A. M. Gonzalez-Angulo

Prognostic impact of discordance between triple-receptor measurements in primary and recurrent breast cancer

BACKGROUND We evaluated discordance in expression measurements for estrogen receptor (ER), progesterone receptor (PR), and HER2 between primary and recurrent tumors in patients with recurrent breast cancer and its effect on prognosis. METHODS A total of 789 patients with recurrent breast cancer were studied. ER, PR, and HER2 status were determined by immunohistochemistry (IHC) and/or FISH. Repeat markers for ER, PR, and HER2 were available in 28.9%, 27.6%, and 70.0%, respectively. Primary and recurrent tumors were classified as triple receptor-negative breast cancer (TNBC) or receptor-positive breast cancer (RPBC, i.e. expressing at least one receptor). Discordance was correlated with clinical/pathological parameters. RESULTS Discordance for ER, PR, and HER2 was 18.4%, 40.3%, and 13.6%, respectively. Patients with concordant RPBC had significantly better post-recurrence survival (PRS) than discordant cases; patients with discordant receptor status had similarly unfavorable survival as patients with concordant TNBC. IHC scores for ER and PR showed weak concordance between primary and recurrent tumors. Concordance of HER2-FISH scores was higher. CONCLUSIONS Concordance of quantitative hormone receptor measurements between primary and recurrent tumors is modest consistent with suboptimal reproducibility of measurement methods, particularly for IHC. Discordant cases have poor survival probably due to inappropriate use of targeted therapies. However, biological change in clinical phenotype cannot be completely excluded.

Pathologic complete response to neoadjuvant chemotherapy with trastuzumab predicts for improved survival in women with HER2-overexpressing breast cancer

BACKGROUND We sought to determine the prognostic value of pathologic response to neoadjuvant chemotherapy with concurrent trastuzumab. PATIENTS AND METHODS Two hundred and twenty-nine women with HER2/neu (HER2)-overexpressing breast cancer were treated with neoadjuvant chemotherapy plus trastuzumab between 2001 and 2008. Patients were grouped based on pathologic complete response (pCR, n = 114) or less than pCR (<pCR, n = 115); as well as by pathologic stage. Locoregional recurrence-free (LRFS), distant metastasis-free (DMFS), recurrence-free (RFS), and overall survival (OS) rates were compared. RESULTS The median follow-up was 63 (range 53-77) months. There was no difference in clinical stage between patients with pCR or <pCR. Compared with patients achieving <pCR, those with the pCR had higher 5-year rates of LRFS (100% versus 95%, P = 0.011), DMFS (96% versus 80%, P < 0.001), RFS (96% versus 79%, P < 0.001), and OS (95% versus 84%, P = 0.006). Improvements in RFS and OS were seen with decreasing post-treatment stage. Failure to achieve a pCR was the strongest independent predictor of recurrence (hazard ratio [HR] = 4.09, 95% confidence interval [CI]: 1.67-10.04, P = 0.002) and death (HR = 4.15, 95% CI: 1.39-12.38, P = 0.011). CONCLUSIONS pCR and lower pathologic stage after neoadjuvant chemotherapy with trastuzumab are the strongest predictors of recurrence and survival and are surrogates of the long-term outcome in patients with HER2-overexpressing disease.

Stability of HER2-positive status in breast carcinoma: a comparison between primary and paired metastatic tumors with regard to the possible impact of intervening trastuzumab treatment

BACKGROUND To date, the stability of human epidermal growth factor receptor 2 (HER2)-positive primary breast carcinomas during disease progression and the role of intervening trastuzumab treatment in the loss of HER2-positive status in paired metastases remain under-investigated. MATERIALS AND METHODS Sixty-six patients with HER2-positive primary carcinoma and paired metastasis were evaluated. We examined the overall agreement of the HER2 status and compared the status agreement between 38 trastuzumab-treated patients and 28 trastuzumab-naive control patients. The impact of chemotherapy, endocrine therapy, metastatic site (locoregional or distant), and time to relapse (≥5 or <5 years) on the HER2 status change was assessed. RESULTS Fifty-six (84.9%) patients had HER2 status agreement between paired tumors; 10 patients had HER2-positive-to-negative conversion. The agreement rate in the trastuzumab-treated group and in the control group was comparable (86.8% versus 82.1%) (P = 0.858). Chemotherapy, endocrine therapy, metastatic site, and time to relapse did not significantly affect HER2 stability in either group. In the discordant tumor pairs, variations in testing methods and borderline scores were common. CONCLUSIONS HER2-positive status remained unchanged in most paired metastases. Loss of HER2-positive status did not seem to be affected by trastuzumab treatment. Differences in testing and interpretation may account for the discordance in some cases.

Multifocality and multicentricity in breast cancer and survival outcomes

BACKGROUND The clinicopathological characteristics and the prognostic significance of multifocal (MF) and multicentric (MC) breast cancers are not well established. PATIENTS AND METHODS MF and MC were defined as more than one lesion in the same quadrant or in separate quadrants, respectively. The Kaplan-Meier product limit was used to calculate recurrence-free survival (RFS), breast cancer-specific survival (BCSS), and overall survival (OS). Cox proportional hazards models were fit to determine independent associations of MF/MC disease with survival outcomes. RESULTS Of 3924 patients, 942 (24%) had MF (n = 695) or MC (n = 247) disease. MF/MC disease was associated with higher T stages (T2: 26% versus 21.6%; T3: 7.4% versus 2.3%, P < 0.001), grade 3 disease (44% versus 38.2%, P < 0.001), lymphovascular invasion (26.2% versus 19.3%, P < 0.001), and lymph node metastases (43.1% versus 27.3%, P < 0.001). MC, but not MF, breast cancers were associated with a worse 5-year RFS (90% versus 95%, P = 0.02) and BCSS (95% versus 97%, P = 0.01). Multivariate analysis shows that MF or MC did not have an independent impact on RFS, BCSS, or OS. CONCLUSIONS MF/MC breast cancers were associated with poor prognostic factors, but were not independent predictors of worse survival outcomes. Our findings support the current TNM staging system of using the diameter of the largest lesion to assign T stage.

Functional proteomics characterization of residual triple-negative breast cancer after standard neoadjuvant chemotherapy

BACKGROUND In this study, we used functional proteomics to determine the molecular characteristics of residual triple receptor-negative breast cancer (TNBC) patients after neoadjuvant systemic chemotherapy (NCT) and their relationship with patient outcomes in order to identify potential targets for therapy. PATIENTS AND METHODS Protein was extracted from 54 residual TNBCs, and 76 proteins related to breast cancer signaling were measured by reverse phase protein arrays (RPPAs). Univariable and multivariable Cox proportional hazard models were fitted for each protein. Survival outcomes were estimated by the Kaplan-Meier product limit method. Training and cross validation were carried out. The coefficients estimated from the multivariable Cox model were used to calculate a risk score (RS) for each sample. RESULTS Multivariable analysis using the top 25 proteins from univariable analysis at a false discovery rate (FDR) of 0.3 showed that AKT, IGFBP2, LKB1, S6 and Stathmin were predictors of recurrence-free survival (RFS). The cross-validation model was reproducible. The RS model calculated based on the multivariable analysis was -1.1086 × AKT + 0.2501 × IGFBP2 - 0.6745 × LKB1+1.0692 × S6 + 1.4086 × stathmin with a corresponding area under the curve, AUC = 0.856. The RS was an independent predictor of RFS (HR = 3.28, 95%CI = 2.07-5.20, P < 0.001). CONCLUSIONS We found a five-protein model that independently predicted RFS risk in patients with residual TNBC disease. The PI3 K pathway may represent potential therapeutic targets in this resistant disease.

Significant increased recurrence rates among breast cancer patients with HER2-positive, T1a,bN0M0 tumors.

Abstract #701 Background: Controversy surrounds the prognosis of breast cancer patients with T1a,bN0M0 tumors following locoregional therapy and the need for adjuvant systemic therapy, especially for HER2+ disease. The purposes of the study were to determine the recurrence-free survival (RFS), and distant recurrence-free survival (DRFS) in small HER2+ tumors compared with hormone receptor ( HR)+ and triple receptor- (TN) tumors.
 Methods: Stage T1a,bN0M0 breast cancers diagnosed between 1973-2003 were reviewed by dedicated breast pathologists. HER2+ tumors were defined as 3+ by IHC or gene amplification. Patients were categorized into 3 groups:TN (ER-, PR-and HER2-), HER2+ (regardless of HR status) and HR+ (HER2-). RFS and DRFS were estimated by the Kaplan-Meier method and compared with the log-rank test. Cox proportional hazards models were fit to determine the association of each group with the risk of recurrence after adjustment for other characteristics.
 Results: Of the 1796 patients, 427 were excluded from the analysis due to being male (2), lack of receptor information (249), and adjuvant chemotherapy (176) leaving 1369 pts for analysis. Median age was 57 years,(range, 26-88). There were 381(28%) T1a and 988(72%) T1b tumors; HR+ 68%, TN 23%, HER2+ 9%. Patients who had HER2+ breast cancer tended to be younger,(p=0.001); have more T1a tumors, (p=0.001); and have higher nuclear grade,(p Conclusions: Breast cancer patients with HER2+ T1a,bN0M0 tumors have a significant risk of relapse and should be considered candidates for adjuvant systemic therapy including anti-HER2 agents.
 Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 701.

Functional proteomics characterization of residual breast cancer after neoadjuvant systemic chemotherapy

BACKGROUND The purpose of this study was to determine the functional proteomic characteristics of residual breast cancer and hormone receptor (HR)-positive breast cancer after neoadjuvant systemic chemotherapy, and their relationship with patient outcomes. METHODS Reverse phase protein arrays of 76 proteins were carried out. A boosting approach in conjunction with a Cox proportional hazard model defined relapse predictors. A risk score (RS) was calculated with the sum of the coefficients from the final model. Survival outcomes and associations of the RS with relapse were estimated. An independent test set was used to validate the results. RESULTS Test (n = 99) and validation sets (n = 79) were comparable. CoxBoost revealed a three-biomarker (CHK1pS345, Caveolin1, and RAB25) and a two-biomarker (CD31 and Cyclin E1) model that correlated with recurrence-free survival (RFS) in all residual breast cancers and in HR-positive disease, respectively. Unsupervised clustering split patients into high- and low risk of relapse groups with different 3-year RFS (P ≤ 0.001 both). RS was a substantial predictor of RFS (P = 0.0008 and 0.0083) after adjustment for other substantial characteristics. Similar results were found in validation sets. CONCLUSIONS We found models that independently predicted RFS in all residual breast cancer and in residual HR-positive disease that may represent potential targets of therapy in this resistant disease.

Effect of adjuvant trastuzumab among patients treated with anti-HER2-based neoadjuvant therapy

Purpose:To study the impact of adjuvant trastuzumab among patients achieving a pathologic complete response (pCR) after trastuzumab-based neoadjuvant systemic therapy (NST).Patients and methods:Patients with primary HER2-positive breast cancer treated with trastuzumab-based NST were categorised according to adjuvant trastuzumab administration and pCR status. Adjuvant trastuzumab became standard of care in 2006, this was the main reason patients in our cohort did not receive adjuvant trastuzumab. Kaplan–Meier was used to estimate survival. A test for interaction between adjuvant trastuzumab and pCR was completed.Findings:Of 589 patients, 203 (34.5%) achieved a pCR. After surgery, 109 (18.5%) patients in the entire cohort did not receive adjuvant trastuzumab. Among patients achieving a pCR, 31.3% received adjuvant trastuzumab compared with 68.8% among those who did not achieve a pCR (P=0.0006). Among patients achieving pCR, adjuvant trastuzumab did not further improve overall survival (OS) or relapse-free survival (RFS) (P=0.35 and P=0.93, respectively). Any benefit of adjuvant trastuzumab in OS and RFS among patients without a pCR did not achieve statistical significance (P=0.3 and P=0.44, respectively).Conclusions:In this cohort, patients treated with trastuzumab-based NST who achieved a pCR have excellent outcome regardless of whether they received adjuvant trastuzumab.

Addition of GM-CSF to trastuzumab stabilises disease in trastuzumab-resistant HER2+ metastatic breast cancer patients

Background:One of the proposed mechanisms of trastuzumab-induced regression of human epidermal growth factor receptor 2-positive (HER2+) tumours includes facilitation of antibody-dependent cell-mediated cytotoxicity (ADCC). Granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates ADCC. We presented our pilot study of adding GM-CSF to trastuzumab in patients with trastuzumab-resistant HER2+ metastatic breast cancer.Methods:Patients with HER2+ metastatic breast cancer that progressed after trastuzumab +/− chemotherapy were continued on trastuzumab 2 mg kg–1 intravenous weekly and GM-CSF 250 μg m–2 subcutaneous daily. Patients were assessed for response every 8 weeks. Treatment was continued until disease progression or intolerable toxicity.Results:Seventeen patients were evaluable (median age 48 years, range 27–75 years). The median number of metastatic sites was 2 (range 1–3); the most common site was the liver (n=10). The median number of prior regimens for metastatic disease was 2 (range 1–5). No objective disease response was observed, but five patients (29%) had stable disease for a median duration of 15.8 (range 10–53.9) weeks. The most common adverse event was rash at the injection site. No grade 4 or irreversible adverse event was seen.Conclusion:The addition of GM-CSF to trastuzumab alone had a modest clinical benefit and acceptable safety profile in heavily pretreated patients with trastuzumab-resistant HER2+ metastatic breast cancer.

Resistance to Endocrine Therapy in Estrogen Receptor-Positive (ER+) Breast Cancer Is Dependent upon Phosphatidylinositol-3 Kinase (PI3K) Signaling.

ER+ breast cancers typically respond to treatment with aromatase inhibitors (AIs), but a significant fraction exhibit de novo or acquired resistance. To model AI resistance, we cultured four ER+, hormone-dependent human breast cancer cell lines under hormone-depleted conditions for several months until hormone-independent populations emerged (termed long-term estrogen-deprived, LTED). LTED cells outgrew parental counterparts under hormone-depleted conditions. While two LTED lines showed increased ER levels and response to estradiol, two LTED lines showed the opposite. Therefore, ER+ breast cancer cells did not consistently overcome hormone deprivation by increasing sensitivity to low estrogen levels.We analyzed protein lysate arrays with 625 antibodies against signaling proteins to discover common mechanisms of hormone-independent growth. All LTED lines showed increased phosphorylation of p70S6K, an mTOR substrate, compared to parental controls. An siRNA library screen targeting 779 kinases revealed that downregulation of kinases linked to the phosphatidylinositol-3 kinase (PI3K) signaling pathway (serum/glucocorticoid-regulated kinase 1; insulin receptor; p110α/PI3K) inhibited the growth of MCF-7/LTED but not parental MCF-7 cells under hormone-depleted conditions. Immunoblot analysis for P-AKT and P-p70S6K showed that PI3K/AKT/mTOR signaling was increased in all LTED lines. Pathway analysis of gene expression microarray data showed significant alteration of genes involved in insulin-like growth factor-I (IGF-I) signaling in 3/4 LTED lines. Receptor tyrosine kinase array analysis revealed increased activation of receptors for IGF-I (IGF-IR) and/or insulin (InsR) in 3/4 LTED lines. These receptors transduce signals to potently activate PI3K. Treatment with the IGF-IR/InsR kinase inhibitor AEW541 decreased P-AKT in 3/4 LTED lines. Treatment with inhibitors of IGF-IR/InsR (AEW541), PI3K/mTOR (BEZ235), or mTOR (RAD001) suppressed the monolayer and anchorage-independent growth of 3/4, 4/4, and 4/4 LTED lines, respectively, and prevented the emergence of hormone-independent cells. Treatment with PI3K and mTOR inhibitors induced apoptosis in 3/4 LTED lines. Finally, we analyzed levels of PI3K pathway markers (P-AKT, P-S6, P-GSK3α/β) in primary tumor lysates from 65 ER+ breast cancer patients using reverse-phase protein arrays. Hierarchical clustering yielded two groups of tumors with high or low PI3K activation. Following adjuvant anastrozole therapy, PI3K-high patients had significantly shorter disease-free survival compared to PI3K-low patients. With a median follow-up of 9.4 months, recurrence rates were 16% and 0%, respectively ( p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 403.