The association between black race and worse outcomes in operable breast cancer reported in previous studies has been attributed to a higher incidence of more aggressive triple-negative disease, disparities in care, and comorbidities. We evaluated associations between black race and outcomes, by tumor hormone receptor and HER2 expression, in patients who were treated with contemporary adjuvant therapy.
The effect of black race on disease-free and overall survival was evaluated using Cox proportional hazards models adjusted for multiple covariates in a clinical trial population that was treated with anthracycline- and taxane-containing chemotherapy. Categorical variables were compared using the Fisher exact test. All P values are two-sided.
Of 4817 eligible patients, 405 (8.4%) were black. Compared with nonblack patients, black patients had a higher rate of triple-negative disease (31.9% vs 17.2%; P < .001) and a higher body mass index (median: 31.7 vs 27.4 kg/m2; P < .001). Black race was statistically significantly associated with worse disease-free survival (5-year disease-free survival, black vs nonblack: 76.7% vs 84.5%; hazard ratio of recurrence or death = 1.58, 95% confidence interval = 1.19 to 2.10, P = .0015) and overall survival (5-year overall survival, black vs nonblack: 87.6% vs 91.9%; hazard ratio of death = 1.49, 95% confidence interval = 1.05 to 2.12, P = .025) in patients with hormone receptor–positive HER2-negative disease but not in patients with triple-negative or HER2-positive disease. In a model that included black race, hormone receptor–positive HER2-negative disease vs other subtypes, and their interaction, the interaction term was statistically significant for disease-free survival (P = .027) but not for overall survival (P = .086).
Factors other than disparities in care or aggressive disease contribute to increased recurrence in black women with hormone receptor–positive breast cancer.
Obesity has been associated with inferior outcomes in operable breast cancer, but the relation between body mass index (BMI) and outcomes by breast cancer subtype has not been previously evaluated.
The authors evaluated the relation between BMI and outcomes in 3 adjuvant trials coordinated by the Eastern Cooperative Oncology Group that included chemotherapy regimens with doxorubicin and cyclophosphamide, including E1199, E5188, and E3189. Results are expressed as hazard ratios (HRs) from Cox proportional hazards models (HR >1 indicates a worse outcome). All P values are 2-sided.
When evaluated as a continuous variable in trial E1199, increasing BMI within the obese (BMI, ≥30 kg/m2) and overweight (BMI, 25-29.9 kg/m2) ranges was associated with inferior outcomes in hormone receptor-positive, human epidermal growth receptor 2 (HER-2)/neu-negative disease for disease-free survival (DFS; P = .0006) and overall survival (OS; P = .0007), but not in HER-2/neu–overexpressing or triple-negative disease. When evaluated as a categorical variable, obesity was associated with inferior DFS (HR, 1.24; 95% confidence interval [CI], 1.06-1.46; P = .0008) and OS (HR, 1.37; 95% CI, 1.13-1.67; P = .002) in hormone receptor-positive disease, but not other subtypes. In a model including obesity, disease subtype, and their interaction, the interaction term was significant for OS (P = .02) and showed a strong trend for DFS (P = .07). Similar results were found in 2 other trials (E5188, E3189).
In a clinical trial population that excluded patients with significant comorbidities, obesity was associated with inferior outcomes specifically in patients with hormone receptor-positive operable breast cancer treated with standard chemohormonal therapy.
obesity; breast cancer; prognosis; hormone receptor positive; disparity
To perform an exploratory analysis of the relationship between gene expression and recurrence in patients with operable triple negative breast cancer (TNBC) treated with adjuvant doxorubicin-containing chemotherapy.
RNA was extracted from archived tumor samples derived from 246 patients with stage I-III TNBC treated with adjuvant doxorubicin-containing chemotherapy, and was analyzed by quantitative RT-PCR for a panel of 374 genes. The relationship between gene expression and recurrence was evaluated using weighted Cox proportional hazards model score tests.
GRB7 was the only gene for which higher expression was significantly associated with increased recurrence in TNBC (Korn’s adjusted p value=0.04). In a Cox proportional hazards model adjusted for clinicopathologic features, higher GRB7 expression was associated with an increased recurrence risk (HR 2.31, p=0.04 using the median as the split). The 5-year recurrence rates were 10.5% (95% confidence intervals [CI] 7.8%, 14.1%) in the low and 20.4% (95% CI 16.5%, 25.0%) in the high GRB7 groups. External validation in other datasets indicated that GRB7 expression was not prognostic in two adjuvant trials including variable systemic therapy, but in two other trials showed that high GBR7 expression was associated with resistance to neoadjuvant doxorubicin and taxane therapy.
GRB7 was associated with an increased risk of recurrence in TNBC, suggesting that GRB7 or GRB7-dependent pathways may serve as potential biomarkers for therapeutic targets. Therapeutic targeting of one or more factors identified which function as interaction nodes or effectors should also be considered.
Breast cancer; GRB7 RNA expression; recurrence
The cognitive function of breast cancer survivors (BC, n = 52) and individually-matched healthy controls (n = 52) was compared on a battery of sensitive neuropsychological tests. The BC group endorsed significantly higher levels of subjective memory loss and scored significantly worse than controls on learning and delayed recall indices from the Rey Auditory Verbal Learning Test (AVLT). Defining clinically significant impairment as scores at or below the 7th percentile of the control group, the rate of cognitive impairment in the BC sample was 17% for total learning on the AVLT, 17% for delayed recall on the AVLT, and 25% for either measure. Findings indicate that a sizeable percentage of breast cancer survivors have clinically significant cognitive impairment.
breast cancer; neuropsychological test; memory; cognition; mild cognitive impairment
Virtual screening targeting the urokinase receptor (uPAR) led to (3R)-4-cyclohexyl-3-(hexahydrobenzo[d][1,3]dioxol-5-yl)-N-((hexahydrobenzo[d][1,3]dioxol-5-yl)methyl)butan-1-aminium 1 (IPR-1) and 4-(4-((3,5-dimethylcyclohexyl)carbamoyl)-2-(4-isopropylcyclohexyl)pyrazolidin-3-yl)piperidin-1-ium 3 (IPR-69). Synthesis of an analog of 1, namely 2 (IPR-9), and 3 led to breast MDA-MB-231 invasion, migration and adhesion assays with IC50 near 30 μM. Both compounds blocked angiogenesis with IC50 of 3 μM. Compounds 2 and 3 inhibited cell growth with IC50 of 6 and 18 μM and induced apoptosis. Biochemical assays revealed lead-like properties for 3, but not 2. Compound 3 administered orally reached peak concentration of nearly 40 μM with a half-life of about 2 hours. In NOD-SCID mice inoculated with breast TMD-231 cells in their mammary fat pads, compound 3 showed a 20% reduction in tumor volumes and less extensive metastasis was observed for the treated mice. The suitable pharmacokinetic properties of 3 and the encouraging preliminary results in metastasis make it an ideal starting point for next generation compounds.
To perform an exploratory analysis of the relationship between gene expression and recurrence in operable hormone receptor (HR)-positive, HER2-normal breast cancer patients treated with adjuvant doxorubicin-containing chemotherapy.
RNA was extracted from archived tumor samples derived from 378 patients with stage I–III HR-positive, HER2-normal breast cancer and analyzed by RT-PCR for a panel of 374 genes, including the 21 gene Recurrence Score (RS). Patients were randomized to receive adjuvant doxorubicin plus cyclophosphamide or docetaxel in trial E2197, with no difference in recurrence seen in the treatment arms. All available recurrent cases were selected plus a non-recurrent cohort. Cox proportional hazard models were used to identify relationships between gene expression and recurrence.
TOP2A expression exhibited the strongest association with increased recurrence risk (p=0.01), and was significantly associated with recurrence (p=0.008) in a multivariate analysis adjusted for clinicopathological features. Elevated TOP2A expression above the median was associated with a 2.6-fold increase (95% confidence intervals [CI], 1.3, 5.2 p=0.008) in risk of recurrence if the RS was less than 18, and a 2.0-fold increase (95% CI, 1.2, 3.2, p=0.003) if there was an intermediate RS of 18–30.
In patients with HR-positive, HER2-normal breast cancer, a population known to have a low incidence of TOP2A gene alterations thought to be predictive of anthracycline benefit, there is a range of TOP2A RNA expression that is strongly associated with recurrence after adjuvant anthracyclines which provides information complementary to RS, indicating that it merits further evaluation as a prognostic and predictive marker.
Breast cancer; TOP2A RNA expression; recurrence
In contrast to endocrine-sensitive and HER2-positive breast cancer, novel agents capable of treating advanced triple negative breast cancer (TNBC) are lacking. PARP (Poly-(adenosine diphosphate [ADP]-ribose) polymerase) inhibitors are emerging as one of the most promising ‘targeted’ therapeutics to treat TNBC, with the intended ‘target’ being DNA repair. PARP's are a family of enzymes involved in multiple cellular processes including DNA repair. TNBC shares multiple clinico-pathologic features with BRCA-mutated breast cancers which harbor dysfunctional DNA repair mechanisms. Investigators hypothesized PARP inhibition, in conjunction with the loss of DNA-repair via BRCA-dependent mechanisms, would result in synthetic lethality and augmented cell death. This hypothesis has borne out in both preclinical models and in clinical trials testing PARP inhibitors in both BRCA-deficient and TNBC. The focus of this review will include an overview of the preclinical rationale for evaluating PARP inhibitors in TNBC, the presumed mechanism of action of this novel therapeutic class, promising results from several influential clinical trials of PARP inhibition in advanced breast cancer (both TNBC and BRCA-deficient), proposed mechanisms of acquired resistance to PARP inhibitors, and, finally, conclude with current challenges and future directions for the development of PARP inhibitors in the treatment of breast cancer.
We examined associations between tumor characteristics (human epidermal growth factor receptor 2 [HER2] protein expression, HER2 gene and chromosome 17 copy number, hormone receptor status) and disease-free survival (DFS) of patients in the N9831 adjuvant trastuzumab trial.
Patients and Methods
All patients (N = 1,888) underwent chemotherapy with doxorubicin and cyclophosphamide, followed by weekly paclitaxel with or without concurrent trastuzumab. HER2 status was determined by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) at a central laboratory, Mayo Clinic, Rochester, MN. Patients with conflicting local positive HER2 expression results but normal central laboratory testing were included in the analyses (n = 103).
Patients with HER2-positive tumors (IHC 3+, FISH HER2/centromere 17 ratio ≥ 2.0, or both) benefited from trastuzumab, with hazard ratios (HRs) of 0.46, 0.49, and 0.45, respectively (all P < .0001). Patients with HER2-amplified tumors with polysomic (p17) or normal (n17) chromosome 17 copy number also benefited from trastuzumab, with HRs of 0.52 and 0.37, respectively (P < .006). Patients who received chemotherapy alone and had HER2-amplified and p17 tumors had a longer DFS than those who had n17 (78% v 68%; P = .04), irrespective of hormone receptor status or tumor grade. Patients with HER2-normal tumors by central testing (n = 103) seemed to benefit from trastuzumab, but the difference was not statistically significant (HR, 0.51; P = .14). Patients with hormone receptor–positive or –negative tumors benefited from the addition of trastuzumab, with HRs of 0.42 (P = .005) and 0.60 (P = .0001), respectively.
These results confirm that IHC or FISH HER2 testing is appropriate for patient selection for adjuvant trastuzumab therapy. Trastuzumab benefit seemed independent of HER2/centromere 17 ratio and chromosome 17 copy number.
Serum microRNAs have the potential to be valuable biomarkers of cancer. This investigation addresses two issues that impact their utility: a) appropriate normalization controls and b) whether their altered levels persist in patients who are clinically free of the disease.
Sera from 40 age-matched healthy women and 39 breast cancer patients without clinical disease at the time of serum collection were analyzed for microRNAs let-7f, miR-16, miR-21 and miR-155 using quantitative real-time PCR. U6 and 5S, which are transcribed by RNA polymerase III (RNAP-III) and the small nucleolar RNU44 (SNORD44), were also analyzed for normalization. Significant results from the initial study were verified using a second set of sera from 15 healthy patients, 15 breast cancer patients without clinical disease and 15 with metastatic disease, and a third set of 12 healthy and 18 patients with metastatic disease. U6 was further verified in the extended second cohort of 75 healthy and 68 breast cancer patients without clinical disease.
U6:SNORD44 ratio was consistently higher in breast cancer patients with or without active disease (fold change range 1.5-6.6, p value range 0.0003 to 0.05). This increase in U6:SNORD44 ratio was observed in the sera of both estrogen receptor-positive (ER+) and ER-negative breast cancer patients. MiR-16 and 5S, which are often used as normalization controls for microRNAs, showed remarkable experimental variability and thus are not ideal for normalization.
Elevated serum U6 levels in breast cancer patients irrespective of disease activity at the time of serum collection suggest a new paradigm in cancer; persistent systemic changes during cancer progression, which result in elevated activity of RNAP-III and/or the stability/release pathways of U6 in non-cancer tissues. Additionally, these results highlight the need for developing standards for normalization between samples in microRNA-related studies for healthy versus cancer and for inter-laboratory reproducibility. Our studies rule out the utility of miR-16, U6 and 5S RNAs for this purpose.
To determine the safety, and efficacy of the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib in combination with trastuzumab in patients with metastatic HER2-positive metastatic breast cancer.
Patients with HER2-overexpressing breast cancer were treated with trastuzumab 2 mg/kg/week and gefitinib 250 to 500 mg/day. The primary end point of the study was to increase the proportion progression-free from 50% to 65% at 6 months in chemotherapy-naive patients and from 50% to 70% at 3 months in patients previously treated with chemotherapy in the metastatic setting.
In the phase I study, all patients treated with gefitinib 500 mg/day developed grade 3 diarrhea. The phase II study was conducted using trastuzumab and gefitinib 250 mg/day. One patient achieved a complete response, 2 had a partial response, and 6 had stable disease for an overall response rate of 9% and a clinical benefit rate of 28% (9 of 32). Median time to progression (TTP) was 3 months (95% confidence interval, 2.3-4.1) in patients with no prior systemic therapy in the metastatic setting (n = 23). In patients treated with prior systemic therapy (n = 9), the median TTP of 5.3 months (95% confidence interval, 2.8-8.1). Overall median survival was 27 months. TTP was similar in EGFR-positive compared with EGFR-negative patients.
Gefitinib 250 mg/day was the maximal dose that can be safely administered with weekly trastuzumab. Interim analysis of the efficacy suggested that the combination was unlikely to result in clinical benefit compared with trastuzumab alone. These results do not support the use of this combination in patients with HER2-positive breast cancer.
Anti-angiogenic therapies have demonstrated their value in the setting of advanced cancer, and are being explored for use in micrometastatic disease. Recent preclinical studies suggest that adjuvant anti-vascular endothelial growth factor (VEGF) therapies may increase the risk of metastasis. How concerning are these preclinical studies, and should they affect our willingness to explore anti-VEGF therapy in the adjuvant setting?
No biomarkers have been identified to predict outcome with the use of an antiangiogenesis agent for cancer. Vascular endothelial growth factor (VEGF) genetic variability has been associated with altered risk of breast cancer and variable promoter activity. Therefore, we evaluated the association of VEGF genotype with efficacy and toxicity in E2100, a phase III study comparing paclitaxel versus paclitaxel plus bevacizumab as initial chemotherapy for metastatic breast cancer.
Patients and Methods
DNA was extracted from tumor blocks of patients from E2100. Three hundred sixty-three samples were available to evaluate associations between genotype and outcome. Genotyping was performed for selected polymorphisms in VEGF and VEGF receptor 2. Testing for associations between each polymorphism and efficacy and toxicity was performed.
The VEGF-2578 AA genotype was associated with a superior median overall survival (OS) in the combination arm when compared with the alternate genotypes combined (hazard ratio = 0.58; 95% CI, 0.36 to 0.93; P = .023). The VEGF-1154 A allele also demonstrated a superior median OS with an additive effect of each active allele in the combination arm but not the control arm (hazard ratio = 0.62; 95% CI, 0.46 to 0.83; P = .001). Two additional genotypes, VEGF-634 CC and VEGF-1498 TT, were associated with significantly less grade 3 or 4 hypertension in the combination arm when compared with the alternate genotypes combined (P = .005 and P = .022, respectively).
Our data support an association between VEGF genotype and median OS as well as grade 3 or 4 hypertension when using bevacizumab in metastatic breast cancer.
Sensitive measures of neuropsychological function were adapted to a telephone administration format for use in a large survey of quality of life in breast cancer survivors (BCS). Healthy controls (HC) and BCS were recruited from the community and administered the same neuropsychological test battery on two occasions separated by 1 week. Subjects were randomly assigned to conditions, stratified by diagnosis: In-person at Time-1 and In-person at Time-2 (P-P); Telephone at Time-1 and Telephone at Time-2 (T-T); T-P; and P-T. Four cognitive (Rey Auditory Verbal Learning Test, Controlled Oral Word Association, Digit Span, Symbol Digit) and two self-report measures (Squire Memory Self-Report Scale, Center for Epidemiological Studies Depression Scale) were used. The 106 subjects were randomized (54 HC and 52 BCS). Test–retest reliabilities (intraclass correlations) did not differ significantly by condition across the cognitive or self-report measures and ranged from moderate to near perfect (r's .43–.93; p's < .05). Mean scores at Time-1, practice effects (Time-1 to Time-2), and standard errors of measurement were comparable between In-person and Telephone administration formats. Results suggest that memory, attention, information processing speed, verbal fluency, and self-report of mood and memory can be measured reliably and precisely over the telephone.
Mass screening; Neuropsychological test; Cognition; Memory; Mild cognitive Impairment; Diagnosis; Dementia
We compared the efficacy of two different taxanes, docetaxel and paclitaxel, given either weekly or every 3 weeks, in the adjuvant treatment of breast cancer.
We enrolled 4950 women with axillary lymph node–positive or high-risk, lymph node–negative breast cancer. After randomization, all patients first received 4 cycles of intravenous doxorubicin and cyclophosphamide at 3-week intervals and were then assigned to intravenous paclitaxel or docetaxel given at 3-week intervals for 4 cycles or at 1-week intervals for 12 cycles. The primary end point was disease-free survival.
As compared with patients receiving standard therapy (paclitaxel every 3 weeks), the hazard ratio for disease-free survival was 1.27 among those receiving weekly paclitaxel (P = 0.006), 1.23 among those receiving docetaxel every 3 weeks (P = 0.02), and 1.09 among those receiving weekly docetaxel (P = 0.29) (with a hazard ratio >1 favoring the groups receiving experimental therapy). As compared with standard therapy, weekly paclitaxel was also associated with improved survival (hazard ratio, 1.32; P = 0.01). An exploratory analysis of a subgroup of patients whose tumors expressed no human epidermal growth factor receptor type 2 protein found similar improvements in disease-free and overall survival with weekly paclitaxel treatment, regardless of hormone-receptor expression. Grade 2, 3, or 4 neuropathy was more frequent with weekly paclitaxel than with paclitaxel every 3 weeks (27% vs. 20%).
Weekly paclitaxel after standard adjuvant chemotherapy with doxorubicin and cyclophosphamide improves disease-free and overall survival in women with breast cancer. (ClinicalTrials.gov number, NCT00004125.)
Adjuvant! is a standardized validated decision aid that projects outcomes in operable breast cancer based on classical clinicopathologic features and therapy. Genomic classifiers offer the potential to more accurately identify individuals who benefit from chemotherapy than clinicopathologic features.
Patients and Methods
A sample of 465 patients with hormone receptor (HR) –positive breast cancer with zero to three positive axillary nodes who did (n = 99) or did not have recurrence after chemohormonal therapy had tumor tissue evaluated using a 21-gene assay. Histologic grade and HR expression were evaluated locally and in a central laboratory.
Recurrence Score (RS) was a highly significant predictor of recurrence, including node-negative and node-positive disease (P < .001 for both) and when adjusted for other clinical variables. RS also predicted recurrence more accurately than clinical variables when integrated by an algorithm modeled after Adjuvant! that was adjusted to 5-year outcomes. The 5-year recurrence rate was only 5% or less for the estimated 46% of patients who have a low RS (< 18).
The 21-gene assay was a more accurate predictor of relapse than standard clinical features for individual patients with HR-positive operable breast cancer treated with chemohormonal therapy and provides information that is complementary to features typically used in anatomic staging, such as tumor size and lymph node involvement. The 21-gene assay may be used to select low-risk patients for abbreviated chemotherapy regimens similar to those used in our study or high-risk patients for more aggressive regimens or clinical trials evaluating novel treatments.
These studies were designed to determine whether ritonavir inhibits breast cancer in vitro and in vitro and, if so, how.
Ritonavir effects on breast cancer cell growth were studied in the estrogen receptor (ER)-positive lines MCF7 and T47D and in the ER-negative lines MDA-MB-436 and MDA-MB-231. Effects of ritonavir on Rb-regulated and Akt-mediated cell proliferation were studied. Ritonavir was tested for inhibition of a mammary carcinoma xenograft.
ER-positive estradiol-dependent lines (IC50, 12–24 µmol/L) and ER-negative (IC50, 45 µmol/L) lines exhibit ritonavir sensitivity. Ritonavir depletes ER-α levels notably in ER-positive lines. Ritonavir causes G1 arrest, depletes cyclin-dependent kinases 2, 4, and 6 and cyclin D1 but not cyclin E, and depletes phosphorylated Rb and Ser473 Akt. Ritonavir induces apoptosis independent of G1 arrest, inhibiting growth of cells that have passed the G1 checkpoint. Myristoyl-Akt, but not activated K-Ras, rescues ritonavir inhibition. Ritonavir inhibited a MDA-MB-231 xenograft and intratumoral Akt activity at a clinically attainable serum Cmax of 22 ± 8 µmol/L. Because heat shock protein 90 (Hsp90) substrates are depleted by ritonavir, ritonavir effects on Hsp90 were tested. Ritonavir binds Hsp90 (KD, 7.8 µmol/L) and partially inhibits its chaperone function. Ritonavir blocks association of Hsp90 with Akt and, with sustained exposure, notably depletes Hsp90. Stably expressed Hsp90α short hairpin RNA also depletes Hsp90, inhibiting proliferation and sensitizing breast cancer cells to low ritonavir concentrations.
Ritonavir inhibits breast cancer growth in part by inhibiting Hsp90 substrates, including Akt. Ritonavir may be of interest for breast cancer therapeutics and its efficacy may be increased by sustained exposure or Hsp90 RNA interference.
Formalin-fixed paraffin-embedded (FFPE) breast tumor tissues are readily available and represent a largely untapped, vast resource for molecular profiling of clinical samples with long-term follow-up data. We have optimized the conditions and parameters that result in the preparation of total RNA that is of the necessary quality for use in the DASL (cDNA-mediated annealing, selection, extension and ligation) assay in which expression of 502 genes are analyzed simultaneously using as little as 100 ng of input RNA.
FFPE; RNA isolation; DASL; genomics; extraction; expression profiling; microarray
As an alternative to DNA microarrays, mass spectrometry based analysis of proteomic patterns has shown great potential in cancer diagnosis. The ultimate application of this technique in clinical settings relies on the advancement of the technology itself and the maturity of the computational tools used to analyze the data. A number of computational algorithms constructed on different principles are available for the classification of disease status based on proteomic patterns. Nevertheless, few studies have addressed the difference in the performance of these approaches. In this report, we describe a comparative case study on the classification accuracy of hepatocellular carcinoma based on the serum proteomic pattern generated from a Surface Enhanced Laser Desorption/Ionization (SELDI) mass spectrometer.
Nine supervised classification algorithms are implemented in R software and compared for the classification accuracy.
We found that the support vector machine with radial function is preferable as a tool for classification of hepatocellular carcinoma using features in SELDI mass spectra. Among the rest of the methods, random forest and prediction analysis of microarrays have better performance. A permutation-based technique reveals that the support vector machine with a radial function seems intrinsically superior in learning from the training data since it has a lower prediction error than others when there is essentially no differential signal. On the other hand, the performance of the random forest and prediction analysis of microarrays rely on their capability of capturing the signals with substantial differentiation between groups.
Our finding is similar to a previous study, where classification methods based on the Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry are compared for the prediction accuracy of ovarian cancer. The support vector machine, random forest and prediction analysis of microarrays provide better prediction accuracy for hepatocellular carcinoma using SELDI proteomic data than six other approaches.
classification; hepatic carcinoma; random forest; SELDI; support vector machine
Histologic classification of thymomas has significant limitations with respect to both subtype definitions and consistency. In order to better understand the biology of the disease processes, we performed whole genome gene expression analysis. RNA was extracted from fresh frozen tumors from 34 patients with thymomas and followup data was available. Using the Illumina BeadStudio® platform and Human Ref-8 Beadchip, gene expression data was analyzed with Partek Genomics Suite®, and Ingenuity Pathways Analysis (IPA). Unsupervised clustering of gene expression data, representing one of the largest series in literature, resulted in identification of four molecular clusters of tumors (C1–C4), which correlated with histology (P = 0.002). However, neither histology nor clusters correlated with clinical outcomes. Correlation of gene expression data with clinical data showed that a number of genes were associated with either advanced stage at diagnosis or development of recurrence or metastases. The top pathways associated with metastases were amino acid metabolisms, biosynthesis of steroids and glycosphingolipids, cell cycle checkpoint proteins and Notch signaling. The differential expression of some of the top genes related to both metastases and stage was confirmed by RT-PCR in all cases of metastases and matched nonmetastatic cases. A number of potential candidates for therapeutics were also identified.
Findings from the human epidermal growth factor receptor 2 (HER2) –positive National Surgical Adjuvant Breast and Bowel Project (NSABP) B31 trial suggested that MYC/HER2 coamplification (> 5.0 copies/nucleus) was associated with additional benefit from adjuvant trastuzumab in patients with early-stage breast cancer. To further explore this relationship, we investigated associations between MYC amplification and disease-free survival (DFS) in a similar adjuvant trastuzumab HER2-positive breast cancer trial—North Central Cancer Treatment Group (NCCTG) N9831.
Patients and Methods
This analysis included 799 patients randomly assigned to receive chemotherapy alone or with concurrent trastuzumab on N9831. Fluorescence in situ hybridization (FISH) was performed by using a dual-probe mixture for MYC and centromere 8 (MYC:CEP8) on tissue microarrays. MYC amplification was prespecified as MYC:CEP8 ratio > 2.2 or average MYC copies/nucleus > 5.0. Exploratory variables included polysomy 8.
In comparing DFS (median follow-up, 4.0 years) between treatments, patients with MYC:CEP8 ratio ≤ 2.2 (n = 618; 77%) and > 2.2 (n = 181; 23%) had hazard ratios (HRs) of 0.46 (P < .001) and 0.67 (P = .33), respectively (interaction P = .38). Patients with MYC copies/nucleus ≤ 5.0 (n = 534; 67%) and > 5.0 (n = 265; 33%) had HRs of 0.52 (P = .002) and 0.48 (P = .02), respectively (interaction P = .94). Patients with MYC:CEP8 ratio < 1.3 with normal chromosome 8 copy number (n = 141; 18%) and ≥ 1.3 or < 1.3 with polysomy 8 (n = 658; 82%) had HRs of 0.66 (P = .28) and 0.44 (P < .001), respectively (interaction P = .23). Patients with MYC copies/nucleus < 2.5 (n = 130; 16%) and ≥ 2.5 (n = 669; 84%) had HRs of 1.07 (P = .87) and 0.42 (P < .001), respectively (interaction P = .05).
We did not confirm the B31 association between MYC amplification and additional trastuzumab benefit. Exploratory analyses revealed potential associations between alternative MYC/chromosome 8 copy number alterations and differential benefit of adjuvant trastuzumab.
The epothilone B analogue, ixabepilone, binds to β-tubulin, is effective for taxane-refractory metastatic breast cancer (MBC), and may be given every 3 weeks or weekly. We evaluated the efficacy of weekly ixabepilone (I) plus trastuzumab (T) and carboplatin (C) as first line therapy in HER2 + MBC. Patients with HER2+ (3+ by IHC or FISH amplified) MBC received I (15 mg/m2 IV) and C (area under the curve, AUC = 2 IV) on days 1, 8, and 15 of a 28-day cycle for a maximum of 6 cycles, plus weekly T (4 mg/kg loading dose then 2 mg/kg IV) during chemotherapy then every 3 weeks (6 mg/kg IV) until disease progression. The primary objective was to determine whether the combination was associated with a response rate (RR) of at least 75%. Fifty-nine patients were treated, and 39 had HER2 overexpression confirmed in a central lab (cHER2+). For all treated patients, objective response occurred in 26 patients (44%; 95% CI 31–58%), median time to progression was 8.2 months (95% CI 6.3–9.9), and median overall survival was 34.7 months (95% CI 25.7 to [not reached]). Results were comparable for cHer2+ cancers. Grade 3–4 adverse events included neutropenia (49%), thrombocytopenia (14%), fatigue (12%), nausea (7%), diarrhea (7%), and neuropathy (7%). One patient died from treatment complications during cycle 1. Weekly ixabepilone and carboplatin plus trastuzumab have an acceptable toxicity profile, but are not likely to be associated with an RR of 75% in HER2+ MBC. Efficacy appears comparable to paclitaxel, carboplatin, and trastuzumab.
Weekly ixabepilone; Carboplatin; Trastuzumab; Breast cancer
The 21-gene Recurrence Score assay (RS) is prognostic for women with node-negative, estrogen receptor (ER)-positive breast cancer (BC) treated with tamoxifen. A low RS predicts little benefit of chemotherapy. For node-positive BC, we investigated whether RS was prognostic in women treated with tamoxifen alone and whether it identified those who might not benefit from anthracycline-based chemotherapy, despite higher recurrence risks.
The phase III trial S8814 for postmenopausal women with node-positive, ER-positive BC showed that CAF chemotherapy prior to tamoxifen (CAF-T) added survival benefit to tamoxifen alone. Optional tumor banking yielded specimens for RS determination by RT-PCR. We evaluated the effect of RS on disease-free survival (DFS) by treatment group (tamoxifen versus CAF-T) using Cox regression adjusting for number of positive nodes.
There were 367 specimens (40% of parent trial) with sufficient RNA (tamoxifen, 148; CAF-T, 219). The RS was prognostic in the tamoxifen arm (p=0.006). There was no CAF benefit in the low RS group (logrank p=0.97; HR=1.02, 95% CI (0.54,1.93)), but major DFS improvement for the high RS subset (logrank p=.03; HR=0.59, 95% CI (0.35, 1.01)), adjusting for number of positive nodes. The RS-by-treatment interaction was significant in the first 5 years (p=0.029), with no additional prediction beyond 5 years (p=0.58), though the cumulative benefit remained at 10 years. Results were similar for overall survival and BC-specific survival.
In this retrospective analysis, the RS is prognostic for tamoxifen-treated patients with positive nodes and predicts significant CAF benefit in tumors with a high RS. A low RS identifies women who may not benefit from anthracycline-based chemotherapy despite positive nodes.