Trial Information

Prospective Evaluation of Topoisomerase II Alpha Gene Amplification and Protein Overexpression as Markers Predicting the Efficacy of Epirubicin in the Primary Treatment of Breast Cancer Patients

Clinical evaluation of topo II a as a predictive marker: Preliminary results from a clinical
study suggest that complete remission after treatment with anthracyclines for advanced
breast cancer is observed only in case of topo II a gene amplification (7 complete
remissions, all in patients with topo II a gene amplified tumors, no complete remissions in
patients with a normal or deleted topo II a gene).

Moreover, our group has analyzed the predictive value of topo II a in a population of
node-positive breast cancer patients randomly treated either with anthracyclines or with CMF
(Belgian cooperative trial). In a first study, topo II a was evaluated by
immunohistochemistry, which allows the detection of topo II a protein expression. The
results of this study suggested that patients deriving the highest benefit from
anthracyclines were those in which topo II a protein is immunostained in more than 10% of
tumor cells. The main findings of this study should be seen as hypothesis-generating
because of the limited number of patients evaluated (about fifty in each study arm) and
because topo II a protein levels depend on gene amplification as well as on tumor
proliferation rate. Therefore, topo II a protein expression does not necessarily reflect
topo II a gene status.

The second study run by our group was based on the same series of patients evaluated in the
first study, but, this time, both HER-2 and topo II a genes were evaluated by fluorescence
in-situ hybridization (FISH), which allows the detection of gene aberrations. The main
findings of the second study were quite consistent with the pre-clinical data suggesting
that only HER-2 amplified/topo II a amplified tumours show great sensitivity to
anthracyclines while the efficacy of these same agents in HER-2 amplified/topo II a
non-amplified tumors is comparable to the efficacy of other drugs or regimens like CMF.

Nevertheless, although the results reported in this study bring some additional support to
the hypothesis of topo II a as a marker predicting the efficacy of anthracyclines, no
definitive conclusions can be drawn because of the fairly limited number of patients
evaluated, and the retrospective nature of the analyses.

The present study protocol: Supported by "in-vitro" and preliminary "in-vivo" data, briefly
summarized above, this study is designed to test prospectively the value of topo II alpha
gene amplification and protein overexpression in predicting the efficacy of anthracyclines.
To our knowledge this is the only prospective trial worldwide which is attempting to
prospectively clarify the predictive value of this interesting biological marker. This
study could have important practical implications in the daily clinical management of early
breast cancer patients because, if the trial confirms that topo II a gene amplification
and/or protein overexpression are associated with high efficacy of anthracyclines, while
topo II a normal/deleted gene and low protein content are associated with modest efficacy,
an important step forward in the direction of anthracycline "tailoring" would be
accomplished.

The practical advantage of this approach would be to use anthracyclines primarily in
patients who are supposed to derive the largest benefit, thus sparing the long-term
anthracycline-related toxicity (i.e. secondary acute myeloid leukemia, cardiac dysfunction,
and amenorrhea/sterility in case of fertile women) to those patients for whom no significant
gain in antitumor activity is anticipated.

To reach this ambitious aim, early breast cancer patients with tumors of at least 2 cm
(defined by breast ultrasound) will be evaluated for topo II a gene and protein expression.
For this purpose, a pre-treatment biopsy (tru-cut) will be performed and topo II a gene will
be evaluated on fixed samples by FISH. The use of a triple probe will allow the concomitant
evaluation of the HER-2 gene status. Topo II a protein will be evaluated by
immunohistochemistry (IHC). Afterwards, all patients, independently of the topo II a gene
and protein status, will be treated with single-agent epirubicin Eligibility criteria will
allow the participation of patients for whom the use of an anthracycline-based adjuvant
therapy would have been most probably proposed after breast cancer surgery, mainly because
of estrogen receptor (ER) negativity. Therefore, no overtreatment with anthracyclines will
occur in this group of patients. Pathological complete response (pCR) to epirubicin will be
correlated with the topo II a gene and protein status. The study has two biological
hypotheses, one for the subgroup of patients with ER negative/HER-2 amplified tumors, the
other one for the subgroup of patients with ER negative/HER-2 non amplified tumors.

1. st hypothesis: Patients with ER negative/HER-2 amplified tumors: In this subgroup of
patients, topo II a gene will be amplified in about 40% of cases. We hypothesize that
in topo II a amplified tumors a three-fold increase in pCR rate will be observed, as
opposed to the pCR rate in tumors with topo II a normal or deleted gene.

2. nd hypothesis : Patients with ER negative/HER-2 non amplified tumors: In this subset of
patients, almost no topo II a gene aberrations will be found based on previous data
discussed above. However, recent data reported by C. Sotiriou et al using cDNA
microarrays, suggest that in this subset of ER-negative HER-2 negative tumors, also
defined as the basal-like subset, two distinct subgroups can be identified (i.e.
basal-like 1 and 2). While basal-like 1 tumors show a high proliferation rate and high
levels of topo II a RNA, basal-like 2 tumors have a moderate-low proliferation rate and
normal levels of topo II a RNA. We hypothesize that the topo II a RNA overexpression
in basal-like 1 tumors is not related to topo II a gene amplification because no
concomitant HER-2 gene amplification is reported in this subset of tumors. The second
study hypothesis is that in ER negative/HER-2 non amplified tumors with topo II a
protein overexpression, a 2.5 fold increase in pCR rate will be observed, as opposed to
the pCR rate in tumors with low topo II a protein content.

A tumor sample drawn at the time of pre-treatment biopsy will be frozen and used to perform
oligonucleotide based microarrays (Affymetrix). This technique allows the evaluation of
thousands of genes and ultimately provides us with the tumor genetic profile. Homogeneous
genetic profiles (genetic clusters) that might be identified, will be correlated with the
efficacy of single-agent epirubicin. This correlation will allow us to address the
secondary end-point of this study, which is the identification of other genes or eventually
a genetic profile playing a role in the determination of sensitivity to anthracyclines.
Among the genes that could interfere with sensitivity to anthracyclines, p-53 seems to
deserve special attention. Indeed, "in-vitro" data suggest that at least some p-53 mutated
tumors are poorly sensitive to anthracyclines, primarily because anthracycline-induced
apoptosis is prevented. Interestingly, p-53 mutated tumors display frequently HER-2 gene
amplification and therefore topo II a gene amplification (23). Accordingly, p-53 mutations
could hamper response to anthracyclines even in tumors carrying topo II a gene
amplification. This hypothesis will also be explored in the present study, because p-53
mutations will be evaluated by DNA sequencing, and the efficacy of epirubicin in topo II a
amplified and non-amplified tumors will be correlated with p-53 status.

Additionally, patients with inflammatory breast cancer will be treated with dense
administration of epirubicin (100 mg/m2/2 weeks). We keep the same drug as for early breast
cancer but we use a slightly more aggressive regimen with a higher dose-density. The
feasibility of the administration of epirubicin 100 mg/m2 every two weeks with
granulocyte-growth factor support has been shown in the neoadjuvant, metastatic and adjuvant
settings with acceptable toxicity. This neoadjuvant epirubicin regimen may be completed by
adjuvant chemotherapy, such as taxane-based regimens, since the sequential approach
(anthracyclines and taxanes) has been suggested superior to anthracyclines regimen in LABC.