Springer 2006 Familial Cancer (2006) 5:135–142 DOI 10.1007/s10689-005-2832-5 BRCA1 and BRCA2: chemosensitivity, treatment outcomes and prognosis William D. Foulkes Program in Cancer Genetics, Departments of Oncology and Human Genetics, McGill University, Montreal, Quebec, Canada, H2W 1S6 Received 27 August 2004; accepted in revised form 9 February 2005 Key words: chemotherapy, hereditary breast and ovarian cancer, oncology, survival Abstract BRCA1 and BRCA2 are important breast and ovarian cancer susceptibility genes, and mutations in these two genes confer lifetime risks of breast cancer of up to 80% and ovarian cancer risks of up to 40%. Clinico-pathological studies have identiﬁed features that are speciﬁc to BRCA1-related breast cancer, but this has been more diﬃcult for BRCA2-related breast cancer. Ovarian cancers due to BRCA1 or BRCA2 mutations cannot usually be distinguished from their non-hereditary counterparts on morphological grounds, but micro-array data suggest that diﬀerences do exist. Prognostic studies have shown that breast cancer in a BRCA1 mutation carrier is likely to have a similar, or worse, outcome than that occurring in a BRCA2- or non-carrier of the same age. By contrast, most studies indicate that women developing a BRCA1/2-related ovarian cancer have an improved survival compared with non-carriers, particularly if they receive platinum-based therapy. In support of this, in vitro chemo-sensitivity studies have found that human cells lacking BRCA1 may be particularly sensitive to cisplatinum and to other drugs that cause doublestrand breaks in DNA. Nevertheless, in breast cancer, little is known regarding clinically important diﬀerences in response to chemotherapy between BRCA1/2 mutation carriers and non-carriers, and between diﬀerent chemotherapeutic regimens within existing series of BRCA1/2 mutation carriers. There are no published prospective studies. It is hoped that, in the near future, randomised controlled trials will be started with the aim of answering these important clinical questions. Introduction BRCA1 and BRCA2 are important breast cancer and ovarian susceptibility genes, accounting for 2–4% of all breast cancer and 5–10% of ovarian cancer worldwide. A considerably higher percentage of mutation carriers are found in some populations, particularly those where religious, cultural or geographical factors may have limited the ﬂow of genes [1]. Identifying mutations in these genes in women who are at risk of breast/ovarian cancer, or who already developed a cancer, is now part of routine clinical cancer genetics. Indeed, in the Canadian province of Ontario, a large part of the practice of medical genetics is now devoted to breast and ovarian cancer risk assessment [2], and most cancer centres have units devoted to hereditary cancer and/or cancer risk assessment. The purpose of this brief and selective review is to highlight research into the effect of the presence or absence of BRCA1 and BRCA2 mutations on (a) the sensitivity in vitro to certain chemotherapeutic drugs; and (b) the response to chemotherapy in patients with breast or ovarian cancer. Clinical response to radiotherapy or hormonal therapy will not be discussed. I hope to show how the presence of BRCA1/2 mutations may come to inﬂuence the practice of oncology, speciﬁcally the choice of chemotherapeutic agents. Most oncologists do not use mutation status to guide therapy, although there are early suggestions that this information can be useful, particularly if the decision whether or not to give chemotherapy is a diﬃcult one (i.e. on the basis of tumour size and nodal status alone) [3]. Increasingly it is accepted that events early in the life of a breast cancer can determine the long-term outcome [4–6] and that BRCA1-related breast cancers have a speciﬁc gene expression proﬁle that can distinguish such cancers from both BRCA2-related and non-hereditary cancers [5, 7]. Additionally, gene expression proﬁles may Correspondence to: William D. Foulkes ; Fax: +514-9348273; E-mail: William.Foulkes@mcgill.ca 136 determine response to certain chemotherapeutic agents [8]. Therefore, it seems plausible that BRCA1 and BRCA2-related breast cancers may possess gene expression proﬁles that will render these tumours particularly susceptible to one type of chemotherapeutic regimen, whereas they may be resistant to another. This is not perhaps very diﬀerent from the situation in nonBRCA1/2-related breast cancer, where similar analyses will soon be possible, but the diﬀerence with hereditary breast cancer is that the pre-existing inherited mutation may favour a speciﬁc initial molecular eﬀect (for example, impaired double-strand DNA repair) or pathological pathway (expression of basal, rather than luminal keratins) which is unlikely to be present in a large sub-set of non-hereditary cancers. This could result in class-based therapy, which may be more ﬁnancially and logistically practical than the much-discussed ‘individualized chemotherapy’. Clinicopathological features of BRCA1/2-related breast cancers The purpose of this section is to illustrate the key differences between three types of breast cancer: BRCA1-related, BRCA2-related and non-hereditary. This will serve as an introduction to the sections on the in vitro and in vivo data that relate speciﬁcally to response to chemotherapeutic agents. Diﬀerences in responses that are observed are likely to be determined, in part, by diﬀerences in gene expression proﬁles, which are reﬂected in the diﬀerent clinico-pathological features of these three groups of breast cancers. BRCA1-related breast cancers tend to be high-grade [9], lymph node negative [10] tumours that do not express estrogen receptors (ER), HER2 [11] or p27Kip1 [12], but do express p53 [13], cyclin E [14] and cytokeratin (CK) 5/6 [15, 16]. A marker of tumour vascularity known as glomeruloid microvascular proliferation (GMP), a feature of glioblastoma multiforme, is signiﬁcantly more likely to be present in BRCA1-related cancers than in other types of breast cancer [17]. In addition, MYC ampliﬁcation appears to be much more frequent in BRCA1-related breast cancer than in nonhereditary breast cancer [18, 19], and this knowledge, when combined with TBX2 status, could possibly used to predict the presence of a BRCA1 mutation [19]. Many of the features of BRCA1-related cancer discussed above are shared with breast cancers that have a basal epithelial phenotype [20, 21] but the true signiﬁcance of this phenotype is not yet known, although most studies have shown that the basal phenotype is associated with an inferior outcome [14, 22–24]. Whether or not this phenotype can be used to determine what type of chemotherapeutic regimen should be employed is an intriguing but unanswered question. The situation for BRCA2 is more complicated: six years ago, the Breast Cancer Linkage Consortium published an analysis of a large number of BRCA2- W. D. Foulkes related breast cancers and showed that, compared with controls, these cancers had (1) a higher score for tubule formation (fewer tubules) (P = 0.0002), (2) a higher proportion of the tumour perimeter with a continuous pushing margin (P = 0.0001), and (3) a lower mitotic count (P = 0.003) [25]. More recent immunohistochemical analyses by the same group showed that is diﬃcult to distinguish BRCA2-related from non-hereditary breast cancer using ER, PR, p53 or erbB-2/HER2 [9], and an analysis of relationship between tumour size and the number of positive axillary lymph nodes also indicates that BRCA2-related breast cancers are more like non-hereditary cancers than are BRCA1-related tumours [10]. Cyclin D1 is more likely to be expressed in BRCA2-related breast cancer (and in sporadic breast cancer) than in BRCA1-related breast cancer [7, 26], and this could be a helpful marker in situations where distinguishing which gene is involved is important. A recent phylogenetic re-analysis of previously published expression array data [7] demonstrated that a distinct branch on the classiﬁcation tree was occupied by BRCA2-positive cancers: this was quite distinct from the BRCA1-related cancer branch. Interestingly, socalled sporadic cancers appeared to be widely spread, and were relatively distant from the path between the BRCA1 and BRCA2 clusters [27]. Chemosensitivity: in vitro results (Table 1) Some of the functions of BRCA1 and BRCA2 proteins, such as the role of BRCA1/2 in DNA repair [39] or apoptosis [30, 40] could be directly involved in response to cytotoxic agents. Mouse and human cell lines deﬁcient in BRCA1 or BRCA2 display an increased sensitivity to agents causing double-strand DNA breaks [41, 42]. In breast and ovarian cell lines, over-expression of wild-type BRCA1 enhanced the apoptotic response to a variety of stimuli, including exposure to ionizing radiation and paclitaxel [30], and absence of BRCA2 protein renders some cell lines particularly sensitive to these agents as well as cisplatin and camptothecin [33, 43]. Other studies demonstrated this hypersensitivity for mitoxantrone, amsacrine, etoposide, doxorubicin and cisplatin with a subsequent increased level of apoptosis, although results have not been entirely consistent [34–36]. Diﬀerences in drug sensitivity might be explained by diﬀering levels of down-stream eﬀectors such as anti-apoptotic BCL-2 [44] or pro-apoptotic caspases in some hereditary breast cancers. Indeed, more recent studies have suggested that BRCA1 acts as a modulator of chemotherapy-induced apoptosis [29]. As noted previously, there are diﬀerent eﬀects depending on which agents are studied: the presence of BRCA1 induces a 10–1000fold increase in resistance to drugs that introduce DNA double-strand breaks, such as etoposide and bleomycin, whereas increased sensitivity for spindle poisons such as paclitaxel was noted. Very similar results were seen in another study, but in this case, BRCA1 null 137 BRCA1/2, chemotherapy and outcome Table 1. Summary of evidence for diﬀerential chemosensitivity in human breast/ovarian/pancreatic or murine cell lines with deﬁned BRCA1/2 levels Drug Wt BRCA1 over-expressed BRCA1 mutated (loss of function) Paclitaxel/ docetaxel Ref. [28] only) vinorelbine (Ref. [29] only) Increased apoptosis [29–31] Cisplatinum Decreased sensitivity [32, 34] Wt BRCA2 over-expressed BRCA2 mutated (loss of function) Comments Decreased apoptosis [29–32]; No eﬀect [28] Increased sensitivity [33] Similar eﬀects seen in one non-BRCA1/2 mutated ovarian cancer cell line (Ref. 33). In Ref. 28, mouse cells null for p53 that were proﬁcient or deﬁcient for Brca1 were used. Increased sensitivity [28, 32, 34, 35] Increased sensitivity [33] In Ref. 33, similar eﬀects seen in one non- BRCA1/2 mutated ovarian cancer cell line Amsacrine Etoposide (topo II poison) Doxorubicin (topo II poison) Increased sensitivity [36] Increased resistance [32] Increased sensitivity [32] Increased sensitivity [36] Decreased sensitivity [32]; Increased sensitivity [28] Mitoxantrone (topo II poison) Increased resistance [36] Bleomycin Increased resistance [29] Mitomycin c Increased resistance [37] Increased sensitivity [37] 5FU, anti-metabolites No change [28] Topotecan/ Camptothecin (topo I poison) Increased sensitivity [28] Busulfan Increased sensitivity [28] Increased sensitivity [36] Increased sensitivity [38] NB checkpoint activation/apoptotic mechanisms were unaﬀected [38] Increased sensitivity [33] Blank cells – no data identiﬁed, NB references in parentheses. cells (HCC1937) were also insensitive to doxorubicin, and this eﬀect was reversed when wild-type BRCA1 was expressed [32]. There are two inferences from these studies: (1) absence of BRCA1 results in an inverse of the chemoresponsive phenotype seen when BRCA1 is present; (2) More controversially, unlike the clinical situation for most breast cancers, those occurring in BRCA1 carriers are more likely to be resistant to paclitaxel than to cisplatinum or etoposide. However, in vitro and in vivo responses to chemotherapy may not be closely correlated. To confuse matters, these results with paclitaxel were contradicted by Zhou et al. who found that the ovarian cancer cell line, SNU-251, that contains a nonsense mutation in BRCA1, had increased cellular sensitivity to ionizing radiation and paclitaxel [31]. This sensitivity was reversed when wild-type BRCA1 was expressed. This cell line is derived from an ovarian, not a breast cancer, as in the previous experiments [29]. It would be instructive to repeat the experiments of Quinn et al. [29] using the same ovarian cancer cell line. To further complicate the picture, when compared with Brca1+/), p53)/) cells, murine embryo ﬁbroblasts lacking both Brca1 and p53 proteins had increased sensitivity to topotecan, doxorubicin, mitoxantrone, etoposide and platinum-containing compounds, but no eﬀect was seen on sensitivity to 5-ﬂuorouracil, gemcitabine, docetaxel or paclitaxel [28]. As shown previously, much of the increased chemosensitivity was revealed by analysis of apoptotic pathways. Again, repeating these experiments using inducible system and human cancer cell lines would be of value, particularly as it known that DNA repair systems, for example, diﬀer between rodents and humans [45]. To emphasize this diﬀerence, increased mRNA levels of BRCA1 predicted a favourable response to anthracycline-containing chemotherapy in 138 W. D. Foulkes a study of 51 patients with breast cancer [46]: the results from the experiments in mice described above would have predicted the reverse eﬀect [28] whereas some previous experiments using the breast cancer line HCC1937 are supportive of these ﬁndings [32]. Interestingly, mRNA levels of p53, erbB-2 and BRCA2 had no eﬀect on response to the cyclophosphamide/ epirubicin regimens used [46]. Methylation of the promotor of BRCA1 could also result in low mRNA levels [47, 48], and it is likely that methylation-based gene silencing could underlie diﬀerences in response to chemotherapy in breast cancer, as has been observed in glioma [49], where a better response to carmustine was observed in individuals with hypermethylation of the O6-methylguanine-DNA methyltransferase promoter than in those without hypermethylation. Interestingly, this hypermethylation is itself a poor prognostic marker, and perhaps a similar poor prognosis/good response combination of eﬀects might exist for mutations that prevent or limit accurate DNA repair, such as BRCA1 or BRCA2, particularly when treatments that cause double-strand breaks in DNA are used. This interpretation is complicated by TP53 data: here certain mutations seem to be both markers of poor prognosis and poor response [50, 51]. On the other hand, the main eﬀect on chemo-responsiveness in cancer cells with TP53 mutations may not operate through the DNA repair pathway, and the complex eﬀects of loss of TP53 on cellular functions may obscure pathwayspeciﬁc diﬀerences. A central role for RAD51 in DNA repair processes has been noted, but the signiﬁcance of the reported association between RAD51 expression levels and resistance to chemotherapy remains uncertain, particularly as studies provide conﬂicting data [52]. Interestingly, several studies have suggested that single nucleotide polymorphisms in RAD51 can modify cancer risk in BRCA2 (but not BRCA1) mutation carriers [53–55]. Forcing cells to repair artiﬁcially created doublestrand breaks by the use of homologous recombination (HR) could lead to cell death in cells that lack intact HR pathways. The premise that BRCA1 and BRCA2 null cells might be especially prone to respond in this fashion was borne out by two recent studies [56,57], that used an inhibitor of the enzyme, poly (ADP-ribose) polymerase (PARP). In these studies, selective inhibitors of PARP1 were tested in mouse and human BRCA1 and BRCA2 null cell lines. BRCA1 or BRCA2 dysfunction profoundly sensitized cells to the inhibition of PARP enzymatic activity. This resulted in chromosomal instability, followed by cell cycle arrest and ﬁnally cell death by apoptosis. This very exciting laboratory work suggests that clinical studies focused on the targeted inhibition of particular DNA repair pathways could be a new and effective way to treat cancers arising in BRCA1 or BRCA2 mutation carriers. Chemosensitivity: in vivo results (Table 2) There have been no prospective randomized controlled trials of different chemotherapeutic regimens in BRCA1/ 2 carriers. The closest to this ideal has been provided by retrospective analysis of completed studies where treatment was not based on mutation status, but was randomly assigned. A differential response to neo-adjuvant chemotherapy for breast cancer on the basis of germ-line BRCA1/2 mutation status may exist. After 3 or 4 cycles of neoadjuvant chemotherapy, a complete clinical response (cCR) was recorded in 10 of 11 BRCA1/2 carriers compared with 8 of 27 non-carriers (P = 0.0009) [58]. Notably, 4 (2 BRCA1 carriers and 2 BRCA2 carriers) of 9 evaluable BRCA1/2 carriers had no residual tumour in the breast and the axillary lymph nodes (a complete pathological response, or pCR), whereas only one case of pCR (4%) was noted among the non-carriers (P = 0.009). When the cases were matched 1:1 to controls on precise TNM stage, the signiﬁcance of the eﬀect of mutation status on complete clinical response rate was slightly less marked. Overall, in this very small retrospective study, BRCA1/2 carriers demonstrated a better clinical response rate to neo-adjuvant chemotherapy than did non-carriers. Importantly, the clinical and pathological responses to adjuvant treatment observed in BRCA1/2 non-carriers were concordant with what has been reported previously, and such responses are not stage-dependent [65]. Support for this observation was provided by a case report from Warner and colleagues [59], who treated a 49-year old woman with neo-adjuvant anthracycline- Table 2. Summary of evidence for diﬀerential response to chemotherapeutic agents in BRCA1- and BRCA2-related breast and ovarian cancers: clinical studies Drug Wt BRCA1 or high levels of wt BRCA1 mRNA BRCA1 mutation or loss of function BRCA2 mutation or loss of function Comments Anthracycline-containing regimens Increased sensitivity [46] Increased sensitivity [58–60] No eﬀect of BRCA2 mRNA levels on response [46] Increased sensitivity [61–64] Increased sensitivity [61–64] Breast cancer studied: small studies, retrospective cohort studies and case reports only Ovarian cancer studied: no diﬀerences between BRCA1 and BRCA2 observed Cisplatinum 139 BRCA1/2, chemotherapy and outcome containing chemotherapy because of a 3 cm invasive ductal breast carcinoma, associated with an ipsilateral 3.5 cm axillary mass. By the middle of the second cycle, no masses could be identiﬁed clinically, by magnetic resonance imaging or by mammography. She was subsequently identiﬁed as a carrier of an Ashkenazi Jewish founder mutation in BRCA1. At deﬁnitive surgery, the total mastectomy specimen was free of cancer. These ﬁndings have been indirectly supported by a recent study that showed that ‘‘basal’’(i.e BRCA1-like) and HER2- positive cancers were the only types of breast cancer which responded well to neo-adjuvant chemotherapy (66). In both groups, 45% had a pCR, exactly the same numbers are seen in the study of Chappuis et al [58], suggesting strongly that the basal pathway is a key factor in the response observed by that group. Another study, also from M.D. Anderson, showed that low levels of BCL-2 are associated with a good response to adjuvant chemotherapy [67], but was not associated with a better long-term survival (BCL-2 positive (56%) vs BCL-2 negative (48%), P = 0.58) Notably, BRCA1-related breast cancers often exhibit low levels of BCL-2 (44). Other basal-associated oncoproteins such a aB-crystallin are also associated with a poor prognosis [68] and the presence of a woundresponse signature, over-represented in basal breast cancers, is also seen in breast cancers that have an adverse outcome [69]. The apparent paradox is that despite this excellent initial response to chemotherapy (as demonstrated by high pCR rates in basal and BRCA1-related breast cancer), cancer is more likely to recur early in basalrelated than in other types of breast cancer (and in our data set, this is true for BRCA1-related cancers too). It appears that although basal breast cancers may respond well to adjuvant chemotherapy [70], response at time of ﬁrst relapse is poor [71], and this could, in part, explain the poor overall prognosis. Well controlled studies of both basal and non-basal forms of BRCA1-related cancer are required to establish the independence of the initial response to chemotherapy in BRCA1 mutation carriers from the basal phenotype. Furthermore, the unsustained nature of the response, in at least some studies, suggests that urgent attempts to identify biological treatments are justiﬁed. The single historical cohort study including a multivariable analysis showed that it is only among affected women not receiving chemotherapy that the presence of a BRCA1 mutation is associated with an adverse prognosis [58]. This provides supporting evidence for a role for BRCA1 in determining response to chemotherapy, but other interpretations of the data are possible, and given then lack of randomization, it is risky to infer too much from such data. The only other study, where the data were not divided by germ-line BRCA1/2 mutation status, but by mRNA levels in the cancers [46], found that, in contrast to the above studies, that increased mRNA levels of BRCA1 predicted a favourable response to anthracycline-containing chemotherapy. As discussed in the previous section, there are conﬂicting data from cell line experiments [28, 32] Several in vitro studies showed increased sensitivity of some ovarian cell lines carrying mutated BRCA1 alleles to various chemotherapeutic agents [30]. Supporting these data, the unbiased historical cohort study by Boyd and colleagues demonstrated that ovarian cancer among Ashkenazi Jewish BRCA1/2 mutation carriers had a better outcome when compared with ovarian cancer in Ashkenazi Jewish non-carriers [61]. Interestingly, although the hereditary and sporadic cancers presented with pathological and treatment (cisplatin-based regimens) characteristics that were remarkably similar, the BRCA1/2-associated cancers were more likely to be optimally cytoreduced at primary surgery and hereditary cases had a signiﬁcantly longer disease-free interval following primary chemotherapy (P = 0.001). These data are compatible with the hypothesis of a more favourable response to chemotherapy among hereditary ovarian cancer cases [61], and have been conﬁrmed by other studies [62–64], although survival diﬀerences between familial and non-familial ovarian cancer (without referring to mutation data) are less apparent [72–74]. There do not appear to be important survival diﬀerences between BRCA1 and BRCA2 mutation carriers. The Fanconi-BRCA ovarian cancer connection The mechanism by which platinum-based drugs produce their effect on ovarian cancer is of substantial interest. It has been observed that Fanconi anemia (FA) cells have increased chromosome breakage and radial formation after cellular exposure to mitomycin C (MMC) [75]. Murine cells lacking Brca1 or Brca2 can also develop a Fanconi anemia-like phenotype, particularly after MMC exposure [37, 38], and FANCD1 has recently been found to be BRCA2 [76]. The BRCA1, BRCA2 and FA gene products interact intimately [77], and normal functioning of this pathway is required for a normal cellular response to DNA cross-linking drugs such as cisplatinum. Recently, it has been suggested that about one-ﬁfth of primary ovarian carcinomas have some disruption of the FANC-BRCA pathway, resulting from biallelic methylation of FANCF [78]. The BRCA1/2 mutation status of the women was not reported. Reversion in one ovarian cancer cell line to cisplatinum resistance was associated with demethylation of FANCF. Therefore cisplatinum sensitivity could, in part, be explained by somatic inactivation of the FANC-BRCA pathway. Hypermethylation of the BRCA1 (and to a lesser extent, BRCA2) promoter is seen in non-hereditary ovarian cancers [48, 79], so it is plausible that the mechanism of cisplatinum sensitivity reported by Taniguchi et al. [78] could have a wider signiﬁcance if methylation of BRCA1 could functionally substitute 140 for methylation of FANCF. Could a similar eﬀect be present in BRCA1/2-related breast cancer? Prognosis in hereditary breast cancer This subject has been extensively reviewed elsewhere and has been alluded to in the sections above. Sufﬁce it to say that most studies ﬁnd that the prognosis for BRCA1-related breast cancer is either similar or worse than age-matched controls. For BRCA2, there are less data, but no substantial diﬀerences in outcome have emerged [3, 80, 81]. Non-BRCA1/2-hereditary breast cancers have a less aggressive proﬁle than BRCA1/2related cancers [82], but the prognostic implication of this has not been studied, for the obvious reason that the genes are not known, and no one single gene or mutation, apart perhaps for CHEK2:1100delC in Finland or the Netherlands, has attained a suﬃcient frequency to permit such an analysis [83]. Surprisingly, other breast cancer susceptibility genes, such as TP53, PTEN and STK11 have not been subjected to survival analyses [81]. Clinical implications and future directions Identifying germ-line BRCA1/2 mutations before deﬁnitive treatment (whether surgical, medical or radiotherapeutic) is beginning to become part of the management of women with breast cancer. Certainly, mutation status inﬂuences surgical choices: women with mutations are much more likely to undergo bilateral mastectomy at primary diagnosis than are non-carriers [84], and this will in turn inﬂuence decisions regarding the use of radiotherapy. As yet, there is only a sense that knowledge of mutation status might inﬂuence the decision to administer chemotherapy to women with small, nodenegative, high-grade breast cancers, insofar as those with BRCA1 mutations may be more likely to beneﬁt than those without [3]. But since many such women probably will receive chemotherapy anyway, this is a marginal contribution. What is more important is if certain regimens are found to be more eﬀective than others in BRCA1/2 carriers. The burning question at the moment is whether platinum-containing compounds will be more eﬀective than taxanes in breast cancer. This conjecture is supported by the literature discussed above. A trial of this nature, in metastatic breast cancer, has just commenced in the U.K. with plans for worldwide enrollment. For anthracyclines, the data are much less clear: most clinical data showing hints of increased responses in BRCA1/2 carriers are based on anthracycline-containing regimens, but the laboratory data are much less encouraging, and the only way to satisfactorily answer these questions will be randomized clinical trials in aﬀected BRCA1/2 mutation carriers. With increasing availability of gene expression data, it may be that clinicians will move straight to the somatic data, without the need to consider germ-line mutation status, and this may obviate the need for such W. D. Foulkes studies in primary breast cancer. 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