Breast-Cancer Subtyping in Clinical Practice: Clinicopathologic Features and Outcomes

Abstract

Background: The main molecular markers associated with breast cancer that classify patients into subtypes are estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2/neu) proteins. Based on ER, PR and HER2/neu analysis, tumors can be divided into the categories of basal-like, luminal, or HER2/neu positive. Objectives: The aim of this survey was to elucidate the efficacy of the prognostic significance of this classification scheme and to compare the clinicopathologic features and survival in the abovementioned subtypes defined by immunohistochemistry (IHC) in association with a variety of clinical and surgical parameters. Materials and Methods: For this prospective study, 163 women, ages 28–87, were registered consecutively. In each case in the database ER, PR, and HER2/neu levels were obtained through standard clinical testing, using IHC for ER and PR and the HercepTest for HER2/neu. Results: The statistical analysis of this sample showed that patients who were characterized as ER(-) and PR(-) had the worst survival, compared to HR(+) women (p<0.001). In addition, there seemed to be strong relationship between a positive HER2/neu immunohistochemical profile and long-term survival (p<0.001). Furthermore, a higher proportion of triple-negative patients experienced distant recurrence and their mean time to disease progression was significantly shorter (p<0.001). Thus triple-negative classification was an independent predictor of disease aggressiveness or metastatic potential. Conclusions: Patients with triple-negative breast cancer have an increased likelihood of distant recurrence and death, compared to women with other types of breast cancer. Furthermore, the patterns of relapse in the two subgroups are completely different. (J GYNECOL SURG 30:260)

Introduction

Breast cancer is the most common cancer and is the second leading cause of cancer death in women.^1,2 This type of cancer is considered to be a multifaceted disease and is characterized by a wide spectrum of clinical, pathologic, and molecular characteristics.³ The heterogenous nature of these tumors present with variations in responses to therapy and outcomes.⁴

Routine clinical variables have recently been complemented by molecular profiling in an attempt to offer a therapeutic approach with increased efficacy and low toxicity.⁵ The main molecular markers associated with breast cancer that classify patients into subtypes are estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2/neu) proteins. Immunohistochemistry (IHC) provides both therapeutic and prognostic clarification.⁶ Based on ER, PR and HER2/neu analysis, tumors can be divided into the categories of basal-like, luminal (ER/PR receptor–positive that generally have a favorable prognosis) or HER2/neu positive.^7,8 Basal-like carcinomas are negative for both hormone receptors (HR) and HER2/neu expression and are specified as triple-negative breast carcinomas.⁹ They usually are overrepresented in African American women and in BRCA1 mutation carriers. Women with triple-negative breast cancer have poor prognoses and an increased likelihood of distant recurrences and death within 5 years of diagnosis. Although clinicians routinely use conventional ER, PR, and HER2/neu status to characterize patients as triple-negative for clinical decision making, there are limited databases evaluating locoregional control in conservatively managed women.¹⁰

The aim of this survey was to elucidate the efficacy of the prognostic significance of this classification scheme and to compare the clinicopathologic features and survival in the abovementioned subtypes defined by IHC in association with a variety of clinical and surgical parameters.

Materials and Methods

For this prospective study, 163 women, ages 28–87, were registered consecutively. These patients were women who attended the Breast Clinic of Attikon University Hospital of Athens from January 1, 2005 to December 31, 2008. This investigation was approved by an institutional review board, but, because written consent was obtained in each case, no ethical committee approval was necessary.

A database was established to record data systematically on clinical presentations, treatments, and outcomes. Data entry into the central database was double-checked by data-verifiers and managers. Patient information was recorded at accrual and included details about age at diagnosis, tumor grade, lymph-node status, and treatment (surgery, chemotherapy or radiation). In each case in the database ER, PR, and HER2/neu data were obtained through standard clinical testing, using IHC for ER and PR and the HercepTest for HER2/neu.¹¹ Lesions were considered ER- or PR-positive when 10%–100% of all related tumor-cell nuclear staining was documented. HER2/neu status was routinely determined for patient treatment during the course of this study. Scores of 0 and 1 were considered to be negative, while a score of 3 was registered as positive.¹² Patients characterized as HER2/neu 2 were also tested by fluorescent in situ hybridization (FISH) for further clarification.

Ki-67 is a nuclear antigen expressed in G1, S, and G2 phases but not in the G0 or resting phase of cell cycle. Ki-67 has become established as a proliferation marker in breast cancer. Normal p53 expression was recorded when 1%–50% of the tumor cells were noted to be positive when they were stained. Abnormal expression was detected in the absence of neoplastic cells, or in case of positive staining in 51%–100% of the tumor cells.^13,14 The surgical management of these patients included either conservative interventions, such as lumpectomy, or more-extensive procedures, such as mastectomy combined with axillary lymphadenectomy. In the majority of the cases, a combination of chemotherapy and radiotherapy was administered postoperatively, while the rest of the women received preoperative chemotherapy in an attempt to reduce tumor size and produce subsequent downstaging. Recommendations for using adjuvant therapy are based on an individual patient's risk and the balance between absolute benefit and associated toxicity. Anthracycline-based regimens with the addition of taxanes were preferred. Follow-up was maintained by the database coordinator by reviewing clinical charts and also by contacting patients.

Statistical analysis of the above parameters was performed using an SPSS statistical analysis program (Version 17.0). This was a multivariate Cox regression analysis, taking into account margin status, nodal status, T-stage, type of surgical management, use of adjuvant therapy, and the triple-negative breast cancer subtype as an independent predictor factor of survival. A p-value more than 0.05 was reported as being not statistically significant (NS).

Results

During the study period 37/161 women (23%) were enrolled as triple-negative breast cancer participants. The average age was 63.68 years (range: 45–84) for the triple-negative group and 64.71 years for the rest of the patients. Therefore, statistically significant differences with regard to the ages of the patients and the immunohistochemical profiles of their tumors were not documented. All of the survey participants had been followed actively for at least 5 years. A higher proportion of patients with strong family histories—defined as at least one first-degree relative with breast cancer—among the triple-negative group was noted. Furthermore, 9 patients who presented with metastatic breast cancer during the study period were not operated because they had documented advanced disease. Triple-negative breast cancer cases with concomitant outcomes and potential recurrences are shown in Table 1. Failed cases were also detected.

Table 1.

Triple Negative or Breast-Cancer Cases with Potential Aggressiveness or Recurrence

PT #	Age	Operation	T	N	M	Stage	Grade	ER%	PR%	CerbB-2	Ki-67%	p53%	Recurrence
1	61	TM	1	0	0	I	3	0	5	−	30	10	0
2	78	BCS+ALND	2	0	0	IIA	3	0	0	+	8	0	0
3	62	BCS+ALND	2	2	1	IV	3	0	0	+++	50	0	1
4	82	MRM	2	2	1	IV	3	0	0	+++	2	0	1
5	71	TM	4	N/A	1	IV	2	0	5	+	1	0	1
6	51	BCS+ALND	1	2	0	IIIA	3	0	0	+	2	0	0
7	77	MRM	2	0	0	IIA	3	0	0	+	2	0	0
8	72	TM	2	1	0	IIB	3	0	0	−	0	90	0
9	58	MRM	1	1	0	IIA	3	0	0	+++	40	70	0
10	74	TM	4	N/A	0	N/A	3	0	0	+	30	90	1
11	52	MRM	2	1	0	IIB	3	0	0	+	70	0	0
12	84	MRM	2	1	0	IIB	3	0	0	−	40	60	0
13	55	MRM	4	1	0	IIIB	3	0	0	+++	30	40	1
14	48	MRM	1	0	0	I	3	0	5	−	80	0	0
15	71	MRM	2	0	0	IIA	2	0	0	+	30	0	0
16	64	BCS+ALND	2	0	0	IIA	3	1	0	−	5	80	0
17	77	MRM	2	1	0	IIB	2	1	5	+	1	0	0
18	56	MRM	2	0	0	IIA	3	0	0	+	20	0	0
19	66	MRM	2	0	0	IIA	3	0	0	−	30	90	0
20	55	MRM	3	2	0	IIIA	3	2	10	+	8	0	1
21	66	BCS+ALND	2	1	0	IIB	3	0	0	+	20	0	0
22	62	MRM	3	2	0	IIIA	3	0	0	+	0	0	1
23	47	BCS+ALND	2	0	0	IIA	3	0	0	+	30	0	0
24	68	BCS+ALND	2	1	0	IIB	3	0	0	+	30	50	0
25	45	MRM	2	0	0	IIA	3	0	0	+	45	0	0
26	78	MRM	2	0	0	IIA	3	0	0	+	75	80	0
27	63	MRM	1	0	0	I	3	0	0	−	10	95	0
28	76	MRM	2	1	0	IIB	3	0	0	++	>50	>50	0
29	56	BCS+ALND	3	1	0	IIIA	3	0	0	+	20	0	0
30	59	BCS+ALND	1	0	0	I	3	0	0	−	10	0	0
31	67	MRM	2	1	0	IIB	3	0	0	+	85	90	0
32	59	BCS+ALND	1	1	0	IIA	3	0	0	−	80	70	0
33	68	MRM	1	0	0	I	3	0	5	+++	70	0	0
34	62	MRM	2	0	0	IIA	3	0	0	++	70	60	0
35	67	MRM	2	0	0	IIA	3	0	0	+	40	80	0
36	48	MRM	2	2	0	IIIA	3	0	0	+	90	0	1
37	51	BCS+ALND	1	0	0	I	3	0	0	+++	80	0	0

PT, patient; T, tumor; N, number of lymph nodes; M, metastasis; ER, estrogen receptor; PR, progesterone receptor; TM, total mastectomy; BCS+ALND, breast-conserving surgery+axillary lymph node dissection; MRM, modified radical mastectomy; N/A, not applicable.

The statistical analysis of the study sample showed that patients who were characterized as ER(-) and PR(-) had worst survival, compared to HR(+) women (p<0.001). In addition, there seemed to be strong relationship between a positive HER2/neu immunohistochemical profile and long-term survival (p<0.001). Furthermore, a higher proportion of triple-negative patients experienced distant recurrence, and their mean time to disease progression was significantly shorter (p<0.001). Thus triple-negative classification was an independent predictor of disease aggressiveness or metastatic potential. Nevertheless, triple-negative patients had similar rates of local recurrence to that of the rest of study group. Neoadjuvant chemotherapy (NACT) was administered in 1 case in the triple-negative breast cancer group.

Cox regression analysis showed that triple-negative women had the worst survival rates and an increased likelihood of breast cancer–related death within 5 years of diagnosis after adjustment for age, tumor size, margin status, grade, nodal infiltration, and therapeutic approach (p<0.001). Moreover, the majority of triple-negative women had a significant probability of early distant recurrence and an associated lower relative risk of disease relapse thereafter. Among the other participants, there seemed to be a steady risk of neoplastic progression throughout the entire follow-up period. Although pathologic nodal status was evenly distributed among the three breast-cancer subtypes, the triple-negative category was associated with a higher pathologic T stage. Additional significant differences among breast-cancer subtypes with regard to proliferation activity, p53 expression, bcl-2 status, or Ki67 profile was not reported.

When other parameters of the investigation were examined, no statistically significant difference emerged between triple-negative women and the rest of the study population, depending on the type of surgery (mastectomy, lumpectomy) and the rate of node positivity (p=0.968). In addition, only 4/37 (10.9%) triple-negative women had a local lesion prior to a distal recurrence and were treated with breast-conserving surgical procedures. Nevertheless, in the proportional hazard model, loco–regional disease progression was an independent predictor of later distal metastasis in the other breast-cancer categories. A relevant association was not documented in the triple-negative group.

Discussion

This study examined the short-term and long-term outcomes of patients with triple-negative breast cancer within the context of specific well-documented prognostic factors. It is common knowledge that patients with triple-negative breast cancer have an increased likelihood of distant recurrences and death, compared to women with other types of cancer.¹⁵ Furthermore, the pattern of relapse in the two subgroups is completely different. Therefore, these patients have high rates of recurrence only in the period from 1–4 years after diagnosis. Relative risk declines rapidly thereafter.¹⁶ In the current study, it also appears that patients with triple-negative breast cancer have the worst survival rate within a 5-year follow-up period. Moreover, a higher proportion of triple-negative patients had distant recurrence, and their mean time to disease progression was significantly shorter. Thus triple-negative classification was an independent predictor of disease aggressiveness or metastatic activity.¹⁷

Classification of breast cancer into basal-type (triple-negative), luminal (ER/PR-positive), and Her2/neu has recently been proposed as a significant parameter with regard to gene-expression profiles, prognostic value, and response to therapy.¹⁸ Worse prognosis and survival is also implicated in the first category. According to recent surveys, these patients appeared with relatively locally extended disease and higher rate of node positivity. As tumor size increases, the proportion of affected lymph nodes is augmented.

Nevertheless, such an association was not reported in the current retrospective analysis.^19,20 Pathologic nodal status was commonly distributed among the three breast-cancer subtypes, whereas the triple-negative category was related to a higher pathologic T stage. Moreover, triple-negative patients had similar rates of local recurrence with the rest of the study participants, despite recent observations of a more-aggressive loco–regional relapse profile.^21,22 Finally, triple-negative patients were more likely to be detected through clinical examination than by imaging with mammography and ultrasound. This situation may indicate that tumors may grow rapidly in relation to the screening interval or may be related to differences in the density of breast tissue in this subgroup of patients.²³

Separating patients into groups depending on their predicted tumor responses may contribute to several clinical advantages in their overall management. Therefore, a variety of proven and putative markers, including oncogenes and growth factors, have been evaluated in previous studies.²⁴ Despite various immunohistochemical parameters being investigated to identify basal-like differentiation, universally agreed criteria or consensus are absent. Ki-67 has become established as proliferation evidence in breast-cancer samples and as a possible predictive factor for a potential response to preoperative chemotherapy. Additional significant differences among breast-cancer subtypes with regard to proliferation activity, Ki67 profile, p53 expression, or bcl-2 status was not reported in the current study patients. Recent surveys support that confirmation of high molecular weight cytokeratins or epidermal growth factor receptor (EGFR) could suggest the presence of a basal-like breast-cancer type.^25,26 Nevertheless, until the elucidation of these criteria, staining for ER, PR and HER2/neu could be used to help classify the majority of basal-like breast cancers correctly.²⁷

One potential weakness of the current study is the absence of a more-detailed molecular profiling and a hierarchical cluster analysis. Nevertheless, HER2/neu data were obtained both through immunohistochemical testing and FISH, ensuring the data's accuracy. In each case, all three markers were available to validate the prognostic utility of the above proposed classification scheme.

The treatment of these patients includes either surgical procedures or neoadjuvant chemotherapy followed by surgical therapeutic interventions.²⁸ Surgical management includes either lumpectomy or mastectomy combined with axillary node biopsy. Significant differences in the prognosis of the patients based on treatment were not documented in the current study.

A multivariant regression analysis showed that triple-negative breast-cancer patients, even among a group of relatively early stage cancers treated with breast-conserving therapy and radiation, share a poor prognosis.²⁹ It is reassuring that these patients do not seem to have a higher rate of local relapse and, therefore, should be considered potential candidates for restricted surgical procedures.³⁰ In the current retrospective survey, rates of local recurrence were nearly identical in the triple-negative cohort and other study participants. In addition, statistically significant differences with respect to patients' ages and immunohistochemical profiles of the tumors were not detected. This is of particular importance, given the fact that triple-negative patients have been noted to be younger and have tumors that are relatively sensitive to radiation therapy.^31,32 It is also notable that, despite the use of adjuvant systematic chemotherapy, triple-negative patients to have unfavorable prognoses.³³ A reduced, distant metastasis-free survival rate was also identified in the current study.

Conclusions

As alternative chemotherapy programs have been considered for triple-negative patients, further analyses evaluating the effects of adjuvant systemic therapy are warranted. By definition of a lack of receptors for ER, PR and Her2/neu, patients with triple-negative tumors are excluded from adjuvant hormonal therapy or trastuzumamb.³⁴ Extensive subtyping through molecular profiling may help identify patients with a potential to respond to combinations of specific regimens. Whether or not, alternative types of chemotherapy associated with a variety of biologic modifiers or targeted antibodies will prove to be superior can only be clarified by accurately designed prospective studies.

Footnotes

Disclosure Statement

No financial conflicts of interest exist.

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