Prediction of Late Disease Recurrence by H/I Biomarker
Prediction of Late Disease Recurrence by H/I Biomarker
The NCIC Clinical Trials Group MA.17 study was a prospective, randomized, double-blind, placebo-controlled, phase III trial of letrozole as extended adjuvant therapy for 5 years in postmenopausal breast cancer patients who remained disease-free after having completed approximately 5 years of standard adjuvant tamoxifen treatment (Figure 1A). Details of this study have been reported previously. Briefly a total of 5157 eligible postmenopausal women with hormone receptor–positive (ER+ and/or PR+) tumors were randomly assigned to receive either letrozole or placebo after completing approximately 5 years of standard adjuvant tamoxifen therapy. In accordance with trial protocol, the MA.17 trial was unblinded in 2003 after the first interim analysis (median follow-up of 30 months) demonstrated a statistically significant disease-free survival benefit and a trend toward survival advantage in patients who received letrozole. Patients on the placebo arm were offered letrozole for a planned period of 5 years.
(Enlarge Image)
Figure 1.
The overall MA.17 trial design (A), and the sample consort of the MA.17 biomarker study (B).
Because of the practical obstacles to retrospectively obtain primary tumor blocks from MA.17 participants, a nested case-control design using all available recurrent case subjects with a planned ratio of 1:2 was used to study the ability of H/I to predict late recurrence and the benefit from extended therapy with letrozole. Case subjects included patients with local, regional, or distant recurrence for which primary tumor tissue blocks were available. For each case subject, two matched control subjects were selected in which the control subject had been recurrence-free for a period of time longer than the case subject. All patients with disease recurrence and with available formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks were included in this study. Detailed methods for case-control selection are described in Supplementary Methods (available online). The prespecified objectives of this study were to determine whether H/I groups [based on a prespecified and validated cut point] would predict late disease recurrence and responsiveness/benefit from extended letrozole therapy after 5 years tamoxifen treatment. The primary endpoint was any breast cancer recurrence, including breast, chest wall, nodal, or metastatic site. Informed consent for the use of tumor specimens for correlative science studies were obtained from each subject. The Massachusetts General Hospital Institutional Review Board approved the use of the MA.17 tumor samples for this study.
Tumor size and grade, lymph node status, and ER and PR status were assessed using local institutional pathology laboratory criteria. Central tumor grade was assessed using the Elston and Ellis modified version of the Bloom and Richardson method by a single subspecialty-trained breast pathologist (D.C. Sgroi). Central review for ER, PR, and HER-2 expression by immunohistochemistry (IHC) and HER gene amplification by fluorescence in situ hybridization was scored by two pathologists (D.C. Sgroi and A.K. Bhan) using two 1.0-mm tissue microarray cores using previously described methods summarized in the Supplementary Material (available online).
Gene expression analysis of formalin-fixed paraffin-embedded specimens was performed blinded to clinical outcome by bioTheranostics Inc, as previously described. Briefly, hematoxylin and eosin–stained slides were reviewed to guide manual microdissection for tumor enrichment if necessary. After RNA extraction from three to six 8-µm unstained tissue sections and DNase treatment, total RNA content was measured and the absence of DNA contamination verified as previously described. Total RNA was reverse transcribed, and the resulting cDNA was preamplified by performing eight rounds of PCR using the PreAmp Master Mix Kit (Applied Biosystems, Foster City, CA) before being analyzed by TaqMan RT-PCR. H/I was calculated as previously described. High and low H/I groups were determined using a prespecified cutpoint (0.06) that was determined and validated in our previous studies. Reportable results were generated using RNA derived from all 249 formalin-fixed paraffin-embedded tumor samples submitted for testing.
Because of the nature of matched case-control study, unadjusted conditional logistic regression that takes matching set into consideration was used to examine whether H/I was prognostic of late disease recurrence and whether H/I predicts benefit from extended letrozole therapy by including an interaction term between the treatment and predefined H/I groups in the model and testing with statistical contrasts. Conditional logistic regression was also performed in an adjusted analysis that also included the following prespecified covariates: age, tumor size, tumor grade, lymph node status, and ER, PR, and HER2 status. Similarly, the predictive analyses in clinically relevant patient subgroups was performed by conditional logistic regressions with a three-way interaction term that included the clinical variable of interest in addition to the treatment and H/I groups. Odds ratios (ORs) and the 95% confidence intervals (CIs) were estimated from conditional logistic regressions.
The absolute recurrence-free survival (RFS) at 5 years and the corresponding 95% CI were estimated using methods developed by Langholz and Borgan for nested case-control study by taking into consideration the number of patients at risk from the entire MA.17 trial at the time of each recurrence in the case-control study. Details on the method are summarized in the Supplementary Methods (available online).
Statistical significance of the interaction between extended letrozole treatment and H/I index as a continuous variable was assessed by a likelihood ratio test that compared a reduced model without H/I index by treatment interaction with the competing full model, which included the interaction. Linearity of H/I index was tested by a likelihood ratio test that compared a model including the H/I index transformed by restricted cubic spline with a model including only the linear H/I index. If a significant nonlinearity existed, then all analyses involving continuous H/I were based on H/I index transformed by restricted cubic spline. All P values were two-sided, and a P value less than .05 was considered statistically significant. All analyses were performed using R statistical package (version 2.12.2, http://www.r-project.org), except for absolute risk estimation for which SAS software (SAS Institute Inc, Cary, NC) was used. All statistical tests were two-sided.
Methods
Patients, Case-control Selection, Study Design, and Endpoints
The NCIC Clinical Trials Group MA.17 study was a prospective, randomized, double-blind, placebo-controlled, phase III trial of letrozole as extended adjuvant therapy for 5 years in postmenopausal breast cancer patients who remained disease-free after having completed approximately 5 years of standard adjuvant tamoxifen treatment (Figure 1A). Details of this study have been reported previously. Briefly a total of 5157 eligible postmenopausal women with hormone receptor–positive (ER+ and/or PR+) tumors were randomly assigned to receive either letrozole or placebo after completing approximately 5 years of standard adjuvant tamoxifen therapy. In accordance with trial protocol, the MA.17 trial was unblinded in 2003 after the first interim analysis (median follow-up of 30 months) demonstrated a statistically significant disease-free survival benefit and a trend toward survival advantage in patients who received letrozole. Patients on the placebo arm were offered letrozole for a planned period of 5 years.
(Enlarge Image)
Figure 1.
The overall MA.17 trial design (A), and the sample consort of the MA.17 biomarker study (B).
Because of the practical obstacles to retrospectively obtain primary tumor blocks from MA.17 participants, a nested case-control design using all available recurrent case subjects with a planned ratio of 1:2 was used to study the ability of H/I to predict late recurrence and the benefit from extended therapy with letrozole. Case subjects included patients with local, regional, or distant recurrence for which primary tumor tissue blocks were available. For each case subject, two matched control subjects were selected in which the control subject had been recurrence-free for a period of time longer than the case subject. All patients with disease recurrence and with available formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks were included in this study. Detailed methods for case-control selection are described in Supplementary Methods (available online). The prespecified objectives of this study were to determine whether H/I groups [based on a prespecified and validated cut point] would predict late disease recurrence and responsiveness/benefit from extended letrozole therapy after 5 years tamoxifen treatment. The primary endpoint was any breast cancer recurrence, including breast, chest wall, nodal, or metastatic site. Informed consent for the use of tumor specimens for correlative science studies were obtained from each subject. The Massachusetts General Hospital Institutional Review Board approved the use of the MA.17 tumor samples for this study.
Sample Preparation and Pathological Evaluation
Tumor size and grade, lymph node status, and ER and PR status were assessed using local institutional pathology laboratory criteria. Central tumor grade was assessed using the Elston and Ellis modified version of the Bloom and Richardson method by a single subspecialty-trained breast pathologist (D.C. Sgroi). Central review for ER, PR, and HER-2 expression by immunohistochemistry (IHC) and HER gene amplification by fluorescence in situ hybridization was scored by two pathologists (D.C. Sgroi and A.K. Bhan) using two 1.0-mm tissue microarray cores using previously described methods summarized in the Supplementary Material (available online).
Reverse-transcription Polymerase Chain Reaction (RT-PCR) Analysis and Calculation of H/I
Gene expression analysis of formalin-fixed paraffin-embedded specimens was performed blinded to clinical outcome by bioTheranostics Inc, as previously described. Briefly, hematoxylin and eosin–stained slides were reviewed to guide manual microdissection for tumor enrichment if necessary. After RNA extraction from three to six 8-µm unstained tissue sections and DNase treatment, total RNA content was measured and the absence of DNA contamination verified as previously described. Total RNA was reverse transcribed, and the resulting cDNA was preamplified by performing eight rounds of PCR using the PreAmp Master Mix Kit (Applied Biosystems, Foster City, CA) before being analyzed by TaqMan RT-PCR. H/I was calculated as previously described. High and low H/I groups were determined using a prespecified cutpoint (0.06) that was determined and validated in our previous studies. Reportable results were generated using RNA derived from all 249 formalin-fixed paraffin-embedded tumor samples submitted for testing.
Statistical Analysis
Because of the nature of matched case-control study, unadjusted conditional logistic regression that takes matching set into consideration was used to examine whether H/I was prognostic of late disease recurrence and whether H/I predicts benefit from extended letrozole therapy by including an interaction term between the treatment and predefined H/I groups in the model and testing with statistical contrasts. Conditional logistic regression was also performed in an adjusted analysis that also included the following prespecified covariates: age, tumor size, tumor grade, lymph node status, and ER, PR, and HER2 status. Similarly, the predictive analyses in clinically relevant patient subgroups was performed by conditional logistic regressions with a three-way interaction term that included the clinical variable of interest in addition to the treatment and H/I groups. Odds ratios (ORs) and the 95% confidence intervals (CIs) were estimated from conditional logistic regressions.
The absolute recurrence-free survival (RFS) at 5 years and the corresponding 95% CI were estimated using methods developed by Langholz and Borgan for nested case-control study by taking into consideration the number of patients at risk from the entire MA.17 trial at the time of each recurrence in the case-control study. Details on the method are summarized in the Supplementary Methods (available online).
Statistical significance of the interaction between extended letrozole treatment and H/I index as a continuous variable was assessed by a likelihood ratio test that compared a reduced model without H/I index by treatment interaction with the competing full model, which included the interaction. Linearity of H/I index was tested by a likelihood ratio test that compared a model including the H/I index transformed by restricted cubic spline with a model including only the linear H/I index. If a significant nonlinearity existed, then all analyses involving continuous H/I were based on H/I index transformed by restricted cubic spline. All P values were two-sided, and a P value less than .05 was considered statistically significant. All analyses were performed using R statistical package (version 2.12.2, http://www.r-project.org), except for absolute risk estimation for which SAS software (SAS Institute Inc, Cary, NC) was used. All statistical tests were two-sided.
