REVIEW URRENT C OPINION
Triple-negative breast cancer: molecular subtypes and targeted therapy Kim M. Hirshfield and Shridar Ganesan
Purpose of review Triple-negative breast cancers (TNBCs), lacking estrogen receptor expression and human epidermal growth factor receptor 2 amplification, have no effective targeted therapy. Large-scale comprehensive genomic analyses have allowed stratification of TNBCs by molecular features. We will review the recent data regarding the classification of these poor prognosis cancers and the associated potential targeted treatment approaches. Recent findings TNBCs are a heterogeneous set of cancers characterized by a diverse set of gene-expression patterns and underlying genomic changes. Mutations in p53 are the only genomic alteration present in the majority of TNBCs. Other potential targetable alterations are only present in small subsets of TNBCs, and include defects in DNA repair present in BRCA1-mutant TNBCs and some sporadic TNBCs. Antiandrogens may be effective for TNBCs that express the androgen receptor and have luminal-like gene-expression features. PI3KCA pathway inhibitors and HSP90 inhibitors may also be effective in a small fraction of TNBCs. Summary Robust methods to functionally classify TNBCs to determine vulnerable pathways are urgently needed to guide the development of clinical trials. It is quite possible that TNBCs, like non-small cell lung cancer, will be stratified into many individually rare cancer classes, each requiring a distinct treatment approach. Keywords androgen receptor, BRCA1, triple-negative breast cancer
INTRODUCTION Breast cancer is a collection of distinct diseases with different natural histories and differing response to treatment, driven by distinct underlying molecular alterations. At present, most oncologists use expression of estrogen receptor and progesterone receptor, and presence of human epidermal growth factor receptor 2 (HER2) amplification to biologically characterize newly diagnosed breast cancers into luminal (ERþ, HER2-), HER2þ (HER2 amplified, either ERþ or ER-), and triple-negative (ER-, PR-, HER2-) categories (see Fig. 1). Use of histologic grade, markers of proliferation, and gene-expression-based assays such as OncotypeDx Recurrence Score (ODxRS), further separates the luminal cancers into luminal A (ERþ, HER2-, low grade, or low ODxRS) and luminal B (ERþ, high grade, or high ODxRS) [1]. This classification drives adjuvant treatment approaches for early-stage breast cancers. HER2þ cancers are treated with HER2-targeted combination therapy. Luminal A cancers are treated primarily with hormonal therapy, whereas luminal B cancers www.co-obgyn.com
have demonstrated benefit from the addition of adjuvant chemotherapy to hormonal therapy. Triple-negative breast cancers (TNBCs) are the only class treated with chemotherapy alone. TNBCs represent a significant treatment challenge as they have a relatively poor prognosis and have no effective targeted therapy. There is a pressing need to better understand the biology of these aggressive cancers in order to develop more effective therapy. Although TNBCs are often discussed as a distinct subset of breast cancers, they are themselves a very heterogeneous group of cancers. In some ways, the category of TNBCs is much like ‘non-Hodgkin’s lymphoma’ or ‘non-small cell lung cancer’; it merely Rutgers Cancer Institute of New Jersey, Rutgers University, New Brunswick, New Jersey, USA Correspondence to Shridar Ganesan, MD, PhD, Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903, USA. Tel: +1 732 235 5211; e-mail: [email protected] Curr Opin Obstet Gynecol 2014, 26:34–40 DOI:10.1097/GCO.0000000000000038 Volume 26 Number 1 February 2014
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Tri ple-negative breast cancer Hirshfield and Ganesan
KEY POINTS TNBCs are a heterogeneous collection of cancers with diverse histology, gene-expression profiles, and patterns of genomic changes. BRCA1-mutant TNBCs have a defect in DNA repair that is therapeutically targetable. A subset of BRCA1wildtype TNBCs may also harbor similar repair defects. The androgen receptor is expressed in a small subset of TNBCs and is a promising therapeutic target currently undergoing clinical evaluation. Methods to functionally classify TNBCs are urgently needed to efficiently guide the use of both standard chemotherapy and novel targeted therapy.
identifies what the cancer is not, gives little insight into either the underlying biology or the effective treatment approaches, and masks the underlying complexity.
TRIPLE-NEGATIVE BREAST CANCERS AND ‘BASAL-LIKE’ CLASS OF BREAST CANCERS Unsupervised gene-expression profiling has identified a robust class of breast cancers labeled ‘basallike’ breast cancer [2,3]. As almost all basal-like cancers have a triple-negative phenotype, the labels TNBCs and basal-like are often used interchangeably. However, it is clear that basal-like cancers only represent a subset of TNBCs and that not all cancers
that profile as basal-like are truly TNBCs. Over 85% of TNBCs with an invasive ductal histology will profile as basal-like by gene-expression analysis. However, a small subset of TNBCs will profile as other intrinsic subclasses. In a recent analysis of 466 breast cancers by the Cancer Genome Atlas (TCGA), there were 75 TNBCs (by clinical estrogen receptor, progesterone receptor, and HER2 testing), 85 cancers that profiled as basal-like by gene expression, and 65 that were both TNBCs and basal-like [4 ]. What proportion of these nonconcordant assignments were because of error rates in clinically defining estrogen receptor, progesterone receptor, and HER2 status; errors associated with gene-expression normalization, and computational methods to assign subtype; reflection of heterogeneity in tissue sampling for the respective assays; or reflection of the true underlying biology, is not certain. At present, the term TNBC is still used in many publications, as the estrogen receptor, progesterone receptor, and HER2 assays are relatively standardized and used universally. There remains no gold standard yet for identifying basal-like cancers by gene-expression profiling, although several geneexpression profiling assays, such as PAM50 and Mammaprint, are being investigated clinically [5]. &&
SUBCLASSES OF TRIPLE-NEGATIVE BREAST CANCERS Various methods have been used to further stratify TNBCs. This includes classification by histology, by gene-expression profiling, and by patterns of
Newly diagnosed breast cancer ER/PR IHC
ER+
ER–/PR– HER2 IHC/FISH ER–/PR– HER2– TNBC Chemo Rx
HER2 IHC/FISH
HER2+ ER–
HER2+ ER+
ER+/HER2– Grade; Ki67; ODx, etc
HER2+ HER2–targeted Rx Chemo Rx +/– Hormonal Rx
(ER+/HER2–/ low grade)
Luminal B (ER+/HER2–/ high grade)
Luminal A
Luminal B
Hormonal Rx
Chemo Rx Hormonal Rx
FIGURE 1. Schema of classification of breast cancer. Clinical testing for ER, PR, and HER2 status is used to stratify breast cancer into TNBC, HER2, and luminal subclasses. Grade, assays of proliferation, or gene-expression assays further divide luminal cancers into luminal A and luminal B subclasses. ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; PR, progesterone receptor; TNBC, triple-negative breast cancer. 1040-872X ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
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genomic alterations. These will be reviewed briefly below.
Histologic classification of triple-negative breast cancers Cancers that arise in the breast and have a triplenegative phenotype include a diverse set of histologic classes. Although almost all (>80%) of TNBCs are invasive ductal carcinomas, rare histologic variants are also present. This includes the metaplastic carcinomas, characterized by areas showing differentiation to a mesenchymal phenotype, ‘medullary’ cancers characterized by extensive lymphocytic infiltration, apocrine cancers, and adenoid cystic carcinomas. These variants are included in the datasets of TNBCs and contribute to the clinical and biologic heterogeneity of this group.
Gene-expression-based classification of triple-negative breast cancers There have been multiple attempts to define subclasses of TNBCs using gene-expression profiling. Lehmann et al. [6 ] performed a meta-analysis of 21 public breast cancer gene-expression datasets to identify a set of more than 500 TNBCs. When analyzed using unsupervised consensus clustering, these segregated into six distinct stable subclasses. This included two ‘basal-like’ classes, BL1 and BL2, which were characterized by high expression of proliferation-associated and DNA repair genes. BL2 was also noted for the enrichment of pathways associated with growth factor receptors, such as EGFR and MET. The other classes were labeled as immunomodulatory subtype, mesenchymal (M), mesenchymal stem-like (MSL), and a luminal androgen receptor (LAR) subtype based on gene-ontology classification of enriched genes. This method was used to classify breast cancer cell lines and identify some potential drug sensitivities. Most BRCA1mutant breast cancer cell lines fell into the BL1 and BL2 subclasses, whereas the cell lines that expressed the androgen receptor tended to classify as LAR and were sensitive to treatment with antiandrogens and HSP90 inhibitors [6 ]. &&
Triple-negative breast cancers
Histology
Invasive ductal
Gene expression
Medullary
BA2
BA1
IM
Metaplastic
M
MSL
Apocrine, rare histology
LAR
Claudin-low
?
BRCA1 Recurrent gene alterations
?
?
p53
Targeted therapy
Platinum, PARPi
?Src inhibitors? ?PI3K inhibitors?
Antiandrogens
FIGURE 2. Stratification of triple-negative breast cancer by histology, gene-expression signatures, and patterns of genomic changes. Potential targeted therapy strategies are shown below some subclasses. BA1, basal 1; BA2, basal 2; IM, immunomodulatory; M, mesenchymal; MSL, mesenchymal stem-like; LAR, luminal androgen receptor. &&
[6 ,7]. The gene sets enriched in M and MSL subclasses include the genes that largely overlap with those previously described for the ‘claudin-low’ subset of TNBCs and metaplastic cancers [8,9]. Similarly, the LAR subclass has significant overlap with the previously described molecular apocrine cancers subclass, which are enriched in ER- cancers with apocrine histology [10]. Molecular subclasses and clinical outcome Analysis of a set of 130 TNBCs treated with neoadjuvant chemotherapy showed a significant correlation between the gene-expression-based subtype obtained on pretreatment biopsy and the pathologic complete response (pCR) rate [11 ]. The BL1 class had the highest rate of pCR (52%), whereas the lowest pCR rate was observed in BL2 (0%) and the LAR (10%). There was no significant association between subtype and either overall survival or distant metastasis-free survival. Of note, the LAR type had the highest overall survival, despite the low pCR rate [11 ,12 ]. &
&
&
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Correlation between gene expression and histology These subtypes have significant correlation with the previously described gene-expression signatures and histological features present in TNBCs (see Fig. 2). The gene-expression signature associated with immunomodulatory subtype has a set of immune signaling genes that are also largely present in the signatures associated with medullary breast cancer 36
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Genomic alterations in triple-negative breast cancers Genomic alterations are critical to the molecular cause of cancers and can also identify actionable vulnerabilities. A key early observation was that almost all breast cancers that arise in women with germline BRCA1 mutations have a triple-negative phenotype and cluster with the basal-like group by gene-expression profiling [3]. Cancers arising in the setting of germline BRCA1 mutation account for Volume 26 Number 1 February 2014
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approximately 10% of TNBCs, and are enriched in the BL1 and BL2 subclasses of TNBCs. However, the vast majority of TNBCs are sporadic cancers that arise in the setting of wildtype BRCA1. Mutations in p53 are present in 60–70% of TNBCs and are mostly truncations and deletions [4 ]. PIK3CA mutations are present in 11% of TNBCs. However, there are no other highly prevalent driver mutations in TNBCs. Instead, there are a large set of gene mutations and genomic rearrangements each found at low frequency [4 ,13 ]. Lack of high-frequency driver mutations has hampered the attempts to develop rational targeted therapy for TNBCs. &&
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TARGETS FOR THERAPY IN TRIPLENEGATIVE BREAST CANCERS Multiple potential targeted therapies are currently being investigated for the treatment of TNBCs. It is likely that each of these targets will only be relevant in a subset of TNBCs. Some of the promising targets are reviewed below.
BRCA1 mutation BRCA1-mutant cancers are a clearly identifiable subset of TNBCs. Current clinical guidelines recommend germline BRCA1 testing for any woman diagnosed with TNBCs who is less than 60 years of age, regardless of the family history. Several studies have found that up to 10% of TNBCs will harbor BRCA1 mutations. BRCA1 plays a critical role in homologous recombination-mediated DNA double-strand break repair, and BRCA1-mutant cancers have a profound defect in this critical DNA repair pathway. This DNA repair defect renders BRCA1-mutant cancers highly vulnerable to certain classes of DNA-damaging agents, such as cisplatin and mitomycin C that induce DNA lesions which are normally resolved by homologous recombination-mediated repair [14]. BRCA1/2-mutant cancers are also highly sensitive to other agents that stress homologous recombination-mediated repair pathways such as poly ADP ribose polymerase (PARP) inhibitors [15]. The underlying DNA repair defect present in BRCA1-mutant cancers has been therapeutically exploited in the clinic. Neoadjuvant platinum-based therapy leads to very high pCR rates in BRCA1mutant cancer [16]. Similarly, single-agent PARP inhibitors have significant response rates in BRCA1-mutant breast and ovarian cancers [17 ]. There are multiple ongoing trials investigating the clinical utility of platinum agents and PARP inhibitors in BRCA1-mutant cancers. This includes trials &&
such as the INFORM (information for patients) trial (NCT01670500), a randomized phase II trial looking at whether neoadjuvant cisplatin therapy will lead to higher pCR rates when compared to standard doxorubicin/cyclophosphamide regimen in women with germline BRCA1/2 mutations. Similarly, multiple PARP inhibitors, including olaparib and velaparib, are in clinical trials targeting BRCA1/2mutant cancers, both as single agents and in combination with DNA damaging agents. Given exciting preclinical data showing synergistic activity of PARP inhibitors with PI3K inhibitors, this combination may also be tested [18 ]. Hopefully, these studies will establish whether the use of these agents will improve outcome in BRCA1/2-mutant cancers, including BRCA1-mutant TNBCs. &
Identifying ‘BRCA1-like’ triple-negative breast cancer with underlying defects in homologous recombination The association of germline BRCA1 mutations with TNBCs has suggested that the underlying defects in the BRCA1-associated homologous recombinationmediated DNA repair (HDR) pathway may be a feature of a subset of sporadic TNBCs. Support for this comes from the data demonstrating that neoadjuvant therapy with modest dose of single-agent cisplatin led to pCR rates of approximately 22% in sporadic TNBCs [19]. The key problem is how to identify TNBCs that have ongoing defects in HDR, which may render them exquisitely sensitive to PARP inhibitors or cross-linking agents. Several methods to assay repair capacity in tumor samples are under development. One method is to functionally assay repair function by measuring the induction of RAD51 repair foci by DNA damaging agents in viable tumor specimens [20]. Although potentially highly specific, this approach is very technically demanding and it is not clear whether it can translate into a robust clinical assay. Other approaches include identifying patterns of alterations, including loss of heterozygosity (LOH), in genomic tumor DNA that would indicate an underlying defect in HDR. This approach has the advantage of being performed in standard archived diagnostic formalin-fixed specimens. Several assays that measure the frequency of certain classes of LOH events associated with defects in homologous recombination have been developed and shown to correlate with both presence of BRCA1 mutation, and with response to platinum-based chemotherapy in TNBCs [21 ,22 ]. Another promising assay looks at the pattern of copy number changes as assayed by comparative genomic hybridization (CGH) [23 ]. A BRCA1-like CGH signature was developed and used
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to evaluate samples from a completed randomized trial that compared standard anthracycline-based chemotherapy with high-dose platinum-based chemotherapy as adjuvant treatment for high-risk, early-stage breast cancers. Cancers identified as ‘BRCA1-like’ by CGH analysis had significant benefit from high-dose platinum-based therapy, whereas those without a BRCA1-like signature had no benefit [23 ]. These approaches are encouraging and hopefully will lead to a practical clinical assay that can identify patients who most benefit from agents that target DNA repair defects. However, it is likely that these will represent only a small fraction of TNBCs with intact BRCA1. &
Targeting p53 p53 mutations are the most common genomic alteration in TNBCs [4 ]. Most p53 mutations in TNBCs are nonsense or frame-shift mutations that result in loss of function. Hotspot missense mutations with gain-of-function activity are less frequent. Several studies have identified G2/M checkpoint inhibitors, including CHK1 inhibitors, WEE1 inhibitors, and ATR inhibitors, as being synthetic lethal with p53 loss of function [24–26]. Intriguingly, hot-spot conformational mutations of p53 may be targeted by small molecules that can specifically ‘reactivate’ these mutant p53 alleles [25,27]. These approaches to treating p53-mutant cancers are being pursued in animal models and in early phase clinical trials. &&
PI3K pathway Activating mutations in PI3KCA are the second most common mutation in TNBCs, being present in approximately 9% of the cancers in the TCGA [4 ]. However, evidence of activation of the PI3KCA pathway by protein and gene expression was frequent in TNBCs [4 ]. Other mechanisms of activating PI3K pathway present in TNBCs include loss of PTEN, loss of INPP4B, translocation involving AKT3, and amplification of PI3KCA [13 ,28 ]. PI3KCA pathway mutations appear to be enriched in the mesenchymal and LAR subclasses. There are now multiple potential therapeutic interventions that target the PI3KCA pathway. This includes PI3KCA inhibitors, AKT inhibitors, MTOR inhibitors, and dual PI3KCA/MTOR inhibitors. Multiple early phase trials targeting TNBCs with these agents, either alone or in combinations with chemotherapy, are currently underway. Some of these trials are either using PI3KCA alterations as entry criteria or are evaluating them as biomarkers. Ultimately, rational development of biomarkers will &&
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be needed to identify which set of TNBCs will benefit most from these drugs and which combinations of these inhibitors will be most effective.
Receptor tyrosine kinases Several receptor tyrosine kinases, including epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), and vascular endothelial growth factor receptor (VEGFR), have been identified as potential targets in TNBCs. Although high expression of EGFR is a common feature of TNBCs, activating mutations in EGFR are rare [4 ]. Two trials of EGFR antibody cetuximab showed little activity either alone or in combination with platinum in unselected TNBCs [29,30]. This finding suggests that, similar to what is seen in lung cancer, EGFR-targeted therapy may only have activity in the setting of rare molecular alterations. Amplification of FGFR1 and FGFR2 has been found in 9 and 2% of TNBCs, respectively, and may be driver mutations [31]. Both small molecule inhibitors of FGFR and antibodies targeting FGFR are under clinical development at present and some have shown clinical activity in breast cancer harboring FGFR amplification. VEGFR has been identified as a potential target in TNBCs, although it is rarely amplified or mutated. Trials with the small molecule VEGFR inhibitors unitinib or VEGF antibody bevacizumab did not show significant benefit when added to chemotherapy in the treatment of TNBCs. Thus, biomarkers need to be identified to determine which TNBCs, if any, would benefit from inhibition of VEGF. Loss of the PTPN12 tyrosine phosphatase has been identified in TNBCs and can result in activation of multiple receptor tyrosine kinases [32 ]. Similarly, although mutations in other RTKs, such as MET, are rare, some data suggest evidence of MET activation in TNBCs. These findings suggest rationale for combination inhibition of multiple RTKs and downstream signaling in some settings. &&
&
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Src family tyrosine kinases Activation of Src family kinases, including SRC, LYN, and YES1, is present in subsets of TNBCs, although genomic alterations are rare [33–35]. Dasatinib, a tyrosine kinase inhibitor whose targets include Src family kinases, showed promising activity in TNBC cell lines, including those that fall into the M and MSL subtypes [6 ,36]. A phase II trial of dasatinib in metastatic TNBCs showed modest activity (4.7% partial response and 9% stable disease); however, one of the partial responses lasted over 1 year [37]. Ongoing trials are studying the &&
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biomarkers to predict activity of dasatinib in breast cancer.
HSP90 inhibitors HSP90 is a chaperone for many signaling kinases and inhibition of HSP90 can lead to degradation of many critical signaling molecules. Although initially investigated in the treatment of HER2þ breast cancer, there is increasing evidence that some subsets of TNBCs may respond to HSP90 inhibitors [38 ]. Cell lines that profiled as LAR showed response to HSP90 inhibitors in vitro, suggesting this subset of TNBCs may be appropriate targets [6 ]. The oral HSP90 inhibitor ganetespib is being tested for activity in both HER2þ breast cancer and TNBC in an ongoing clinical trial (NCT01677455) and has some encouraging preliminary reports. &
&&
Androgen receptor The LAR class of TNBCs is characterized by the expression of luminal genes, such as GATA, and expression of androgen receptor, and tend not to respond to standard chemotherapy. Antiandrogen therapy may target this subset of TNBCs [6 ]. A phase II trial was recently reported that identified androgen receptor expression in approximately 12% of ER-/PR- metastatic breast cancers [39 ]. Treatment with the antiandrogen bicalutamide led to a clinical benefit rate of 19% and was very well tolerated. This result suggests that antiandrogen therapy may have clinical utility in a subset of TNBCs (see Fig. 2). &&
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CONCLUSION TNBC is a heterogeneous collection of cancers with varying histology, gene-expression features, and patterns of genomic alterations (see Fig. 2). BRCA1mutant TNBCs have a clear underlying DNA repair defect that can be therapeutically targeted. Other promising treatment approaches include antiandrogen therapy for LAR subtype, and specific kinase inhibitors for TNBC harboring genomic alterations in PIK3CA, PTEN, and FGFR pathway. Robust, clinically tractable methods to functionally classify TNBCs to determine the presence of DNA repair defects and addiction to specific signaling pathways are urgently needed. It is quite possible that TNBCs, like non-small cell lung cancer, will be stratified into many individually rare cancer classes, each requiring a distinct treatment approach. Acknowledgements None.
Conflicts of interest None. K.H. receives support from the BCRF. S.G. receives support from the NCI, the Triple Negative Breast Cancer Foundation, and Hugs for Brady Foundation.
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Triple-negative breast cancer: molecular subtypes and targeted therapy Kim M. Hirshfield and Shridar Ganesan
Purpose of review Triple-negative breast cancers (TNBCs), lacking estrogen receptor expression and human epidermal growth factor receptor 2 amplification, have no effective targeted therapy. Large-scale comprehensive genomic analyses have allowed stratification of TNBCs by molecular features. We will review the recent data regarding the classification of these poor prognosis cancers and the associated potential targeted treatment approaches. Recent findings TNBCs are a heterogeneous set of cancers characterized by a diverse set of gene-expression patterns and underlying genomic changes. Mutations in p53 are the only genomic alteration present in the majority of TNBCs. Other potential targetable alterations are only present in small subsets of TNBCs, and include defects in DNA repair present in BRCA1-mutant TNBCs and some sporadic TNBCs. Antiandrogens may be effective for TNBCs that express the androgen receptor and have luminal-like gene-expression features. PI3KCA pathway inhibitors and HSP90 inhibitors may also be effective in a small fraction of TNBCs. Summary Robust methods to functionally classify TNBCs to determine vulnerable pathways are urgently needed to guide the development of clinical trials. It is quite possible that TNBCs, like non-small cell lung cancer, will be stratified into many individually rare cancer classes, each requiring a distinct treatment approach. Keywords androgen receptor, BRCA1, triple-negative breast cancer
INTRODUCTION Breast cancer is a collection of distinct diseases with different natural histories and differing response to treatment, driven by distinct underlying molecular alterations. At present, most oncologists use expression of estrogen receptor and progesterone receptor, and presence of human epidermal growth factor receptor 2 (HER2) amplification to biologically characterize newly diagnosed breast cancers into luminal (ERþ, HER2-), HER2þ (HER2 amplified, either ERþ or ER-), and triple-negative (ER-, PR-, HER2-) categories (see Fig. 1). Use of histologic grade, markers of proliferation, and gene-expression-based assays such as OncotypeDx Recurrence Score (ODxRS), further separates the luminal cancers into luminal A (ERþ, HER2-, low grade, or low ODxRS) and luminal B (ERþ, high grade, or high ODxRS) [1]. This classification drives adjuvant treatment approaches for early-stage breast cancers. HER2þ cancers are treated with HER2-targeted combination therapy. Luminal A cancers are treated primarily with hormonal therapy, whereas luminal B cancers www.co-obgyn.com
have demonstrated benefit from the addition of adjuvant chemotherapy to hormonal therapy. Triple-negative breast cancers (TNBCs) are the only class treated with chemotherapy alone. TNBCs represent a significant treatment challenge as they have a relatively poor prognosis and have no effective targeted therapy. There is a pressing need to better understand the biology of these aggressive cancers in order to develop more effective therapy. Although TNBCs are often discussed as a distinct subset of breast cancers, they are themselves a very heterogeneous group of cancers. In some ways, the category of TNBCs is much like ‘non-Hodgkin’s lymphoma’ or ‘non-small cell lung cancer’; it merely Rutgers Cancer Institute of New Jersey, Rutgers University, New Brunswick, New Jersey, USA Correspondence to Shridar Ganesan, MD, PhD, Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903, USA. Tel: +1 732 235 5211; e-mail: [email protected] Curr Opin Obstet Gynecol 2014, 26:34–40 DOI:10.1097/GCO.0000000000000038 Volume 26 Number 1 February 2014
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Tri ple-negative breast cancer Hirshfield and Ganesan
KEY POINTS TNBCs are a heterogeneous collection of cancers with diverse histology, gene-expression profiles, and patterns of genomic changes. BRCA1-mutant TNBCs have a defect in DNA repair that is therapeutically targetable. A subset of BRCA1wildtype TNBCs may also harbor similar repair defects. The androgen receptor is expressed in a small subset of TNBCs and is a promising therapeutic target currently undergoing clinical evaluation. Methods to functionally classify TNBCs are urgently needed to efficiently guide the use of both standard chemotherapy and novel targeted therapy.
identifies what the cancer is not, gives little insight into either the underlying biology or the effective treatment approaches, and masks the underlying complexity.
TRIPLE-NEGATIVE BREAST CANCERS AND ‘BASAL-LIKE’ CLASS OF BREAST CANCERS Unsupervised gene-expression profiling has identified a robust class of breast cancers labeled ‘basallike’ breast cancer [2,3]. As almost all basal-like cancers have a triple-negative phenotype, the labels TNBCs and basal-like are often used interchangeably. However, it is clear that basal-like cancers only represent a subset of TNBCs and that not all cancers
that profile as basal-like are truly TNBCs. Over 85% of TNBCs with an invasive ductal histology will profile as basal-like by gene-expression analysis. However, a small subset of TNBCs will profile as other intrinsic subclasses. In a recent analysis of 466 breast cancers by the Cancer Genome Atlas (TCGA), there were 75 TNBCs (by clinical estrogen receptor, progesterone receptor, and HER2 testing), 85 cancers that profiled as basal-like by gene expression, and 65 that were both TNBCs and basal-like [4 ]. What proportion of these nonconcordant assignments were because of error rates in clinically defining estrogen receptor, progesterone receptor, and HER2 status; errors associated with gene-expression normalization, and computational methods to assign subtype; reflection of heterogeneity in tissue sampling for the respective assays; or reflection of the true underlying biology, is not certain. At present, the term TNBC is still used in many publications, as the estrogen receptor, progesterone receptor, and HER2 assays are relatively standardized and used universally. There remains no gold standard yet for identifying basal-like cancers by gene-expression profiling, although several geneexpression profiling assays, such as PAM50 and Mammaprint, are being investigated clinically [5]. &&
SUBCLASSES OF TRIPLE-NEGATIVE BREAST CANCERS Various methods have been used to further stratify TNBCs. This includes classification by histology, by gene-expression profiling, and by patterns of
Newly diagnosed breast cancer ER/PR IHC
ER+
ER–/PR– HER2 IHC/FISH ER–/PR– HER2– TNBC Chemo Rx
HER2 IHC/FISH
HER2+ ER–
HER2+ ER+
ER+/HER2– Grade; Ki67; ODx, etc
HER2+ HER2–targeted Rx Chemo Rx +/– Hormonal Rx
(ER+/HER2–/ low grade)
Luminal B (ER+/HER2–/ high grade)
Luminal A
Luminal B
Hormonal Rx
Chemo Rx Hormonal Rx
FIGURE 1. Schema of classification of breast cancer. Clinical testing for ER, PR, and HER2 status is used to stratify breast cancer into TNBC, HER2, and luminal subclasses. Grade, assays of proliferation, or gene-expression assays further divide luminal cancers into luminal A and luminal B subclasses. ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; PR, progesterone receptor; TNBC, triple-negative breast cancer. 1040-872X ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
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genomic alterations. These will be reviewed briefly below.
Histologic classification of triple-negative breast cancers Cancers that arise in the breast and have a triplenegative phenotype include a diverse set of histologic classes. Although almost all (>80%) of TNBCs are invasive ductal carcinomas, rare histologic variants are also present. This includes the metaplastic carcinomas, characterized by areas showing differentiation to a mesenchymal phenotype, ‘medullary’ cancers characterized by extensive lymphocytic infiltration, apocrine cancers, and adenoid cystic carcinomas. These variants are included in the datasets of TNBCs and contribute to the clinical and biologic heterogeneity of this group.
Gene-expression-based classification of triple-negative breast cancers There have been multiple attempts to define subclasses of TNBCs using gene-expression profiling. Lehmann et al. [6 ] performed a meta-analysis of 21 public breast cancer gene-expression datasets to identify a set of more than 500 TNBCs. When analyzed using unsupervised consensus clustering, these segregated into six distinct stable subclasses. This included two ‘basal-like’ classes, BL1 and BL2, which were characterized by high expression of proliferation-associated and DNA repair genes. BL2 was also noted for the enrichment of pathways associated with growth factor receptors, such as EGFR and MET. The other classes were labeled as immunomodulatory subtype, mesenchymal (M), mesenchymal stem-like (MSL), and a luminal androgen receptor (LAR) subtype based on gene-ontology classification of enriched genes. This method was used to classify breast cancer cell lines and identify some potential drug sensitivities. Most BRCA1mutant breast cancer cell lines fell into the BL1 and BL2 subclasses, whereas the cell lines that expressed the androgen receptor tended to classify as LAR and were sensitive to treatment with antiandrogens and HSP90 inhibitors [6 ]. &&
Triple-negative breast cancers
Histology
Invasive ductal
Gene expression
Medullary
BA2
BA1
IM
Metaplastic
M
MSL
Apocrine, rare histology
LAR
Claudin-low
?
BRCA1 Recurrent gene alterations
?
?
p53
Targeted therapy
Platinum, PARPi
?Src inhibitors? ?PI3K inhibitors?
Antiandrogens
FIGURE 2. Stratification of triple-negative breast cancer by histology, gene-expression signatures, and patterns of genomic changes. Potential targeted therapy strategies are shown below some subclasses. BA1, basal 1; BA2, basal 2; IM, immunomodulatory; M, mesenchymal; MSL, mesenchymal stem-like; LAR, luminal androgen receptor. &&
[6 ,7]. The gene sets enriched in M and MSL subclasses include the genes that largely overlap with those previously described for the ‘claudin-low’ subset of TNBCs and metaplastic cancers [8,9]. Similarly, the LAR subclass has significant overlap with the previously described molecular apocrine cancers subclass, which are enriched in ER- cancers with apocrine histology [10]. Molecular subclasses and clinical outcome Analysis of a set of 130 TNBCs treated with neoadjuvant chemotherapy showed a significant correlation between the gene-expression-based subtype obtained on pretreatment biopsy and the pathologic complete response (pCR) rate [11 ]. The BL1 class had the highest rate of pCR (52%), whereas the lowest pCR rate was observed in BL2 (0%) and the LAR (10%). There was no significant association between subtype and either overall survival or distant metastasis-free survival. Of note, the LAR type had the highest overall survival, despite the low pCR rate [11 ,12 ]. &
&
&
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Correlation between gene expression and histology These subtypes have significant correlation with the previously described gene-expression signatures and histological features present in TNBCs (see Fig. 2). The gene-expression signature associated with immunomodulatory subtype has a set of immune signaling genes that are also largely present in the signatures associated with medullary breast cancer 36
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Genomic alterations in triple-negative breast cancers Genomic alterations are critical to the molecular cause of cancers and can also identify actionable vulnerabilities. A key early observation was that almost all breast cancers that arise in women with germline BRCA1 mutations have a triple-negative phenotype and cluster with the basal-like group by gene-expression profiling [3]. Cancers arising in the setting of germline BRCA1 mutation account for Volume 26 Number 1 February 2014
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approximately 10% of TNBCs, and are enriched in the BL1 and BL2 subclasses of TNBCs. However, the vast majority of TNBCs are sporadic cancers that arise in the setting of wildtype BRCA1. Mutations in p53 are present in 60–70% of TNBCs and are mostly truncations and deletions [4 ]. PIK3CA mutations are present in 11% of TNBCs. However, there are no other highly prevalent driver mutations in TNBCs. Instead, there are a large set of gene mutations and genomic rearrangements each found at low frequency [4 ,13 ]. Lack of high-frequency driver mutations has hampered the attempts to develop rational targeted therapy for TNBCs. &&
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TARGETS FOR THERAPY IN TRIPLENEGATIVE BREAST CANCERS Multiple potential targeted therapies are currently being investigated for the treatment of TNBCs. It is likely that each of these targets will only be relevant in a subset of TNBCs. Some of the promising targets are reviewed below.
BRCA1 mutation BRCA1-mutant cancers are a clearly identifiable subset of TNBCs. Current clinical guidelines recommend germline BRCA1 testing for any woman diagnosed with TNBCs who is less than 60 years of age, regardless of the family history. Several studies have found that up to 10% of TNBCs will harbor BRCA1 mutations. BRCA1 plays a critical role in homologous recombination-mediated DNA double-strand break repair, and BRCA1-mutant cancers have a profound defect in this critical DNA repair pathway. This DNA repair defect renders BRCA1-mutant cancers highly vulnerable to certain classes of DNA-damaging agents, such as cisplatin and mitomycin C that induce DNA lesions which are normally resolved by homologous recombination-mediated repair [14]. BRCA1/2-mutant cancers are also highly sensitive to other agents that stress homologous recombination-mediated repair pathways such as poly ADP ribose polymerase (PARP) inhibitors [15]. The underlying DNA repair defect present in BRCA1-mutant cancers has been therapeutically exploited in the clinic. Neoadjuvant platinum-based therapy leads to very high pCR rates in BRCA1mutant cancer [16]. Similarly, single-agent PARP inhibitors have significant response rates in BRCA1-mutant breast and ovarian cancers [17 ]. There are multiple ongoing trials investigating the clinical utility of platinum agents and PARP inhibitors in BRCA1-mutant cancers. This includes trials &&
such as the INFORM (information for patients) trial (NCT01670500), a randomized phase II trial looking at whether neoadjuvant cisplatin therapy will lead to higher pCR rates when compared to standard doxorubicin/cyclophosphamide regimen in women with germline BRCA1/2 mutations. Similarly, multiple PARP inhibitors, including olaparib and velaparib, are in clinical trials targeting BRCA1/2mutant cancers, both as single agents and in combination with DNA damaging agents. Given exciting preclinical data showing synergistic activity of PARP inhibitors with PI3K inhibitors, this combination may also be tested [18 ]. Hopefully, these studies will establish whether the use of these agents will improve outcome in BRCA1/2-mutant cancers, including BRCA1-mutant TNBCs. &
Identifying ‘BRCA1-like’ triple-negative breast cancer with underlying defects in homologous recombination The association of germline BRCA1 mutations with TNBCs has suggested that the underlying defects in the BRCA1-associated homologous recombinationmediated DNA repair (HDR) pathway may be a feature of a subset of sporadic TNBCs. Support for this comes from the data demonstrating that neoadjuvant therapy with modest dose of single-agent cisplatin led to pCR rates of approximately 22% in sporadic TNBCs [19]. The key problem is how to identify TNBCs that have ongoing defects in HDR, which may render them exquisitely sensitive to PARP inhibitors or cross-linking agents. Several methods to assay repair capacity in tumor samples are under development. One method is to functionally assay repair function by measuring the induction of RAD51 repair foci by DNA damaging agents in viable tumor specimens [20]. Although potentially highly specific, this approach is very technically demanding and it is not clear whether it can translate into a robust clinical assay. Other approaches include identifying patterns of alterations, including loss of heterozygosity (LOH), in genomic tumor DNA that would indicate an underlying defect in HDR. This approach has the advantage of being performed in standard archived diagnostic formalin-fixed specimens. Several assays that measure the frequency of certain classes of LOH events associated with defects in homologous recombination have been developed and shown to correlate with both presence of BRCA1 mutation, and with response to platinum-based chemotherapy in TNBCs [21 ,22 ]. Another promising assay looks at the pattern of copy number changes as assayed by comparative genomic hybridization (CGH) [23 ]. A BRCA1-like CGH signature was developed and used
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to evaluate samples from a completed randomized trial that compared standard anthracycline-based chemotherapy with high-dose platinum-based chemotherapy as adjuvant treatment for high-risk, early-stage breast cancers. Cancers identified as ‘BRCA1-like’ by CGH analysis had significant benefit from high-dose platinum-based therapy, whereas those without a BRCA1-like signature had no benefit [23 ]. These approaches are encouraging and hopefully will lead to a practical clinical assay that can identify patients who most benefit from agents that target DNA repair defects. However, it is likely that these will represent only a small fraction of TNBCs with intact BRCA1. &
Targeting p53 p53 mutations are the most common genomic alteration in TNBCs [4 ]. Most p53 mutations in TNBCs are nonsense or frame-shift mutations that result in loss of function. Hotspot missense mutations with gain-of-function activity are less frequent. Several studies have identified G2/M checkpoint inhibitors, including CHK1 inhibitors, WEE1 inhibitors, and ATR inhibitors, as being synthetic lethal with p53 loss of function [24–26]. Intriguingly, hot-spot conformational mutations of p53 may be targeted by small molecules that can specifically ‘reactivate’ these mutant p53 alleles [25,27]. These approaches to treating p53-mutant cancers are being pursued in animal models and in early phase clinical trials. &&
PI3K pathway Activating mutations in PI3KCA are the second most common mutation in TNBCs, being present in approximately 9% of the cancers in the TCGA [4 ]. However, evidence of activation of the PI3KCA pathway by protein and gene expression was frequent in TNBCs [4 ]. Other mechanisms of activating PI3K pathway present in TNBCs include loss of PTEN, loss of INPP4B, translocation involving AKT3, and amplification of PI3KCA [13 ,28 ]. PI3KCA pathway mutations appear to be enriched in the mesenchymal and LAR subclasses. There are now multiple potential therapeutic interventions that target the PI3KCA pathway. This includes PI3KCA inhibitors, AKT inhibitors, MTOR inhibitors, and dual PI3KCA/MTOR inhibitors. Multiple early phase trials targeting TNBCs with these agents, either alone or in combinations with chemotherapy, are currently underway. Some of these trials are either using PI3KCA alterations as entry criteria or are evaluating them as biomarkers. Ultimately, rational development of biomarkers will &&
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be needed to identify which set of TNBCs will benefit most from these drugs and which combinations of these inhibitors will be most effective.
Receptor tyrosine kinases Several receptor tyrosine kinases, including epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), and vascular endothelial growth factor receptor (VEGFR), have been identified as potential targets in TNBCs. Although high expression of EGFR is a common feature of TNBCs, activating mutations in EGFR are rare [4 ]. Two trials of EGFR antibody cetuximab showed little activity either alone or in combination with platinum in unselected TNBCs [29,30]. This finding suggests that, similar to what is seen in lung cancer, EGFR-targeted therapy may only have activity in the setting of rare molecular alterations. Amplification of FGFR1 and FGFR2 has been found in 9 and 2% of TNBCs, respectively, and may be driver mutations [31]. Both small molecule inhibitors of FGFR and antibodies targeting FGFR are under clinical development at present and some have shown clinical activity in breast cancer harboring FGFR amplification. VEGFR has been identified as a potential target in TNBCs, although it is rarely amplified or mutated. Trials with the small molecule VEGFR inhibitors unitinib or VEGF antibody bevacizumab did not show significant benefit when added to chemotherapy in the treatment of TNBCs. Thus, biomarkers need to be identified to determine which TNBCs, if any, would benefit from inhibition of VEGF. Loss of the PTPN12 tyrosine phosphatase has been identified in TNBCs and can result in activation of multiple receptor tyrosine kinases [32 ]. Similarly, although mutations in other RTKs, such as MET, are rare, some data suggest evidence of MET activation in TNBCs. These findings suggest rationale for combination inhibition of multiple RTKs and downstream signaling in some settings. &&
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Src family tyrosine kinases Activation of Src family kinases, including SRC, LYN, and YES1, is present in subsets of TNBCs, although genomic alterations are rare [33–35]. Dasatinib, a tyrosine kinase inhibitor whose targets include Src family kinases, showed promising activity in TNBC cell lines, including those that fall into the M and MSL subtypes [6 ,36]. A phase II trial of dasatinib in metastatic TNBCs showed modest activity (4.7% partial response and 9% stable disease); however, one of the partial responses lasted over 1 year [37]. Ongoing trials are studying the &&
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biomarkers to predict activity of dasatinib in breast cancer.
HSP90 inhibitors HSP90 is a chaperone for many signaling kinases and inhibition of HSP90 can lead to degradation of many critical signaling molecules. Although initially investigated in the treatment of HER2þ breast cancer, there is increasing evidence that some subsets of TNBCs may respond to HSP90 inhibitors [38 ]. Cell lines that profiled as LAR showed response to HSP90 inhibitors in vitro, suggesting this subset of TNBCs may be appropriate targets [6 ]. The oral HSP90 inhibitor ganetespib is being tested for activity in both HER2þ breast cancer and TNBC in an ongoing clinical trial (NCT01677455) and has some encouraging preliminary reports. &
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Androgen receptor The LAR class of TNBCs is characterized by the expression of luminal genes, such as GATA, and expression of androgen receptor, and tend not to respond to standard chemotherapy. Antiandrogen therapy may target this subset of TNBCs [6 ]. A phase II trial was recently reported that identified androgen receptor expression in approximately 12% of ER-/PR- metastatic breast cancers [39 ]. Treatment with the antiandrogen bicalutamide led to a clinical benefit rate of 19% and was very well tolerated. This result suggests that antiandrogen therapy may have clinical utility in a subset of TNBCs (see Fig. 2). &&
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CONCLUSION TNBC is a heterogeneous collection of cancers with varying histology, gene-expression features, and patterns of genomic alterations (see Fig. 2). BRCA1mutant TNBCs have a clear underlying DNA repair defect that can be therapeutically targeted. Other promising treatment approaches include antiandrogen therapy for LAR subtype, and specific kinase inhibitors for TNBC harboring genomic alterations in PIK3CA, PTEN, and FGFR pathway. Robust, clinically tractable methods to functionally classify TNBCs to determine the presence of DNA repair defects and addiction to specific signaling pathways are urgently needed. It is quite possible that TNBCs, like non-small cell lung cancer, will be stratified into many individually rare cancer classes, each requiring a distinct treatment approach. Acknowledgements None.
Conflicts of interest None. K.H. receives support from the BCRF. S.G. receives support from the NCI, the Triple Negative Breast Cancer Foundation, and Hugs for Brady Foundation.
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