Subtyping Breast Cancer: Genome Analysis Opens Door to New Era in Targeted Therapies

Charles M. Perou, PhD

Current Treatment Paradigms

The clinical subtyping of breast cancer appears to be coming into a golden era as an increased understanding of subtyping promises to help advance treatments and outcomes for patients. A comprehensive review published this year by Matthew J. C. Ellis, MB BChir, PhD, and colleagues with The Cancer Genome Atlas (TCGA) Research Network has provided new information about these subtypes in addition to identifying a new classification of HER2-positive disease, and has also suggested a rationale for new treatment options in each subtype. The study builds on one of the seminal works in breast cancer subtyping, published in 2000 by Charles M. Perou, PhD, and colleagues, which at the time provided a much more detailed understanding of gene signatures in malignant breast tissue than had previously existed, although the initial characterization was limited due to the technique of choice (DNA microarray) and sample size.Currently, clinicians recognize the following forms of disease: luminal A and luminal B, two cancer subtypes that express the estrogen and progesterone receptors (ER and PR, respectively); human epidermal growth factor receptor 2 (HER2)-positive tumors, which express the HER2 protein; and basal-like tumors, which lack HER2, ER, and PR (often referred to as triple-negative breast cancers [TNBCs], but approximately 25% of TNBCs fall into other micro-RNA subtypes).

Surgery and radiation represent frontline options for patients with breast cancer. Typically, a chemotherapeutic regimen including a taxane is then given to systemically suppress the regrowth of the tumor and prevent metastasis. During initial diagnosis, hormone receptor and HER2 expression are also determined. When the ER and PR status is positive, anti-hormone drugs such as tamoxifen are often indicated. Trastuzumab (Herceptin), a monoclonal antibody against HER2, can be used in HER2-positive disease. In addition, targeted therapeutics have improved the prognosis of patients with these specific cancer subtypes.

An Expanded Analysis of Breast Cancer Subtypes

Despite the advances in targeted therapy, significant limitations persist. Anti-hormone compounds in hormone receptor-positive disease are able only to suppress the growth of cancer cells. They do not kill these cells, and this gives the tumor time to circumvent hormone signaling and resume unchecked growth, often leading to relapse and resistance. Anti-HER2 therapy is successful in treating a large number of patients, but the development of resistance is a common feature of HER2-positive disease. In addition, roughly half of HER2-positive patients with previously untreated metastatic disease do not respond to trastuzumab when used in combination with chemotherapy . The reason for this lack of response is unclear. No approved targeted therapies yet exist for the basal-like breast cancers, since they do not express any known targetable proteins. This leaves only the nonspecific chemotherapy option for patients with this disease.The TCGA network, through its pivotal new work, ultimately may help to expand the treatment options for breast cancer by increasing the understanding of the subtypes. The network, launched in 2006 through a collaboration of the National Cancer Institute and the National Human Genome Research Institute, brings together researchers from approximately 15 institutions, including centers at Washington University in St. Louis, Missouri, where Ellis is based.

In the breast cancer study, the researchers combined a variety of techniques using tumor samples from 825 patients in an effort to definitively characterize the breast cancer subtypes. They also hoped to elucidate specific pathways of disease to improve the current standards of care. Six features of genes across the entire exome were analyzed, including DNA copy number, mutation, DNA methylation, mRNA expression, micro-RNA expression, and protein expression.

Snapshot of Mutated Genes in Breast Cancer Subtypes

Nature

The TCGA study included an analysis of the significantly mutated genes in 498 tumor samples correlated with genomic subtype. Adapted from Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours [published online ahead of print September 23, 2012]. . 2012;490(7418):61-70. doi: 10.1038/nature11412.

New Findings in HER2-Positive Breast Cancer

Overall, the team concluded that diverse genetic and epigenetic alterations in breast cancers could be classified into the four major biological subtypes, and that “much of the clinically observable plasticity and heterogeneity occurs within, and not across,” those subtypes.The study elucidated distinct subtypes of HER2-positive cancer. Nearly half of the samples revealed the classically-defined HER2-positive disease where mRNA and protein levels were elevated. In the other half, the gene expression signature indicated strong similarity to the luminal subtype.

Given that nearly half of the cases of HER2-positive disease show little or no effect, response to trastuzumab treatment has been difficult to predict. Breast cancer expressing HER2 but with the luminal gene signature may account for this phenomenon. Indeed, the luminal gene signature for HER2-positive disease may prove to be a useful predictor of trastuzumab efficacy.

Table. Potential Therapeutic Targets

Gene-Associated Targets

Drug Candidate

Tyrosine Kinases

EGFR

Gefitinb, cetuximab, others

ERBB2

Lapatinib, trastuzumab, others

ERBB3

MM-121, MM-111, AMG888

ERBB4

Lapatinib, neratinib

KIT PDGFRA PDGFRB

Imatinib, masitinib (AB1010)

ABL1 ABL2

Imatinib, dasatinib, nilotinib

DDR2 DDR1

Imatinib, nilotinib, bafetinib (INNO-406)

FGFR2 FGFR3 FGFR4

Dovitinib (TKI258), AZD4547

JAK1 JAK2 JAK3 TYK2

Tofacitinib (CP-690550), ruxolitinib (INCB018424 or INC424)

ALK ROS1 LTK

Crizotinib

MET MST1R

Foretinib, tivantinib (ARQ 197), MetMAb (PRO143966)

Serine/Threonine Kinases

AKT1 AKT2 AKT3

MK2206

BRAF

Vemurafenib

CDK4 CDK6 Cyclin D1

PD0332991

PIK3CA Pathway

PIK3CA PIK3R1 PIK3R3 PTEN

BKM120, GDC-0941, and others

BRCA1 or BRCA2 Mutations

BRCA1 or 2 germline

BRCA1 or 2 somatic

Cisplatin, carboplatin, PARP inhibitors

New Findings in Luminal Breast Cancer

In the HER2-overexpressing disease, a marked upregulation of other receptor tyrosine kinase-related phenomena were noted. This provides more rationale for the targeting of both HER2 and other receptors such as the epidermal growth factor receptor (EGFR). Small molecules such as lapatinib (Tykerb) could be used in this context along with trastuzumab to potentially increase the efficacy of treatment. The high rate of PIK3CA mutations in this subtype also suggests that AKT pathway inhibition may be a viable route of therapy for HER2-positive disease.Many features of the luminal subtype were confirmed upon analysis of the gene signature results. The ER signaling pathway was intact in both types, for example. In addition, the luminal subtypes were given the novel distinction of being “reactive” through analysis of protein expression. This referred to the expression of proteins likely provided by the tumor microenvironment. The finding implies that luminal tumors are especially sensitive to changes in the ecosystem of the disease.

New Clues to Treating Basal-Like Breast Cancer

The study also highlighted several potential drug targets in addition to the current standard of care (Table). Mutations in the PIK3CA gene were common in many luminal tumors, a sign that drugs affecting the AKT signaling pathway could prove useful to hormone-positive breast cancer treatment. The luminal B subtype, which relies less on hormone signals to grow, was also identified as having a significant number of TP53 mutations, along with elevated MDM2 copy number. Both of these observations suggest an interference with the p53 tumor suppressor pathway in luminal B that helps to explain its more aggressive phenotype. Treating with inhibitors of the p53-MDM2 interaction could therefore help reactivate this tumor suppressive pathway in luminal B cancers and improve prognosis.Because no targeted therapeutics have been approved for the basal-like phenotype, there is a critical need to identify new drug targets for this subgroup in particular. The study found a strong correlation between p53 pathway suppression and basal-like breast cancer. The AKT pathway was also strongly upregulated in basal-like disease.

Translating Research to the Clinic

Most surprisingly, the gene signature of basal-like breast cancer was found to be highly similar to serous ovarian carcinoma. The work confirmed that other subtypes, by contrast, did not share the similarity. Furthermore, the BRCA1 gene is inactivated in both basal-like breast cancer and serous ovarian carcinoma alike. Platinum-based drugs may prove effective in treating basal-like disease. In addition, upregulation of receptor tyrosine kinase signaling was observed and suggests that targeting receptors other than HER2 or EGFR could prove useful in the treatment of basal-like disease.Although the TCGA study findings are not intended for direct translation to drug development, researchers hope that the comprehensive mapping of the genomic characteristics of breast cancer (and at least 20 other tumor types) eventually will advance the practice of personalized medicine. The treatment of breast cancer has been moving toward more personalized approaches since 1977, when tamoxifen became the first molecularly targeted therapeutic to gain FDA approval. Tamoxifen acts as an ER modulator that suppresses hormone signaling in breast tissue and is approved for metastatic breast cancer, ductal carcinoma in situ, and systemic inhibition of node growth following radiation and surgery.

The next breakthrough came in 1998 with the approval of Herceptin for use in women with metastatic breast cancer who have tumors that overexpress the HER2 protein. Herceptin is a monoclonal antibody that targets the HER2 protein extracellular domain. Subsequently, the armamentarium of targeted therapeutics both as monotherapy and in various combinations has been expanded with aromatase inhibitors, lapatinib, and, in recent months, pertuzumab (Perjeta) and everolimus (Afinitor).

Everolimus targets the mammalian target of rapamycin (mTOR) protein, one of the downstream targets of the PI3K/AKT pathway. Interestingly, the TCGA study found that, in each breast cancer subtype, the PI3K/AKT pathway was activated in some way. Therefore, the use of AKT inhibitors may be an effective secondary therapeutic strategy to prevent cells from engaging compensatory signaling pathways that help them survive the primary treatment.

Furthermore, activation of receptor tyrosine kinases such as fibroblast growth factor receptor (FGFR) has not typically been associated with breast cancer, but recent studies have highlighted the importance of this protein in breast cancer. This work, in particular, found that FGFR signaling was upregulated in each subtype, providing an attractive, novel candidate for therapeutic intervention.

Key Research

Nature

Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours [published online ahead of print September 23, 2012 . 2012;490(7418):61-70. doi: 10.1038/nature11412.

Herceptin [prescribing information]. South San Francisco, CA: Genentech, Inc; 2010.

National Cancer Institute. Targeted cancer therapies [fact sheet]. http://www.cancer.gov/cancertopics/factsheet/Therapy/targeted. Reviewed October 15, 2012. Accessed November 1, 2012.

Nature

Perou CM, Sørlie T, Eisen MB, et al. Molecular portraits of human breast tumors. . 2000;406(6797):747-752.

Slamon D, Leyland-Jones B, Shak S, et al. Addition of Herceptin (humanized anti-HER2 antibody) to first line chemotherapy for HER2 overexpressing metastatic breast cancer (HER2+/MBC) markedly increases anticancer activity: a randomized, multinational controlled phase III trial. Presented at: 34th Annual Meeting of the American Society of Clinical Oncology; May 16-19, 1998; Los Angeles, CA. Abstract 377.