Potential Clinically Useful Prognostic Biomarkers in Triple-negative Breast Cancer: Preliminary Results of a Retrospective Analysis
Breast Cancer: Targets and Therapy
Background: Triple-negative breast cancer (TNBC) has a poor prognosis, even in its early stages. In the absence of postoperative targeted treatments, intensive adjuvant chemotherapy regimens are proposed. For those favorable histologies, such as apocrine and adenoid cystic carcinoma, which frequently belong to TNBC, aggressive treatments are unnecessary.
Patients and methods: We retrospectively analyzed 631 cases of breast cancer, primary operated curatively, and followed up at our institution for at least 36 months to identify the biomarkers assessable by immunohistochemistry, to be proposed as prognostic score for tailoring adjuvant treatment to TNBC patients.
Results: The triple-negative phenotype was found in 85 patients (13.5%). Over a mean follow-up of 55.7 months, relapses occurred in 106 patients (16.8%), of which 18 (2.8%) were TNBC. Recurrence was directly correlated with Ki67 and cytokeratin 5/6 (CK5/6) immunoreactivity in all breast cancer patients (P=0.005), but only marginally with CK5/6 and epithelial cadherin (E-cad) expression in TNBC patients (P=0.07). Mean event-free survival (EFS) in TNBC patients was 85.52 months compared with 100.4 months in non-TNBC patients (P=0.228). The EFS of CK5/6-negative triple-negative patients was 68.84 months compared with 98.84 months in those who were CK5/6 positive (HR =5.08; P=0.038). EFS differed among patients identified as double-positive for E-cad and CK5/6 (83.87 months), those expressing E-cad or CK5/6 (64.23 months), and those negative for both biomarkers (39.64 months).
Conclusion: These preliminary results suggest that CK5/6 and E-cad are possible core biomarkers for a cost-effective prognostic evaluation of primary operable TNBC patients.
Keywords: prognosis, immunohistochemical biomarkers, triple-negative breast cancer, CK5/6, E-cadherin
Breast cancer, the leading cause of cancer-related death in women, comprises a heterogeneous group of diseases that vary regarding morphology, biology, and prognosis. The different subtypes of breast cancer are based on the expression of certain receptors on the surfaces of tumor cells.1,2
One of the first classification systems reported in 2001 was based on microarray mRNA quantitative measurement. Five molecularly distinct intrinsic subtypes were identified according to genes' clusters from two main types of cells in human mammary glands (luminal and basal or myoepithelial) and human epidermal growth factor receptor 2 (HER2) and hormone receptor genes expression. However, microarray-based gene expression profiling technologies remain impractical for the routine analysis of tumor specimens due to the high cost and complexity associated with sample preparation, assays, and data analysis.3–5
Closely following the early invasive breast cancer classification system of Voduc et al, which is based on the immunohistochemical (IHC) semiquantitative analysis of the estrogen receptor (ER), progesterone receptor (PR), Ki67 (nuclear antigen, marker of proliferation), and HER2 expressions, a four-surrogate breast cancer classification system similar to that of the intrinsic subtypes was adopted and recommended by international guidelines.6,7 Triple-negative breast cancer (TNBC) represents the phenotype that expresses very low levels of ER, PR, and HER2 and accounts for 10%–20% of all invasive breast cancers, representing over 170,000 of the one million cases diagnosed annually.8 TNBC is the IHC surrogate for the microarray-based profiling of basal-like breast cancer. According to recommendations by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP), TNBC is defined by <1% of invasive cells staining for ER and PR expression, by either a 0–1+ or 2+ expression score as determined by IHC, and by testing negative in situ hybridization (ISH) for ERBB2 (gene encoding HER2 not amplified).9,10
TNBC is associated with young age, high proliferative index, poor prognosis, premature relapse (2–3 years), high rate of visceral metastasis (primarily lung, liver, and brain), and short median time to death (9 months).11–14 Even in very early stages, such as T1N0–1, the 5-year relapse-free survival (RFS) rate is poor (85%–89%).15 The presumption of an aggressive clinical course and the absence of targeted adjuvant treatments for TNBC have resulted in the overuse of intensified postoperative chemotherapy; for example, the European and North American guidelines recommend starting adjuvant chemotherapy at a 0.6-cm pathological diameter and preferably using dose-dense regimens.16–19 Nevertheless, in one retrospective study, two-thirds of early-stage TNBC patients (N=117) treated conservatively by surgery remained disease-free after 5 years without any adjuvant chemotherapy.20 A proportion of favorable histologies, such as medullary or adenoid cystic carcinoma, frequently belongs to the triple-negative phenotype, for which chemotherapy is not necessary.21 To identify aggressive or nonaggressive subtypes, the gene-profiling microarray technique would be desirable; yet, pecuniary and feasibility reasons mean that an assessment by IHC methods is more convenient for everyday practice.
Gene expression analysis has revealed that TNBC comprises several subtypes displaying different targets and outcomes, such as a mesenchymal-like cell subtype with phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit alpha mutations in almost 50% of cases, phosphatase and tensin homolog deficiencies, or a luminal androgen receptor (LAR) subtype with overactivated PI3K/mammalian target of rapamycin pathway signaling. RFS was significantly lower in the LAR subtype than in the basal-like (HR =2.9), and when compared with the mesenchymal stem-like subtype (HR =10.5; P<0.05).22 However, unlike other breast cancer subtypes, the classical prognostic signatures based on gene modules known to regulate or execute cell proliferation did not show any value in highly proliferative hormone-negative breast cancers.23
Staining by IHC techniques is the surrogate for the assessment of many aberrant proteins that result from gene mutations or deletions, although the specificity compared with that of a gene assay is ~80%.24 Most analyses that attempt to characterize this phenotype use formalin-fixed paraffin-embedded tissue specimens.25
Lately, a plethora of biomarkers has been associated with the basal-like or triple-negative phenotype. However, only some of them have been validated in independent series, whereas very few have been compared with gene expression and no study has compared a large number of these candidate biomarkers in parallel.26 The prognostic roles of several biomarkers in TNBC have been studied, including assessments of correlated biomarker clusters, with a view to create a prognostic score.27
Considering everyday clinical practice needs, we analyzed several biomarkers assessable by IHC according to literature findings and institutional laboratory assessments to provide a prognostic stratification for patients diagnosed with upfront operable localized TNBC that could be used as a means of adapting adjuvant treatment.