Circulating oncoproteins
HER2/neu, EGFR and CAIX (MN)
as novel cancer biomarkers
Walter P Carney
Siemens Medical Solutions Diagnostics, Cambridge, MA 02142, USA Tel.: +1 617 441 4409 Fax: +1 617 492 8438 walter.carney@siemens.com KEYWORDS:
biomarker, breast cancer, CAIX, CA9, cancer, EGFR, HER1, HER2/neu, MN, oncoprotein, personalized medicine, targeted therapy
Pharmaceutical companies have developed targeted therapies such as trastuzumab and lapatinib for human epidermal growth factor receptor (HER)2/neu-positive tumors, while others have developed antiepidermal growth factor receptor (EGFR) therapies, such as tarceva and erbitux for EGFR-positive tumors. A drug called rencarex is targeted to an oncoprotein designated carbonic anhydrase IX (CAIX), which is being evaluated in renal cell carcinoma patients. Based on these targeted therapeutic approaches, this review describes clinical research studies performed with enzyme-linked immunosorbent assays specific for the circulating oncoproteins, HER2/neu, EGFR and CAIX. These circulating biomarkers have the potential to be used in conjunction with the specific targeted therapies for patient selection, monitoring and management. With the variety of new therapeutic options, the major challenge ahead will be to select the appropriate therapy or combinations of therapies for each patient. Specific biomarker tests, either alone or in panels, will be needed at the appropriate time in the course of disease to ensure that patients receive the right drug at the right time. These tests will also be valuable in monitoring the efficacy of the targeted therapies. A circulating biomarker such as serum HER2/neu may be able to specifically identify patients with progressing HER2/neu-positive disease and provide the information needed by physicians to choose from the variety of HER2/neu-targeted therapies that will soon be available to cancer patients.
Expert Rev. Mol. Diagn. 7(3), 309–319 (2007)
Monitoring of HER2/neu oncoproteins levels Studies have demonstrated that the extra-cellular domain (ECD = p97 – 115KDa) of the human epidermal growth factor receptor (HER)2/neu p185 is cleaved from cancer cells, and can be measured in serum of normal indi-viduals and cancer patients [1]. Zhou and col-leagues have reported that ECD cleavage is mediated by disintegrin and metalloproteases (ADAMs). ADAM 17 is thought to be the major ErbB ligand sheddase and that ADAMs regulate the availability of ErbB ligands by lib-erating the soluble forms that activate ErbB signaling [2]. The test known as serum HER2/neu, is the only test cleared by the US FDA for monitoring changes in serum HER2/neu levels in patients with metastatic breast cancer (MBC), and is used clinically for the management and monitoring of patients
with MBC [3–7]. Numerous studies have estab-lished that serum HER2/neu values under 15 ng/ml are normal, while levels greater than 15 ng/ml are considered elevated and may be a way to track clinical changes in patients with HER2/neu-positive tumors. Many publica-tions have demonstrated that monitoring serial changes in serum HER2/neu levels in MBC patients paralleled the clinical course of dis-ease, regardless of whether the therapy was hormone therapy, chemotherapy or the combi-nation of trastuzumab and chemotherapy [1,3–7]. Patients with MBC who were treated with trastuzumab-based therapies (trastuzu-mab plus various combinations of chemother-apy) had serial changes in serum HER2/neu levels that correlated with clinical course of disease [5,6,8]. FIGURE 1 is a typical example of a MBC patient treated with trastuzumab-based CONTENTS
Monitoring of HER2/neu oncoproteins levels Serum HER2/neu levels under 15 ng/ml are associated with improved clinical outcome Circulating EGFR oncoprotein as a cancer biomarker Circulating CAIX oncoprotein as a cancer biomarker Expert commentary Five-year view Key issues References Affiliation
therapy and the changes in serum HER2/neu as the patient responded or progressed during therapy. Many studies have demonstrated that increasing levels reflect progression, while decreasing levels reflect response to therapy. Therefore, the serum HER2/neu test may be specifically tailored to monitoring patients with HER2/neu-positive disease, and with the develop-ment and approval of such anti-HER2/neu therapies such as lapatinib (also known as Tykerb®, GlaxoSmithKline, PA, USA), the test can also provide real-time information with respect to the patient’s response or progression. Since increasing levels reflect progression, monitoring levels may provide the physi-cian with reason to consider alternative HER2/neu-directed therapies in conjunction with traditional therapies.
Serum HER2/neu levels under 15 ng/ml is associated with improved clinical outcome
Some studies suggest that clinical outcomes can be improved by maintaining serum HER2/neu levels under 15 ng/ml. Schip-pinger reported that, in 286 MBC patients, those with contin-uously elevated (>15 ng/ml) serum HER2/neu levels had a sig-nificantly poorer survival after disease recurrence compared with patients with levels continuously or temporarily under 15 ng/ml. A decrease of serum HER2/neu levels to less than 15 ng/ml or levels continuously under 15 ng/ml during the course of disease correlated with significantly longer survival [4]. In a 2005 report by Lipton and colleagues, it was demonstrated that conversion to HER2/neu levels over 15 ng/ml at the time of disease progression on first-line hormone therapy correlated with significantly shortened survival. The median survival from initiation of therapy was 47.8 months for patients with HER2/neu levels continuously less than 15 ng/ml. In patients who had an increase in serum HER2/neu at the time of disease
progression, the median survival was 26.5 months. The survival of patients who changed from less than 15 ng/ml to greater than 15 ng/ml did not differ significantly from the survival of patients had levels over 15 ng/ml from the start of therapy (median = 20.8 months). Survival from the time of disease pro-gression was longest (28.1 months) for patients whose levels remained under 15 ng/ml, and shortest (12.5 months), for patients whose levels remained elevated (>15 ng/ml). However, patients who converted from less than 15 ng/ml to greater than 15 ng/ml had a median survival of 15.2 months [9].
In the pooled analysis of over 250 MBC patients from seven sites presented at the 2006 American Sociey for Clinical Oncol-ogy (ASCO) meeting, patients were divided into two groups (TABLE 1)[10]. Group 1 consisted of 156 MBC patients who had pretreatment serum HER2/neu levels greater than 15 ng/ml and continued to have serum HER2/neu levels greater than 15 ng/ml at first follow-up (between 16 and 120 days). The median time to progression (TTP) for this group was 224 days. Group 2 consisted of 147 patients who had either pretreatment samples of greater than or less than 15 ng/ml. In group 2, if serum HER2/neu remained less than 15 ng/ml or dropped to less than 15 ng/ml at first follow-up, the median TTP was sig-nificantly higher at 334 days (p = 0.0003). With respect to median survival (TABLE 2), the 121 patients who were initially greater than 15 ng/ml at baseline and who remained greater than 15 ng/ml at first follow-up had a median survival of 618 days. In contrast, the 111 patients who had a pretreatment value of greater than 15 ng/ml or less than 15 ng/ml and who either remained less than 15 ng/ml or dropped to less than 15 ng/ml had significantly (p = 0.003) longer median survival of 1030 days. In summary, patients who remained less than 15 ng/ml or who decreased to less than 15 ng/ml during ther-apy had a significantly longer median TTP and longer median survival. In a poster presented at the 2006 San Antonio Breast Cancer Conference, Dinocca and col-leagues measured serum HER2/neu in 145 MBC patients, and demonstrated that the median survival for patients greater than 15 ng/ml was 14.5 months while median survival for patients less than 15 ng/ml was 35.5 months (p = 0.001) [11].
Collectively, these four studies suggest that maintaining serum HER2/neu levels less than 15 ng/ml may be a target for managing patients with HER2/neu-posi-tive breast cancer that can lead to better clinical outcomes [4,9,10,11].
Decline in serum HER2/neu levels predicts response to treatment
In a report by Koestler and colleagues, serum HER2/neu samples were measured before and 30 days after initiation of trastuzumab and chemotherapy. Koestler Figure 1. Represents a metastatic breast cancer patient monitored for 93 weeks in which serum
HER2/neu levels were plotted against the patients’ clinical course of disease.
HER: Human epidermal growth factor receptor.
0 10 20 30 40 50 60 70 80 90 100 0 25 50 75 100 125 150 175 200 Recurrence
Time from start of herceptin and chemotherapy (weeks)
Serum HER2/neu (ng/ml)
Responding Stable
and colleagues demonstrated that a significant decrease in serum HER2/neu from the pretreatment or baseline sample to the post-treatment sample at 30 days was an early predictor of outcome to trastuzumab therapy [8]. Koestler and colleagues identified the kinetics of serum HER2/neu levels as the only factor that enabled the accurate prediction of response. Koestler and colleagues also reported that serial changes in serum HER2/neu levels paralleled the clinical course of disease and preceded clinical changes, which enabled a significant predic-tion of response, clinical benefit and progression-free survival in the early weeks of trastuzumab-based treatment [8]. Sub-sequent reports by Esteva and colleagues [12], Fornier and col-leagues [13], Bethune-Volter and colleagues [14], Tse and col-leagues [15], Ali and colleagues [10] and Kashiwaha and colleagues [16] strongly support the observation that a signifi-cant decrease in serum HER2/neu from the pretreatment level to a 30-day post-treatment level is a predictor of outcome to trastuzumab-based therapy.
At the 2006 ASCO meeting, Ali and colleagues also dis-cussed the multicenter (seven clinical sites) multinational study of 307 MBC patients who were monitored for changes in serum HER2/neu levels during trastuzumab-based thera-pies. Serum HER2/neu levels at baseline were compared with serum HER2/neu levels 30 days after therapy initiation. Patients with more than 20% decreased serum HER2/neu had significantly better response rates, duration of response, TTP and overall survival [10]. The MBC patients with a less than 20% drop in serum HER2/neu had a lower response rate, shorter duration of response, shorter TTP and decreased over-all survival. The 20% cut-off point was confirmed in a 2006 study presented by Kashiwaha and colleagues at the San Anto-nio Breast Cancer conference; however, the investigators did not indicate the number of patients in the study [16]. Ali and colleagues concluded that patients who do not drop by at least 20% by day 30 may be the patients who could benefit from additional anti-HER2/neu therapies. Currently, there are at least a dozen new tyrosine kinase inhibitors, such as lapatinib, in clinical development.
Monitoring MBC patients receiving lapatinib
Lapatinib is an oral dual tyrosine kinase inhibitor that targets HER2/neu- and epidermal growth factor receptor (EGFR)-positive tumors in MBC. At the 2004 ASCO meeting, Black-well and colleagues reported that a decrease in serum HER2/neu levels at 4 and 8 weeks after initiation of lapatinib in MBC patients correlated with therapy response and was an early indicator of clinical response to lapatinib [17]. In a poster presented at the 2006 San Antonio Breast Cancer Conference, serum HER2/neu decreases at 4, 8 and 12 weeks in HER2/neu-amplified MBC patients were associated with clini-cal response to lapatinib and may also be a biomarker for increased response to lapatinib therapy [18]. Currently, serum HER2/neu monitoring studies are ongoing in many of the lap-atinib clinical trials in both the adjuvant and MBC setting with and without trastuzumab.
Pretreatment serum HER2/neu levels associated with poor response to therapies
In 1989, it was demonstrated by immunohistochemistry (IHC) that MBC patients with HER2/neu overexpression had a decreased response rate to first-line hormone therapy [19]. Since that report, several studies have shown that pretreatment serum HER2/neu levels over 15 ng/ml are associated with a signifi-cantly lower response rate to endocrine therapy [20–22]. In stud-ies by Lipton and colleagues, MBC patients with pretreatment serum HER2/neu levels greater than 15 ng/ml had a shorter duration of response, shorter TTP and shorter overall survival when compared with patients with serum HER2/neu levels with less than 15 ng/ml [21]. Sandri and colleagues reported that pretreatment levels of serum HER2/neu greater than 15 ng/ml were associated with patients that had more aggressive tumors and less response to chemotherapy [23]. Colomer and colleagues demonstrated that patients with pretreatment levels over 15 ng/ml were less responsive than patients with serum HER2/neu under 15 ng/ml [24]. Several additional publica-tions demonstrate that elevated pretreatment serum HER2/neu levels are associated with poor clinical outcome [1].
Frequency of MBC patients with elevated serum HER2/neu levels
To determine the prevalence of MBC patients with elevated serum HER2/neu levels, we reviewed publications that reported elevated levels of HER2/neu [1]. However, the draw-back to reviewing many older reports of HER2/neu levels is that many studies used invalidated, homebrew assays or assays that were not standardized according to diagnostic procedures. Results with such assays have contributed a considerable amount of confusion in the literature about the clinical value of measuring and monitoring serum HER2/neu levels.
Table 1. Serum HER2/neu and median time to progression. Group n Baseline serum level (ng/ml) First follow-up (ng/ml) Median time to progression (days) 1 156 ≥15 >15 224 2 147 <15 or >15 <15 304* *p = 0.0003.
Table 2. Serum HER2/neu and median survival. Group n Baseline serum
level (ng/ml) First follow-up (ng/ml) Median survival (days) 1 121 ≥15 >15 618 2 111 <15 or >15 <15 1030* *p = 0.003.
We reviewed 50 publications representing 5044 patients with MBC and found that, on average, 48.6% (range: 23–80%) had elevated HER2/neu levels, whereas 18.1% (0–30.8%) of patients with primary breast cancer (PBC) had elevated serum HER2/neu levels. Recently, using the FDA-cleared and stand-ardized serum HER2/neu assay, Manouni and colleagues dem-onstrated that 28.2% of patients with PBC had elevated serum HER2/neu levels [25]. In over 40% of the publications, 50% or more of the MBC patients had elevated serum HER2/neu levels [1]. Several publications report over 60% of MBC patients can have an elevated serum HER2/neu level of greater than 15 ng/ml [7,26–28]. The broad variability observed in circulating serum HER2/neu levels may be due to the use of assays that are not validated or standardized, and employed uncharacterized antibodies in the assays.
HER2/neu status of the primary breast cancer
Current treatment of MBC patients with HER2/neu-positive tumors is based on the HER2/neu status derived from IHC or fluorescent in situ hybridization (FISH) analysis of the primary tumor, which is generally removed and stored for many years. In a report by Zidan and colleagues, it was noted that HER2/neu status of the primary tumor may not accurately reflect the HER2/neu status of MBC and that this should be taken into account when making treatment decisions [29]. This report demonstrated 14% discordance between primary and metastatic tumors by IHC. A total of 12% (seven of 58) of the patients were HER2/neu positive in the metastatic tumor and negative in the corresponding primary tumor. Interestingly, three of the seven patients negative in primary, but positive in the metastatic lesion, responded to trastuzumab-based therapy. The Zidan publication reviewed several other references that clearly confirmed the existence of HER2/neu-negative primary breast tumors with a HER2/neu-positive metastatic tumor from the same patient [30–33]. Current medical practice only allows patients with a HER2/neu-positive tumor by IHC or FISH to be candidates for trastuzumab. The importance of the Zidan and colleagues paper is that it clearly illustrates that patients HER2/neu negative in the primary tumor should either have the primary tumor re-evaluated at the time of MBC or have a metastatic lesion tested by IHC or FISH. If either sample is positive, then the patient may be a candidate for trastuzumab and chemotherapy.
Thus, the recommendation by Zidan and colleagues is that the discordance in HER2/neu status between primary and meta-static tumor should be considered in treatment decisions and should be taken seriously [29].
Comparison of serum HER2/neu levels in MBC
In several reports, the HER2/neu status of the primary breast tumor was compared with serum HER2/neu levels in the same patients who developed MBC. Many reports [34–36], but not all reports [14,37], have shown a strong concordance between patients with a positive HER2/neu status of the pri-mary breast tumor (by IHC and FISH) with elevated (>15 ng/ml)
HER2/neu levels in the same patients when they developed MBC. Similar results have been reported by Tse and colleagues [35], Mueller and colleagues [36] and more recently by Kong and colleagues [28]. Kong and colleagues reported a strong correla-tion between serum HER2/neu levels in MBC patients and HER2/neu status of the primary tumor, as determined by IHC or FISH. In the Kong and colleagues publication, it was also demonstrated that the HER2/neu tissue-negative group had a median serum HER2/neu value of 22.2 ng/ml, while the tis-sue-positive group had a median serum HER2/neu value of 363 ng/ml [28]. It appears from recent data that there is approx-imately an 80–90% correlation between a positive HER2/neu tissue status and elevated serum HER2/neu levels greater than 15 ng/ml. Based on these results, patients with HER2/neu pos-itivity in the primary tumor might be excellent candidates to be monitored for increasing serum HER2/neu levels post surgery as a means of detecting early breast cancer recurrence.
Conversion of a negative HER2/neu status to positive
Several publications have found populations of patients with PBC who were designated HER2/neu negative by tissue test-ing, but who developed elevated serum HER2/neu levels in MBC. This may indicate the presence of HER2/neu-positive tumors [1]. For example, Andersen and colleagues demonstrated that 28 of the 82 patients (34%) who had IHC-negative PBC developed elevated serum HER2/neu levels during MBC [38]. Fehm and colleagues also reported that 34% of patients desig-nated HER2/neu-negative by tissue testing had elevated serum HER2/neu levels at the time of MBC [27]. The observation that a population of patients with a HER2/neu-negative primary tumor can develop elevated serum levels at MBC is consistent with Zidan and colleagues, who demonstrated that a popula-tion of patients exists with a negative HER2/neu status on the primary tumor, but a positive HER2/neu status on the corre-sponding metastatic tumor [29]. Therefore, MBC patients with an elevated serum HER2/neu, but a negative HER2/neu status by tissue testing, may benefit from a re-analysis of their primary breast tumors by IHC and FISH. However, if the re-test of the original tumor is still HER2/neu negative, then a metastatic lesion could be evaluated by IHC or FISH to determine HER2/neu positivity.
To date, studies have not been performed based on an ele-vated serum HER2/neu from a MBC patient and a normal HER2/neu tissue test. However, as reported by Zidan and col-leagues, three of the seven patients who had a negative HER2/neu status on the primary breast tumor, but a positive metastatic lesion, responded to trastuzumab. It seems likely that once serum HER2/neu is more widely accepted as a surrogate marker for HER2/neu-positive tumors, then studies will be conducted to clearly determine whether serum HER2/neu can be used as a marker to select patients for HER2/neu-directed therapies. However, it should be clearly stated that the use of the serum HER2/neu test is not approved for placing patients on trastuzumab, nor should it be proposed until sufficient data are available to support such an indication.
HER2/neu is more than another tumor marker
Several reports suggest that serum HER2/neu levels are associ-ated with tumor burden, HER2/neu overexpression and receptor activation. Molina and colleagues have demonstrated that cleav-age of the ECD leads to increased phosphorylation of the intra-cellular tyrosine kinase. This observation suggests that circulating HER2/neu levels are not only a marker of tumor overexpression, but also indicative of the degree of receptor activation [39].
Ali and colleagues [40] and Fehm and colleagues [41] examined the question of whether serum HER2/neu reflects tumor aggressiveness or is simply a surrogate marker of disease bulk. In the Ali and colleagues report, pretreatment samples were tested in 566 MBC patients for serum HER2/neu levels as well as levels of the surrogate marker of tumor burden, cancer anti-gen (CA)15-3. The Ali and colleagues report concluded that the serum HER2/neu level is a significant independent predic-tive and prognostic factor in hormone receptor-posipredic-tive MBC patients, even when adjusted for tumor burden using CA15-3. In addition, Fehm and colleagues, using a multivariate analysis, also concluded that serum HER2/neu was still an independent marker of tumor aggressiveness, when serum HER2/neu results were adjusted for tumor load with CA15-3 and reflects the bio-logical behavior of the tumor [41]. Several other studies have also concluded that the combination of serum HER2/neu and CA15-3 identifies a subset of patients with a worse prognosis when compared with one marker alone. However, it has also been reported several times that there is a weak correlation between serum HER2/neu and CA15-3, but each is providing important information about the biology of the tumor [42]. Monitoring serum HER2/neu for the detection of
early recurrence
Several publications have demonstrated that monitoring serum HER2/neu levels after surgery in patients with HER2/neu-posi-tive tissue may be beneficial in the earlier detection of breast can-cer recurrence. Overall, a search of the literature found approxi-mately 12 publications that reported an increase in serum HER2/neu levels that could be measured 2–9 months before the actual clinical diagnosis of recurrent breast cancer [42]. Therefore, it is possible that monitoring serum HER2/neu levels in all tissue-positive patients might be a way of detecting breast cancer recurrence earlier.
Fehm and colleagues reported that 27% of breast cancer patients with recurrence had elevated serum HER2/neu levels 6 months before clinical signs or clinical diagnosis [27]. This number increased to 50% of the patients who had elevated serum HER2/neu levels at 3 months before clinical diagnosis. Isola and colleagues reported that 37% of patients could be predicted to have recurrent breast cancer on the basis of increasing serum HER2/neu levels 6 months before the actual clinical diagnosis [43]. Clinical value of serum HER2/neu levels in MBC patients HER2/neu status is an important piece of information that can guide therapy of patients with HER2/neu-positive breast cancer, and therefore accurate determination of HER2/neu status by
IHC or FISH is essential to providing appropriate treatment. In addition to tissue testing, the serum HER2/neu test may benefit patients in the following ways:
• An elevated serum HER2/neu, but a negative HER2/neu in the tumor tissue, may be reason to re-assess the original tumor or a metastatic lesion for HER2/neu positivity. If either is IHC positive or FISH amplified, it is sufficient evi-dence to consider the patient for trastuzumab-based therapy; • Serum HER2/neu may be used to monitor MBC patients receiving HER2/neu-directed therapies for response or lack of response;
• Since serum HER2/neu measures the circulating ECD, it may also serve as a specific biomarker for HER2/neu positive disease. Circulating EGFR oncoprotein as a cancer biomarker
The EGFR oncogene encodes a transmembrane tyrosine kinase receptor that demonstrates extensive homology with the HER2/neu oncoprotein. EGFR is a 170-kDa membrane-bound protein expressed on the surface of epithelial cells with an ECD (p110 kDa) that is shed from the surface of normal and cancer cells and shown to circulate in normal people and in cancer patients [44]. In a 1999 report by Baron and colleagues, a specific EGFR immunoassay was used to measure circulating levels of p110 EGFR in women with epithelial ovarian cancer (EOC) [45]. The studies demonstrated that women with Stage III and IV EOC had significantly lower levels of p110 than age-matched controls.
To facilitate clinical research, we developed and commercial-ized an enzyme-linked immunosorbent assay (ELISA) for research use only to measure circulating EGFR levels in clinical samples. In a report presented in 2002, we established normal levels of circulating EGFR p110 in both males (61 ng/ml) and females (62 ng/ml) [46]. From our studies, we were able to estab-lish 45 ng/ml as the lower limit of normal and 78 ng/ml as the upper limit of normal (ULN). We used this ELISA to study p110 levels in samples from several cancer types.
FIGURE 2 illustrates the circulating EGFR levels in women with benign ovarian conditions or various stages of ovarian cancer. In the benign ovarian sera, 8% had levels below the normal range, while 29% of the Stage I ovarian cancer sera samples were below normal, 50% of the Stage II samples were below normal, 74% of the Stage III samples below normal, while Stage IV ovarian cancer sera showed 78% of the samples below normal. These results show that, as the stage of ovarian cancer increased, the percentage of serum samples that were less than normal increased, reflecting a correlation of increased stage of ovarian cancer with a decrease in serum EGFR levels.
We also evaluated samples from patients with advanced cancers (TABLE 3) [45–46]. Possible explanations for diminished levels of EGFR levels in advanced cancers could be to the for-mation of homo- or heterodimers of erbB family members that mask the epitopes recognized by the EGFR assay [2]. It is also possible that autoantibodies complexed with the EGFR ECD
may mask epitopes recognized by the EGFR assay. It is also possible that, wih advanced cancer, ECD EGFR levels decrease due to lower membrane levels of the EGFR.
In a report by Gregorc and colleagues, circulating EGFR levels were evaluated in advanced non-small-cell lung cancer patients treated with the anti-EGFR inhibitor known as gefit-inib (Iressa®, Astra Zeneca). They reported that baseline EGFR levels and serum changes in EGFR levels during therapy were associated with response to gefitinib and progression-free sur-vival. In fact, during gefitinib therapy, 80% of responders had a decrease in EGFR serum levels, while 88% of patients with pro-gressive disease had an increase in EGFR levels. Gregoric and colleagues reported that early changes in serum EGFR levels at day 28 of treatment appeared to be an early indicator of response versus resistance. These data suggest that monitoring serum EGFR levels may be informative for patients receiving anti-EGFR-targeted therapies, such as gefitinib. According to Gregorc and colleagues, patients with higher pretreatment EGFR serum levels responded more frequently, suggesting that circulating EGFR may reflect the need of functional activation of the EGFR pathway in responding patients. Although base-line EGFR serum levels appeared to be associated with response, a clinically relevant cut-off was not reported in this study [47].
Additional studies must be performed to determine whether circulating p110 levels have clinical utility in patients receiving anti-EGFR therapies, such as tarceva, or monoclonal-based EGFR therapies, such as erbitux.
In a report by Marx and colleagues, pre-treatment serum samples were evaluated for EGFR levels in 265 postmenopausal MBC patients treated with hormone ther-apy [48]. Normal controls consisted of two groups of females. Group 1 consisted of 46 premenopausal women with a mean p110 level of 79.54 ± 9.87 ng/ml, with a range (mean ± 2 standard deviation [SD]) of 59.80–99.28 ng/ml. Group 2 consisted of 13 postmenopausal women with a mean p110 value of 89.87 ± 16.40 ng/ml, with a range (mean ± 2 SD) of 57.07–122.67 ng/ml. For the control groups, the mean serum p110 level was significantly higher in postmeno-pausal females (i.e., 89.87 ± 16.40 ng/ml; n = 13) compared with the mean p110 level in premenopausal females (i.e., 79.54 ± 9.87 ng/ml; p = 0.006; n = 46). Analysis of the serum EGFR levels of the 265 postmenopausal cancer patients revealed a mean serum EGFR level of 66.2 ng/ml, which was significantly lower than that of the postmenopausal female control group (n = 13; p 0.00001). Using a cut-off point of 57.07 ng/ml (mean ± 2 SD) from the postmenopausal female control group, 71 of 265 of the cancer patients (26.8%) had a decreased serum EGFR levels, compared with none of the 13 controls. The clini-cal benefit (i.e., complete response plus partial response plus sta-ble decrease at 24 weeks) was significantly less in patients with a decreased serum p110 level (p = 0.04). Patients with decreased serum EGFR p110 levels had a shorter TTP (median 3.5 months) compared with patients with normal serum EGFR p110 levels (median 6.4 months; p = 0.04). In addition, the patients with decreased serum EGFR p110 levels (median 21.8 months) trended toward a shorter overall survival com-pared with patients with normal serum EGFR (27.8 months; p = 0.06). These results demonstrate that pretreatment serum EGFR p110 levels were significantly decreased in MBC patients compared with healthy controls, and patients with decreased serum EGFR p110 levels had reduced clinical benefit, TTP and overall survival compared with patients with normal serum EGFR p110 levels (FIGURE 3).
In the Mueller and colleagues study, serum samples were col-lected from 110 MBC patients before and after treatment with first-line chemotherapy, which included epirubicin and either cyclophosphamide or paclitaxel. In this report, the lower level of normal was shown to be 45 ng/ml and consistent with our 2002 report. Serum EGFR serum levels were below 45 ng/ml in 15 of the 110 MBC patients (11%). Patients with EGFR serum levels below 45 ng/ml showed a trend of shorter overall survival (median 11.7 vs 15.4 months; p = 0.08), which was more pronounced and significant in patients with estrogen receptor-positive primary
Table 3. Percentages of cancer patients with elevated or reduced epidermal growth factor receptor levels. Cancer type Reduced EGFR
levels (%) Elevated EGFR levels (%) Ref. Lung 42 0 Bladder 56 0 Prostate 44 7
Breast (Stage IV) 32 2
Colon 67 0 [45]
EGFR: Epidermal growth factor receptor.
Figure 2. Serum epidermal growth factor receptor levels were evaluated patients with benign ovarian disease or patients with various stages of ovarian cancer.
8 29 50 74 78 0 10 20 30 40 50 60 70 80 90
Benign Stage I Stage II Stage III Stage IV
tumors (median 9.6 vs 15.4 months; p = 0.022). Patients with low serum EGFR levels and elevated serum HER2/neu levels (>15 ng/ml) demon-strated a shorter overall survival than those with normal values (7.1 vs 15.4 months; p = 0.03). Again, the difference was higher in patients with estrogen receptor-positive tumors (4.6 vs 15.4 months; p < 0.0001). Low EGFR serum levels were associated with a decreased overall survival, espe-cially in patients with estrogen receptor-positive tumors. When these investiga-tors used a cut-off value in the context of prognostic impact, they found that the EGFR serum value of 44 ng/ml was the best discriminating value [49].
Since there are clinical studies using a combination of EGFR and HER2/neu-directed therapies in the same patients, Muller and colleagues evaluated a com-bined analysis of serum EGFR and serum HER2/neu levels in MBC patients. They
reported that patients with a low serum EGFR level and elevated serum HER2/neu level (>15 ng/ml) had a significant lower median survival than patients with normal EGFR and HER2/neu levels. In patients with estrogen receptor-positive primary tumors, this difference was again more pronounced and significant. In a similar manner, Souder and colleagues reported the combined use of EGFR and HER2/neu at the 2005 ASCO meeting and recently published their findings [50]. In this report, serum EGFR levels from 117 healthy
post-menopausal female controls measured 64.1 ± 13.3 ng/ml (mean ± SD), with a range of 39.5–117.1 ng/ml. Similar to those of Muller and colleagues, a cut-off point of 44.1 ng/ml was reported for the control group (5% nonparametric method). Their data, which were very similar to Mueller and colleagues, demonstrated that 62 of 521 patients (11%) had decreased serum EGFR levels. However, patients with decreases in serum EGFR had no signifi-cant difference in objective response rate, clinical benefit rate (CBR), TTP or time to treatment failure (TTF) compared to first-line hormone therapy, but did have signifi-cantly reduced survival compared with patients with normal serum EGFR levels (median survival 22.8 vs 30.4 months; p = 0.006). Combined analysis of pretreat-ment serum EGFR and HER2 yielded no additional predictive information for over-all response rate, clinical benefit, TTP and TTF compared with serum HER2 alone.
However, combined serum EGFR and HER2 analysis identified a subgroup of patients with decreased serum EGFR and normal serum HER2 (n = 38 of 521; 7.3%) who had significantly reduced survival compared with patients with normal serum levels of both EGFR and HER2 (median survival 22.9 vs 39.9 months; p = 0.007). In a multivariate analysis, decreased serum EGFR level remained a significant independent prognos-tic factor for decreased survival (hazard ratio [HR] = 1.59;
Figure 4. Serum HER2/neu and EGFR levels were measured metastatic breast cancer patients and then classified by low, high or normal levels.
EGFR: Epidermal growth factor receptor; HER: Human epidermal growth factor receptor.
0 365 730 1095 1460 0.00 0.25 0.50 0.75 1.00 Time (days)
Probability of progression from start of therapy
Normal EGFR and HER2 Low EGFR and normal HER2 Normal EGFR and elevated HER2 Low EGFR and elevated HER2
Figure 3. Serum EGFR levels were measured in metastatic breast cancer patients and stratified in patients with normal EGFR levels of levels below normal.
EGFR: Epidermal growth factor receptor.
Time (months) 0 12 24 36 48 0.00 0.25 0.50 0.75 1.00 Probability of progression p = 0.04 EGFR normal (n = 196) EGFR decreased (n = 69)
p = 0.005). Other significant variables for decreased survival included the presence of liver metastasis (HR = 2.31; p≤0.0001) and elevated serum HER2/neu (HR = 1.80; p≤0.0001), while positive hormone receptor status lowered the risk of mortality demonstrated that, in MBC patients, decreased serum EGFR and normal serum HER2/neu pre-dicted shorter survival compared with normal levels of serum EGFR and HER2/neu [48]. The survival of the decreased serum EGFR/normal serum HER2/neu patients was similar to that of patients with elevated serum HER2/neu (FIGURE 4).
Studies by Gregorc and colleagues [47], Mueller and col-leagues [49] and Souder and colleagues [50] suggest that combin-ing serum EGFR and HER2/neu information may enable the selection patients for combined EGFR and HER2/neu-tar-geted therapies. Additional studies must be performed to understand the changes in serum EGFR levels that could occur in patients receiving the variety of anti-EGFR therapies, such as gefitinib, tarceva and erbitux. Changes in serum EGFR levels may be clinically useful in a similar way that serum HER2/neu is with conventional (hormone therapy or chemotherapy) or trastuzumab-based therapies.
Circulating CAIX oncoprotein as a cancer biomarker
The transmembrane protein MN, also termed CAIX, is a mem-ber of the large family of enzymes known as carbonic anhy-drases, which share the ability to catalyze the reversible hydra-tion of carbon dioxide to carbonic acid, leading to a decrease in pH. Upregulation of CAIX gene expression occurs under hypoxic conditions and is believed to be involved in sensing and maintaining the acidic environment of hypoxic cells, par-ticularly in hypoxic regions within tumors. Normal expression of CAIX protein is restricted to primarily gastric, intestinal and liver mucosa. Using IHC and reverse-transcriptase PCR,
significant levels of CAIX protein have been detected in a vari-ety of tumors, including those of the kidney, cervix, lung, blad-der, colon, breast, liver, gall bladder and pancreas [51–58]. How-ever, studies have shown that the CAIX oncoprotein can be detected in bodily fluids. Using a western blot assay, Zavada and colleagues, were able to detect CAIX protein in preconcen-trated serum and urine at approximately 35 pg/ml in controls and 20–3600 pg/ml in renal cell carcinoma patients [59]. How-ever, until now, an ELISA has not been commercially available for measuring circulating levels of the CAIX oncoproteins or for examining the clinical value of CAIX levels in cancer patients. Therefore, to explore the clinical potential of circulat-ing CAIX oncoprotein levels, we have developed an ELISA for research use only that specifically measures the CAIX onco-protein in human samples. The ELISA is a typical sandwich assay in a microtiter format that uses two monoclonal anti-bodies directed at different CAIX epitopes and a recombinant human CAIX protein for assay standards. After confirming that the analytical performance criteria were met for this ELISA, we used this research product to investigate levels of the circulating CAIX in clinical samples. We have evaluated samples from nor-mal individuals and samples from a variety of cancer types. Our preliminary studies show a mean level of the circulating MN oncoprotein of 221 pg/ml in 50 normal females and 289 pg/ml in 50 normal males. We observed circulating levels in a variety of cancers. FIGURE 5 illustrates results from normal as well as ovarian and renal cell cancer patients. These results demon-strate that there is a normal level of the CAIX circulating pro-tein and that some cancer patients have elevated levels of the CAIX oncoprotein.
In an abstract presented at the American Association of Cancer Research by Leitzel and colleagues, pretreatment serum CAIX levels were measured in 147 MBC patients enrolled in a Phase III second-line hormone therapy trial and in 50 healthy postmenopausal female controls [60]. Serum CAIX levels from the 50 controls had a mean ± SD of 244 ± 131 pg/ml. The ULN was defined as the mean ± 2 SD (506.5 pg/ml). Only three of 50 control subjects (6%) had elevated serum MN levels above the ULN. Pretreatment serum CAIX levels were elevated above the ULN in 32 of 147 (22%) of MBC patients. Serum CAIX levels were also significantly elevated in the MBC patients compared with the control group (p < 0.0001).
The measurement of serum CAIX levels deserves further study to determine its predictive and prognostic biomarker potential in MBC patients. Future studies should focus on determining if circulating CAIX levels have clinical value when examined in the context of such parameters as TTP and overall survival. Studies will also investigate whether CAIX levels can be used to identify patients with CAIX-positive tumors as well as to select patients for CAIX-directed therapy. This ELISA may also have value in monitoring the efficacy of CAIX-directed therapies. If such a clinical utility is proved, then one can envi-sion not only combining HER2/neu-, EGFR- and MN-directed therapies, but combining these tests for personalized management of such targeted therapies.
Figure 5. Circulating levels of carbonic anhydrase IX measured in normal males, normal females, ovarian cancer patients and patients with renal cell carcinoma. Normal male Normal female Ovarian cancer Renal cell 0 2000 4000 6000 7000 10000 13000 Carbonic anhydrase IX (pg/ml)
Expert commentary
As we move closer to personalized treatment of tumors based on molecular mechanisms, the need for specific biomarker tests related to new medicines will grow dramatically. One can envision the use of biomarkers at all stages of cancer to manage treatment. The ideal situation is to have detectable and meas-urable biomarkers based on biochemical mechanisms of the drug that can be used for early detection of the primary cancer as well as early detection of recurrence. Circulating biomarkers may be available for neoadjuvant and adjuvant treatment for high-risk recurrence as well as treatment of patients with meta-static disease. The goal should be to bring new therapies such as trastuzumab to HER2/neu-positive patients at the earliest possible time and to attack the smallest possible tumors with the most specific therapies available before the tumor has chance to stimulate angiogenesis and spread. To achieve this, specific tests that can be used with blood, serum or plasma are needed. Circulating biomarkers will provide real-time infor-mation about the patient and the patient host response to the tumor, but, more importantly, these tests will fit nicely into the medical setting. The most commonly performed tests involve a blood sample. The advantage of a blood test is that it is real time and generally very simple to obtain and analyze. As the pharmaceutical industry strives to develop more specific therapies, diagnostic companies need to work with pharma-ceutical companies to develop the necessary specific tests to implement personalized cancer treatment. Diagnostic compa-nies and pharmaceutical compacompa-nies also must work closely to develop win–win scenarios, not only for their respective com-panies, but more importantly for the cancer patient. Early
detection may lead to earlier therapeutic intervention, which will hopefully benefit patients by longer survival and an improved quality of life.
Five-year view
At present, patients are selected for targeted therapies based on analysis of the primary tumor. However, the majority if not all new cancer medicines are evaluated in patients with metastatic disease. In order to increase the efficacy of each new medicine, we will need to stratify and select patients with the highest prob-ability of response to the new drug. Currently, patients are selected for therapies such as trastuzumab based on analysis of the tumor tissue removed years earlier. The ideal situation will be to draw a blood sample and to use circulating biomarkers (DNA, RNA, protein or cells ) to tailor the best therapy for the patient. In the next 5 years, I believe the use of circulating biomarkers will increase at the time of therapy initiation to select patients and to monitor response or lack of response of the therapy. Cir-culating biomarkers may even provide predictive value in real time rather than depending on old tissues stored for years. The advantage to having real-time biomarker analysis is that it should enable physicians to get the most up-to-date understanding of the effectiveness of the therapy and to change therapy, if neces-sary. Since there will be a variety of targeted therapies, physicians will have several choices within a class of drugs such as tyrosine kinase inhibitors. If circulating biomarkers can indicate effective-ness or lack of effectiveeffective-ness, the physician will have alternative drugs to consider. Rather than waiting while the tumor contin-ues to grow, earlier insight into progressive disease will allow earlier intervention and hopefully longer survival.
Key issues
• Linking diagnostic biomarkers with targeted therapies will be essential to the success of personalized medicine.
• Circulating biomarkers to specific oncoproteins, such as human epidermal growth factor receptor (HER)2/neu, epidermal growth factor receptor (EGFR) and carbonic anhydrase IX should be investigated for their value with corresponding targeted therapies.
• Circulating biomarkers may be valuable in selecting patients for therapy and monitoring patients for response or progression as well as value as predicitive tests. Circulating biomarkers can provide a real-time assessment of the biomarker in question. • Serum HER2/neu is a biomarker for monitoring response or lack of response in patients with HER2/neu-positive
breast cancer.
• Circulating biomarkers, such as serum HER2/neu and serum EFGR, may be used as single tests or in combination to identify patients at high risk of breast cancer recurrence.
• Determining the HER2/neu status of a primary tumor with immunohistochemistry and fluorescent in situ hybridization may not always reflect the HER2/neu status of the metastatic tumor. Since most new medicines are evaluated in the metastatic setting, there should be tests employed in patients with metastatic disease to determine whether they are candidates for those medicines.
• Tissue tests should be used in conjunction with circulating biomarkers to fully understand the HER2/neu status of metastatic tumors. An elevated serum HER2/neu level in metastatic breast cancer patients with a HER2/neu-negative tumor should be suitable justification to re-evaluate the original tumor or metastatic tumor to determine whether the patient can be a candidate for HER2/neu-targeted therapy.
References
Papers of special note have been highlighted as: • of interest
•• of considerable interest
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• Provides a good overview of extracellular domain cleavage from cancer cells.
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• Describes the decline in serum human epidermal growth factor receptor (HER)2/neu and predicting outcome from trastuzumab therapy.
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• Nicely reviews the concept and provides considerable proof that primary breast tumors can be HER2/neu negative, while the metastatic tumor from the same patient can be HER2/neu positive.
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• Good review of the serum HER2/neu literature.
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• First paper to describe the observation that serum epidermal growth factor receptor (EGFR) levels are decreased in cancer.
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47 Gregorc V, Ceresoli GL, Floriani I. Effects of gefitinib on serum epidermal growth factor receptor and HER2 in patients with advanced non-small cell lung cancer. Clin. Cancer Res. 10, 6006–6012 (2004).
• Good study that montitored serum EGFR levels in non-small-cell lung cancer patients receiving Iressa®.
48 Marx J, Leitzel K, Al S et al. Serum EGFR in metastatic breast cancer patients. Proc. Am. Assoc. Cancer Res. 21, 436 (2002) (Abstract 1743).
49 Mueller V, Witzel I, Pantel K, Krenkel S, Luck HJ. Prognostic and predictive impact of soluble epidermal growth factor receptor (EGFR) protein in the serum of patients treated with chemotherapy for metastatic breast cancer. Anticancer Res. 26, 1479–1488 (2006).
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• Presents data combining two circulating oncoproteins, such as HER2/neu and EGFR, which have the ability to select out
patients with the least favorable outcomes.
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55 Ivanov S, Liao S, Ivanova A et al. Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer. Am. J. Pathol. 158(3), 905–919 (2001).
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• First description of the circulating carbonic anhydrase.
60 Sivendran S, Leitzel K, Ali SM et al. Elevated serum MN (carbonic anhydrase) levels in metastatci breast camcer patient sera. Proc. Am. Assoc. Cancer Res. 48, 636 (Abstract 2666) (2007).
Affiliation
• Walter P Carney, PhD
Head – Oncogene Science, Siemens Medical Solutions Diagnostics, Cambridge, MA 02142, USA
Tel.: +1 617 441 4409 Fax: +1 617 492 8438 walter.carney@siemens.com
