HAGE, the helicase antigen as a biomarker for breast cancer prognosis (WO2013144616)

Abstract

Introduction: Breast cancer (BC) is the most common cancer in women and it ranks second as a cause of cancer death in women (after lung cancer). The receptor-based diagnosis of BC tumors allows application of more individual therapies. Depending on the status of the receptors and other risk markers, like tumor size and lymph node status, patients are assigned to risk classes. Invention of new biomarkers that could improve diagnosis and prognosis of BC patients is thus of an increased need.

Areas covered: The invention estimates the possibility of using the amount of expression of helicase antigen (HAGE) in samples of BC tissue as a biomarker for screening and prognosis. A total of 1650 BC patients were tested for HAGE expression and analyzed for well-characterized prognostic and predictive factors. HAGE expression was found to correlate significantly with aggressive clinicopathological features. A total of 443 triple negative patients were analyzed for therapeutic treatment received and survival. The HAGE expression was identified as a predictor of response to anthracycline treatment. The result was confirmed by comparison with an independent validation group.

Expert opinion: Тo identify patients who could be treated, a more detailed analysis taking into account, the full distribution spectrum of HAGE expression is desirable.

Keywords::

• biomarker
• breast cancer
• helicase antigen
• therapy

1. Introduction

In the recent years, stratification in medicine has led to more individual therapies, especially for cancer diseases. In the concept of the personalized medicine, the use of cancer biomarkers can help not only to differentiate between normal and pathological conditions but also to stratify different patient groups in terms of clinical response.

Breast cancer (BC) is one of the most frequently diagnosed cancers in women. At the same time, BC ranks second as a cause of cancer death in women (after lung cancer) [1]. The primary goal in the modern pharmacological treatment of BC is to accurately define the patient risk categories in order to administer the treatment required for a successful outcome. Nowadays, the most commonly investigated biomarkers are the estrogen receptor (ER), the progesterone receptor and the human epidermal growth factor 2 (HER2). Recently, gene expression signatures have been also used due to the observed evidence in relationship between gene expression and clinical outcome [2-5]. Depending on the status of the receptors and signatures, the patients can benefit from treatment with hormone or other targeted therapies. BC patients whose cancers do not express any of these three receptors belong to the category of the triple-negative breast cancer (TNBC) and are known to have a poor prognosis and least treatment options. Thus, there is an increasing need for new biomarkers that can help in BC risk assessment and in guided selection of the most appropriate pharmacological therapy.

The helicase antigen (HAGE) is a cancer–testis antigen, a member of the DEAD (Asp–Glu–Ala–Asp) box family of ATP-dependent RNA-helicases, and has been first identified in human sarcoma [6]. In normal tissues, the protein is only expressed in testis; at the same time, it is expressed in a broad range of ascites and solid tumors [7]. HAGE is shown to play a crucial role in the RNA metabolism in tumor cells, thus opening opportunities for its application as a tumor biomarker and a target for immunotherapy of cancer in general.

The patent focuses on the use of HAGE for screening BC patients by testing the amount of its expression in samples of BC tissue [8]. Several well-characterized clinicopathological parameters have been measured and based on their statistical analysis, it has been shown that the subjects with positive levels of HAGE expression (HAGE+) have an increased probability for aggressive cancer, and correspondingly, a decreased probability of long-term survival. The inventors demonstrate also the use of HAGE as a potential prognostic marker of outcome as well as a predictor of response to anthracycline treatment of TNBC patients. Considering the limitations of the current chemotherapy, the inventors claim additionally for HAGE as a potential target to treat cancer with a HAGE-specific chemotherapeutic agent antigen or HAGE-specific antibody. In this relation, a HAGE-derived epitope has been suggested for BC vaccination. Thus, the level of HAGE can serve as a biomarker in the context of the modern diagnosis and pharmacological treatment of patients with BC.

2. Results

The patent reports a systematic analysis of a large group of patients with primary invasive BC for HAGE expression and its correlation with a number of well-characterized prognostic and predictive factors to estimate the role of HAGE as a biomarker. The main scientific results of the inventors can be summarized as follows:

• HAGE is overexpressed at protein level in samples of BC tissue.

• A high expression of HAGE correlates significantly with aggressive clinicopathological parameters, like high proliferation, absence of simultaneous expression of ER, progesterone receptor and HER2 (triple negative), overexpression of both EGFR and HER2 and abnormal expression of p53 and p16.

• An increased probability of high levels of HAGE expression is found in the subpopulations of ER and TNBC patients.

• Thus, the significance of HAGE expression is proven at the protein level in a large group of BC patients, including also TNBC, confirming in this way, the prognostic and predictive value of HAGE as a biomarker. The reported data from Kaplan Meier curves for the whole cohort and various subgroups are summarized in Table 1, showing the high hazard ratios associated with high HAGE expression.

Table 1. Summary of the reported survival hazard ratios with 95% CI in parenthesis based on helicase antigen expression level for cancer-specific survival and disease-free survival times for different subpopulations.

Population	HAGE+ tumors	HAGE- tumors	Hazard ratio for cancer-specific survival	Hazard ratio for disease-free survival
Whole cohort	126	1210	2.4 (1.8 – 3.1)	2.5 (2.0 – 3.2)
Low lymph node status	78	836	3.5 (2.5 – 4.9)	3.4 (2.6 – 4.6)
Low-risk, no adjuvant therapy	21	227	4.8 (2.4 – 9.6)	4.0 (2.4 – 6.9)
High-risk, endocrine therapy	49	496	3.0 (2.0 – 4.4)	2.9 (2.0 – 4.1)
High-risk ER-,no chemotherapy	34	181	4.0 (2.6 – 6.5)	3.4 (2.2 – 5.2)
High-risk ER-, chemotherapy	43	227	n.s.*	n.s.
Whole TNBC cohort	77	443	1.3 (1.2 – 1.5)	1.2 (1.1 – 1.4)
No chemotherapy	43	252	1.4 (1.2 – 1.7)	1.3 (1.1 – 1.5)
Chemotherapy	34	191	1.2 (0.9 – 1.47)	1.1 (0.9 – 1.3)

*Not significant, no value reported.

ER: Estrogen receptor; HAGE: Helicase Antigen; TNBC: Triple-negative breast cancer.

When patients with high HAGE expression tumors receive chemotherapy, their hazard ratio is no longer higher than for HAGE patients. This is also true for patients with primary locally advanced BC who received pre-chemotherapy. Thus, HAGE expression may be a tool for the stratification of patients with BC for chemotherapy. These findings are especially important for patients considered to belong to the low-risk group but with HAGE+ tumors, as these could significantly benefit from chemotherapy (4 – 4.8-fold hazard reduction, Table 1). Also, for TNBC patients whose opportunities for pharmacological treatment are very limited, and who are traditionally considered to have a poor prognosis, the lack of HAGE expression is associated with a poor effect of chemotherapy. In a subset of TNBC patients, association of HAGE+ with androgen receptor was observed and this subpopulation might benefit from additional hormone treatment targeting androgen. Moreover, for TNBC patients with HAGE+ residual disease (anthracycline resistant) immunotherapy is suggested as alternative treatment by targeting HAGE along with other chemotherapeutic agents in conjunction with chemotherapy.

3. Expert opinion

The advantage of this study over a few other studies that deal with expression of cancer–testis antigens in TNBC patients is that the HAGE expression has been measured not by mRNA expression but by immunohistochemical staining to define the protein level in BC cells. The survival data confirm the role of HAGE expression as prognostic marker and identify the subgroups of low-risk/HAGE+ and TNBC/androgen receptor positive patients to benefit from additional treatment. High HAGE expression has been defined as the presence of strong cytoplasmic and/or nuclear staining in > 10% of malignant cells. To our opinion, this cut-off needs to be further specified as it considers tumors with very high HAGE levels and neglects those of lower HAGE expression (medium and low levels). Thus, a more detailed risk analysis that takes into account the full distribution spectrum of HAGE expression is desirable. There was a slight tendency for tumors with high HAGE expression to show a breast cancer 1, early onset mutation (46 vs 37 % in HAGE negative tumors) but the relationship was statistically not significant (p = 0.2).

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.