Symposium article. HER2 as a prognostic and predictive marker for breast cancer. T. Cooke, J. Reeves, A. Lanigan & P. Stanton

Transcription

1 Annals of Oncology 12 (Suppl. I): S23-S28, Kluwer Academic Publishers. Printed in the Netherlands. Symposium article HER2 as a prognostic and predictive marker for breast cancer T. Cooke, J. Reeves, A. Lanigan & P. Stanton University Department of Surgery, Royal Infirmary, Glasgow, UK Summary In recent years investigators have looked at the human epidermal growth factor receptor-2 (HER2), which is overexpressed in 20%-30% of breast cancer patients, with regard to its role as a prognostic and predictive factor. Although many studies have suggested that HER2 overexpression may be associated with a poor clinical outcome, other studies have not fully supported this observation. The inconsistencies between studies may be due in part to discrepancies between different HER2 testing methods. To overcome this problem, a radioimmunohistochemical method was developed to quantitatively measure HER2 overexpression levels in breast tumor samples. The application of this method demonstrated that 85% of all breast tumor samples expressed HER2 at levels greater than normal. Of these, 23% expressed HER2 at levels between 45 and 480 times greater than normal, and this was associated with poor clinical outcome. The investigation of HER2 status as a predictor of response to therapy has also yielded many conflicting results. Overall, it appears that HER2 overexpression may correlate with resistance to hormonal therapy, sensitivity to anthracycline-based chemotherapy and resistance to CMF. With the development of targeted anti-her2 therapies, assessment of HER2 status will be important in stratifying patients to the most appropriate treatment regimens. Key words: breast cancer, HER2, prognostic factor, predictor Introduction Hormones control the growth of many breast cancers and the use of hormonal therapy remains the treatment of choice for the majority of metastatic breast cancer patients with hormone-sensitive disease. Sir George Beatson first demonstrated a link between hormones and advanced breast cancer more than 100 years ago. He identified that oophorectomy was associated with regression of breast cancer in pre-menopausal women, although it was a further 20 years before hormones were first described [1]. The mechanism of action was not understood until the 1960s, when estrogen-receptor (ER) overexpression was demonstrated to occur in some breast cancers [2]. Today, ER status and/or progesteronereceptor (PgR) status are useful as prognostic factors, although their importance lies more as predictors of response to endocrine therapy [3]. Patients with ER/PgRpositive tumors are hormone responsive and therefore have a significantly better prognosis compared with patients whose tumors are ER/PgR negative [3]. Breast cancer prognosis can be determined using a number of endpoints including increased risk of aggressive disease, faster relapse, metastasis and shortened disease-free (DFS) or overall survival (OS). DFS and OS are most frequently used to assess breast cancer prognosis. A range of morphology- or molecular-based factors can be used to assess prognosis. Morphologybased variables include tumor type, size and grade, and lymph node status. Non-morphology-based variables include ER and PgR, oncogenes (e.g., myc, ras), tumorsuppressor genes (e.g.,p53), proteases (e.g., cathepsin D), and cell cycle regulators (e.g., cyclins, cyclin-dependent kinases). Overall, the presence of metastases in regional lymph nodes is probably the single most reliable factor for poor prognosis in breast cancer patients [4]. However, the combination of certain individual factors such as tumor grade, lymph-node status and hormone-receptor status carry greater predictive weight for prognosis than any individual factor. This is illustrated by the Nottingham Prognostic Index (NPI), which combines tumor size x 0.2, number of lymph nodes (1 = no nodes, 2=1-3 nodes and 3 = > 3 nodes) and tumor grade (1-3) to give an accurate prediction of prognosis [5]. Patients are classified into three groups: (1) NPI group 1 score 2-3.4; (2) NPI group 2 score ; (3) NPI group 3 score > 5.6. This scoring system allows patients to be stratified according to risk and facilitates choice of therapeutic regimen. Using the NPI on breast cancer patients from Glasgow, the 10-year survival of patients in NPI group 1 was 88% compared with 66% in NPI group 2 and 28% in NPI group 3 (T. Cooke, unpublished data). Many molecular factors have been investigated in an attempt to find a prognostic/predictive entity as significant as ER status. One of the best studied of these putative factors is the human epidermal growth factor receptor-2 (HER2). HER2 belongs to a family of four homologous receptors involved in the tyrosine kinasemediated regulation of normal breast tissue growth and

2 24 development [6-8]. The four transmembrane receptors HER1, HER2, HER3 and HER4 are composed of an extracellular-binding domain, a transmembrane segment and an intracellular protein tyrosine-kinase domain, with the exception of HER3 which lacks the tyrosine kinase domain. The HER receptors form homo- or heterodimers that are stabilized and activated by ligand binding. While ligands have been identified for HER1, HER3 and HER4 [9, 10], no such ligand has been identified for HER2. Instead, it appears that HER2 is the preferred heterodimer partner within the family [11]. HER2 heterodimers show particularly high ligand binding and signal-transduction activity compared with homo- or heterodimers that do not contain HER2 [12, 13]. Preclinical in vitro and in vivo studies indicate that HER2-gene amplification and /or protein overexpression play an important role in oncogenic transformation and tumorigenesis in breast cancer [14-17]. Having identified that HER2 plays a direct role in breast cancer, other studies have focused on HER2 as a prognostic factor and as a predictor of response to therapy. HER2 status as a prognostic factor The significance of HER2 amplification /overexpression in breast cancer was first described by Slamon et al. [18]. HER2 gene amplification was demonstrated to occur in 20%-30% of primary breast tumors and was associated with concurrent HER2 receptor overexpression [18, 19]. In these studies, HER2 amplification was found to be a significant predictor of both overall survival (P = ) and time to disease relapse (P < ) in node-positive tumors. In addition, Slamon et al. suggested that, with the exception of the number of positive lymph nodes, the prognostic impact of HER2 was superior to all other known prognostic factors in the group of patients sampled [19]. Since the original study by Slamon et al., most large studies have found a positive correlation between HER2 amplification/overexpression and poor disease outcome. A retrospective analysis of 47 studies involving more than 15,000 patients demonstrated that in the majority of studies (60%) and in the majority of patients (67%), a HER2-positive status was an independent predictor of prognosis on multivariate analysis [20]. HER2 status in node-positive and -negative breast cancer Patient prognosis and treatment are generally based on regional nodal status, predicated on the well-accepted knowledge that the presence of nodal metastases is the single most reliable factor for poor prognosis [4]. The majority of studies investigating HER2 status in nodepositive tumors found a positive correlation between a HER2-positive status and poor prognosis [18, 19, 21-26]. Although these studies are retrospective and some other studies did not find this correlation [27-29], it is now generally accepted that a HER2-positive status does predict for a poor outcome in node-positive breast cancer patients. Node-negative breast cancer patients have a better prognosis following surgery than node-positive patients. However, 20%-30% of these patients go on to develop distant metastases. It is therefore necessary to identify those patients who have a greater risk of developing metastases. HER2-positive status has been studied extensively with regard to its prognostic impact in nodenegative breast cancer patients, however, many conflicting results have been obtained. Some studies have indicated that HER2 status does predict development of metastases in node-negative patients [24, 25, 29-31], while other studies show no such correlation [28, 32]. HER2 status in ductal carcinoma in situ HER2 status in ductal carcinoma in situ (DCIS) has also been studied in depth. DCIS is frequently detected on mammograms due to the formation of characteristic microcalcifications. If DCIS is treated by mastectomy, breast cancer-specific survival is nearly 100%. However, patients are increasingly being treated with breastconserving therapy after which the risk of recurrence or subsequent invasive disease is increased. It is important to be able to determine characteristics associated with increased risk in order to stratify patients to the appropriate treatment regimens. Various studies have examined the relationship between HER2 status and various histopathologic forms of DCIS (reviewed in Ross & Fletcher [20]). Many of these studies found that HER2-positive status correlated with comedo and high-grade subtypes of DCIS [33-35]. In particular, the majority of comedo DCIS (up to 100%), a particularly aggressive form of the disease, were found to overexpress HER2 [35, 36]. It may be that HER2 overexpression identifies a subgroup of DCIS patients with greater invasive potential who should receive aggressive therapy accordingly [20]. HER2 testing and prognosis Overall, the results from studies investigating HER2 status as a prognostic factor indicate that HER2 status can predict clinical outcome. However, enough variability exists between studies for this to be inconclusive. While the original study by Slamon et al. [18] examined HER2- gene amplification in frozen tissue samples, the majority of subsequent studies looked at protein expression on archival tissue samples using different antibodies and scoring systems. Therefore, conflicting results between studies may be a consequence of discrepancies between different HER2 testing methods. In a review of HER2 as a prognostic factor, the method of determining HER2 status was also investigated [20]. Six of forty-seven studies analyzed did not find an association between HER2 status and poor prognosis [20]. Of these, four studies used immunohisto-

3 25 chemistry (IHC) on paraffin-embedded tissue and two studies used gene-based techniques. Although only four studies used fluorescence in situ hybridization (FISH) to determine HER2 status, all four studies found a positive correlation between HER2 and poor clinical outcome. In summary, it was found that overall HER2 status determined by both amplification and protein overexpression was associated with poor clinical outcome. The most consistent results were, however, obtained when fresh or frozen tissue samples were analyzed. A second review study [37] focused on the use of IHC or FISH to assess HER2 status. Generally, it was found that although IHC did associate HER2-positive status with poor prognosis, there were some inconsistencies. FISH, however, more reliably demonstrates the correlation between HER2 status and prognosis. Inconsistencies within different IHC tests are likely to be a result of different antibodies used and loss of HER2 protein during the tissue sample fixation process. DNA is not affected by fixation and, therefore, FISH can be used with greater accuracy to determine HER2 status on archival tissue. Radioimmunohistochemistry: A quantitative method for determining HER2 status It is apparent that many of the techniques used to determine HER2 status have disadvantages. While IHC is widely available, lack of standardization results in discrepancies between studies; FISH is more objective than IHC, but is not commonly used within pathology laboratories. We have developed a radioimmunohistochemical (rlhc) method that allows quantitative assessment of HER2 status to be made on frozen tissue samples [38]. In this technique, radiolabelled anti-her2 antibody is used to bind to HER2 receptors on the surface of HER2-overexpressing tumor cells in a particular tissue sample. The samples are then dipped into autoradiographic emulsion, exposed, developed and finally counterstained. This results in a tissue sample on which grains of stain are visible. Using computerized image analysis, a particular area of the treated sample can be selected and the number of grains (relating to the number of HER2 receptors) counted. A comparison of a particular breast tumor tissue sample with normal breast tissue samples allows a quantitative analysis to be made on the level of HER2 overexpression in that sample. The rlhc method was applied to 177 frozen breasttumor samples and levels of expression were expressed relative to normal breast tissue samples. It was found that approximately 85% of the tumor samples expressed HER2 at levels higher than normal [38, 39]. Of these, the majority expressed HER2 up to 15 times higher than normal. The remaining 23% of samples expressed HER2 at levels between 45 and 480 times higher than normal and this was consistently associated with concurrent 30 n Unamplified :. Amplified II HER2 x normal 1000 Figure 1. HER2 protein overexpression and gene amplification. 4-1 E 3-1 CO J5 CD rr HER2 expression (x normal) Strong evidence of greater risk with decreasing as well as increasing levels of HER2 (P< 0.01) Figure 2. Hazard function for HER2 alone. HER2-gene amplification (Figure 1). In addition, 15% of the tissue samples analyzed demonstrated lower than normal or no HER2 expression. When the hazard ratios for HER2 expression levels were studied, it was found that HER2 underexpression or high HER2 overexpression resulted in a poor prognosis, compared with HER2 expression at levels up to 15-fold higher than normal (Figure 2). HER2 status, as determined by the rlhc method, was added to the NPI. Combining HER2 with the NPI had an additive effect because HER2 status was found to be statistically independent from conventional prognostic factors, such as tumor grade and size, or nodal status. For example, 10-year survival for patients in NPI group 2 was 66% but addition of HER2-positive status to the index reduced the 10-year survival to 29% (P < ) [CookeT, unpublished data]. HER2 expression levels were also investigated using rlhc in DCIS tissue samples (n - 21), in invasive cancers with evidence of DCIS (n = 47), and in invasive tumor samples (n - 179) [40]. The results of this study demonstrated that the distribution of HER2 expression levels were similar in each disease type from DCIS through to invasive disease. In addition, where DCIS was associated with invasive disease, comparable distri-

4 26 bution of HER2 expression levels were seen in the DCIS and the invasive components (Wilcoxon signed rank test, P = 0.108). Together, these data suggest that alteration of the HER2 receptor occurs before progression of in situ disease to invasive cancer. HER2 status as a predictor of response to therapy Many researchers have investigated HER2 as a predictor of response to therapy. Various endpoints, similar to those used for prognosis, can be used to determine response to therapy including increased risk for aggressive disease, faster relapse, DFS or OS. In addition, other measures such as measurement of drug susceptibility in vitro and in vivo using cell culture and transfection have been used to determine response to therapy according to HER2 status. However, compared with prognostic studies fewer reports of HER2 as a predictive factor have been produced, the number of patients in each study has been lower, and the studies have been less well controlled. The consequence of this is that the results obtained are somewhat ambiguous (reviewed in Ross and Fletcher [20]). HER2 status and hormonal therapy HER2 overexpression has been associated with resistance to hormonal therapy in several studies. In particular a number of these studies found that HER2-positive tumors were specifically resistant to tamoxifen [41-44]. Of particular interest, the recently reported 20-year update of the Naples GUN Trial [41] showed that not only did a HER2-positive status predict resistance to tamoxifen, but that HER2-positive patients treated with tamoxifen had a worse outcome compared with HER2- positive patients who were untreated. However, two studies did not confirm these findings [45, 46]. In the first of these studies, 205 ER-positive patients were examined for HER2 status and response to tamoxifen. The conclusions from this trial were that in ER-positive patients HER2 status did not predict resistance to tamoxifen or a more aggressive disease progression [45]. In the second study involving 1572 ER-positive or -negative/pgr-positive patients, tamoxifen therapy improved DFS and OS compared with no tamoxifen therapy irrespective of HER2 status [46]. HER2 status and chemotherapy The relationship between HER2 status and anthracyclinebased chemotherapy, usually FAC or FEC (5-fluorouracil (5-FU), doxorubicin or epirubicin, cyclophosphamide) has been investigated. The data available suggest that there is a strong interaction between HER2 status and anthracycline-based chemosensitivity [47-50]. Of particular note, the National Surgical Adjuvant Breast and Bowel Project (NSABP) study B-ll examined the addition of doxorubicin to L-phenylalanine mustard plus 5-FU [48]. In HER2-positive patients the addition of doxorubicin improved outcomes to the extent that they were equivalent to those seen in HER2-negative patients. Furthermore, Cancer and Leukemia Group B trial 8541 demonstrated that HER2-positive but not HER2- negative patients benefit significantly from increased anthracycline dose intensity [50]. However, other studies have indicated that HER2 status has no value in predicting response to anthracycline-based chemotherapy [51-54]. It is important to stress that these studies used fewer patients than the studies that did indicate a correlation between HER2 status and chemosensitivity to anthracycline-based chemotherapy. In addition, one of the studies [51] used only one cycle of chemotherapy rather than the standard four or more cycles. Overall, although discrepancies were seen between different studies, it is important to note that none of the studies suggested that HER2 status was a predictor of resistance to anthracyclinebased chemotherapy. HER2 status as a predictor of response to CMF (cyclophosphamide, methotrexate, 5-FU) has also been examined. As with hormone-based therapy and anthracycline-based chemotherapy, controversy exists between different studies. While the majority of studies suggest that HER2-positive patients demonstrate resistance to CMF treatment [22, 30, 43, 55], a recent study has not supported thisfinding [56]. Conclusion The interval between Sir George Beatson's initial observations and the identification of ER overexpression in breast cancer illustrates the difficulty that can occur in identifying molecular factors that play a role in disease prognosis or response to therapy. ER is now well established as a biologic marker that provides prognostic and predictive information as well as a valid target for therapy. In recent years, the development of techniques based on molecular genetics has allowed wide screening of many potential oncogenes. In addition, sophisticated testing methods enable these factors to be identified relatively easily within tumor samples. However, the use of different methods and techniques within different establishments has increased the potential for inconsistencies to occur. Since HER2 was first identified as playing an important role in oncogenic transformation, many studies have investigated the potential of HER2 status to determine disease outcome. Generally, these studies have indicated that HER2 status can identify patients with reduced DFS and OS. However, inconsistencies between studies do exist and this is likely to be due in part to different testing methods used to determine HER2 status. The rlhc HER2 testing method has been developed to allow a quantitative analysis of HER2 overexpression to be made. The application of this method has demonstrated that 85% of breast cancer tumors express HER2

5 27 at levels greater than normal. Of these, 23% overexpress HER2 at levels higher than 15-fold greater than normal and this is associated with concurrent HER2-gene amplification. HER2 overexpression was also found to occur at all stages of the disease from DCIS to invasive disease, indicating that HER2 amplification occurs early in the development of the cancer. Addition of HER2 status, as determined by rlhc, to the NPI was found to be of benefit in predicting disease outcome, particularly in view of the fact that HER2 status is independent of other prognostic factors. At present the data regarding HER2 status as a predictor to therapy are unclear. Inconsistencies between HER2 testing methods and the relatively small patient populations used in different studies have contributed to the discrepancies. Generally, HER2 status has been shown to predict resistance to hormonal therapy and, in particular, tamoxifen therapy. The relationship between HER2 status and chemotherapy is less well defined. Several studies suggest that HER2 status predicts sensitivity to anthracycline-based treatment and resistance to CMF therapy, although other studies have found conflicting results. Although it has been generally accepted that HER2 does correlate with poor prognosis in breast cancer and may predict response to therapy, it will take large, quantitative, prospective studies to verify these hypotheses conclusively. As with ER status, it is likely that in the future the importance of HER2 status will be as a predictor of response to therapy. In addition, HER2 status is essential in stratifying patients to anti-her2 therapy regimens. Furthermore, HER2 receptor expression in invasive disease and DCIS suggests that anti- HER2 therapy could potentially be of therapeutic benefit for the treatment of breast cancer at an early stage in the disease. 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Proc Am Soc Clin Oncol 1999; 18: 69a (Abstr 257). Correspondence to: T. Cooke, MD University Department of Surgery Royal Infirmary Queen Elizabeth Building Alexandra Parade Glasgow G31 2ER UK t.cooke@clinmed.gla.ac.uk