Clinical Outcome of Triple Negative Breast Cancer in BRCA1 Mutation Carriers and Non-carriers

Abstract

BACKGROUND

Women with BRCA1 mutations develop breast cancer with similar pathologic features to sporadic triple negative (TN) breast cancer, a subtype associated with early disease relapse and poor outcome. We compared the clinical outcome of women with and without BRCA1 mutations who had TN breast cancer treated with conventional chemotherapy.

METHODS

Women with stage I-III TN breast cancer who had BRCA1 testing within 36 months of diagnosis and received alkylating chemotherapy were identified from clinical databases and a SPORE specimen bank. BRCA2 mutation carriers were excluded, resulting in a study cohort of 46 BRCA1 carriers and 71 non-carriers. Sites of metastasis, relapse rates and survival were compared among carriers and non-carriers. The median follow-up was 75 months.

RESULTS

BRCA1 carriers were younger at diagnosis (p<0.001) and had smaller tumors (p=0.03) than non-carriers. Freedom from distant metastasis at 5 years was 76% for carriers and 70% for non-carriers (HR 0.79, p=0.5). Sites of distant recurrence did not differ significantly (p=0.15), although BRCA1 carriers had a propensity for brain relapse (58% vs. 24%, p=0.06). Overall survival at 5 years was 82% for carriers and 74% for non-carriers (HR 0.64, p=0.25). Adjusting for age and stage, BRCA1 mutation status was not an independent predictor of survival (HR 0.73, p=0.48).

CONCLUSION

BRCA1 mutation carriers with TN disease have similar survival rates to non-carriers when treated with alkylating chemotherapy. Women with BRCA1-related breast cancer may benefit from novel therapies that target DNA repair, and further study is needed to identify sporadic TN breast cancers with a BRCA-deficient phenotype.

Introduction

Triple negative (TN) breast cancer accounts for 15-20% of all breast cancer subtypes, but a proportionately larger number of breast cancer deaths¹. The TN subtype is characterized by the absence of expression of estrogen receptor (ER), progesterone receptor (PR) and the epidermal growth factor receptor 2 (HER2). TN breast cancers are diagnosed at a younger age and have aggressive biologic behavior with the development of local-regional and distant metastasis within 5 years^{2, 3}. Distant metastases more often involve visceral sites such as the brain and lung, and less likely bone and liver^4-6. Despite systemic therapy, distant-metastasis free survival for patients with localized TN breast cancer is approximately 70% at 5 years, with overall 5-year survival rates ranging from 70-80%^{2, 7}.

The majority of breast cancers that develop in women with germline mutations in the BRCA1 breast cancer susceptibility gene are TN cancers^{8, 9}. BRCA1 has a central function in the repair of DNA double strand breaks, and loss of function mutations in BRCA1 lead to genomic instability and underlie cancer predisposition¹⁰. BRCA1-related TN cancers share several pathologic features with sporadic TN cancers, including low or absent expression of hormone receptors and HER2, high histologic and nuclear grade, and high mitotic index^{8, 11, 12}. By gene expression profiling, BRCA1-related breast cancers, as well as the majority of sporadic TN breast cancers, cluster within the basal subgroup¹³ and express basal cytokeratins and EGFR¹⁴. Recent studies have also shown that a proportion of sporadic basal-like breast cancers may have a dysfunctional BRCA1 pathway due to gene promoter methylation or transcriptional inactivation^{15, 16}.

Despite the significant overlap between the clinical and biologic features of sporadic and BRCA1-related TN breast cancers, the prognostic significance of a deleterious BRCA1 mutation in women with TN breast cancer is not known. Prognostic differences between mutation carriers and non-carriers could influence the treatment decisions of patients and their physicians regarding the use of adjuvant chemotherapy or preventative surgeries, such as prophylactic mastectomy. The goal of this study was to determine whether BRCA1 mutation status was associated with a difference in survival in women with TN breast cancer treated with adjuvant chemotherapy. We analyzed the rates of local-regional and distant relapse, sites of distant metastasis, breast-cancer specific survival and overall survival in BRCA1 mutation carriers and non-carriers diagnosed with invasive TN breast cancer who were treated with alkylating chemotherapy.

Methods

A review of the clinical databases and annotated SPORE specimen bank at Beth Israel Deaconess Medical Center and Dana-Farber Cancer Institute identified 183 women diagnosed with invasive TN breast cancer between January 1, 1996 and December 31, 2004 for whom BRCA1 testing had been performed. BRCA2 mutation carriers were excluded. ER, PR and HER2 status, assessed as part of the routine clinical evaluation, was abstracted from institutional pathology reports. BRCA1 mutation status had previously been established for 120 of these women who had donated blood for research by high thoughput heteroduplex detection from a banked blood sample as described previously¹⁷. Fourteen of these 120 women carried a BRCA1 mutation. The other 63 BRCA1 mutation carriers identified had genetic testing performed by commercial methods in high risk clinics. Inclusion in the cohort required the diagnosis of a first invasive stage I-III TN breast cancer and use of adjuvant or neoadjuvant chemotherapy. Therefore, 15 women were excluded for the following reasons: diagnosis of a second or third primary breast cancer (n=7), stage IV disease (n=1), and no adjuvant or neoadjuvant chemotherapy given (n=7).

To limit the potential survival bias of delayed genetic testing, 19 mutation carriers and 15 non-carriers were excluded because the time to genetic testing was greater than 36 months from breast cancer diagnosis. An additional 12 women were excluded for unsuccessful genotyping, and 5 women had BRCA1 variants of unknown significance. The final study cohort consisted of 117 women, including 46 BRCA1 mutation carriers and 71 non-carriers. Clinical data were abstracted from the medical record, including review of operative notes, pathology reports, and clinic visits with the approval of the Dana Farber/Harvard Cancer Center Institutional Review Board.

Chemotherapy was delivered in the adjuvant setting in 108 patients (92%), and 9 patients received neoadjuvant treatment. The majority of chemotherapy regimens contained an anthracycline backbone (91%), most commonly doxorubicin and cyclophosphamide (AC), doxorubicin, cyclophosphamide, 5-fluoruracil (CAF) or doxorubicin, cyclophosphamide followed by paclitaxel (AC+T). Cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) was used in 9 patients (8%). Docetaxel and cyclophosphamide (TC) was used in 2 patients (2%). Nine women (8%) received hormonal therapy, including tamoxifen in 7 and leuprolide acetate in 2 for chemoprevention and fertility preservation. Mastectomy was performed in 52 patients (44%), and partial mastectomy in 65 (56%). 82 patients (70%) received adjuvant radiation to the breast (48 patients), breast and nodal areas (15 patients), or chest wall and nodal areas (19 patients).

According to institutional practice, patients were seen in routine follow-up every 3-6 months for 3 years after the completion of therapy, and thereafter every 6 months. Follow-up visits included an interval history and physical examination. Surveillance imaging was not routine and performed at the discretion of the treating physician. The site or sites of metastatic disease were documented following re-staging chest, abdomen, and pelvis CT. Brain MRI was performed if clinical history was concerning for metastatic brain disease. Recurrent disease was categorized as local-regional, distant, or a combination of sites. Local-regional recurrence was defined as recurrent disease in the ipsilateral breast, chest wall or draining lymph nodes (axilla, supraclavicular or internal mammary chain). The first sites of distant metastatic involvement were documented after re-staging studies were performed. The diagnosis of a second cancer and incident brain metastasis were also recorded.

Statistical analysis was performed using the statistical software JMP, v. 7.0.1 (SAS Institute). Clinical, pathologic, and treatment characteristics were compared for BRCA1 carriers and non-carriers using a chi-squared approximation, Fisher’s exact test or Wilcoxon test. Actuarial estimates of local-regional failure, freedom from distant metastasis (FFDM), breast-cancer specific survival (BCSS) and overall survival (OS) were calculated by the Kaplan-Meier method and compared by a logrank test. Local-regional recurrence was defined from the date of diagnosis until the date of recurrence involving the ipsilateral breast, chest wall, or regional lymphatics. Patients who had a prophylactic mastectomy after breast conserving therapy (BCT) were censored from the analysis at the date of surgery. Freedom from distant metastasis was defined from the date of diagnosis until the date of first distant metastasis. Breast-cancer specific survival was determined from the date of diagnosis until death due to breast cancer. Overall survival was calculated from the date of diagnosis until death from any cause determined by the medical record or the social security death index. The following covariates were analyzed to identify predictors of FFDM, BCSS and OS: age, AJCC stage, nodal status, tumor size, presence of lymphovascular invasion (LVI) and BRCA1 mutation status. The univariate Cox proportional hazards model was used to analyze continuous covariates. Multivariate analysis was performed using the Cox proportional hazards model to identify independent predictors of DMFS, BCSS and OS. A type 1 error (α) of less than 0.05 was considered statistically significant. Assuming a hazard ratio for breast cancer death of 0.5 for mutation carriers treated with chemotherapy¹⁸ and a median survival of 50 months, 33 carriers and 50 non-carriers were required to achieve 80% power using a 2-sided test and α=0.05.

Results

The median time to genetic testing was 6.0 months for carriers and 3.1 months for non-carriers (Wilcoxon p=0.14). Three carriers had a blood draw prior to the diagnosis of breast cancer at -17.0, -13.5, and -2.2 months. BRCA1 mutation carriers were significantly younger at diagnosis than non-carriers (median age: 39.3 vs. 51.3 years, p<0.001), as shown in Table 1. The most common histologic type was invasive ductal carcinoma among both carriers (96%) and non-carriers (89%). The median tumor size was smaller for mutation carriers compared to non-carriers (1.8 cm vs. 2.0 cm, p=0.03). Carriers and non-carriers had similar rates of high histologic grade (93% vs. 91%), LVI (47% vs. 44%) and nodal involvement (48% vs. 46%). In addition, the TNM and AJCC stage distributions did not significantly differ between the groups (Table 1). The treatment characteristics for the 117 women with invasive TN breast cancer are summarized in Table 2 by mutation status. Mastectomy was more common in mutation carriers (54%) than non-carriers (38%), p=0.08. The chemotherapy regimen most commonly used was doxorubicin and cyclophosphamide (AC) or AC+paclitaxel in both groups. Adjuvant radiation was delivered to 70% of mutation carriers and 77% of non-carriers.

Table 1.

Clinical and pathologic features of 117 patients with triple negative breast cancer.

	BRCA1 carrier N=46	Non-carrier N=71	p value
Age at diagnosis	39.3 years (range, 28.1-73.4)	51.3 years (range, 28.1-75.6)	<0.001
Histology
IDC	44 (96%)	63 (89%)
ILC	1 (2%)	0	0.15
IC, ductal/lobular	1 (2%)	4 (6%)
IC, NOS	-	4 (6%)
Tumor size	1.8 cm (range, 0.4-6.7)	2.0 cm (range, 0.4-15)	0.03
Tumor grade
1	0	0	0.71
2	3 (7%)	6 (9%)
3	42 (93%)	64 (91%)
Presence of LVI	20 (47%)	31 (44%)	0.82
Proportion with LN+	22 (48%)	32 (46%)	0.89
T classification
T1	33 (73%)	36 (51%)	0.054
T2	7 (16%)	27 (38%)
T3	3 (7%)	4 (6%)
T4	2 (4%)	4 (6%)
N classification
N0	24 (52%)	37 (54%)	0.35
N1	16 (35%)	21 (30%)
N2	6 (13%)	8 (12%)
N3	0	3 (4%)
Stage
1	18 (39%)	24 (34%)	0.57
2A	14 (30%)	22 (31%)
2B	3 (7%)	11 (15%)
3A	8 (17%)	8 (11%)
3B	2 (4%)	3 (4%)
3C	1 (2%)	3 (4%)

Abbreviations: IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; IC, ductal/lobular, invasive carcinoma with ductal and lobular features; IC, NOS, invasive carcinoma, not otherwise specified; LVI, lymphovascular invasion; LN+, positive lymph nodes.

Table 2.

Treatment characteristics by surgery type, use of systemic therapy and adjuvant radiation therapy.

	BRCA1 carrier N=46	Non-carrier N=71	p-value
Surgery
Partial mastectomy	21 (46%)	44 (62%)	0.08
Mastectomy	25 (54%)	27 (38%)
Chemotherapy type
CMF	4 (9%)	5 (7%)
Anthracycline-based (AC, CAF)	18 (39%)	29 (41%)	0.54
Anthracyline+taxane (AC+T)	24 (52%)	35 (49%)
Taxane-based (TC)	0	2 (3%)
Hormonal therapy*	2 (4%)	7 (10%)	0.27
Adjuvant RT	32 (70%)	51 (77%)
BCT	17 (55%)	31 (60%)	0.34
BCT/nodal	5 (16%)	10 (20%)
PMRT	9 (29%)	10 (20%)

^*Tamoxifen and leuprolide acetate for chemoprevention and fertility preservation

Abbreviations: CMF, cyclophosphamide, methotrexate, fluorouracil; AC, doxorubicin and cyclophosphamide; CAF, cyclophosphamide, doxorubicin, fluorouracil; T, paclitaxel; TC, paclitaxel and cyclophosphamide; BCT, breast conserving therapy; PMRT, post-mastectomy radiotherapy.

The median follow-up for survivors was 77 months (range, 10-149 months) among BRCA1 mutation carriers and 75 months (range, 13-114 months) for non-carriers. Of the 21 mutation carriers and 44 non-carriers treated with BCT, 2 (9.5%) carriers and 8 (18.2%) non-carriers developed local-regional recurrence at a median of 22.4 months and 17.4 months, respectively. Following mastectomy, 4 of 25 (16.0%) mutation carriers and 5 of 27 (18.5%) non-carriers had a local-regional recurrence at a median of 22.4 and 12.9 months. The 2- and 5-year estimates of local-regional failure did not differ between mutation carriers and non-carriers (Table 3). Of note, 5 of 21 (23.8%) mutation carriers initially treated with BCT had a prophylactic mastectomy at a median of 48 months after diagnosis (range, 24-99 months).

Table 3.

Actuarial estimates of local-regional failure, sites and incidence of distant metastases, and number of salvage chemotherapy regimens by BRCA1 mutation status.

	BRCA1 N=46	Non-carrier N=71	p value
Local-regional failure
Breast conserving therapy*	N=21	N=44
2-year	4.8%	11.5%	0.38
5-year	10.4%	19.8%
Mastectomy	N=25	N=27
2-year	12.7%	15.1%	0.64
5-year	17.1%	20.8%
Site of first distant recurrence	N=12	N=21
Bone	3 (25%)	7 (33%)	0.15
Lung	6 (50%)	15 (71%)
Liver	3 (25%)	7 (33%)
Brain	3 (25%)	0
Other nodal groups	1 (8%)	6 (29%)
Disseminated cutaneous	0	1 (5%)
Overall incidence of brain metastasis	7/12 (58%)	5/21 (24%)	0.06
Median number of salvage chemotherapy regimens	2 (range, 0-6)	2 (range, 0-6)	0.98

^*5 of 21 (23.8%) mutation carriers initially treated with BCT had a prophylactic mastectomy at a median of 48 months after diagnosis.

Twelve mutation carriers and 21 non-carriers developed distant metastatic disease at a median of 22.4 and 19.5 months after diagnosis, respectively. At 5 years, the freedom from distant metastasis was 75.6% for BRCA1 mutation carriers and 69.9% for non-carriers (HR 0.79, logrank p=0.51) (Figure 1). As shown in Table 3, the most common site of first distant metastatic spread in mutation carriers was lung (50%), followed by bone, liver and brain (25% each). For the 21 non-carriers with metastatic disease, the most common site was lung (71%), followed by liver and bone (33%). None of the non-carriers (0 of 21) developed brain metastasis at the time of first distant relapse, compared to 3 of 12 (25%) BRCA1 mutation carriers. The overall incidence of brain metastasis was 58% (7/12) for carriers and 24% (5/21) for non-carriers (p=0.06). The number of metastatic sites (single, 2 or greater than 2) did not differ between the two groups (data not shown). The median number of salvage chemotherapy regimens was 2 (range, 0-6) for both carriers and non-carriers (p=0.98).

Figure 1.

Freedom from distant metastasis (FFDM) by BRCA1 mutation status.

The cause of death was breast cancer for all patients who died during the study period (9 carriers, 19 non-carriers). Breast cancer-specific survival and overall survival at 5 years was 82.1% for BRCA1 mutation carriers and 73.9% for non-carriers (HR 0.64, logrank p=0.25) (Figure 2). On univariate analysis for survival, AJCC stage, nodal involvement, tumor size, and the presence of LVI were significant predictors of FFDM and BCSS (Table 4). On multivariate analysis when adjusted for age and stage, BRCA1 mutation was not an independent predictor of FFDM (HR 0.90, p=0.79) or BCSS (HR 0.73, p=0.48).

Figure 2.

Breast cancer-specific survival (BCSS) by BRCA1 mutation status.

Table 4.

Univariate analysis for freedom for distant metastais (FFDM) and breast cancer-specific survival (BCSS).

	FFDM		BCSS
	Unadjusted HR (95% CI)	p value	Unadjusted HR (95% CI)	p value
BRCA1 mutation carrier	0.79 (0.38-1.58)	0.51	0.58 (0.25-1.25)	0.17
Age	1.01 (0.98-1.04)	0.48	1.01 (0.98-1.04)	0.46
AJCC stage	3.9 (2.4-6.6)	<0.0001	3.7 (2.2-6.6)	<0.0001
Lymph node positive	6.2 (2.7-16.7)	<0.0001	6.3 (2.6-18.7)	<0.0001
Tumor size (per cm)	1.3 (1.1-1.4)	<0.0001	1.3 (1.1-1.4)	0.0002
Presence of LVI	3.4 (1.6-7.0)	0.001	3.1 (1.4-7.7)	0.006

Discussion

This study is the first to report the clinical outcome of women with TN breast cancer treated with conventional chemotherapy according to BRCA1 mutation status. Freedom from distant metastasis and overall survival did not differ significantly between BRCA1 mutation carriers and non-carriers. BRCA1 carriers had a propensity for brain relapse, although the rate and distribution of metastatic sites did not differ significantly by mutation status. When adjusted by age and stage, BRCA1 mutation status was not an independent predictor of outcome, including freedom from distant metastasis, breast cancer-specific and overall survival.

A number of retrospective series have reported a similar or worse prognosis for women with BRCA-related breast cancer compared to patients with sporadic breast cancer. Early clinical reports were limited by a small number of BRCA mutation carriers and heterogeneous treatments. Furthermore, clinical and treatment factors were often imbalanced due to the high grade, estrogen receptor negative nature of the cancers diagnosed in BRCA1 mutation carriers^19-23. Other studies did not differentiate between BRCA1 and BRCA2 mutation carriers^{19, 24-26}. Despite the limitations of earlier reports, a large population-based study from the Israeli National Cancer Registry found no difference in overall mortality or breast cancer-specific mortality for BRCA1 and BRCA2 mutation carriers compared to non-carriers¹⁸. For the 76 BRCA1 and 52 BRCA2 carriers, the hazard ratio for death from any cause and death from breast cancer were not significantly different between carriers and non-carriers after adjusting for age, tumor size, lymph node status and presence of metastasis. BRCA1 mutation carriers were less likely to have estrogen receptor positive tumors than non-carriers (24% vs. 65%), but outcome by hormone receptor status was not reported due to missing data in the majority of cases. Of interest, the 10-year survival rates for women who received adjuvant chemotherapy were 71% for carriers and 46% for non-carriers (HR 0.48, p=0.12). Among women who did not receive chemotherapy, the 10-year survival rates were 75% for carriers and 74% for non-carriers (HR 0.93, p=0.86). The difference in outcome stratified by use of chemotherapy was not seen for BRCA2 mutation carriers. A second study by Robson, et. al. found that BRCA1 mutation status predicted for breast cancer mortality only among women who did not receive chemotherapy (HR 4.8, range 2.0-11.7), while survival rates were similar for carriers and non-carriers treated with chemotherapy (HR 1.5, range 0.7-3.5)²¹. Neither of these reports, however, controlled for biologic subtype, grade or breast cancer histology.

In contrast to the similar outcomes for BRCA1 mutation carriers and non-carriers found in the current study, others have described more clinical heterogeneity among TN breast cancers. Gene expression array data suggest that the TN immunophenotype includes both the basal-like and normal breast subtype^{13, 27, 28}. Supporting this notion, there is only moderate concordance between TN and basal-like breast cancers; 56-84% of TN breast cancers express basal cytokeratins and EGFR^{29, 30}, and conversely, 15-45% of basal-like cancers express ER, PR and/or HER2^{14, 28, 31}. A report from Rakha, et. al. defined basal and non-basal subtypes of TN breast cancers based on a distinct immunohistochemical profile of 3 basal cytokeratin markers (CK5/6, CK17, CK14) and EGFR³². Of 232 TN cases, 71% were positive for at least one of the 3 basal keratins and/or EGFR, and 29% were negative for all 4 basal markers. The basal-like subtype was associated with BRCA1 mutation status (odds ratio 13), improved response to chemotherapy and shorter survival. Among the basal-like cancers, there were more frequent metastases to brain and lung compared to other lymph node groups. Cheang et. al. also found that a 5 marker immunohistochemical panel including CK5/6 and EGFR was a better predictor of outcome than the triple negative definition³³. One possible explanation for the similar clinical outcomes observed in the current study is that a substantial proportion of sporadic TN breast cancers in this cohort have a BRCA1-deficient or basal-like phenotype.

Alternatively, BRCA1 deficiency may not have prognostic significance in BRCA1-related or sporadic TN breast cancer treated with alkylator-based chemotherapy. Our cohort received a combination of alkylating and anthracyline chemotherapy agents, which has been the standard adjuvant chemotherapy backbone for the last decade. Newer platinum containing regimens as well as poly-(ADP) ribose polymerase (PARP-1) inhibitors have shown substantial promise in BRCA-related breast cancers^34-37. In a recent report of 102 women with BRCA1-related breast cancer, the pathologic complete response (CR) rate for 12 patients treated with neo-adjuvant cisplatin on clinical study was 83%, compared to 22% for the 51 BRCA1 mutation carriers who received AC in a retrospective comparison³⁴. It is therefore possible that alkylator or anthracycline-based chemotherapy.does not exploit the unique biologic defects which exist in BRCA1-deficient breast cancers, although direct comparison of these chemotherapeutic agents will require a randomized trial. Nonetheless, identifying the TN breast cancers that share biologic similarities to BRCA1-related breast cancers will likely aid in selecting appropriate patients for these therapies.

In cohort studies, the clinical outcome of mutation carriers may be affected by longevity selection, as the timing of mutation testing may select for long-term survivors of breast cancer. A strength of this study is that all 117 patients included in the cohort had genetic testing within 36 months of diagnosis, either by commercial sequencing or conformation-specific gel electrophoresis. This method was validated at our institution and has a sensitivity of 97.4% in detecting BRCA1 sequence changes¹⁷, although it will not detect complex gene rearrangements or mutations in the BRCA2 gene that could result in misclassification. The limitations of this report include the small sample size and retrospective nature that may introduce bias due to unmeasured confounders and patient and treatment selection. With the current sample size, the study is underpowered to detect the observed effect, a hazard ratio of 0.73 for breast cancer mortality for mutation carriers.

In summary, this study found that the clinical outcome of TN breast cancer is similar in BRCA1 mutation carriers and non-carriers who received conventional alkylating chemotherapy in the adjuvant setting. These findings may support the emerging biologic hypothesis of a BRCAness phenotype in a large proportion of TN breast cancers. Alternatively, alkylating and anthracycline-based chemotherapy may not exploit the biologic differences which may exist between BRCA1-related and sporadic TN breast cancers. Correlative studies are needed to identify women with sporadic TN breast cancer that have a BRCA1-deficient phenotype and may benefit from therapies that target DNA repair pathways. By understanding the heterogeneity of TN breast cancers, we can provide better prognostic information for our patients, and improve our ability to stratify patients for clinical trials based on molecular subtype.