Rationale • Over the past decade, there has been a rapid increase in the number of thera-peutic agents and regimens available for the treatment of breast cancer. Although the expansion in effective treatment options is certainly benefi-cial, it simultaneously introduces ever-increasing levels of complexity into the decision-making process as physicians seek to match each patient with the most appropriate therapy. • In the wake of the human genome sequencing project, there has been an explosion of molecular diagnostics designed to characterize human malig-nancies, identify therapeutic targets, and individualize treatments to maximize effectiveness and/or minimize adverse events. Already, efforts to harness the power of these genetic and proteomic tools have yielded valuable information regarding molecular classification of breast cancer subtypes, the association between genetic polymorphisms and susceptibility to chemotherapy-related toxicities, and selection criteria for tra-ditional and targeted therapies. • At the 2005 San Antonio Breast Cancer Symposium (SABCS), a num-ber of presentations offered new insights into the clinical utility of genomic tools. Some focused on opti-mizing the selection of patients with breast cancer for adjuvant chemo-therapy, and others identified molecu-lar markers that might be useful in determining the relative responsive-ness to commonly used agents such as anthracyclines and trastuzumab. Finally, additional studies investigat-ed estrogen receptor (ER)–negative breast cancers, revealing subclassi-fications at the molecular level that could provide clues to aid in the devel-opment of more effective treatments

for this subset of patients. Results of these studies are summarized herein.

Gene Expression Analysis for Recurrence Risk Assessment One major uncertainty in the area of adjuvant chemotherapy is the question of which women with node-negative early-stage breast cancer will benefit from adjuvant chemotherapy and which have such a low risk of recurrence that they could forego chemotherapy. Although guidelines have been devel-oped to aid in this decision process, they have traditionally been based on prog-nostic criteria such as tumor size, stage, and grade, which might not accurately reflect the underlying tumor biology and thus might not truly predict the need for and effectiveness of therapy for an individual patient. The 21-gene recurrence score assay was developed to aid in the assessment of risk for recurrence, using reverse-transcriptase polymerase chain reac-tion to quantitatively analyze the gene expression profile of a select panel of 16 breast cancer–related genes and 5 refer-ence genes. This panel, which includes genes involved in proliferation, estrogen signaling, HER2 signaling, and invasion, is used in conjunction with an algorithm to calculate a recurrence score. The clinical value of this assay in predicting the likelihood of distant recurrence-free

survival (RFS) was validated in a pro-spectively designed analysis of tamox-ifen-treated patients enrolled in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14 clinical trial.1 In a follow-up study of patients from the NSABP B-14 and B-20 trials, Paik et al demonstrated that patients with low and intermediate recurrence scores gained the greatest benefits from tamoxifen, whereas patients with high recurrence scores received the most benefit from adjuvant chemotherapy.2

At the 2005 SABCS, Mamounas et al presented results of a subsequent study of patients from the B-14 and B-20 trials, this time examining the association between the 21-gene recur-rence score and the risk of locoregional failure in patients with node-negative, ER-positive breast cancer treated with tamoxifen (both trials) or tamoxifen plus chemotherapy (B-20 only).3 The study populations included 895 patients treated with tamoxifen (668 patients from B-14; 227 patients from B-20), 355 patients from the placebo arm of B-14, and 424 patients treated with chemotherapy and tamoxifen on B-20. There was a significant association between the 10-year locoregional failure rate and recurrence score for patients treated with tamoxifen, with a local relapse rate of 4.3% for patients with a recurrence score < 18, 7.2% for those with an intermediate score (18-30), and 15.8% for those with a

Molecular Insights from the 2005 San Antonio Breast Cancer Symposium

Clinical Breast Cancer April 2006 • 29

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Prepared by: Susan Peck, PhDReviewed by: Joyce A. O’Shaughnessy, MD

Table 1: Ten-Year Locoregional Failure Rates by Treatment and Recurrence Score in NSABP B-20 and NSABP B-143

RecurrenceScore < 18

Placebo

Tamoxifen

Chemotherapyplus Tamoxifen

10.8

4.3

1.6

P ValueRecurrenceScore 18-30

20

7.2

2.7

RecurrenceScore > 31

18.4

15.8

7.8

0.022

< 0.0001

0.028

Values are percentages unless otherwise indicated.

Treatment

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high score ≥ 31 (P < 0.0001; Table 1).3 In the placebo-controlled group, locore-gional failure was significantly higher in patients with intermediate or high scores compared with those with scores < 18 (recurrence score < 18, 10.8%; recurrence score 18-30, 20%; recur-rence score ≥ 31, 18.4%; P = 0.022). Finally, locoregional relapse rates were also significantly higher in patients with a recurrence score ≥ 31 (7.8%) who received chemotherapy and tamoxi-fen compared with those with low or intermediate recurrence scores (recur-rence score < 18, 1.6%; recurrence score 18-30, 2.7%; P = 0.028). In a multivariate Cox proportional hazards model analysis, recurrence score, age (≥ 50 years vs. < 50 years), and surgery type (mastectomy vs. lumpectomy plus radiation therapy) were significantly associated with risk of locoregional failure (P = 0.005, P = 0.0002, and P = 0.047 respectively), whereas no association was found with clinical tumor size or grade. This analysis supports the utility of the reverse-transcriptase polymerase chain reaction–based assay in predicting not only distant but also locoregional events, potentially impact-ing clinical decision-making regarding appropriate local therapy as well as systemic treatment. A second genomic approach, which uses a gene chip microarray to measure messenger RNA expression, identified a 76-gene signature with prognostic ability in subclassifying node-nega-tive breast cancers according to risk of distant metastases in a single-center study.4 Results from an independent multicenter validation study designed to assess the prognostic utility of the 76-gene signature on a wider, more diverse

population of patients with breast can-cer were presented by Foekens et al at SABCS 2005 and recently published.5,6 Gene chip microarrays were used to analyze the messenger RNA expression of the 76 genes in 180 patients with node-negative early-stage breast cancer who had not received systemic adjuvant chemotherapy. Forty-three percent of these cancers were associated with a “good” gene signature, based on the previously defined profiles. At a medi-an follow-up of 100 months, 5- and 10-year distant metastases-free survival (DMFS) was significantly higher for patients with “good” compared with “bad” gene profiles (5-year DMFS, 96% vs. 74%; hazard ratio [HR], 7.41; P = 8.5 × 10–6; 10-year DMFS, 94% vs. 65%, respectively; Table 2).5,6 Sensitivity of the assay in predicting 5-year DMFS was 90% with 50% specificity. Overall survival (OS) also significantly favored patients in the good profile group (HR, 5.46; P = 0.002). In addition, prognostic ability for DMFS was maintained in all subgroups examined, including 126 postmenopausal patients (HR, 9.84; P = 0.002), 54 premenopausal patients (HR, 4.84; P = 0.0394), 164 patients with ER-positive disease (HR, 6.62; P = 0.0004), 95 T1 tumors (HR, 4.27; P = 0.0258), and 84 T2 tumors (HR, 13.6; P = 0.0108). Taken together, the accumulating evidence from studies performed using these 2 genetic profil-ing techniques demonstrate the value of using genomic-based approaches to identify patients who stand to receive the most benefit from systemic adju-vant chemotherapy, and future refine-ments will further aid in individualiz-ing treatment.

Molecular Markers as Predictors of Response to Chemotherapy As adjuvant therapy options become more diverse, particularly as new tar-geted agents with their associated costs move from the metastatic setting into the treatment of early-stage disease, it becomes imperative that predictors of response be identified in order to effec-

tively individualize therapy to maxi-mize the cost/benefit ratio, as well as to decrease exposure to unnecessary toxici-ties. At SABCS 2005, 2 groups presented data on molecular markers that are asso-ciated with responsiveness to therapy, which could potentially aid in treatment decision-making in the future. The potential role of the protoonco-gene c-MYC in cancer has long been recognized, since studies demonstrat-ing that deregulated expression leads to apoptosis or, in the presence of a second antiapoptotic signal, prolifera-tion and neoplastic progression.7,8 Kim et al investigated the significance of coamplification of c-MYC and HER2 in predicting response to adjuvant trastu-zumab in combination with chemo-therapy in patients on the NSABP B-31 trial.9 Analysis of patient samples from NSABP B-28 had shown that coam-plification of these 2 genes, assessed by fluorescence in situ hybridization, was associated with a poorer prognosis in patients treated with chemotherapy than when either gene alone was ampli-fied. Fluorescence in situ hybridization results were available from 1549 patients from NSABP B-31, 432 of whom dem-onstrated c-MYC amplification (28%). The addition of trastuzumab to che-motherapy improved RFS in patients without c-MYC amplification (HR, 0.63; P = 0.007), but this improvement was potentiated when c-MYC was amplified

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Table 2: Distant Metastases-Free Survival and 76-Gene Prognostic Signature5,6

5-Year DMFS*

Good

Bad

96

74

10-Year DMFS

94

65

Values are percentages.*Hazard ratio, 7.41 (poor vs. good signature);P = 8.5 × 10–6.

Signature

Table 3: Distant Metastases-Free Survival and 76-Gene Prognostic Signature5,6

Recurrence-Free Survival

c-MYC not amplified

c-MYC amplified

Overall Survival

c-MYC not amplified

c-MYC amplified

HazardRatio*

0.63

0.24

0.99

0.36

P Value

0.007

< 0.0001

0.96

0.012

Signature

*Doxorubicin/cyclophosphamide followed by docetaxel without versus with trastuzumab.

Molecular RIB.indd 4 5/4/06 9:10:28 AM

(HR, 0.24; P < 0.0001; Table 3).9 When all 4 HER2-overexpressing subgroups were compared, patients with ampli-fied c-MYC treated with chemotherapy alone had the worst prognosis, and those with amplified c-MYC who also received trastuzumab fared the best (P value for interaction = 0.001). A similar trend was observed for OS. No OS benefit with trastuzumab has been observed in this early analysis in patients with HER2-overexpressing diesase without ampli-fication of c-MYC (HR, 0.99; P = 0.96), but in those with c-MYC amplification, a significant survival advantage with trastuzumab is already detectable (HR, 0.36; P = 0.012). These results sug-gest that c-MYC coamplification might sensitize HER2-overexpressing cells to the effects of chemotherapy plus trastuzumab, potentially by enhanc-ing the proapoptotic action of c-MYC via inhibition of HER2 signaling. Further research is needed, however, to determine whether this marker has clinical utility in selecting patients for trastuzumab therapy. A second gene that is undergoing investigation for its predictive value is topoisomerase IIα (TOP2A). Response to anthracycline-based therapy has pre-viously been linked to amplification of HER2, but analyses in preclinical mod-els failed to substantiate this link.10-13

However, TOP2A, a cellular target of anthracyclines, is found in close prox-imity to HER2 on chromosome 17q and is frequently coamplified with HER2, providing the rationale for investigat-ing the potential relationship between TOP2A and sensitivity to anthracycline therapy. Press et al used fluorescence in situ hybridization to analyze the TOP2A gene status in samples from patients on Breast Cancer International Research Group 006, which randomized patients with HER2-overexpressing early-stage breast cancer to receive doxorubicin/cyclophosphamide followed by docetax-el without or with trastuzumab, or docetaxel/carboplatin/trastuzumab.14 HER2 and TOP2A were coamplified in 35% of cancers, and this was asso-ciated with greater RFS (P < 0.001)

and improved OS (P = 0.01) compared with patients in whom only HER2 was amplified. Furthermore, patients with TOP2A amplification received greater benefit from anthracycline-based ther-apy in terms of RFS (P < 0.001) and OS (P = 0.00589) when compared with those without TOP2A amplifi-cation. Once again, although further investigation is necessary, these results suggest the determination of TOP2A status might prove valuable in iden-tifying which patients should receive anthracycline-based therapies and which will receive equivalent benefit from non–anthracycline-based regimens.

Estrogen Receptor–Negative Breast Cancer Although the ER and progester-one receptor (PgR) provide prognostic information and are therapeutic targets and thus have been studied extensive-ly, comparatively little is understood about ER/PgR–negative cancers, and consequently, fewer treatment options exist for this subtype. Clinically, ER/PgR–negative tumors exhibit signifi-cant heterogeneity, suggesting that, similar to ER-positive cancers, subsets with distinctive biology and prognosis exist within this group. In an attempt to identify and classify hormone recep-tor–negative breast cancers, Doane et al performed gene expression analysis on 99 primary breast cancer samples and 8 cell lines using an oligonucleotide microarray system.15 Unsupervised cluster analysis revealed ≥ 2 distinct subgroups, designated as Class A, in which expression of 96 genes was > 2-fold higher when compared with other tumor samples grouped in Class B (P < 0.0001). HER2 gene expression did not differentiate between the ER-negative Class A or Class B cancers. A predictive algorithm based on the Class A/Class B gene signature was validated on an independent set of 77 ER/PgR–negative breast cancers. Of note, despite lack of ER or PR expression, the Class A gene expression signature is character-ized by an ER-positive molecular phe-notype, with many of the overexpressed

genes regulated by estrogen or contain-ing estrogen- or androgen-response ele-ments in their promoters. Of note, the androgen receptor (AR) was the only nuclear receptor overexpressed in Class A samples. In vitro analysis of an ER-negative Class A cell line revealed that proliferation was dependent on AR sig-naling promoted by androgens and that AR-regulated genes overlapped with the Class A signature. Of note, the Class A cell line was not stimulated by estrogen and was not inhibited by antiestrogens, whereas antiandrogen therapy blocked androgen-stimulated growth. These results suggest that AR signaling might provide a therapeutic target in a subset of ER/PgR–negative human breast can-cers, warranting further research, and that investigation into the molecular signatures of Class B cancers might allow additional subclassification and yield important information on under-lying mechanisms regulating growth and survival. Gruvberger-Saal et al examined the prognostic value of ER-β in patients with early-stage breast cancer treated with tamoxifen.16 Samples from 353 stage II breast cancers were examined for ER-β expression by immunohisto-chemistry. Thirty percent of the 353 cancers were negative for ER-α expres-sion, the ER that is identified by immu-nohistochemistry in standard clinical pathology practice and that predicts for tamoxifen benefit. Seventy-four percent of all samples were positive for ER-β, and 20% exhibited strong expression. Although ER-β expression was associ-ated with improved distant disease-free survival in all tamoxifen-treated patients (P = 0.01), this association was more dramatic in ER-α–negative cancers (P = 0.003) and was not observed in ER-α–positive cancers (P = 0.49). Estrogen receptor-α had prognostic value only in the absence of ER-β (P = 0.05) and was not predictive of distant dis-ease-free survival in cancers with mod-erate or high levels of ER-β expression (P = 0.4 and P = 0.09, respectively). Importantly, ER-β was associated with an improvement in OS in tamoxifen-

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treated ER-α–negative breast cancers (P = 0.04). When the ER-α–negative, tamoxifen-treated subgroup was exam-ined, lack of ER-β expression was asso-ciated with a poor prognosis compared with strongly positive expression (HR, 14; P = 0.01). Gene expression analysis revealed a distinct molecular signature in ER-β–positive cancers compared with ER-α–positive cancers. These results suggest that factors in addi-tion to ER-α expression might need to be taken into consideration when making decisions regarding endocrine therapy and that further investigation into the role of tamoxifen treatment in ER-α–negative, ER-β–positive breast cancer might reveal an effective strategy for the treatment for this cancer subset.

Clinical Relevance These studies illustrate the expand-ing role that molecular analysis is playing in the individualization of treatment and the identification of new therapeutic approaches. Although most molecular marker–based strate-gies, with the exception of gene expres-sion assays for the prediction of recur-rence risk, are still in the investi-gational stages, these presentations hint at a future in which treatment recommendations can be made based on the biologic characteristics of each patient’s cancer. Ongoing and future trials will evaluate the utility of prom-ising assays and biomarkers in indi-

vidualizing breast cancer treatments, and preclinical research will deepen understanding of the mechanisms of malignant growth and progression, providing new therapeutic targets and diagnostic tools. As molecular data from ongoing and planned trials accu-mulate and new rationally designed agents and therapies become available, further improvements in patient out-comes can be expected.

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assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med 2004; 351:2817-2826.

2. Paik S, Shak S, Tang G, et al. Expression of the 21 genes in the recurrence score assay and prediction of clinical benefit from tamoxifen in NSABP study B-14 and che-motherapy in NSABP study B-20. Breast Cancer Res Treat 2004; 88(suppl 1):s15 (Abstract #24).

3. Mamounas E, Tang G, Bryant J, et al. Association between the 21-gene recurrence score assay (RS) and risk of locoregional failure in node-negative, ER-positive breast cancer: results from NSABP B-14 and NSABP B-20. Breast Cancer Res Treat 2005; 94(suppl 1):S16 (Abstract #29).

4. Wang Y, Klijn JG, Zhang Y, et al. Gene-expression profiles to predict distant metas-tasis of lymph-node-negative primary breast cancer. Lancet 2005; 365:671-679.

5. Foekens JA, Atkins D, Sweep CGJ, et al. Multi-center validation of the 76-gene prognostic sig-nature in lymph node negative (LNN) primary breast cancer. Breast Cancer Res Treat 2005; 94(suppl 1):S15 (Abstract #28).

6. Foekens JA, Atkins D, Zhang Y, et al. Multicenter validation of a gene expres-sion-based prognostic signature in lymph node-negative primary breast cancer. J Clin Oncol 2006; 24:1665-1671.

7. Evan GI, Wyllie AH, Gilbert CS, et al. Induction of apoptosis in fibroblasts by c-myc protein. Cell 1992; 69:119-128.

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of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression. Cell 2002; 109:321-334.

9. Kim C, Bryant J, Horne Z. Trastuzumab sensitivity of breast cancer with co-ampli-fication of HER2 and cMYC suggests pro-apoptotic function of dysregulated cMYC in vivo. Presented at: The 28th Annual San Antonio Breast Cancer Symposium; December 8-10, 2005; San Antonio, TX. Abstract #46.

10. Muss HB, Thor AD, Berry DA, et al. c-erbB-2 expression and response to adju-vant therapy in women with node-positive early breast cancer. N Engl J Med 1994; 330:1260-1266.

11. Paik S, Bryant J, Park C, et al. erbB-2 and response to doxorubicin in patients with axillary lymph node-positive, hormone receptor-negative breast cancer. J Natl Cancer Inst 1998; 90:1361-1370.

12. Dressler LG, Berry DA, Broadwater G, et al. Comparison of HER2 status by fluorescence in situ hybridization and immunohistochemistry to predict benefit from dose escalation of adjuvant doxo-rubicin-based therapy in node-positive breast cancer patients. J Clin Oncol 2005; 23:4287-4297.

13. Pegram MD, Finn RS, Arzoo K, et al. The effect of HER-2/neu overexpression on che-motherapeutic drug sensitivity in human breast and ovarian cancer cells. Oncogene 1997; 15:537-547.

14. Press MF, Bernstein L, Sauter G, et al. Topoisomerase II-alpha gene amplification as a predictor of responsiveness to anthracy-cline-containing chemotherapy in the Cancer International Research Group 006 clinical trial of trastuzumab (herceptin) in the adju-vant setting. Breast Cancer Res Treat 2005; 94(suppl 1):S54 (Abstract #1045).

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