Daniel F. Hayes, M.D.
Breast Oncology Program, University of Michigan Comprehensive Cancer Center
Past data as well as results presented at this years SABCS highlight the importance of optimizing application of adjuvant systemic therapy. If such therapy were free of side effects, life-threatening toxicities, and financial cost, the obvious strategy would be to apply all therapy to all patients with invasive breast cancer. However, such is not the case, and therefore judicious application of the specific therapies most likely to work in those most likely to need it should continue to reduce mortality rates. To do so requires a fundamental understanding of prognostic factors, which permit identification of those patients who are likely to suffer relapse after primary therapy, and predictive factors, which help selection of therapies directed towards each individual’s tumor. While tumor markers have provided some advances in predicting prognosis, the most promising results come from gene expression profiles, including a 70-gene prognostic profile that utilizes microarray technology and the Oncotype DX®, a validated 21-gene profile.
Improved Mortality with Adjuvant Therapy: Background Data and Recent Updates from SABCS
Mortality from breast cancer has been decreasing in the western world over the last 15-20 years.
,,This decline is almost certainly due to widespread application of adjuvant systemic therapy to those patients most likely to have suffered recurrence and death without it. Although screening and early surgical intervention may make some contribution, this strategy was not widely applied in North America or Europe until the late 1980s and early 1990s, and mortality reductions from screening asymptomatic women would not be expected in the population for 10 years or more from the initiation of screening programs. Improved therapy for patients with metastatic disease also appears to improve survival, but the gains are modest and median survival from time of first metastasis remains below 30 months.
Meta-analyses and evaluation of individual clinical trial groups suggest that adjuvant tamoxifen decreases risk of mortality in patients with estrogen-receptor rich tumors by approximately one-fourth over 10-15 years.
,Although survival differences have not yet been reported, published results and updates at this SABCS demonstrate that the aromatase inhibitors are clearly more effective than tamoxifen in preventing recurrences, and one hopes that these advances will result in further reductions in mortality.
The Oxford overview also demonstrates that adjuvant chemotherapy, either alone or in association with endocrine therapy, reduces mortality.
More recent studies have demonstrated further progress, with addition of the taxanes and changes in schedule, using the so-called “dose-dense” approach.
,Indeed, again at this SABCS, investigators from the CALGB, presented consecutive results from a series of chemotherapy trials initiated from 1985 to 1997 that suggest that, in ER-negative patients, overall mortality may be decreased by as much as 50% or more with dose-dense therapy compared to low-dose CAF.
These survival benefits have spilled over to the community, as illustrated by an apparent 20-30% reduction in mortality from breast cancer over 10 years in a population-based study from British Columbia, also presented by Olivetto and colleagues at this year’s SABCS.
Optimizing the Use of Routine Prognostic Factors
Classically, clinical and pathologic staging, tumor grade and/or measures of proliferation are the most widely used and accepted prognostic factors.
,In general, these are accepted because they separate those likely to recur vs. those who will not, independent of treatment, by approximately 1.5 (in the case of grade and proliferation ) to 2.0 (clinical and pathological stage) fold. The only predictive factors that are widely accepted include estrogen receptor (ER) and progesterone receptor (PgR) for prediction of benefit from endocrine treatment and HER2 for prediction of benefit from trastuzumab.15 In the absence of definitive data demonstrating improved outcomes in the adjuvant setting, and pending the results of several large international trials, HER2 is only indicated for selection of trastuzumab in the metastatic setting or identification of patients who might be eligible for participation in the randomized studies. The relative benefit of endocrine therapy in ER-positive patients vs. ER-negative patients, and of trastuzumab in HER2-positive patients vs. HER2-negative patients appears to be four-fold or greater.5,
Adjuvant! These markers have been incorporated into a patient-physician decision aid, designated Adjuvant!, that can be used to estimate an individual’s risk of recurrence in the absence of adjuvant systemic therapy and the absolute odds of her benefiting from it.
At this year’s meeting, further validation of Adjuvant! in a community-based population was also presented by the British Columbia Cancer Agency investigators.14
New Tumor Markers
Clearly, new prognostic and predictive factors are needed. In the wake of the revolution in molecular biology over the last 20 years, several have been proposed. However, they have not been accepted for routine clinical care, either because initially exciting results have not been validated, or because the magnitude of separation in outcomes between “positive” and “negative” patients has not been sufficient to justify differential treatment of the two groups.
Two of the best studied of the newer markers are the presence or absence of bone marrow metastases (BMM), as demonstrated by immunohistochemical techniques, and overexpression of urinary plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1).
,However, the prognostic significance of BMM has not been validated in the group of patients for whom one is most likely to need the information: women with small, node-negative tumors who would otherwise not receive chemotherapy.
The enzyme-linked immunosorbent assay (ELISA) required for UPA/PAI-1 determination can only be performed in fresh/frozen tissue.
Thus, the reasonably large amounts of tissue required for this assay are impractical in North America in the setting of widespread screening and smaller tumors, and the prognostic significance of uPA/PAI-1 determination in core biopsies has not been validated.
Gene Expression Profiles
In retrospect, perhaps it is naïve to hypothesize that a single or small number of markers would be sufficiently powerful to separate “good” from “bad” tumors to the extent that patients would elect to be treated differently based on the results. New technologies and statistical methodologies now permit evaluation of hundreds and even thousands of markers simultaneously, with grouping of samples based on gene expression profiles, or “signatures”.
The most widely studied such technologies so far are based on DNA segments representing different genes arrayed on chips or glass slides to which RNA from a given specimen is hybridized, a technique called microarrays.
A 70-gene Prognosis Profile
Using microarray technology developed by Rosetta Inpharmatics (Seattle, Washington), investigators from the Netherlands Cancer Institute reported remarkable results in two separately published manuscripts.
In their most recent publication, among 295 young patients with breast cancer for whom tissue had been archived, 180 had a poor-prognosis signature and 115 had a good-prognosis signature.27
At 10 years, the probability of remaining free of distant metastases was 50.6±4.5 percent in the group with a poor-prognosis signature and 85.2±4.3 percent in the group with a good-prognosis signature.
The estimated hazard ratio for distant metastases in the group with a poor-prognosis signature, as compared with the group with the good-prognosis signature, was 5.1 (95 percent confidence interval, 2.9 to 9.0; P<0.001).
At this year’s SABCS, a multi-center external validation study of this 70 gene signature was reported by Piccart on behalf of the TRANSBIG Consortium. Researchers studied tissue from 301 patients who were followed for at least 10 years in one of six non-Dutch European centers and one U.S. center.
Although the 70-gene profile signature was strongly prognostic, again outperforming classic prognostic criteria such as those used by the St. Gallen consensus panel, the magnitude of effect was much less than what was previously reported in the Dutch series, with hazard ratios for time to distant metastases = 1.85 [1.14.-3.0] and for overall survival = 2.5 [1.4 – 4.5]. < /SPAN > The European Organization for Research and Treatment of Cancer (EORTC) and the Breast International Group (BIG) are now designing a large, prospective clinical trial to validate the utility of this assay in patients with newly diagnosed breast cancer (the “MINDACT” study).
Oncotype DX™: Utilizing Formalin-Fixed, Paraffin-Embedded Tissue
Since frozen tissue is difficult to collect in the era of screening, a multi-gene assay that can be applied to formalin-fixed, paraffin-embedded (FFPE) tissue would be of great value. Such an assay has been developed by Genomics Health Inc (Redwood City, CA). This assay, called Oncotype DX™, is based on performance of multiple, real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays to quantify expression of several, selected genes in FFPE tissue. Two hundred and fifty candidate genes were selected from published literature, genomic databases, and in-house experiments performed on frozen tissue. Three separate sets of archived tissue from different clinical data sets were then used to test the relation between quantitative analysis of expression of these 250 candidate genes and breast cancer recurrence. From these data, a panel of 16 cancer-related genes and 5 reference genes were used to develop an algorithm to compute a “recurrence score,” ranging from 0-100, that can be used to estimate the odds of recurrence over 10 years from diagnosis.
Of importance, these studies were based on an important NSABP study: clinical trial B20, in which node-negative patients with ER-positive tumors were randomly assigned to tamoxifen alone or with CMF chemotherapy. Only those who took tamoxifen alone, without chemotherapy, were studied in this initial evaluation. A second study of the three test sets was a matched case control study of more than 700 node-negative women treated in the Northern California Kaiser Permanante hospital system. Results from this study were also presented at this years SABCS.
Paik and colleagues reported at last year’s SABCS, and just recently published data indicating that the Oncotype DX® assay is a reproducible and accurate prognostic test in node negative patients with ER-positive breast cancers who received tamoxifen. Patients were categorized into three recurrence score groups using the results from the three separate data sets, including B20:
- Low risk (recurrence score <18)
- Intermediate risk (recurrence score 18-30)
- High risk (recurrence score 31-100).
These data were then validated using archival FFPE tissue samples from NSABP B14, in which patients with node-negative, ER-positive breast cancer were randomly assigned to tamoxifen vs. placebo. Kaplan-Meier estimates of rate of distant recurrence of patients in B14 who took tamoxifen were found to be 6.8% (4.0-9.6%) for low risk, 14.3% (8.3-20.3%) for intermediate risk and 30.5% (23.6-37.4%) for high risk patients.
Evaluation of Oncotype DX® as a predictive factor: At this year’s SABCS, Paik and his NSABP colleagues updated their analysis to include patients in the other arms of B14 and B20.
In this regard, the investigators were now analyzing the 21-gene signature as a predictive factor, as well as a prognostic factor. . Indeed, in a study of neoadjuvant chemotherapy reported at the American Society of Clinical Oncology 2004 meeting, Gianni and colleagues from Milan observed that pathologic complete response to doxorubicin and paclitaxel was statistically more likely in those with higher Oncotype recurrence scores.
The 16 cancer-related genes that were evaluated were not randomly chosen. They included ER, ,PgR, HER2, and various markers of proliferations. ER and PgR are known positive predictive factors for endocrine therapy and probably negative predictive factors for chemotherapy. HER2 is likely a negative predictive factor for tamoxifen, if not all endocrine therapies, and may be a negative predictive factor for benefit from CMF. And the markers of proliferation also probably predict for response to chemotherapy
Not surprisingly, the NSABP investigators found that the Oncotype assay was a strong predictor of benefit from CMF in B20. There was little or no benefit for addition of chemotherapy to tamoxifen for patients with low or intermediate recurrence scores, but substantial benefit for those with high recurrence scores. In the high recurrence score group, 10-year distant recurrence-free survival for tamoxifen-only patients was approximately 65% vs. approximately 90% for those who received CMF (p=0.001; with a test for interaction p-value of 0.037). Conversely, in B14, the benefit from tamoxifen vs. observation was almost totally confined to the low and intermediate-risk categories, with a p-value for interaction of 0.001.< /SPAN>
Taken together, these data suggest that in patients who have an apparently favorable prognosis based on clinical features (negative nodes, positive ER), the Oncotype DX™ assay helps determine those most likely to benefit from tamoxifen only (low recurrence scores) vs. those less likely to benefit from tamoxifen but likely to benefit from chemotherapy (high recurrence scores). The benefits of chemotherapy in the 25% of patients who have intermediate recurrence scores remains uncertain.
Planned prospective randomized trial of Oncotype DX™: These results are the basis of a planned prospective randomized trial that will be performed by the Breast Intergroup (TBIG) of North America (the so-called Program for the Assessment of Cancer Clinical Tests (PAACT) trial), in which patients with low risk will all receive endocrine therapy and will be asked to participate in a registry study for future prognostic assay validation. Those with high recurrence scores will be treated with endocrine therapy and chemotherapy, preferably within other prospective trials. Those with intermediate recurrence scores, for whom we have equipoise regarding the benefits of adjuvant chemotherapy, will be randomly assigned to endocrine therapy only or endocrine and chemotherapy.
This trial takes on even greater importance in light of ongoing progress in the field. It is likely that the overall prognosis of low and intermediate-score patients will be even better in the modern era with application of aromatase inhibitors instead of, or in sequence with, tamoxifen. In contrast, it is also likely that modern chemotherapy, incorporating anthracyclines and taxanes in a dose-dense fashion, will be more effective than the standard CMF used in B20. Thus, especially in the intermediate group, chemotherapy may be needed less (because hormone therapy is better) but more effective, further increasing the clinician’s equipoise for this population.
Other Promising Gene Array Data
Although the Piccart and Paik presentations generated the most interest, other investigators also presented gene array data that might be prognostic or predictive. All of these studies require further validation in more well-defined patient cohorts to determine their precise clinical utility:
Harris and colleagues re-analyzed the Dutch dataset, in association with previously published comparative genomic hybridization datasets, to further refine the signature profile.
Likewise, Baunoch and colleagues, in collaboration with Arcturus Applied Genomics (Carlsbad, CA) reported exciting results from a pilot study of a different gene expression profiling technology (the Gene Expression Profiling Assay) that also works in fixed tissue.
Ross and colleagues from the Comprehensive Cancer Institute of Huntsville have applied a novel screening technique to identify six antibodies against various antigens that are predictive of outcomes in a retrospectively assembled and highly heterogenous group of patients.
Expression of Tau protein: Pusztai and his colleagues from MD Anderson Cancer Center built on previous reports that multi-gene analysis of fine needle aspiration specimens predicts response to neoadjuvant chemotherapy.
At this SABCS, they reported that expression of one of these genes, the microtubule associated protein, Tau, as determined by immunohistochemical studies, was highly associated with failure to achieve a complete pathologic response to paclitaxel-containing neoadjuvant chemotherapy.
Likewise, Cleator and colleagues from the Royal Marsden Hospital have performed gene expression analyses using a cDNA microarray platform on core needle biopsies collected before and at 21 days after doxorubicin and cyclophosphamide neoadjuvant chemotherapy.
In this relatively small set of patients (n=16), they were unable to find any reliable gene signature changes associated with response. Not entirely dissimilar results were reported from the Dana Farber/Harvard Cancer Center from a very small group of patients treated with neoadjuvant trastuzumab and vinorelbine, although there appeared to be three distinct gene cluster groups with differing response patterns.< /SPAN> 
Gene expression and resistance to endocrine treatment: Baseline and follow-up gene expression may also identify mechanisms of resistance to endocrine treatment. In a study of 81 women treated with neoadjuvant letrozole, Miller and colleagues serially sampled primary breast cancers and analyzed gene expression using the Affymetrix HG_U133A chip.
They were able to identify changes in expected genes of hormone sensitivity and in a group of novel candidate genes. Similarly, Daidone and colleagues performed gene expression analysis (using the I.M.A.G.E. Consortium clones, Research Genetics) of tissues from 21 patients who received neoadjuvant toremifene.
They found that 130 genes were associated with clinical response and that certain genes associated with expected cellular responses, such as stress and apoptosis, changed during treatment. Taken together, these fascinating neoadjuvant studies offer great promise for future patient evaluation, but currently must be considered very much in the preliminary phase.
In summary, have we made progress in individualizing patient care for women with newly diagnosed breast cancer? Data from both Europe and the North American cooperative groups are highly promising, but still need prospective validation. Nonetheless, it does appear that, for women who are willing to forego some benefit to avoid toxicity, application of one or another of these assays may be appropriate even in 2005.
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