conference of experts on early-stage breast cancer, nodal status remained the most important prognostic factor for defining risk (Table 7).²³ Interestingly, the intermediate-risk category includes both node-negative and node-positive disease.

The predicted endocrine responsiveness of the resected tumor combined with the risk category (Table 7) and tumor HER-2/neu status serves to direct adjuvant systemic therapy for early breast cancer (Table 8).²³ Positive ER and PR in the resected tumor predicts response to endocrine therapies. If negative for these receptors, the tumor is classified as nonresponsive to endocrine therapy and chemotherapy is the treatment of choice. When endocrine responsiveness is uncertain, the St Gallen conference recommendation is for endocrine therapy or sequential chemotherapy and endocrine therapy, depending on the risk. See reference 4 for specific treatment regimens for pre- and postmenopausal women.

HER-2/neu, Tumor Tissue

Targeted treatment with trastuzumab (Herceptin^®), a humanized monoclonal antibody directed against the extracellular domain of HER-2/neu protein, is included in the St Gallen recommendations for treatment (Table 8). Results from 4 large clinical trials of early-stage, HER-2/neu+ breast cancer found that trastuzumab, when combined with various chemotherapies, improved DFS (HR ranged from 0.48 to 0.61, P ≤0.001).²⁴ As follow-up times were short, only 1 of these studies has reported overall survival data: following chemotherapy, patients treated with trastuzumab showed a survival benefit at 2-years relative to those with no additional treatment (HR = 0.66, 95% CI 0.47–0.91; P = 0.012).²⁵ Thus, trastuzumab treatment of patients with early-stage HER-2/neu+ breast cancer results in an improvement in DFS and OS.

The most important use of tumor HER-2/neu testing is to determine eligibility for treatment with trastuzumab. This targeted therapy is only effective with tumors positive for HER-2/neu; furthermore, trastuzumab is associated with risk of congestive heart failure and should not be given to patients who would not benefit from this therapy.²⁴ Eligibility for the clinical trials described in the previous paragraph required HER-2/neu–positive tumors; these were defined as 3+ IHC, 2+ IHC if FISH-positive, or a FISH ratio of ≥2.0.²⁴ The ASCO/CAP guidelines changed the definition of FISH-positive HER-2/neu to >2.2 with 1.8 to 2.2 reported as equivocal.⁹ Thus, trastuzumab treatment decisions should consider this change.

HER-2/neu testing can also aid in selection of adjuvant chemotherapy. HER-2/neu+ tumors are more likely to respond to anthracycline-based treatments (eg, CAF, [cyclophosphamide, doxorubicin, 5-fluorouracil]) than alkylating-based therapy (eg, CMF, [cyclophosphamide, methotrexate, 5-fluorouracil]).²⁶ Furthermore, patients treated with CAF who are node positive and HER-2/neu+, may benefit from higher doses of doxorubicin.²⁷ Although HER-2/neu expression may help predict which patients are most likely to benefit from anthracycline-based treatment, levels of HER-2/neu should not be used to exclude patients from this type of therapy.²⁸

Selecting Therapy: Local Recurrence and Metastatic Breast Cancer

Selection of therapy for local recurrence or stage IV metastatic breast cancer includes a detailed history and determination of ER, PR, and tumor HER-2/neu, if not previously performed.⁴ Treatment options for patients with local recurrence include surgery, radiation therapy, and/or systemic chemotherapy or endocrine therapy; systemic and sometimes radiation therapy is suggested for patients with metastatic breast cancer.⁴ Candidates for first- or second-line endocrine therapy for either recurrent or metastatic disease include those with ER+ and/or PR+ tumors, those with asymptomatic visceral involvement, and those with only bone or soft tissue involvement.⁴ Chemotherapy should be considered for patients with ER–/PR– tumors  or symptomatic visceral metastasis or those who are refractory to endocrine therapy.⁴

Trastuzumab may be used in combination with chemotherapy for patients with HER-2/neu+ tumors. When trastuzumab was included with docetaxel, overall response rate and median OS were improved vs docetaxel alone.²⁹ The response rate was 61% vs 34% (P <0.001) and median OS was 31.2 months vs 22.7 months (P = 0.033) with and without trastuzumab. These results were consistent with earlier results comparing other first-line chemotherapies alone and with trastuzumab added.³⁰

HER-2, Serum
Preliminary studies used pretreatment serum HER-2 levels to predict response to trastuzumab-based therapy in patients with metastatic breast cancer. In patients with HER-2/neu-positive tumors, elevated HER-2 levels (≥15 ng/mL) were associated with increased response rates (76% vs 33% with HER-2 <15 ng/mL) when patients were treated with trastuzumab and docetaxel.³¹

As mentioned above, endocrine therapy is frequently used for treatment of metastatic breast cancer in patients with ER+ or PR+ tumors. However, patients with elevated pretreatment serum HER-2 levels are relatively resistant to both first- and second-line endocrine therapy (Table 9).

Serum HER-2 levels can in some cases also be used to select the type of endocrine therapy most likely to be efficacious. For example, Lipton et al. reported no advantage of letrozole, an aromatase inhibitor, over tamoxifen in patients with metastatic breast cancer (n = 907) when HER-2 levels were ≥15 ng/mL.³² However, in patients with lower HER-2 levels, letrozole was more effective than tamoxifen (median TTP 12.2 vs 8.5 months, P = 0.002).

Patient Monitoring

CA 15-3, CA 27.29, and Carcinoembryonic Assay (CEA)

Although not tumor-specific, tests for CA 15-3, CA 27.29, and CEA have been used to monitor treatment response and detect recurrence in patients with breast cancer. While data show these markers correlate with treatment response and detect recurrence, the data do not meet ASCO criteria of clinical benefit (eg, improved DFS, OS, and quality of life; decreased toxicity; and cost-effectiveness); therefore, ASCO does not recommend these tumor markers for routine use.²⁸ However, these markers are recommended for monitoring patients with advanced disease in conjunction with imaging and clinical follow-up. Levels are especially useful in the absence of measureable disease.²⁸

CA 15-3 and CA 27.29 assays both measure the MUC-1 mucin glycoprotein, which is a breast cancer -associated antigen. Accordingly, only 1 of these markers should be used when monitoring a particular patient since results are not interchangeable.³⁴

For patients receiving hormone adjuvant therapy, the European Group on Tumor Markers [EGTM] recommends tumor marker testing before every chemotherapy administration and at 3-month intervals.³⁷ A rise in tumor marker concentration is considered clinically significant when 2 criteria are met: 1) ≥25% increase over the previous value and 2) the follow-up value is above the reference interval.³⁴ When the increase is confirmed with another specimen collected within 1 month, results are consistent with disease progression. Similarly, confirmed decreases of ≥50% are consistent with treatment response.³⁴

For early detection of breast cancer recurrence, serial CA 15-3 (or CA 27.29) and CEA measurement are recommended by the EGTM if detection would alter clinical management.³⁴ The recommended testing frequency is every 2 to 4 months for the first 5 years after diagnosis, then every 6 months for the next 3 years, and annually thereafter. Elevated CA 15-3 levels are seen in 50% to 70% of patients with distant metastases, whereas 40% to 50% will have elevated CEA levels.³⁴ The simultaneous use of both CA 15-3 and CEA allows detection of metastases (mainly in bone and liver) in 60% to 80% of patients with breast cancer, but these markers are of limited use in the early detection of locoregional recurrence.³⁴

HER-2, Serum

The use of serum HER-2 to monitor response to trastuzumab-based therapies is in its infancy. Preliminary data suggest that decreasing levels indicate a favorable response even as early as 15 days after initiating treatment (Table 10), whereas increasing levels reflect disease progression.^35,36

CellSearch

In addition to being FDA cleared for predicting survival, CellSearch is also cleared for use as an aid in monitoring patients with metastatic breast cancer. Detection of an elevated CTC count (≥5/7.5 mL blood) at any time during therapy was found to be an accurate indicator of rapid disease progression and shorter survival.³⁷ Furthermore, assessment of CTC count may provide an earlier indication of treatment response and be more reproducible and less subjective than imaging, which is traditionally used to measure effectiveness of therapy.³⁸