Growth factor signalling in clinical breast cancer and its impact on response to conventional therapies: a review of chemotherapy
http://www.100md.com
《肿瘤与内分泌》
Cardiff Breast Unit, Velindre NHS Trust, Cardiff CF14 2 TL, UK
This paper was presented at the 1st Tenovus/AstraZeneca Workshop, Cardiff (2005). AstraZeneca has supported the publication of these proceedings.
Abstract
Adjuvant chemotherapy has been shown to provide survival benefits in patients with breast cancer, but some patients still relapse despite this. There is therefore a need for molecular markers present within the primary tumour that can predict for chemotherapy sensitivity or resistance. Until now, no single marker has emerged into routine clinical practice, but several candidate pathways are being extensively investigated. This paper summarises the current status of growth factor singalling and p53 function in this context. The data on human epidermal growth factor receptor-2, topoisomerase II and p53 expression in a variety of breast cancer treatment settings are discussed.
Background
Breast cancer is a major global health problem, and the incidence of the disease continues to increase steadily. It is the most common malignancy in women accounting for 30% of all female cancers in the UK, with around 41 000 newly diagnosed cases and nearly 13 000 deaths each year (Cancer Research UK website, http://www.cancerresearchuk.org/). Despite recent advances in the management of early disease, breast cancer remains a major clinical problem, causing considerable morbidity and mortality and making considerable demands on health-care resources.
The use of adjuvant systemic chemotherapy has been clearly shown to provide survival benefit to all groups of patients regardless of menopausal, nodal or hormonal receptor status. The 1998 polychemotherapy (cyclophosphamide methotrexate 5-fluorouracil (CMF) anthracycline-based chemotherapy) overview by the Early Breast Cancer Trialists’ Collaborative Group (1998) reported a 23.5% (S.D.±2.1%) reduction in the annual odds of cancer recurrence and a 15.3% (S.D.±2.4%) reduction in the odds of death. Although benefits were greater in women under 50 years of age, a significant reduction in odds of recurrence and death was found in women aged 50–69. The proportional effects in women with node-positive and node-negative disease were similar (Early Breast Cancer Trialists’ Collaborative Group 1998).
Anthracycline-based chemotherapy is now considered the standard approach by many oncologists for women with node-positive disease and high-risk node-negative disease. Although a number of earlier studies failed to show a benefit for the addition of anthracyclines, the recent overview analysis confirms a useful additional survival benefit for anthracyclines (reduction in the odds of death by 11%; S.D.±5%). The combination of doxorubicin and cyclophosphamide (AC) has been a commonly used standard for many years in the USA, following the NSABP B-15 trial which confirmed that four cycles of AC were as effective as six cycles of CMF in node-positive patients (Fisher et al. 1990), but in Europe at least six or more cycles of epirubicin-based therapy (e.g. 5-fluorouracil epirubicin cyclophosphamide (FEC)) remains the most common standard used in clinical trials and is considered superior (Adjuvant Therapy for Breast Cancer 2000).
The taxanes, paclitaxel (TaxolTM; Bristol-Myers Squibb) and docetaxel (TaxotereTM; Sano.-Aventis), have been shown to have impressive activity in patients with metastatic breast cancer resistant to anthracyclines with response rates of the order of 40–50% (Lister-Sharp et al. 2000). These drugs are now established as standard agents in patients with metastatic breast cancer patients, especially after failure of anthracycline-based chemotherapy. More recently it has been postulated that the sequential administration of non-cross-resistant agents (such as anthracyclines and taxanes) or even combinations of these drugs may be superior to standard combination chemotherapy in the adjuvant setting, an idea reinforced by data from CALGB study 9344, which showed a small but significant survival advantage for the paclitaxel sequential combination (Henderson et al. 2003), and from the PACS 01 study which showed similar results using docetaxel (Roché et al. 2004).
Mechanisms of sensitivity and resistance
As discussed above, several different regimens are used to improve the prognosis from breast cancer, and all are acceptable for this purpose but differ in their durations, toxicities and costs. Generally, these regimens are used across the board for groups of patients considered to be at risk of recurrence (in the adjuvant setting) or who may benefit with symptom reduction (in the metastatic setting), but without tailoring to any particular clinico-pathological features of the patients. This is because until now there have not been reliable markers of predictive benefit for chemotherapy, let alone for distinguishing one particular drug regimen from another for individual patients. This concept would be the ideal, however, as it is likely that some shorter and less-toxic regimen may be sufficient for some very responsive tumours, whereas other patients may require more intensive therapy or different drugs (Pusztai et al. 2004).
Unfortunately, although it is true that oestrogen receptor (ER)-negative status predicts a slightly higher response rate to chemotherapy, this parameter does not have sufficient positive predictive value to be used in clinical practice, unlike its establishment as a standard predictive marker for hormone therapy in ER-positive breast cancer. In other words, both ER-positive and ER-negative patients can benefit from chemotherapy, and we must look to other candidate markers for a way forward.
Molecular markers for chemotherapy response
At the present time, no single predictive biological marker has become established in routine practice in breast cancer to assess clinical benefit from chemotherapy. The mechanisms of cytotoxic drug action are complex, and involve several intracellular pathways. There is increasing recognition and clinical evidence that these involve apoptotic pathways predominantly (Ellis et al. 1998), with contributions from proliferation mechanisms and drug-metabolism pathways. Certain drugs have fairly well-defined actions, such as interference with topoisomerase II (TOPO II) (anthracyclines, etoposide) or stabilisation of microtubules (taxanes). The former is also an example of a DNAdamaging agent leading to apoptosis and is therefore a p53-dependent mechanism (Aas et al. 1996), whereas the latter is an example of an agent interfering with mitosis, and is therefore independent of p53 actions (Kandioler-Eckersberger et al. 2000), but may be affected by proliferation mechanisms such as overexpression of human epidermal growth factor receptor-2 (HER2). This review will concentrate on the three most studied biomarkers and will review the clinical data available for HER2, TOPO II and p53 with respect to response to chemotherapy in metastatic, and (neo)adjuvant settings (Table 1). It should be noted, of course, that HER2 itself has become a standard predictive marker for the benefit of therapy with the humanised monoclonal antibody trastuzumab (Herceptin; Ellis et al. 2000), but this review will concentrate on its role as predictive marker for chemotherapy benefit.
HER2 as a marker for chemotherapy response
Metastatic breast cancer and anthracycline-based chemotherapy
Overall, HER2 has been studied extensively as a possible predictive marker for a variety of chemotherapy regimens. In metastatic disease however, studies examining response rate in relation to anthracycline-containing regimens such as 5-fluorouracil adriamycin cyclophosphamide (FAC)/FEC or AC/epirubicin cyclophosphamide (EC) are not numerous. In a recent study from Belgium, 59 patients were selected form a database of 350 patients with locally advanced or metastatic breast cancer and divided into cases receiving anthracycline-based chemotherapy and controls receiving taxane-based therapy. HER2 status was determined by fluorescence in situ hybridisation (FISH) using archival tumour samples. No association was found between HER2 gene amplification and response to anthracycline-based chemotherapy (five out of 14 (36%) HER2-positive tumours had a complete response and five out of 17 (29%) had progressive disease with this therapy; Cardoso et al. 2004). A second study from the same group looked at 108 patients randomised to either single-agent doxorubicin or single-agent docetaxel, with determination of HER2 status. Whereas HER2-positive status seemed to be correlated with overall response to chemotherapy, the numbers were too small to establish any difference in the anthracycline group (Durbecq et al. 2004a). These studies are illustrative of the data in metastatic disease with small patient numbers, lack of randomised trials and inconsistent results. Other problems encountered are the different methodologies employed for measurement of HER2, and a lack of standard cut-offs for positivity in relation to chemotherapy prediction.
Neoadjuvant anthracycline-based chemotherapy
Several studies have been carried out in the neoadjuvant setting, where an advantage is that the primary tumour remains in situ, allowing assessment of tumour response. Unfortunately, all of these studies are also small and confounded with the same variables as in the metastatic setting. Another advantage, however, is the prospective nature of these studies avoiding selection bias and in some cases the availability of serial testing of the tumour tissue during the course of the chemotherapy treatment. Thus Penault-Llorca et al.(2003) studied 115 patients receiving induction anthracycline-based chemotherapy, with biopsies of tumour tissue taken before and after competition of chemotherapy and tested for HER2 by immunohistochemistry (IHC). In this study, patients with HER2-negative tumours had a 9% rate of pathological complete response (CR) rising to 39% with HER2-positive disease. Interestingly, they were also able to confirm the finding from other studies which demonstrates that HER2 expression remains fairly constant during chemotherapy, and indeed during the metastatic process (Vincent-Salomon et al. 2002, Burcombe et al. 2005). In another older French study among 79 patients treated with either low-dose FEC (epirubicin 50 mg/m2) or with FEC100 (epirubicin 100 mg/m2), there was no difference in the response rate with either regimen in HER2-negative tumours, but a much higher response in HER2-positive patients treated with FEC100 compared with the lower-dose regimen, suggesting a dose--response relationship for epirubicin in HER2-positive disease (Petit et al. 2001). However, recently Martin-Richard et al.(2004) have shown no correlation between HER2 status by IHC and response to anthracycline-based chemotherapy (FAC/FEC) in 41 patients receiving neoadjuvant therapy. Furthermore, Bonnefoi et al.(2003) have also shown no correlation between HER2 status and treatment outcome for 187 patients receiving epirubicin-based neoadjuvant chemotherapy for locally advanced breast cancers. Therefore, the data on HER2 and response to neoadjuvant anthracycline-based chemotherapy are very conflicting and cannot be used currently to determine treatment options.
Adjuvant breast cancer and anthracycline-based chemotherapy
There have been several well-conducted, and relatively large, randomised, controlled trials that have evaluated the role of HER2 and survival in relation to chemotherapy benefit. The focus has been on anthracycline-based chemotherapy as with the previous studies in metastatic and neoadjuvant therapy, but there are also some data on chemotherapy with CMF-type regimen (reviewed extensively in Ravdin 2001, Yamouchi et al. 2001).
There are at least six trials with over 4000 patients in total where anthracycline-based adjuvant chemotherapy was compared in a randomised fashion to either a CMF-like regimen or no chemotherapy. In terms of disease-free and overall survival, only two of the studies show a statistically significant interaction with HER2-positivity, although most of the studies show a trend towards a relative risk reduction for HER2-positive patients receiving anthracycline-based chemotherapy (Table 2).
The conclusion since the publication of these studies has been that the results are not sufficient for HER2 to be used in routine clinical practice to determine the type of chemotherapy to recommend to patients. The problems associated with HER2 measurement include which antibody to use for IHC, the differing methodologies for HER2 measurement (IHC or FISH), the lack of a positive prediction in metastatic disease and finally the lack of a credible mechanism by which HER2 itself could exert this effect. These shortcomings have led to the evaluation of another associated marker, TOPO II, which has emerged as a strong candidate as a predictive biomarker for anthracycline-based chemotherapy. The apparent association with HER2 may be explained by their close proximity on chromosome 17q21. This will be discussed below.
TOPO II as a marker for anthracyclinebased chemotherapy response
The hypothesis
The topoisomerases are key enzymes in DNA replication that change the topology of DNA by facilitating strand passage. The HER2 gene is located on chromosome 17q21 near the locus encoding TOPO II, and co-amplifications of both genes have been found to occur commonly (Jarvinen et al. 1999, Coon et al. 2002)Anthracyclines are believed to act by interfering with TOPO II, and furthermore the levels of this enzyme seem to correlate well with chemosensitivity to anthracyclines in vitro (Harris et al. 2001). Therefore, there has been intense research activity around this molecule, which is discussed below.
Metastatic and locally advanced breast cancer
Several studies in metastatic or locally advanced patients have suggested a consistent relationship between TOPO II gene over-expression or amplification and enhanced benefit from anthracycline-based chemotherapy. These studies are summarised in Table 3.
In all of these studies, the results are presented in small patient numbers, with different methods of analysis. However, a similar picture had begun to emerge. Firstly, that there was less consistent evidence for a correlation between HER2 over-expression and response to anthracyclines. Secondly, TOPO II gene amplification generally only occurs in the presence of HER2 amplification (although in some subsequent studies low level TOPO II gene deletions have been seen in HER2 normal tumours). Thirdly, TOPO II over-expression could be measured by IHC or FISH and both seemed to predict for anthracycline sensitivity, although this was less convincing for the protein expression. One study found that TOPO II protein expression increased after chemotherapy in tumours less responsive to anthracycline-based chemotherapy (Burcombe et al. 2002) The Finnish group have also found in their series that TOPO II gene deletions also occur in HER2-amplified tumours (Jarvinen et al. 1999), and that this rendered the tumours resistant to anthracyclines (Di Leo & Isola 2003), but this remains to be confirmed by other groups.
Adjuvant breast cancer studies and TOPO II expression
As yet there have been few adjuvant studies reported in the literature. Di Leo et al.(2002) recently reported the results from an adjuvant trial in 777 post-surgical breast cancer patients randomised to chemotherapy with CMF or two EC regimens. In the predictive marker study 354 tumour samples were analysed for HER2 by FISH. Of these, 73 (21%) were amplified for HER2. Only these samples were then analysed for TOPO II gene alterations and 38% were found to be TOPO II-amplified, with another eight cases having deletions. The overall rate of TOPO II gene alteration in this population of node-positive breast cancer patients was estimated to be about 6%. Statistically significant results were not possible with these small numbers of HER2-positive patients, but there was a trend to suggest a greater benefit for anthracycline-based chemotherapy versus CMF in only those tumours with both HER2 and TOPO II amplifications (Di Leo et al. 2002). This study illustrates the difficulty with predictive marker studies: that of incomplete collection of pathological material from the total study patients, and the relative infrequency of genetic alteration in the putative markers. In Europe, a meta-analysis is underway to evaluate HER2 and TOPO II expression in over 4000 tumours from patients participating in randomised trials evaluating the benefits of anthracycline-based therapy (J Bartlett, personal communication).
The status of TOPO II as a putative marker for chemotherapy response is further complicated by a recent study that examined TOPO II amplification and protein expression in 103 HER2-amplified tumours. Interestingly, HER2 amplification levels (as determined by copy number) did not predict for TOPO II amplification, and in this study the TOPO II protein levels in TOPO II-amplified tumours were only slightly higher than in TOPO II-non-amplified tumours. This is in complete contrast to HER2 amplification where it is almost invariably associated with protein over-expression. Furthermore, the correlation between TOPO II amplification and protein expression was more impressive in tumours also showing positive staining for Ki67, the proliferation marker. As TOPO II expression is restricted to S-phase and is tightly linked to proliferation, this should be taken into account when evaluating TOPO II expression in relation to chemotherapy sensitivity (Durbecq et al. 2004b).
The role of TOPO II amplification and overexpression is now being tested prospectively in a randomised phase III clinical trial in breast cancer patients in the neoadjuvant setting in Europe (TOP Trial).
p53 as a marker for chemotherapy response
Any discussion of predictive markers for chemotherapy sensitivity and response must include a discussion of the role of the p53 protein. Experimental and clinical studies have demonstrated that chemotherapy agents exert many of there effects through apoptosis (Kerr et al. 1994, Ellis et al. 1998). p53 is a key regulatory protein that acts as a transcription factor regulating progression through the cell cycle, and influences those genes involved in G1 arrest. Intact (wild-type) p53 can induce apoptosis in response to ionizing radiation and chemotherapy, whereas mutated p53 and loss of function can lead to resistance because the insult is tolerated and apoptosis is reduced (Lowe et al. 1993; Figure 1). In relation to the differing actions of chemotherapy drugs, it has been postulated that DNA-damaging agents rely on an intact p53 pathway to exert their effect through apoptosis (p53-dependent), whereas drugs affecting the microtubules during mitosis, such as the taxanes, do not require functioning p53 to the same extent (p53-independent; Table 4) (Kandioler-Eckersberger et al. 2000).
Thus in clinical breast cancer, one might expect DNA-damaging agents such as anthracyclines to be less effective in tumours with mutant p53, whereas microtubule agents such as paclitaxel and docetaxel (acting during mitosis) may actually be more effective due to the lack of G1 arrest and higher mitotic rate due to p53 deficiency.
Older studies of p53 expression in clinical breast cancer
Most of the literature in the past decade reported the results of p53 IHC staining in relation to its prognostic value. The results were contradictory, with several studies supporting its role as a prognostic marker (Barnes et al. 1993, Silvestrini et al. 1993, MacGrogan et al. 1995, Silvestrini et al. 1996, Thor et al. 1998, Chappuis et al. 1999) and many failing to show this relationship (Elledge et al. 1995, Sjogren et al. 1996, Clahsen et al. 1998, Broet et al. 1999, Penault-Llorca et al. 2003, Martin-Richard et al. 2004). However, as is well known, the differing methodologies and antibodies used in these studies, and the failure to detect unstable p53 mutants, suggests that assessment of p53 status should be based on gene-sequencing methods for a proper evaluation of its role.
Neoadjuvant breast cancer studies and p53 mutations
Several recent studies in neoadjuvant breast cancer patients have employed modern DNA techniques to evaluate p53 status in relation to different chemotherapy regimens (Table 4). These studies are very small but are more consistent in their findings. Thus, the presence of p53 mutations led to a reduction in the response rate to chemotherapy in most cases. The one exception is the first study in which single-agent paclitaxel had an enhanced effect. One possible explanation for this has already been described; the absence of p53 function leads to loss of G1 arrest, greater S-phase activity and increased effect of paclitaxel on microtubules.
Clearly there is a need to clarify these suggestive data. A European initiative is currently underway to recruit 1440 patients with locally advanced or large operable breast cancer into a randomised, prospective phase III trial comparing a taxane (docetaxel) versus FEC (EORTC Breast group 10994/BIG 00-01). All patients within the trial will have histological samples taken, which are snap-frozen and analysed for p53 status using a novel functional assay in yeast. The primacy objective of this trial is to compare progression-free survival between the two chemotherapy regimen within the two p53 subgroups. This approach avoids the pitfalls of previous studies and, it is to be hoped, will provide definitive information on the role of p53 as a predictive marker in chemotherapy for human breast cancer.
The future
The single marker-gene studies discussed above represent, in some ways, the standard approach to molecular prediction in human cancers. However, these methods can now be enhanced considerably by the newer techniques employing gene-expression profiling of breast tumours. Several groups have recently measured a large number of genes at the mRNA level, using a variety of ‘platforms’, and have identified geneexpression profiles that could separate, in their small series, chemotherapy-sensitive tumours from chemotherapy-resistant ones (reviewed in Pusztai & Gianni 2004). This new approach is now being studied extensively and randomised phase III clinical trials in the adjuvant setting are being planned. It is likely that such profiles will be combined with the results of the single marker-gene studies, to finally bring to the clinic the necessary tools to guide clinicians and patients on the best treatments
Acknowledgements
The author declares that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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This paper was presented at the 1st Tenovus/AstraZeneca Workshop, Cardiff (2005). AstraZeneca has supported the publication of these proceedings.
Abstract
Adjuvant chemotherapy has been shown to provide survival benefits in patients with breast cancer, but some patients still relapse despite this. There is therefore a need for molecular markers present within the primary tumour that can predict for chemotherapy sensitivity or resistance. Until now, no single marker has emerged into routine clinical practice, but several candidate pathways are being extensively investigated. This paper summarises the current status of growth factor singalling and p53 function in this context. The data on human epidermal growth factor receptor-2, topoisomerase II and p53 expression in a variety of breast cancer treatment settings are discussed.
Background
Breast cancer is a major global health problem, and the incidence of the disease continues to increase steadily. It is the most common malignancy in women accounting for 30% of all female cancers in the UK, with around 41 000 newly diagnosed cases and nearly 13 000 deaths each year (Cancer Research UK website, http://www.cancerresearchuk.org/). Despite recent advances in the management of early disease, breast cancer remains a major clinical problem, causing considerable morbidity and mortality and making considerable demands on health-care resources.
The use of adjuvant systemic chemotherapy has been clearly shown to provide survival benefit to all groups of patients regardless of menopausal, nodal or hormonal receptor status. The 1998 polychemotherapy (cyclophosphamide methotrexate 5-fluorouracil (CMF) anthracycline-based chemotherapy) overview by the Early Breast Cancer Trialists’ Collaborative Group (1998) reported a 23.5% (S.D.±2.1%) reduction in the annual odds of cancer recurrence and a 15.3% (S.D.±2.4%) reduction in the odds of death. Although benefits were greater in women under 50 years of age, a significant reduction in odds of recurrence and death was found in women aged 50–69. The proportional effects in women with node-positive and node-negative disease were similar (Early Breast Cancer Trialists’ Collaborative Group 1998).
Anthracycline-based chemotherapy is now considered the standard approach by many oncologists for women with node-positive disease and high-risk node-negative disease. Although a number of earlier studies failed to show a benefit for the addition of anthracyclines, the recent overview analysis confirms a useful additional survival benefit for anthracyclines (reduction in the odds of death by 11%; S.D.±5%). The combination of doxorubicin and cyclophosphamide (AC) has been a commonly used standard for many years in the USA, following the NSABP B-15 trial which confirmed that four cycles of AC were as effective as six cycles of CMF in node-positive patients (Fisher et al. 1990), but in Europe at least six or more cycles of epirubicin-based therapy (e.g. 5-fluorouracil epirubicin cyclophosphamide (FEC)) remains the most common standard used in clinical trials and is considered superior (Adjuvant Therapy for Breast Cancer 2000).
The taxanes, paclitaxel (TaxolTM; Bristol-Myers Squibb) and docetaxel (TaxotereTM; Sano.-Aventis), have been shown to have impressive activity in patients with metastatic breast cancer resistant to anthracyclines with response rates of the order of 40–50% (Lister-Sharp et al. 2000). These drugs are now established as standard agents in patients with metastatic breast cancer patients, especially after failure of anthracycline-based chemotherapy. More recently it has been postulated that the sequential administration of non-cross-resistant agents (such as anthracyclines and taxanes) or even combinations of these drugs may be superior to standard combination chemotherapy in the adjuvant setting, an idea reinforced by data from CALGB study 9344, which showed a small but significant survival advantage for the paclitaxel sequential combination (Henderson et al. 2003), and from the PACS 01 study which showed similar results using docetaxel (Roché et al. 2004).
Mechanisms of sensitivity and resistance
As discussed above, several different regimens are used to improve the prognosis from breast cancer, and all are acceptable for this purpose but differ in their durations, toxicities and costs. Generally, these regimens are used across the board for groups of patients considered to be at risk of recurrence (in the adjuvant setting) or who may benefit with symptom reduction (in the metastatic setting), but without tailoring to any particular clinico-pathological features of the patients. This is because until now there have not been reliable markers of predictive benefit for chemotherapy, let alone for distinguishing one particular drug regimen from another for individual patients. This concept would be the ideal, however, as it is likely that some shorter and less-toxic regimen may be sufficient for some very responsive tumours, whereas other patients may require more intensive therapy or different drugs (Pusztai et al. 2004).
Unfortunately, although it is true that oestrogen receptor (ER)-negative status predicts a slightly higher response rate to chemotherapy, this parameter does not have sufficient positive predictive value to be used in clinical practice, unlike its establishment as a standard predictive marker for hormone therapy in ER-positive breast cancer. In other words, both ER-positive and ER-negative patients can benefit from chemotherapy, and we must look to other candidate markers for a way forward.
Molecular markers for chemotherapy response
At the present time, no single predictive biological marker has become established in routine practice in breast cancer to assess clinical benefit from chemotherapy. The mechanisms of cytotoxic drug action are complex, and involve several intracellular pathways. There is increasing recognition and clinical evidence that these involve apoptotic pathways predominantly (Ellis et al. 1998), with contributions from proliferation mechanisms and drug-metabolism pathways. Certain drugs have fairly well-defined actions, such as interference with topoisomerase II (TOPO II) (anthracyclines, etoposide) or stabilisation of microtubules (taxanes). The former is also an example of a DNAdamaging agent leading to apoptosis and is therefore a p53-dependent mechanism (Aas et al. 1996), whereas the latter is an example of an agent interfering with mitosis, and is therefore independent of p53 actions (Kandioler-Eckersberger et al. 2000), but may be affected by proliferation mechanisms such as overexpression of human epidermal growth factor receptor-2 (HER2). This review will concentrate on the three most studied biomarkers and will review the clinical data available for HER2, TOPO II and p53 with respect to response to chemotherapy in metastatic, and (neo)adjuvant settings (Table 1). It should be noted, of course, that HER2 itself has become a standard predictive marker for the benefit of therapy with the humanised monoclonal antibody trastuzumab (Herceptin; Ellis et al. 2000), but this review will concentrate on its role as predictive marker for chemotherapy benefit.
HER2 as a marker for chemotherapy response
Metastatic breast cancer and anthracycline-based chemotherapy
Overall, HER2 has been studied extensively as a possible predictive marker for a variety of chemotherapy regimens. In metastatic disease however, studies examining response rate in relation to anthracycline-containing regimens such as 5-fluorouracil adriamycin cyclophosphamide (FAC)/FEC or AC/epirubicin cyclophosphamide (EC) are not numerous. In a recent study from Belgium, 59 patients were selected form a database of 350 patients with locally advanced or metastatic breast cancer and divided into cases receiving anthracycline-based chemotherapy and controls receiving taxane-based therapy. HER2 status was determined by fluorescence in situ hybridisation (FISH) using archival tumour samples. No association was found between HER2 gene amplification and response to anthracycline-based chemotherapy (five out of 14 (36%) HER2-positive tumours had a complete response and five out of 17 (29%) had progressive disease with this therapy; Cardoso et al. 2004). A second study from the same group looked at 108 patients randomised to either single-agent doxorubicin or single-agent docetaxel, with determination of HER2 status. Whereas HER2-positive status seemed to be correlated with overall response to chemotherapy, the numbers were too small to establish any difference in the anthracycline group (Durbecq et al. 2004a). These studies are illustrative of the data in metastatic disease with small patient numbers, lack of randomised trials and inconsistent results. Other problems encountered are the different methodologies employed for measurement of HER2, and a lack of standard cut-offs for positivity in relation to chemotherapy prediction.
Neoadjuvant anthracycline-based chemotherapy
Several studies have been carried out in the neoadjuvant setting, where an advantage is that the primary tumour remains in situ, allowing assessment of tumour response. Unfortunately, all of these studies are also small and confounded with the same variables as in the metastatic setting. Another advantage, however, is the prospective nature of these studies avoiding selection bias and in some cases the availability of serial testing of the tumour tissue during the course of the chemotherapy treatment. Thus Penault-Llorca et al.(2003) studied 115 patients receiving induction anthracycline-based chemotherapy, with biopsies of tumour tissue taken before and after competition of chemotherapy and tested for HER2 by immunohistochemistry (IHC). In this study, patients with HER2-negative tumours had a 9% rate of pathological complete response (CR) rising to 39% with HER2-positive disease. Interestingly, they were also able to confirm the finding from other studies which demonstrates that HER2 expression remains fairly constant during chemotherapy, and indeed during the metastatic process (Vincent-Salomon et al. 2002, Burcombe et al. 2005). In another older French study among 79 patients treated with either low-dose FEC (epirubicin 50 mg/m2) or with FEC100 (epirubicin 100 mg/m2), there was no difference in the response rate with either regimen in HER2-negative tumours, but a much higher response in HER2-positive patients treated with FEC100 compared with the lower-dose regimen, suggesting a dose--response relationship for epirubicin in HER2-positive disease (Petit et al. 2001). However, recently Martin-Richard et al.(2004) have shown no correlation between HER2 status by IHC and response to anthracycline-based chemotherapy (FAC/FEC) in 41 patients receiving neoadjuvant therapy. Furthermore, Bonnefoi et al.(2003) have also shown no correlation between HER2 status and treatment outcome for 187 patients receiving epirubicin-based neoadjuvant chemotherapy for locally advanced breast cancers. Therefore, the data on HER2 and response to neoadjuvant anthracycline-based chemotherapy are very conflicting and cannot be used currently to determine treatment options.
Adjuvant breast cancer and anthracycline-based chemotherapy
There have been several well-conducted, and relatively large, randomised, controlled trials that have evaluated the role of HER2 and survival in relation to chemotherapy benefit. The focus has been on anthracycline-based chemotherapy as with the previous studies in metastatic and neoadjuvant therapy, but there are also some data on chemotherapy with CMF-type regimen (reviewed extensively in Ravdin 2001, Yamouchi et al. 2001).
There are at least six trials with over 4000 patients in total where anthracycline-based adjuvant chemotherapy was compared in a randomised fashion to either a CMF-like regimen or no chemotherapy. In terms of disease-free and overall survival, only two of the studies show a statistically significant interaction with HER2-positivity, although most of the studies show a trend towards a relative risk reduction for HER2-positive patients receiving anthracycline-based chemotherapy (Table 2).
The conclusion since the publication of these studies has been that the results are not sufficient for HER2 to be used in routine clinical practice to determine the type of chemotherapy to recommend to patients. The problems associated with HER2 measurement include which antibody to use for IHC, the differing methodologies for HER2 measurement (IHC or FISH), the lack of a positive prediction in metastatic disease and finally the lack of a credible mechanism by which HER2 itself could exert this effect. These shortcomings have led to the evaluation of another associated marker, TOPO II, which has emerged as a strong candidate as a predictive biomarker for anthracycline-based chemotherapy. The apparent association with HER2 may be explained by their close proximity on chromosome 17q21. This will be discussed below.
TOPO II as a marker for anthracyclinebased chemotherapy response
The hypothesis
The topoisomerases are key enzymes in DNA replication that change the topology of DNA by facilitating strand passage. The HER2 gene is located on chromosome 17q21 near the locus encoding TOPO II, and co-amplifications of both genes have been found to occur commonly (Jarvinen et al. 1999, Coon et al. 2002)Anthracyclines are believed to act by interfering with TOPO II, and furthermore the levels of this enzyme seem to correlate well with chemosensitivity to anthracyclines in vitro (Harris et al. 2001). Therefore, there has been intense research activity around this molecule, which is discussed below.
Metastatic and locally advanced breast cancer
Several studies in metastatic or locally advanced patients have suggested a consistent relationship between TOPO II gene over-expression or amplification and enhanced benefit from anthracycline-based chemotherapy. These studies are summarised in Table 3.
In all of these studies, the results are presented in small patient numbers, with different methods of analysis. However, a similar picture had begun to emerge. Firstly, that there was less consistent evidence for a correlation between HER2 over-expression and response to anthracyclines. Secondly, TOPO II gene amplification generally only occurs in the presence of HER2 amplification (although in some subsequent studies low level TOPO II gene deletions have been seen in HER2 normal tumours). Thirdly, TOPO II over-expression could be measured by IHC or FISH and both seemed to predict for anthracycline sensitivity, although this was less convincing for the protein expression. One study found that TOPO II protein expression increased after chemotherapy in tumours less responsive to anthracycline-based chemotherapy (Burcombe et al. 2002) The Finnish group have also found in their series that TOPO II gene deletions also occur in HER2-amplified tumours (Jarvinen et al. 1999), and that this rendered the tumours resistant to anthracyclines (Di Leo & Isola 2003), but this remains to be confirmed by other groups.
Adjuvant breast cancer studies and TOPO II expression
As yet there have been few adjuvant studies reported in the literature. Di Leo et al.(2002) recently reported the results from an adjuvant trial in 777 post-surgical breast cancer patients randomised to chemotherapy with CMF or two EC regimens. In the predictive marker study 354 tumour samples were analysed for HER2 by FISH. Of these, 73 (21%) were amplified for HER2. Only these samples were then analysed for TOPO II gene alterations and 38% were found to be TOPO II-amplified, with another eight cases having deletions. The overall rate of TOPO II gene alteration in this population of node-positive breast cancer patients was estimated to be about 6%. Statistically significant results were not possible with these small numbers of HER2-positive patients, but there was a trend to suggest a greater benefit for anthracycline-based chemotherapy versus CMF in only those tumours with both HER2 and TOPO II amplifications (Di Leo et al. 2002). This study illustrates the difficulty with predictive marker studies: that of incomplete collection of pathological material from the total study patients, and the relative infrequency of genetic alteration in the putative markers. In Europe, a meta-analysis is underway to evaluate HER2 and TOPO II expression in over 4000 tumours from patients participating in randomised trials evaluating the benefits of anthracycline-based therapy (J Bartlett, personal communication).
The status of TOPO II as a putative marker for chemotherapy response is further complicated by a recent study that examined TOPO II amplification and protein expression in 103 HER2-amplified tumours. Interestingly, HER2 amplification levels (as determined by copy number) did not predict for TOPO II amplification, and in this study the TOPO II protein levels in TOPO II-amplified tumours were only slightly higher than in TOPO II-non-amplified tumours. This is in complete contrast to HER2 amplification where it is almost invariably associated with protein over-expression. Furthermore, the correlation between TOPO II amplification and protein expression was more impressive in tumours also showing positive staining for Ki67, the proliferation marker. As TOPO II expression is restricted to S-phase and is tightly linked to proliferation, this should be taken into account when evaluating TOPO II expression in relation to chemotherapy sensitivity (Durbecq et al. 2004b).
The role of TOPO II amplification and overexpression is now being tested prospectively in a randomised phase III clinical trial in breast cancer patients in the neoadjuvant setting in Europe (TOP Trial).
p53 as a marker for chemotherapy response
Any discussion of predictive markers for chemotherapy sensitivity and response must include a discussion of the role of the p53 protein. Experimental and clinical studies have demonstrated that chemotherapy agents exert many of there effects through apoptosis (Kerr et al. 1994, Ellis et al. 1998). p53 is a key regulatory protein that acts as a transcription factor regulating progression through the cell cycle, and influences those genes involved in G1 arrest. Intact (wild-type) p53 can induce apoptosis in response to ionizing radiation and chemotherapy, whereas mutated p53 and loss of function can lead to resistance because the insult is tolerated and apoptosis is reduced (Lowe et al. 1993; Figure 1). In relation to the differing actions of chemotherapy drugs, it has been postulated that DNA-damaging agents rely on an intact p53 pathway to exert their effect through apoptosis (p53-dependent), whereas drugs affecting the microtubules during mitosis, such as the taxanes, do not require functioning p53 to the same extent (p53-independent; Table 4) (Kandioler-Eckersberger et al. 2000).
Thus in clinical breast cancer, one might expect DNA-damaging agents such as anthracyclines to be less effective in tumours with mutant p53, whereas microtubule agents such as paclitaxel and docetaxel (acting during mitosis) may actually be more effective due to the lack of G1 arrest and higher mitotic rate due to p53 deficiency.
Older studies of p53 expression in clinical breast cancer
Most of the literature in the past decade reported the results of p53 IHC staining in relation to its prognostic value. The results were contradictory, with several studies supporting its role as a prognostic marker (Barnes et al. 1993, Silvestrini et al. 1993, MacGrogan et al. 1995, Silvestrini et al. 1996, Thor et al. 1998, Chappuis et al. 1999) and many failing to show this relationship (Elledge et al. 1995, Sjogren et al. 1996, Clahsen et al. 1998, Broet et al. 1999, Penault-Llorca et al. 2003, Martin-Richard et al. 2004). However, as is well known, the differing methodologies and antibodies used in these studies, and the failure to detect unstable p53 mutants, suggests that assessment of p53 status should be based on gene-sequencing methods for a proper evaluation of its role.
Neoadjuvant breast cancer studies and p53 mutations
Several recent studies in neoadjuvant breast cancer patients have employed modern DNA techniques to evaluate p53 status in relation to different chemotherapy regimens (Table 4). These studies are very small but are more consistent in their findings. Thus, the presence of p53 mutations led to a reduction in the response rate to chemotherapy in most cases. The one exception is the first study in which single-agent paclitaxel had an enhanced effect. One possible explanation for this has already been described; the absence of p53 function leads to loss of G1 arrest, greater S-phase activity and increased effect of paclitaxel on microtubules.
Clearly there is a need to clarify these suggestive data. A European initiative is currently underway to recruit 1440 patients with locally advanced or large operable breast cancer into a randomised, prospective phase III trial comparing a taxane (docetaxel) versus FEC (EORTC Breast group 10994/BIG 00-01). All patients within the trial will have histological samples taken, which are snap-frozen and analysed for p53 status using a novel functional assay in yeast. The primacy objective of this trial is to compare progression-free survival between the two chemotherapy regimen within the two p53 subgroups. This approach avoids the pitfalls of previous studies and, it is to be hoped, will provide definitive information on the role of p53 as a predictive marker in chemotherapy for human breast cancer.
The future
The single marker-gene studies discussed above represent, in some ways, the standard approach to molecular prediction in human cancers. However, these methods can now be enhanced considerably by the newer techniques employing gene-expression profiling of breast tumours. Several groups have recently measured a large number of genes at the mRNA level, using a variety of ‘platforms’, and have identified geneexpression profiles that could separate, in their small series, chemotherapy-sensitive tumours from chemotherapy-resistant ones (reviewed in Pusztai & Gianni 2004). This new approach is now being studied extensively and randomised phase III clinical trials in the adjuvant setting are being planned. It is likely that such profiles will be combined with the results of the single marker-gene studies, to finally bring to the clinic the necessary tools to guide clinicians and patients on the best treatments
Acknowledgements
The author declares that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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