TP53 has been implicated in regulation of the cell cycle,DNA repair, and apoptosis. We studied, in primary breast tumors through direct cDNA sequencing of exons 2–11, whether TP53 gene mutations can predict response in patients with advanced disease to either first-line tamoxifen therapy (202 patients, of whom 55%responded) or up-front (poly)chemotherapy (41 patients, of whom 46%responded). TP53 mutations were detected in 90 of 243(37%) tumors, and one-fourth of these mutations resulted in a premature termination of the protein. The mutations were observed in 32% (65 of 202) of the primary tumors of tamoxifen-treated patients and in 61% (25 of 41) of the primary tumors of the chemotherapy patients. TP53 mutation was significantly associated with a poor response to tamoxifen [31% versus 66%;odds ratio (OR), 0.22; 95% confidence interval (CI), 0.12–0.42; P < 0.0001]. Patients with TP53 gene mutations in codons that directly contact DNA or with mutations in the zinc-binding domain loop L3 showed the lowest response to tamoxifen (18% and 15% response rates, respectively). TP53 mutations were related, although not significantly, to a poor response to up-front chemotherapy (36%versus 63%; OR, 0.34; 95% CI, 0.09–1.24). In multivariate analysis for response including the classical parameters age and menopausal status, disease-free interval, dominant site of relapse, and levels of estrogen receptor and progesterone receptor, TP53 mutation was a significant predictor of poor response in the tamoxifen-treated group (OR, 0.29; 95% CI, 0.13–0.63; P = 0.0014). TP53-mutated and estrogen receptor-negative (<10 fmol/mg protein) tumors appeared to be the most resistant phenotype. Interestingly, the response of patients with TP53 mutations to chemotherapy after tamoxifen was not worse than that of patients without these mutations(50% versus 42%; OR, 1.35, nonsignificant). The median progression-free survival after systemic treatment was shorter for patients with a TP53 mutation than for patients with wild-type TP53 (6.6 and 0.6 months less for tamoxifen and up-front chemotherapy, respectively). In conclusion, TP53 gene mutation of the primary tumor is helpful in predicting the response of patients with metastatic breast disease to tamoxifen therapy. The type of mutation and its biological function should be considered in the analyses of the predictive value of TP53.

TP53, a tumor suppressor gene, has been shown to have prognostic value in patients with breast cancer. A few studies with conflicting results on its predictive value have been published. Endocrine treatment and chemotherapy improve the survival of women with (primary or advanced) breast cancer. The antiestrogen tamoxifen is one of the major compounds used for the endocrine treatment of breast cancer. It is effective in about one-third of all patients with metastatic disease and in about 50% of those breast cancer patients that are ER3positive. Like chemotherapeutic agents, tamoxifen may cause DNA damage(1). TP53, by regulating response to DNA damage, is a key element in DNA repair. This tumor suppressor gene has also been implicated in the regulation of normal cell growth and division, gene transcription, genomic stability, apoptosis, and senescence. Mutations in the TP53 gene (also known as P53) are the most frequent genetic changes found in human breast cancer. Mutation is often accompanied by deletion of the second allele, resulting in the elimination of wild-type TP53 activity. P53 protein accumulation, which often results from TP53 gene mutation, can be measured with immunological techniques, i.e., IHC or LIA/ELISAs on tumor extracts.

In two studies on patients with metastatic breast cancer (including 92 and 205 patients, respectively), no significant relation between immunohistochemically assessed P53 expression and response to endocrine treatment was observed (2, 3), whereas in another study(on 17 patients), P53 overexpression was associated with a poor response to endocrine therapy (4). Recently, we have shown a significant relation between P53 accumulation, as measured by a LIA,and poor response to tamoxifen therapy in a series of 401 patients(5). Moreover, the existing data on TP53 do not clarify its capability to predict resistance to chemotherapy for advanced or recurrent breast cancer (6, 7, 8, 9). Furthermore, the data on its predictive role in the adjuvant setting are conflicting as well(10, 11, 12, 13, 14, 15, 16). Studies on the predictive value of TP53 are hampered by a number of methodological issues including immunological versus molecular biological analyses, use of different cutoff levels in the immunohistochemical analyses, or selection of patient groups.

About 20% of the TP53 gene mutations do not result in p53 protein accumulation, whereas, on the other hand, p53 accumulation may also occur without a gene mutation. Therefore we have studied TP53 gene mutations in a relatively large series of 243 patients with advanced disease. Analysis of the various mutations allowed us to investigate the biological significance of particular mutations, which may eventually aid in selecting those aberrations that could be of predictive value in breast cancer. We show that TP53 gene mutations predict a poor response to first-line tamoxifen therapy and probably to chemotherapy in advanced breast cancer. In an exploratory subset analysis, we also observed that mutations in one zinc-binding domain, i.e., loop L3, or in codons that directly contact DNA were related with an even poorer response to tamoxifen.

Assays of Cell Biological Factors

Sequence-based Analysis of TP53.

cDNA-based sequencing was used essentially as described by Bergh et al.(14), Sjögren et al.(17), Kressner et al.(19)and Falette et al.(20). Frozen untreated primary tumor samples were verified histologically for the presence of tumor cells, and only those samples containing more than 20% tumor cells were included in our study. mRNA was prepared from the frozen samples under stringent conditions to avoid degradation and contamination, and cDNA was generated. TP53 was amplified from the tumor cDNA by PCR using four sets of overlapping primers (one primer in each pair was biotin-labeled) to cover the complete protein coding region (exons 2–11) of the TP53 cDNA. Solid-phase sequencing was carried out using Autoload solid-phase sequencing combs and T7 DNA polymerase (Amersham Pharmacia Biotech). Samples were analyzed on an automated laser fluorescence DNA sequencer (Amersham Pharmacia Biotech). Sequence gels and sequences were evaluated with Sequence Evaluator and Mutation Analyzer software (Amersham Pharmacia Biotech). All mutations were confirmed by reamplifying the relevant cDNAs and sequencing the new PCR products.

Assays of ER and PgR.

ER and PgR levels were determined in cytoplasmic extracts (cytosols)prepared routinely according to procedures recommended by the European Organization for Research and Treatment of Cancer Breast Cancer Cooperative Group with ligand binding assays or enzyme immunoassays(ER-EIA and PgR-EIA; Abbott Laboratories, IL), as described previously (20).

To evaluate the clinical significance of TP53 gene alterations in advanced disease, 265 primary breast tumor specimens were analyzed. Sequence data (protein coding exons 2–11 of the TP53 gene) were successfully obtained from 243 tumors (92%;Table 1). In five cases, the sequence analysis was incomplete, although a mutation was observed; these samples were included. For nine cases, the sequence analysis was incomplete, but no gene mutation was observed;these nine samples were not included because mutations could not be excluded. In 13 cases (5%), no PCR product could be generated (in two separate experiments). Consequently, the analysis with respect to TP53 mutation and response to systemic therapy was performed on 243 breast cancer patients who developed recurrent disease. The patient group in the present study is not identical to the population described previously, i.e., 165 patients were described previously (5). A total of 202 patients (83%) received first-line tamoxifen therapy, whereas 41 (17%) patients, especially younger women with ER-negative tumors, received first-line chemotherapy for advanced disease.

First-Line Tamoxifen Treatment.

Between 1979 and 1991, 202 patients underwent resection of their primary tumors (breast-conserving therapy, 67 patients; modified mastectomy, 132 patients; and biopsy, 3 patients) and were selected according to the criteria described previously(21). In the first-line tamoxifen-treated group, 20 patients were stage I, 110 patients were stage II, 45 patients were stage III, and 22 patients were stage IV. With respect to stage, data on five patients are unknown. Twenty-seven patients (13%) received systemic adjuvant chemotherapy (CMF, 18 patients; CAF, 9 patients). Twenty-three patients (11%) were diagnosed with metastatic disease(M1) at the time of primary surgery. All patients were treated with 40 mg of tamoxifen daily as a first-line hormonal therapy after relapse. All patients were tamoxifen naïve and had not received prior chemotherapy for advanced disease. The median age of these 202 patients at the start of first-line tamoxifen therapy was 62 years (range, 28–85 years). Additional characteristics are listed in Table 2. The median follow-up of patients who are still alive is 81 months(range, 51–123 months) from primary surgery and 44 months (range,8–80 months) from the start of tamoxifen treatment. A total of 163 patients have died (median survival time, 20 months). Tumor progression occurred in most patients (190 of 202 patients, 94%) during follow-up. Median time to progression was 7.5 months. After tumor progression on first-line tamoxifen treatment, 70% of the patients were treated with one or more additional endocrine agents (mostly high-dose progestins),whereas 117 patients were subsequently treated with one or more regimens of chemotherapy after development of hormone resistance.

First-Line (Up-Front) Chemotherapy.

Forty-one patients underwent resection of their primary tumors(breast-conserving lumpectomy, 22 patients; modified mastectomy, 19 patients) between 1983 and 1991. In the up-front chemotherapy group, 6 patients were stage I, 22 patients were stage II, 9 patients were stage III, and 4 patients were stage IV. After primary surgery, six patients received adjuvant chemotherapy, and six patients received adjuvant hormonal therapy. Four patients (10%) were diagnosed with metastasis at the time of surgery for their primary tumor. As a first-line treatment for advanced disease, 22 of these 41 patients (54%) received CMF, 16 of 41 patients (39%) received CAF, 1 of 41 patients received Adriamycin weekly, and 2 of 41 patients received a platinum-containing chemotherapy. The median age at start of chemotherapy was 50 years(range, 29–74 years). Additional characteristics are listed in Table 4. Median follow-up was 33 months (range, 5–107 months) from the date of primary surgery and 12 months (range, 1–75 months) from the start of chemotherapy for recurrent disease. Tumor progression occurred in all but one of the patients (98%) during follow-up after the start of chemotherapy. Median time to progression was 4 months. After 12 months,19% of the patients were alive without progression. Response to systemic treatment was defined by standard Union Internationale Contre le Cancer criteria as a patient having either CR or PR or prolonged stable disease of more than 6 months (21, 22).

The associations of TP53 with continuous variables (ER, PgR, age,and disease-free interval) were studied with a nonparametric test. Mutation subsets were analyzed with a Wilcoxon rank-sum test. Two-sided Ps < 0.05 were considered statistically significant. Logistic regression analysis was used for the analysis of response to treatment. Cox regression analysis was used for the analysis of time to treatment failure and overall survival after start of treatment. Survival curves were generated using the method of Kaplan and Meier (23). TP53 gene status and the following factors were evaluated in the multivariate regression analysis: (a) age; (b) menopausal status;(c) adjuvant therapy; (d) disease-free interval;(e) the dominant site of relapse (in case of multiple sites,the site with the worst prognosis was considered dominant);(f) ER; and (g) PgR. TP53 was added to this model.

TP53 Gene Mutations.

We have sequenced the entire open reading frame of the TP53gene (exons 2–11). Mutations were detected in 90 of the 243 (37%)samples, 9 of which (10%) were observed outside the sequence-specific DNA binding domain (codons 102–292). Fifty of these 90 mutations(56%) were restricted to the conserved region. Within the zinc-binding domain regions L2 and L3 (residues 163–195 and 236–251,respectively), 31 (34%) mutations were found. Moreover, we observed 15 mutations in four of the seven amino acids important in direct DNA binding (i.e., codons 241, 248, 273, and 280). As expected,the majority of the mutations were transitions, and the amino acid arginine was altered most frequently. Of all 90 mutations, 62 (69%)were missense mutations, including 3 complex mutations and 1 tandem mutation. In total, these amount to 42 different changes. The remaining 28 mutations (referred to as non-missense mutations) were 5 in-frame deletions/insertions, 11 nonsense mutations leading to a stop codon,and 12 out-of-frame deletions/insertions causing a premature stop codon. These latter “null mutations” were localized mainly in exon 6. These results imply that one-fourth of the mutations detected in this study would not result in an increased expression of the TP53 protein, i.e., false negatives.

Patient and tumor characteristics are summarized in Table 1. The prevalence of TP53 gene mutations is highest in tumors from younger women (<40 years of age), in patients who experienced a shorter disease-free interval, and in ER- or PgR-negative (<10 fmol/mg protein) tumors. The median ER levels are about 10 times lower in the tumors with mutations than in those without a TP53 gene mutation (15 versus 144 fmol/mg protein, respectively). Menopausal status and prior adjuvant therapy were not significantly related to TP53 gene mutation. Interestingly, if the primary tumor had a TP53 mutation, the metastases more often developed in soft tissue (50%) or visceral tissues (46%) than in bone(26%) as the dominant site of disease. In an exploratory analysis, we observed a relatively low ratio between missense and nonmissense mutations in the primary tumor of patients who developed a relapse in soft tissue (ratio, 1.4) when compared with the ratios in patients who relapsed to bone or visceral tissues (ratios, 3 and 3.5, respectively)as the first site of relapse.

TP53 Gene Mutations and Response to Tamoxifen.

We observed a mutation in 65 of 202 (32%) primary tumor samples,and this resulted in a premature termination of the protein in 13 of these 65 samples (20%). Of the 202 patients, 55% responded to first-line tamoxifen therapy (10 CRs, 25 PRs, and 76 cases of stable disease). Very young patients (<40 years) tended to have a lower response rate (33%; Table 2). Furthermore, the presence of bone or visceral metastasis, a short disease-free interval after primary surgery, and low ER and PgR levels were significantly associated with low response rates to tamoxifen, whereas prior adjuvant therapy showed no relation with response (Table 2). Sixty-six percent of the patients without a TP53 mutation responded to tamoxifen, whereas only 31% of the patients with a TP53 mutation responded to tamoxifen therapy (OR,0.22; P < 0.0001; Table 2). There was no significant difference between the response percentages of either missense or nonmissense mutations (29% and 38%, respectively). The TP53 gene mutations were stratified according to the type of mutation, i.e., mutations in the zinc-binding domains L2 and L3 of the protein, or by residues that directly contact DNA in an exploratory analysis. Five of 12 (42%) patients with mutations in L2 but only 2 of 13 (15%) patients with mutations in L3 or 2 of 11(18%) patients with mutations in codons that directly contact DNA showed a response compared to a response of 66% in patients with a wild-type TP53 gene.

Multivariate Analysis for Response to Tamoxifen.

The independent relationship of the variables with response to tamoxifen therapy for advanced disease was studied using multivariate logistic regression analysis. TP53 gene mutation status (as a dichotomized variable) was added to the classical variables (see Table 2). Disease-free interval, dominant site of relapse, and ER and TP53 status were significant in this multivariate analysis. There was no statistically significant interaction between TP53 mutation and ER levels.

Response in Clinically Relevant Subsets of Patients Stratified by ER and PgR Status.

We explored the association of TP53 gene status with response to tamoxifen therapy in clinically relevant subgroups. Subsets of tumors from patients with low ER levels (<10 fmol/mg protein,median survival of the patients = 16 months; see Table 2), intermediate ER levels (≥10 fmol/mg protein but <75 fmol/mg protein, median survival of the patients = 23 months),and high ER levels (>75 fmol/mg protein, median survival of the patients = 30 months) were created. In all three subgroups, according to ER status, the response rate was better in patients with wild-type TP53 in their primary tumors. The worst overall response was observed in patients with ER-negative and TP53-mutated tumors (n = 23), of whom only 22% responded. The best response was observed in the patients with high ER levels and wild-type TP53 in their primary tumors (n = 90), of whom 73% responded.

Relationship Between P53 Mutation and PFS after the Start of Treatment.

Duration of response is perhaps as important as time to treatment failure because the duration of response contributes directly to the quality and duration of life. Using Cox univariate regression analysis,we observed that the presence of a TP53 gene mutation was significantly associated with a shorter duration of response (16 versus 11 months; P = 0.0006) in 111 patients who responded to tamoxifen. However, TP53mutation was not significantly associated with survival after the start of treatment (40 versus 31 months) in the responders.

The median PFS was shorter for patients with a TP53 mutation as compared with those with wild-type TP53 (3.0 versus 9.6 months). Patients with TP53 gene mutation in their primary tumors experienced a shorter PFS and an earlier death (relative hazard rate, 2.61 and 1.99, respectively, both P < 0.001) than those with TP53 wild-type tumors (see Fig. 1, ALL). In addition (see Table 2), younger patients(age < 40 years) and those with visceral metastasis, a short disease-free interval, low ER or PgR levels, or TP53 mutation showed a shorter median overall postrelapse survival after the start of tamoxifen treatment. In the multivariate analysis for survival after the start of tamoxifen treatment, the presence of a TP53 mutation was associated with decreased survival (RHR, 1.39; 95% CI,0.94–2.07; P = 0.10).

We next explored the predictive value of TP53 mutations in subgroups with low, intermediate, and high ER levels. The relationship between TP53 status and PFS and overall survival after the start of treatment was most apparent in the high ER and intermediate ER (Fig. 1, ER-medium) subsets of patients but absent in the ER-negative subset (Fig. 1, bottom panels).

TP53 Gene Mutations and Response to Chemotherapy.

Of the 113 patients that were evaluable for chemotherapy after tamoxifen therapy in this study, 51 (45%) responded to chemotherapy (1 CR, 19 PRs, and 31 cases of stable disease). When compared with bone metastasis and soft tissue, patients with visceral metastasis had the worst response to chemotherapy after tamoxifen treatment (OR, 0.66). As shown in Table 3, patients with TP53 mutations did not respond differently to chemotherapy after tamoxifen therapy than did patients without mutations. Stratification according to domain L3 of the protein or by residues that directly contact DNA (see above) revealed that three of five patients with mutations in L3 and two of seven patients with mutations in codons that directly contact DNA showed a response when compared with an overall response of 42% of patients with the wild-type TP53 gene.

Of the 41 patients studied with respect to up-front (first-line)chemotherapy, 19 (46%) responded (4 CRs, 11 PRs, and 4 cases of stable disease). A relatively high prevalence (61%) of TP53 gene mutation was observed, i.e.,TP53 gene mutation was observed in 25 of 41 primary tumors. In 10 of these 25 samples(40%), this resulted in a premature termination of the protein. As shown in Table 3, only 36% percent of the 25 patients with a TP53 mutation responded to first-line chemotherapy, in contrast to 63% of the 16 patients without a TP53 mutation (OR, 0.34). In this exploratory study on a small number of patients, only menopausal status was significantly correlated with response to up-front chemotherapy; none of the other patient and tumor characteristics studied were significantly correlated with response to up-front chemotherapy. The median PFS was 3.0 months for those patients with TP53-mutated tumors versus 3.6 months for patients without TP53 mutation.

The onset and progression of breast cancer is accompanied by multiple genetic alterations that result in quantitative and qualitative changes in the expression of these genes. Mutations in the TP53 gene are the most frequent genetic changes in human cancer, and, depending on the method of detection, the frequencies of TP53 mutations reported in invasive breast cancer range from 12–46% (24). In recent years, numerous reports have appeared on the relation between TP53 status and (disease-free)survival, and conflicting conclusions were reached on the prognostic value of TP53 in breast cancer. The lack of unanimity between authors may be explained by: (a) differences in techniques used for the analyses of p53 status (e.g., immunohistochemical analyses with different antibodies on frozen or paraffin-embedded tissues using different cutoff levels, ELISA/LIA, PCR-single-strand conformational polymorphism/constant denaturing gel electrophoresis analyses of primarily exons 5–8, or cDNA sequencing of the entire gene); (b) patient sample size;(c) subset analyses; (d) retrospective nature of the studies; (e) different (adjuvant) treatments of the patient population; (f) different (modern) prognostic covariates used in the multivariate analyses; (g) the subjectivity inherent to some approaches; and (h)publication bias.

Moreover, the relapse tumor could be dissimilar to the primary tumor because it was shown that TP53 mutations found in the primary tumors can be absent in the metastasis.

In the present study, we evaluated the predictive value of TP53 gene mutations, as estimated through cDNA sequencing of the entire coding sequence of the TP53 gene, in a relatively large series of breast cancer patients who were treated with tamoxifen or chemotherapy for advanced disease. We observed TP53 gene mutations in 32% of the 202 primary tumors of tamoxifen-treated patients and in 61% of the 41 patients (with mainly ER-negative tumors) treated with up-front chemotherapy. The observed prevalence is slightly higher than the mean percentage of 25% [range, 15–71%;examined in 1425 breast tumor samples worldwide; reviewed by Hartmann et al.(25)] and a prevalence of 29% [range,15–71%; examined in 16 populations by Soussi (26)]. This difference can be explained by the fact that the entire coding sequence of TP53 was investigated and that only those patients who developed advanced disease were included in the present study. TP53 gene mutation was related with a poor response to up-front chemotherapy in our small series of patients (OR, 0.34) and was similarly but significantly related with a poor response to first-line tamoxifen treatment in both univariate analysis (OR, 0.22; P < 0.0001) and multivariate analysis. In contrast, patients with mutated TP53 in their primary tumors may respond better to chemotherapy (50% versus 42%) after tamoxifen treatment, but they did show a shorter PFS and duration of response.

When evaluating only those reports that have covered TP53 alterations in relationship with the efficacy of various treatments in breast cancer, we noticed that there is no agreement on the significance of the predictive value of TP53. The summary of these 26 studies on almost 6000 breast cancer patients is listed in Table 4. In 15 reports, there was no significant relation between TP53 status and type of response (2, 3, 6, 7, 8, 9, 10, 11, 12, 15, 28, 30, 31, 32, 33),7 reports described resistance (Refs. 5, 6, 14, 16, 27 and29 and this study), whereas the other 4 studies reported sensitivity (13, 34, 35, 36). However, this survey indicates that the TP53 genotype, not the immunohistochemical results, is predictive of response in breast cancer patients: all 5 studies that used DNA analysis showed a predictive value of TP53, whereas this was only true in 6 of the 21 immunohistochemical studies, i.e., 2 studies showed sensitivity, and 4 showed resistance. Both studies on radiotherapy showed sensitivity to this form of treatment (34, 36).

The mechanisms that underlie the relationship of p53 alterations and a poor response to tamoxifen are not clear. Tamoxifen acts as an antiestrogen via the ER, but some of its effects are thought to be mediated through the activation of transforming growth factor β and by decreasing plasma insulin-like growth factor I levels. Previous data suggest that the mutant forms of TP53 inhibit the antiproliferative effect of transforming growth factor β by interfering with its signaling pathway. In addition, wild-type TP53 can repress the insulin receptor and the insulin-like growth factor I receptor promotor in other cell types. Although the exact mechanisms are unclear, this complex interplay provides a link between the TP53 gene and signaling pathways in breast cancer cells (discussed in Ref.5). Our finding on the relationship between TP53gene mutation and poor response to tamoxifen treatment of advanced disease is well in line with our previous study on p53 protein levels by LIA in 401 patients with advanced disease (5) but disagrees with the studies of Archer et al.(2)and Elledge et al.(3), who assessed p53 expression immunohistochemically and found no relationship with response to endocrine treatment. Bergh et al.(14) and Silvestrini et al.(32)showed that tamoxifen also appears to be of less benefit in patients with TP53 gene mutations or overexpression, respectively, in the adjuvant setting (see Table 4). The relationship of TP53gene status with PFS is most apparent in the high ER (>10 fmol/mg protein) subgroups of our patients. In contrast to our findings,Elledge et al.(3), who studied the accumulation of p53 using IHC in mainly ER-positive metastatic breast cancer, did not observe a relation between p53 status and response to tamoxifen therapy, although they stated that breast tumors with altered p53 protein are inherently more aggressive, even after they have metastasized. These conflicting outcomes may again be explained by the different techniques and study designs.

It is known that some chemotherapeutic agents and ionizing radiation act by inducing apoptosis in tumor cells. Lowe et al.(37) showed that cells expressing mutant TP53 were totally resistant to apoptosis on treatment, whereas cells expressing the wild-type gene were sensitive to these therapeutic agents. In the present study, TP53 gene mutations were related, although not significantly, to a poor response to up-front chemotherapy (36%versus 63%; OR, 0.34). However, the number of patients receiving first-line chemotherapy was small, the regimen is heterogeneous, and the follow-up was relatively short. Based on this,it is not possible to draw firm conclusions. Furthermore, we suspect a better response to chemotherapy after tamoxifen treatment.

Three reported molecular analyses also revealed that TP53gene mutations predict a response to chemotherapy or adjuvant radiotherapy in breast cancer patients (6, 33, 35). In contrast, all 4 reports on metastatic disease and 7 of 10 reports in the adjuvant setting revealed no predictive value of TP53 when using immunohistochemical analyses.

Various mutations can alter the TP53 protein distinctly and lead to different biological characteristics and tumorigenic potential(38, 39). L2 and L3 loops of the TP53 gene contain residues involved in direct DNA contact as well as protein stabilization. In the present study, those patients with TP53 gene mutations in codons that directly contact DNA or with mutations in the zinc-binding domain loop L3 showed the lowest response to tamoxifen (18% and 15% response rates, respectively). Survival analyses showed a significantly reduced survival rate for patients with mutations affecting the zinc-binding domains L2 and L3 compared with patients with mutations outside these regions or with no mutations (40) or for patients with mutations affecting the direct DNA contact (41). Moreover, Aas et al.(6) showed that mutants affecting the L3 loop were significantly associated with de novo resistance to doxorubicin monotherapy in small subsets of patients. Extending the analysis of these various mutations allows us to focus attention on the biological significance of particular mutations that may assist the selection of residues that could be of predictive value in breast cancer.

IHC is not able to detect every TP53 alteration. In the present study,we have shown that one-fourth of TP53 mutations found are“null mutations” that do not lead to TP53 accumulation. Furthermore, IHC does not us allow to distinguish the heterogeneity of TP53 mutants. Based on the data from the literature and the present results, we conclude that the presence of mutation and the type of mutation have predictive value for response to tamoxifen treatment. Therefore, we reason that, if tissue resources permit, the TP53 mutation status should preferably be included in the daily practice of treatment of metastatic breast cancer patients.

The main conclusion of this study is that TP53 mutation is significantly associated with a poor response to tamoxifen treatment in patients with advanced breast cancer. Based on the present results and the data reviewed, it is tempting to hypothesize that the TP53 genotype in particular and, to a lesser degree, the immunohistochemical analysis are predictive of response to therapy in breast cancer. Prospective studies should be performed to support this conclusion. Besides mutation analyses, a direct functional assay (42) or the measurement of downstream components of the TP53 pathway such as p21 or MDM2 may confirm the TP53 integrity.

Fig. 1.

PFS (left) and survival after the start of tamoxifen treatment (right) as a function of TP53 gene status in all patients (ALL),patients with high ER (>75 fmol/mg protein; ER-high),patients with intermediate ER (10 > ER <75 fmol/mg protein; ER-medium), and patients with low ER (<10 fmol/mg protein; ER-low). The number of patients below the X axis represents the number at risk over total patients in the wild-type and mutated subgroups, respectively.

Fig. 1.

PFS (left) and survival after the start of tamoxifen treatment (right) as a function of TP53 gene status in all patients (ALL),patients with high ER (>75 fmol/mg protein; ER-high),patients with intermediate ER (10 > ER <75 fmol/mg protein; ER-medium), and patients with low ER (<10 fmol/mg protein; ER-low). The number of patients below the X axis represents the number at risk over total patients in the wild-type and mutated subgroups, respectively.

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1

Supported in part by Grant DDHK 96-1234 from the Dutch Cancer Society.

3

The abbreviations used are: ER, estrogen receptor; PgR, progesterone receptor; OR, odds ratio, CI, confidence interval; LIA, luminometric assay; CMF, cyclophosphamide, methotrexate,and 5-fluorouracil; CAF, cyclophosphamide, Adriamycin, and 5-fluoro-uracil; CR, complete remission; PR, partial remission;PFS, progression-free survival; IHC, immunohistochemistry.

Table 1

Patient and tumor characteristics and TP53 mutation

TP53 gene status
No. of patients% mutatedP
All patients 243 37  
Menopausal statusa    
Premenopausal 70 41  
Postmenopausal 173 35 0.37 
Age (yr)a    
<40 25 64  
40–55 79 39  
55–65 58 29  
>65 81 32 0.01c 
Dominant site of relapse    
Soft tissue 34 50  
Bone 115 26  
Visceral 94 46 0.003 
Disease-free interval (mo)    
<12 90 49  
>12 153 30 0.003 
Prior adjuvant therapy    
No 204 35  
Yes 39 46 0.20 
ER levels (fmol/mg protein)b    
<10 63 67  
10–75 58 36  
>75 121 22 <0.0001c 
PgR levels (fmol/mg protein)b    
<10 82 57  
10–75 68 35  
>75 90 19 0.0001c 
TP53 gene status
No. of patients% mutatedP
All patients 243 37  
Menopausal statusa    
Premenopausal 70 41  
Postmenopausal 173 35 0.37 
Age (yr)a    
<40 25 64  
40–55 79 39  
55–65 58 29  
>65 81 32 0.01c 
Dominant site of relapse    
Soft tissue 34 50  
Bone 115 26  
Visceral 94 46 0.003 
Disease-free interval (mo)    
<12 90 49  
>12 153 30 0.003 
Prior adjuvant therapy    
No 204 35  
Yes 39 46 0.20 
ER levels (fmol/mg protein)b    
<10 63 67  
10–75 58 36  
>75 121 22 <0.0001c 
PgR levels (fmol/mg protein)b    
<10 82 57  
10–75 68 35  
>75 90 19 0.0001c 
a

Age and menopausal status at start therapy.

b

ER data are missing for one patient, but PgR data are present. PgR data are missing on three patients for whom ER data are available.

c

Test for trend.

Table 2

Patient and tumor characteristics and response rate to first-line tamoxifen therapy of recurrent disease

No. of patientsResponse rate (%)UnivariateMultivariateSurvival after start of therapy (median)
PORCIPORCI
All patients 202 55       24 mo 
Menopausal status          
Premenopausal 47 45     30 mo 
Postmenopausal 155 58 0.11 1.71 0.89–3.31 0.67 1.28 0.41–4.01 23 mo 
Age (yr)a          
<40 15 33     18 mo 
40–55 61 51  2.07 0.63–6.76  1.33 0.31–5.68 29 mo 
55–65 51 57  2.64 0.79–8.82  1.44 0.24–8.78 25 mo 
>65 75 61 0.21 3.17 0.98–10.22 0.55 2.47 0.41–15.01 20 mo 
Dominant site of relapse          
Soft tissue 24 79     28 mo 
Bone 105 53  0.30 0.10–0.87  0.12 0.03–0.44 28 mo 
Visceral 73 49 0.03 0.26 0.09–0.76 0.001 0.11 0.03–0.43 19 mo 
Disease-free interval (mo)          
<12 70 37     18 mo 
>12 132 64 0.0002 3.06 1.68–5.59 0.0027 2.95 1.44–6.07 29 mo 
Prior adjuvant therapy          
No 175 54     24 mo 
Yes 27 59 0.63 1.22 0.54–2.79 0.23 1.95 0.65–5.84 25 mo 
ER levels (fmol/mg protein)b          
<10 37 30   16 mo 
10–75 47 47  2.08 0.84–5.16  1.84 0.57–5.92 23 mo 
>75 117 66 0.00025 4.55 2.04–10.14 0.03 3.82 1.27–11.51 30 mo 
PgR levels (fmol/mg protein)b          
<10 53 43   19 mo 
10–75 61 48  1.18 0.56–2.48  0.70 0.27–1.82 21 mo 
>75 85 68 0.0054 2.80 1.38–5.70 0.28 1.33 0.52–3.41 36 mo 
TP53 gene status          
Wild-type 137 66     29 mo 
Mutated 65 31 <0.0001 0.22 0.12–0.42 0.0014 0.29c 0.13–0.63 20 mo 
No. of patientsResponse rate (%)UnivariateMultivariateSurvival after start of therapy (median)
PORCIPORCI
All patients 202 55       24 mo 
Menopausal status          
Premenopausal 47 45     30 mo 
Postmenopausal 155 58 0.11 1.71 0.89–3.31 0.67 1.28 0.41–4.01 23 mo 
Age (yr)a          
<40 15 33     18 mo 
40–55 61 51  2.07 0.63–6.76  1.33 0.31–5.68 29 mo 
55–65 51 57  2.64 0.79–8.82  1.44 0.24–8.78 25 mo 
>65 75 61 0.21 3.17 0.98–10.22 0.55 2.47 0.41–15.01 20 mo 
Dominant site of relapse          
Soft tissue 24 79     28 mo 
Bone 105 53  0.30 0.10–0.87  0.12 0.03–0.44 28 mo 
Visceral 73 49 0.03 0.26 0.09–0.76 0.001 0.11 0.03–0.43 19 mo 
Disease-free interval (mo)          
<12 70 37     18 mo 
>12 132 64 0.0002 3.06 1.68–5.59 0.0027 2.95 1.44–6.07 29 mo 
Prior adjuvant therapy          
No 175 54     24 mo 
Yes 27 59 0.63 1.22 0.54–2.79 0.23 1.95 0.65–5.84 25 mo 
ER levels (fmol/mg protein)b          
<10 37 30   16 mo 
10–75 47 47  2.08 0.84–5.16  1.84 0.57–5.92 23 mo 
>75 117 66 0.00025 4.55 2.04–10.14 0.03 3.82 1.27–11.51 30 mo 
PgR levels (fmol/mg protein)b          
<10 53 43   19 mo 
10–75 61 48  1.18 0.56–2.48  0.70 0.27–1.82 21 mo 
>75 85 68 0.0054 2.80 1.38–5.70 0.28 1.33 0.52–3.41 36 mo 
TP53 gene status          
Wild-type 137 66     29 mo 
Mutated 65 31 <0.0001 0.22 0.12–0.42 0.0014 0.29c 0.13–0.63 20 mo 
a

Age (years) at the time of the primary tumor.

b

ER information on one patient and PgR information for three patients are missing.

c

The increment in χ2 is 10.2. Significant data are shown in bold.

Table 3

Patient and tumor characteristics and response to chemotherapy of patients with recurrent disease

No. of patientsUp-front chemotherapyNo. of patientsChemotherapy after tamoxifen
ResponseUnivariateResponseUnivariate
(%)POR95% CI(%)POR95% CI
All patients 41 46    113 45    
Menopausal status           
Premenopausal 23 61   29 48   
Postmenopausal 18 28 0.03 0.25 0.07–0.93 84 44 0.69 0.84 0.36–1.97 
Age (yr)           
<40 10 50   10 50   
40–55 18 56  1.25 0.27–5.89 39 49  0.95 0.24–3.81 
55–65 43  0.75 0.11–5.24 34 47  0.89 0.22–3.64 
>65 17 0.38 0.20 0.02–2.39 30 37 0.75 0.58 0.14–2.46 
Dominant site of relapse           
Soft tissue 10 40    4a 50   
Bone 10 60  2.25 0.38–13.47 46 52  1.09 0.14–8.42 
Visceral 21 43 0.60 1.13 0.24–5.21 63 40 0.42 0.66 0.09–4.98 
Disease-free interval (mo)           
<12 20 55   39 44   
>12 21 38 0.28 0.50 0.14–1.75 74 46 0.81 1.10 0.50–2.40 
Prior adjuvant therapy           
No 29 48   96 45   
Yes 12 42 0.70 0.77 0.20–2.98 17 47 0.86 1.10 0.39–3.08 
ER levels (fmol/mg protein)           
<10 26 46   21 43   
10–75 11 55  1.40 0.34–5.76 31 45  1.10 0.36–3.35 
>75 25 0.59 0.39 0.04–4.25 60 47 0.95 1.17 0.43–3.18 
PgR levels (fmol/mg protein)           
<10 29 48   29 41   
10–75 43  0.80 0.15–4.25 40 43  1.05 0.40–2.76 
>75 40 0.92 0.71 0.10–4.93 42 50 0.71 1.42 0.55–3.68 
TP53 gene status           
Wild type 16 63   73 42   
Mutated 25 36 0.10 0.34 0.09–1.24 40 50 0.44 1.35 0.62–2.94 
No. of patientsUp-front chemotherapyNo. of patientsChemotherapy after tamoxifen
ResponseUnivariateResponseUnivariate
(%)POR95% CI(%)POR95% CI
All patients 41 46    113 45    
Menopausal status           
Premenopausal 23 61   29 48   
Postmenopausal 18 28 0.03 0.25 0.07–0.93 84 44 0.69 0.84 0.36–1.97 
Age (yr)           
<40 10 50   10 50   
40–55 18 56  1.25 0.27–5.89 39 49  0.95 0.24–3.81 
55–65 43  0.75 0.11–5.24 34 47  0.89 0.22–3.64 
>65 17 0.38 0.20 0.02–2.39 30 37 0.75 0.58 0.14–2.46 
Dominant site of relapse           
Soft tissue 10 40    4a 50   
Bone 10 60  2.25 0.38–13.47 46 52  1.09 0.14–8.42 
Visceral 21 43 0.60 1.13 0.24–5.21 63 40 0.42 0.66 0.09–4.98 
Disease-free interval (mo)           
<12 20 55   39 44   
>12 21 38 0.28 0.50 0.14–1.75 74 46 0.81 1.10 0.50–2.40 
Prior adjuvant therapy           
No 29 48   96 45   
Yes 12 42 0.70 0.77 0.20–2.98 17 47 0.86 1.10 0.39–3.08 
ER levels (fmol/mg protein)           
<10 26 46   21 43   
10–75 11 55  1.40 0.34–5.76 31 45  1.10 0.36–3.35 
>75 25 0.59 0.39 0.04–4.25 60 47 0.95 1.17 0.43–3.18 
PgR levels (fmol/mg protein)           
<10 29 48   29 41   
10–75 43  0.80 0.15–4.25 40 43  1.05 0.40–2.76 
>75 40 0.92 0.71 0.10–4.93 42 50 0.71 1.42 0.55–3.68 
TP53 gene status           
Wild type 16 63   73 42   
Mutated 25 36 0.10 0.34 0.09–1.24 40 50 0.44 1.35 0.62–2.94 
a

Dominant site of relapse at start of chemotherapy after tamoxifen treatment. Significant data are shown in bold.

Table 4

Predictive relevance of TP53 in breast cancer

AuthorYearNo.Stage of diseaseTechnique% abnormalTreatmentPredictive
Endocrine therapy  1435      
Bergh 1995 298 Primary cDNA sequencing 22% Adjuvant TAMa and RT Resistant 
Archer 1995 92 Advanced IHC 58% Hormonal No 
Silvestrini 1996 240 Primary IHC 14% Adjuvant TAM Resistant 
Elledge 1997 202 Metastatic IHC 20% TAM No 
Berns 1998 401 Metastatic LIA Median TAM Resistant 
Berns 2000 202 Metastatic cDNA sequencing 32% TAM Resistant 
 (this study)       
Chemoendocrine therapy  341      
Elledge 1995 261 Primary IHC 38% Adjuvant chemo (CMF & prednisone) No 
Markris 1997 80 Primary IHC 39% Neoadjuvant chemo and TAM No 
Chemo  3428      
Jacquemier 1994 81 Primary IHC 39% Adjuvant chemo No 
Faille 1994 38 Loc advanced PCR-SSCP sequencing 36% Chemo Resistant 
Muss 1994 394 Primary IHC 35% Adjuvant chemo No 
Stål 1995 139 Primary IHC 14% Adjuvant chemo Sensitive 
Aas 1996 63 Loc advanced IHC ??? Doxorubicin monotherapy No 
Aas 1996 63 Loc advanced CDGE/cDNA sequencing 29% Doxorubicin monotherapy Resistant 
MacGrogan 1996 125 Primary IHC 27% Neoadjuvant chemo No 
Linn 1997 70 Primary IHC 65%/30% Neoadjuvant chemo high dose & GM-CSF No 
Dublin 1997 277 Primary IHC 21% Adjuvant CMF No 
Niskanen 1997 103 Metastatic IHC 16% FEC No 
Bonetti 1998 43 Metastatic IHC 51% CMF/CA-EF No 
Rozan 1998 329 Primary IHC 38% Neoadjuvant chemo No 
Degeorges 1998 277 Primary IHC 24% Adjuvant chemo No 
Clahsen 1998 440 Primary IHC 18% FAC, one course perioperative Resistant 
Järvinen 1998 55 Advanced IHC 33% Epirubicin monotherapy No 
Thor 1998 994 Primary IHC 33% Adjuvant CAF dose intensive Sensitive 
Radiotherapy  781      
Jansson 1995 168 Primary cDNA-based sequence 18% Adjuvant locoregional RT Sensitive 
Silvestrini 1997 613 Primary IHC 18% Adjuvant RT Sensitive 
AuthorYearNo.Stage of diseaseTechnique% abnormalTreatmentPredictive
Endocrine therapy  1435      
Bergh 1995 298 Primary cDNA sequencing 22% Adjuvant TAMa and RT Resistant 
Archer 1995 92 Advanced IHC 58% Hormonal No 
Silvestrini 1996 240 Primary IHC 14% Adjuvant TAM Resistant 
Elledge 1997 202 Metastatic IHC 20% TAM No 
Berns 1998 401 Metastatic LIA Median TAM Resistant 
Berns 2000 202 Metastatic cDNA sequencing 32% TAM Resistant 
 (this study)       
Chemoendocrine therapy  341      
Elledge 1995 261 Primary IHC 38% Adjuvant chemo (CMF & prednisone) No 
Markris 1997 80 Primary IHC 39% Neoadjuvant chemo and TAM No 
Chemo  3428      
Jacquemier 1994 81 Primary IHC 39% Adjuvant chemo No 
Faille 1994 38 Loc advanced PCR-SSCP sequencing 36% Chemo Resistant 
Muss 1994 394 Primary IHC 35% Adjuvant chemo No 
Stål 1995 139 Primary IHC 14% Adjuvant chemo Sensitive 
Aas 1996 63 Loc advanced IHC ??? Doxorubicin monotherapy No 
Aas 1996 63 Loc advanced CDGE/cDNA sequencing 29% Doxorubicin monotherapy Resistant 
MacGrogan 1996 125 Primary IHC 27% Neoadjuvant chemo No 
Linn 1997 70 Primary IHC 65%/30% Neoadjuvant chemo high dose & GM-CSF No 
Dublin 1997 277 Primary IHC 21% Adjuvant CMF No 
Niskanen 1997 103 Metastatic IHC 16% FEC No 
Bonetti 1998 43 Metastatic IHC 51% CMF/CA-EF No 
Rozan 1998 329 Primary IHC 38% Neoadjuvant chemo No 
Degeorges 1998 277 Primary IHC 24% Adjuvant chemo No 
Clahsen 1998 440 Primary IHC 18% FAC, one course perioperative Resistant 
Järvinen 1998 55 Advanced IHC 33% Epirubicin monotherapy No 
Thor 1998 994 Primary IHC 33% Adjuvant CAF dose intensive Sensitive 
Radiotherapy  781      
Jansson 1995 168 Primary cDNA-based sequence 18% Adjuvant locoregional RT Sensitive 
Silvestrini 1997 613 Primary IHC 18% Adjuvant RT Sensitive 
a

TAM, tamoxifen; RT, radiotherapy; chemo,chemotherapy; GM-CSF, granulocyte macrophage colony-stimulating factor; FEC, 5-fluorouracil, epirubicin, and cyclophosphamide;CA-EF, cyclophosphamide Adriamycin-epirubicin 5-fluorouracil;FAC, 5-fluorouracil, doxorubicin, and cyclophosphamide; Loc,local; SSCP, single strand conformational polymorphism; CDGE, constant denaturing gel electrophoresis.

We thank Esfir Lôfman and Nigel Tooke (Amersham Pharmacia Biotech) and J. Bolt-de Vries, S. E. Binnendijk-Noordegraaf,and H. Peters (Division of Endocrine Oncology, University Hospital Rotterdam, Rotterdam, the Netherlands) for excellent technical assistance.

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