High-risk Early Breast Cancer Patients Research Articles

Dose-dense adjuvant chemotherapy in HER2-positive early breast cancer patients before and after the introduction of trastuzumab: Exploratory analysis of the GIM2 trial.

Dose-dense adjuvant chemotherapy is standard of care in high-risk early breast cancer patients. However, its role in HER2-positive patients is still uncertain. In this exploratory analysis of the GIM2 trial, we investigated the efficacy of dose-dense chemotherapy in HER2-positive breast cancer patients with or without exposure to trastuzumab. In the GIM2 trial, node-positive early breast cancer patients were randomized to receive four cycles of (fluorouracil)epirubicin/cyclophosphamide followed by four cycles of paclitaxel administered every 2 (dose-dense) or 3 (standard-interval) weeks. After approval of adjuvant trastuzumab, protocol was amended in April 2006 to allow use of trastuzumab for 1 year after chemotherapy completion in HER2-positive patients. The efficacy of dose-dense chemotherapy in terms of disease-free survival (DFS) and overall survival (OS) was assessed according to HER2 status and trastuzumab use. Out of 2,003 breast cancer patients, HER2 status was negative/unknown in 1,551 patients; among the 452 patients with HER2-positive breast cancer, chemotherapy alone or followed by trastuzumab was given to 320 and 132 patients, respectively. Median follow-up was 8.1 years. No significant interaction between HER2 status, trastuzumab use and chemotherapy treatment was observed for both DFS (p = 0.698) and OS (p = 0.708). Nevertheless, there was no apparent benefit in the HER2-positive group treated with trastuzumab (DFS: HR, 0.99; 95% CI 0.52-1.89; OS: HR, 0.95; 95% CI 0.37-2.41). Although dose-dense chemotherapy was associated with a significant survival improvement in high-risk breast cancer patients, its benefit appeared to be smaller (if any) in patients with HER2-positive disease who received adjuvant trastuzumab.

Role of dose-dense chemotherapy in high-risk early breast cancer.

Adjuvant chemotherapy for breast cancer significantly reduces the risk of recurrence and improves overall survival (OS). The purpose of the current article is to review available evidence on dose-dense chemotherapy, also focusing on special population, including premenopausal women and those with HER2-positive disease. A recent patient-level meta-analysis showed that the use of dose-dense chemotherapy is associated with significant reduction in disease recurrence, breast cancer mortality and improvement in OS. The benefit of dose-dense chemotherapy is irrespective from HER2 status, although women with HER2-positive disease enrolled in trials included in the meta-analysis did not receive the current standard of treatment with anti-HER2 agents. Among premenopausal women, dose-dense chemotherapy improved OS, and thus should be considered standard of care for them. In conclusion, high-risk early stage breast cancer patients should be treated with (neo)adjuvant dose-dense anthracycline-based chemotherapy followed by paclitaxel. In the era of trastuzumab, the benefit of dose-dense chemotherapy is still unclear for patients with HER2-positive breast cancer.

Abstract PD2-01: Personalized serial circulating tumor DNA (ctDNA) analysis in high-risk early stage breast cancer patients to monitor and predict response to neoadjuvant therapy and outcome in the I-SPY 2 TRIAL

Abstract ctDNA analysis offers a non-invasive approach for monitoring response and resistance to treatment. Serial ctDNA testing during neoadjuvant therapy (NAT) may provide early indicators of emerging resistance and disease progression. In this study, we analyzed ctDNA from high-risk early breast cancer patients who received NAT and definitive surgery in the I-SPY 2 TRIAL (NCT01042379). We hypothesize that (1) assessment of ctDNA levels early in treatment will improve the performance of molecular and imaging-based predictors of pathologic complete response (pCR) to NAT; and (2) levels of ctDNA after NAT are associated with residual cancer burden and recurrence [distant recurrence free survival (DRFS)]. Methods: ctDNA analysis was performed in 84 high-risk stage II and III breast cancer patients randomized to neoadjuvant investigational agent (n=52), AKT inhibitor MK-2206 (M) in combination with paclitaxel (T) followed by doxorubicin and cyclophosphamide (AC) (M+T->AC), or standard-of-care (T->AC) (n=32). HER2+ patients also received trastuzumab (H). Serial plasma was collected before NAT (T0), early treatment (3 weeks, T1), between regimens (12 weeks, T2), and after NAT prior to surgery (T3). Mutational profiles derived from pretreatment tumor biopsy and normal matched DNA whole exome sequencing were used to design personalized assays targeting 16 patient-specific somatic variants to detect ctDNA in serial plasma. Results: Of the 84 patients in this study, 43% were HR-/HER2- (TNBC), 35% HR+/HER2-, and 23% HER2+. In total, 74% (61 of 83), 35% (28 of 79), 14% (9 of 65), and 8% (5 of 61) were positive for ctDNA at timepoints T0, T1, T2, and T3, respectively. At T0, ctDNA positivity and levels (average number of mutant molecules detected per mL) were significantly associated with increased tumor burden (by clinical and MRI examination), more aggressive tumor biology (as reflected in higher Mammaprint scores and grade) and subtype (HER2+ and TNBC). Twenty-seven percent (27%) of the 84 patients achieved a pCR and all patients who were ctDNA-positive at T3 (n=5) did not achieve a pCR. Currently, data are being collected to: (1) assess the relationship of ctDNA and MRI imaging in predicting tumor response to therapy; (2) examine the relationship of ctDNA levels before and after NAT with 3-year DRFS and event-free survival (EFS). The results of these analyses will be presented at the SABCS 2018 meeting. Conclusions: Our study provides a platform to evaluate the clinical significance of ctDNA for serial monitoring of response to NAT. Accurate and early response prediction by highly sensitive ctDNA analysis can facilitate a timely and judicious change in treatment to improve patients' chances of achieving a pCR. Finally, personalized ctDNA testing may complement imaging and pathologic evaluation of tumor response to fine-tune pCR as a surrogate endpoint for improved DRFS and EFS. Citation Format: Magbanua MJM, Brown-Swigart L, Hirst GL, Yau C, Wolf D, Ma AA, Bergin E, Venters S, Sethi H, Wu H-T, Salari R, Tin T, Sawyer S, Louie M, Zimmermann B, Lin C-HJ, Keats J, Liang WS, Cuyugan L, Enriquez D, Tripathy D, Chien AJ, Forero A, DeMichele A, Liu M, Delson AL, Asare S, Esserman L, van't Veer L, I-SPY 2 Consortium. Personalized serial circulating tumor DNA (ctDNA) analysis in high-risk early stage breast cancer patients to monitor and predict response to neoadjuvant therapy and outcome in the I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-01.

Genomic Alterations Associated with Recurrence and TNBC Subtype in High-Risk Early Breast Cancers

The identification of early breast cancer patients who may benefit from adjuvant chemotherapy has evolved to include assessment of clinicopathologic features such as tumor size and nodal status, as well as several gene-expression profiles for ER-positive, HER2-negative cancers. However, these tools do not reliably identify patients at the greatest risk of recurrence. The mutation and copy-number landscape of triple-negative breast cancer (TNBC) subtypes defined by gene expression is also largely unknown, and elucidation of this landscape may shed light on novel therapeutic opportunities. The USO01062 phase III clinical trial of standard chemotherapy (with or without capecitabine) enrolled a cohort of putatively high-risk patients based on clinical features, yet only observed a 5-year disease-free survival event rate of 11.6%. In order to uncover genomic aberrations associated with recurrence, a targeted next-generation sequencing panel was used to compare tumor specimens from patients who had a recurrence event with a matched set who did not. The somatic mutation and copy-number alteration landscapes of high-risk early breast cancer patients were characterized and alterations associated with relapse were identified. Tumor mutational burden was evaluated but was not prognostic in this study, nor did it correlate with PDL1 or CD8 gene expression. However, TNBC subtypes had substantial genomic heterogeneity with a distinct pattern of genomic alterations and putative underlying driver mutations. IMPLICATIONS: The present study uncovers a compendium of genomic alterations with utility to more precisely identify high-risk patients for adjuvant trials of novel therapeutic agents.

Abstract GS1-06: Extended adjuvant bisphosphonate treatment over five years in early breast cancer does not improve disease-free and overall survival compared to two years of treatment: Phase III data from the SUCCESS A study

Abstract Background: A recent meta-analysis has reported benefits of adjuvant bisphosphonate treatment in early breast cancer patients with improved breast cancer survival and reduced rate of breast cancer recurrences in the bone, especially in postmenopausal patients. However, a comparison between bisphosphonate treatments duration is lacking. Therefore, we examined 2 and 5 years of zoledronate treatment following adjuvant chemotherapy in the SUCCESS A trial. Methods: The SUCCESS A trial is a randomized, open-label, 2x2 factorial design Phase III study in high-risk early breast cancer patients that were randomized to adjuvant chemotherapy treatment with 3 cycles of FEC followed by either 3 cycles of docetaxel or 3 cycles of gemcitabine-docetaxel. After chemotherapy, patients were subject to a second randomization of 5 years of zoledronate treatment (4 mg i.v. every 3 months for 2 years, followed by 4 mg i.v. every 6 months for 3 years) vs. 2 years of zoledronate treatment (4 mg i.v. every 3 months for 2 years). Outcome was analyzed using adapted disease-free survival (DFS) and adapted overall survival (OS), with survival times measured as of 2 years after the start of zoledronate treatment. Maximal observation time was set to 4 years. Median observation time was 2.95 years for DFS and 3 years for OS. Results: Overall, 3421 of the 3754 patients randomized in the SUCCESS A study received at least one zoledronate dose. 434 patients had a DFS event or were lost to follow up in the first two years after the start of zoledronate treatment; thus, 2987 patients were available for analysis (1540 and 1447 patients in the 5-year and 2-year zoledronate treatment arm, respectively). Both adapted DFS and adapted OS did not differ between the two treatment arms (5 vs. 2 years) as shown by multivariate cox regressions adjusted for patient and tumor characteristics as well as chemotherapy (DFS: hazard ratio [HR] 0.97, 95% confidence interval [CI] 0.75 – 1.25, p = 0.81; OS: HR 0.98, 95% CI 0.67 – 1.42, p = 0.90). In addition, subgroup analyses according to menopausal status revealed no difference in DFS or OS between the two treatment arms in premenopausal women (DFS: HR 1.21, 95% CI 0.81 – 1.81, p = 0.35; OS: HR 0.93, 95% CI 0.57 – 1.53, p = 0.78) or postmenopausal women (DFS: HR 0.85, 95% CI 0.62 – 1.16, p = 0.30; OS: HR 0.96, 95% CI 0.67 – 1.39, p = 0.84). Conclusions: Our study showed no difference in DFS or OS between 5-years and 2-years of adjuvant zoledronate treatment in early breast cancer patients, irrespectively of menopausal status. Thus, our results indicate that extended treatment with zoledronate does not improve DFS or OS in high-risk early breast cancer patients. Citation Format: Janni W, Friedl TWP, Fehm T, Müller V, Lichtenegger W, Blohmer J, Lorenz R, Forstbauer H, Bauer E, Fink V, Bekes I, Jückstock J, Schneeweiss A, Tesch H, Mahner S, Brucker SY, Heinrich G, Beckmann MW, Coleman R, Rack B. Extended adjuvant bisphosphonate treatment over five years in early breast cancer does not improve disease-free and overall survival compared to two years of treatment: Phase III data from the SUCCESS A study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS1-06.

German Adjuvant Intergroup Node-positive Study (GAIN): a phase III trial comparing two dose-dense regimens (iddEPC versus ddEC-PwX) in high-risk early breast cancer patients

Dose-dense (dd) regimens are one of the preferred options for the adjuvant treatment of breast cancer patients with intermediate to high risk. The German Adjuvant Intergroup Node-positive trial aimed at optimizing intense dd (idd) strategies by evaluating drug combinations and the addition of capecitabine. Women (aged 18 years and biologically <65 years) with histologically involved axillary lymph nodes were randomly assigned to receive three courses each of epirubicin (E) 150 mg/m2, paclitaxel (P) 225 mg/m2 and cyclophosphamide (C) 2500 mg/m2 (reduced to 2000 mg/m2 after recruitment of 1200 patients) q2w intravenously (i.v.) (iddEPC-regimen) or ddEC (E 112.5 mg/m2 + C 600 mg/m2, i.v. q2w for 4 cycles) followed by paclitaxel weekly (Pw 67.5 mg/m2 i.v. q8d for 10 weeks) plus capecitabine (X 2000 mg/m2 p.o. days 1-14, q22 for 4 cycles) (ddEC-PwX-regimen). Further randomization assigned patients to ibandronate for 2 years versus observation and to pegfilgrastim day 2 versus 4. From June 2004 to August 2008, 2994 patients were randomized to either iddEPC (N = 1498), or ddEC-PwX (N = 1496) and started treatment. Median age was 50 years; pN1 (37.8%), pN2 (35.3%); pN3 (26.9%); 46.4% were G3 tumors; 76.9% hormone receptor-positive and 22% HER2-positive. After a median follow-up of 74 months, 645 events and 383 deaths were recorded. Hematological adverse events grades 3-4 were more common with iddEPC (P < 0.001), nonhematological with ddEC-PwX (P = 0.04), even if the toxicity profile of the two regimens was different. At 5 years, estimated disease-free survival rates for ddEC-PwX and iddEPC were 81.7% [95% confidence interval (CI) 79.5-83.6] versus 80.2% (95% CI 78.0-82.2). Hazard ratio (HR)=0.95 (95% CI 0.81-1.11, log-rank P = 0.49). Five-year overall survival rates were 89.4% for ddEC-PwX (95% CI 87.7-91.0) and 89.0% for iddEPC (95% CI 87.2-90.6), HR = 0.85 (95% CI 0.69-1.04, log-rank P = 0.10). Adding capecitabine to ddEC-Pw did not improve outcome in comparison to iddEPC but increased toxicity and should not be recommended for further use.

Abstract P1-01-03: Dynamics of circulating tumor cells during the course of chemotherapy and prognostic relevance across molecular subtypes in high-risk early breast cancer patients – Results from the adjuvant SUCCESS A trial

Abstract Background: The presence of circulating tumor cells (CTCs) before chemotherapy is known to be associated with reduced disease free survival (DFS) and overall survival (OS) in early breast cancer (EBC). In addition, recent findings suggest that CTCs persisting after adjuvant chemotherapy indicate poor prognosis. In an explorative analysis of the SUCCESS A trial, we evaluated the prognostic relevance of changes in CTC counts during the course of adjuvant chemotherapy across molecular subtypes to assess whether the prognostic role of persisting CTCs varies according to tumor biology. Methods: The SUCCESS A trial is a phase III study, where patients with high-risk EBC (stage pN1-3 or pT2-4 or grade 3 or age ≤ 35 or hormone-receptor negative) were randomized to adjuvant chemotherapy with 3 cycles of epirubicin-fluorouracil-cyclophosphamide followed by either 3 cycles of docetaxel or 3 cycles of gemcitabine-docetaxel. CTC enumeration was performed before and after chemotherapy using the FDA-approved CellSearch® System (Janssen Diagnostics, LLC), and CTC positivity was defined as ≥ 1 CTC in 23 ml blood. Molecular subtypes were defined as luminal A like (hormone-receptor positive, grading 1 or 2), luminal B like (hormone-receptor positive, grading 3), triple-negative or HER2-positive. Patient outcome in terms of DFS and OS was analyzed using univariate log-rank tests and Cox regression models (median follow-up time 65.2 months). Results: Data on both molecular subtypes and CTC status before and after chemotherapy were available for 1485 (39.6%) of 3754 patients randomized. This cohort contained 577 (38.9%) luminal A like, 236 (15.9%) luminal B like, 379 (25.5%) HER2-positive and 293 (19.7%) triple negative tumors. Overall, 917 (61.8%) patients were CTC negative before and after chemotherapy (neg/neg), 260 (17.5%) patients had a negative CTC status before and a positive CTC status after chemotherapy (neg/pos), 229 (15.4%) patients converted from positive to negative CTC status (pos/neg), and 79 (5.3%) patients were positive for CTCs at both time points (pos/pos). There were significant differences in DFS and OS among these four groups in patients with luminal A like tumors (log rank test, both p &amp;lt; 0.003) and patients with luminal B like tumors (log rank test, both p &amp;lt; 0.001). In both patients with luminal A like or luminal B like tumors, persistently CTC positive patients had the worst outcome (relative to persistently CTC-negative patients) in terms of DFS and OS. In contrast to luminal-like tumors, no significant differences with regard to DFS or OS were found among the four groups (neg/neg, neg/pos, pos/neg, pos/pos) in patients with HER2-positive or triple-negative tumors (log rank test, all p &amp;gt; 0.13). Conclusion: The presence of CTCs both before and after adjuvant chemotherapy was associated with poor survival in luminal A like and luminal B like tumors, but not in HER2-positive or triple-negative tumors. Further research is needed to evaluate the effect of chemotherapy on CTC prevalence in different molecular subtypes of EBC. Citation Format: Tzschaschel MLJ, Rack B, Andergassen U, Friedl TWP, Schneeweiss A, Mueller V, Tanja F, Pantel K, Gade J, Lorenz R, Rezai M, Tesch H, Soeling U, Polasik A, Alunni-Fabbroni M, Trapp EK, Mahner S, Schindlbeck C, Lichtenegger W, Beckmann MW, Fasching PA, Janni W. Dynamics of circulating tumor cells during the course of chemotherapy and prognostic relevance across molecular subtypes in high-risk early breast cancer patients – Results from the adjuvant SUCCESS A trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-03.

Abstract P4-07-01: Profiling miRNA in high risk post-operative early breast cancer patients to detect possible association to correlation with immune system antitumor response

Abstract Aims: Identifying which patient will be the one who developes metastatis or relapses is still an unsolved problem in breast cancer research. Circulating free nuclei acids such as microRNA (miRNA) are very promising cancer biomarkers to detect those patients. Aim of this study was to unravel an association among whole blood miRNA profiling, circulating tumor cells (CTCs) and clinical outcome in early breast cancer (EBC) patients and to test the utility of miRNAs as prognostic and diagnostic markers in post-operative patients. Methods: 48 high risk EBC patients, a subsample of the German SUCCESS A trial, were included in this retrospective study together with 18 aged-matched healthy donors (HDs) as controls. Peripheral blood was drawn post-operatively before (T0), after (T1) and two years after (T2) chemotherapy directly in Tempus Blood RNA Tubes (ThermoFischer Scientific) to stabilize and isolate total RNA. miRNAs were quantified by qPCR and their relative expression was calculated according to the 2-DCt method. CTCs were detected by immunocytochemistry. Data were analyzed using non-parametric statistical tests and p-values below 0.05 were considered significant. Results: 8 different miRNAs, either with oncogenic (miR-10a, -19a, -21, -22) or tumor suppressive (miR-20a, -127, -155, -200b) properties, in most of the cases linked to immune system regulation (miR-19a, -20a, -21, -127 and -155) were tested. miR-19a, miR-21, miR-22 and miR-127 showed the most promising results as prognostic markers in our tests. They could differentiate patients from HDs (Patients vs. HD: miR-19a at T0, p=0.004), T1 ( p&amp;lt;0.0001), miR-21 at T0 (p=0.001), T1 (p=0.004), miR-22 at T1 (p=0.012), T2 (p=0.034)). Additionally they correlated with the post-operative detection of CTCs (miR-127) and the late development of metastasis (miR-19a). Kaplan-Meier analysis indicated a borderline association between a longer progression free survival and higher miR-19a levels (HR= 2.808, 95% confidence interval [CI] =0.904, p=0.074.) Conclusion: We found a panel of miRNAs that could discriminate post-operative patients from HDs and could predict patients' outcome. We suggest that whole blood miRNA profiling could unravel a correlation with the activation of the immune system, leading to an efficient antitumor response and a better clinical outcome. These findings could open new perspectives in immune oncology and personalized therapy already in the early phase of the disease. Citation Format: Tzschaschel MLJ, Rack B, Majunke L, Trapp EK, Mahner S, Fasching PA, Fehm T, Schneeweiss A, Friedl TWP, Polasik A, Janni W, Alunni-Fabbroni M. Profiling miRNA in high risk post-operative early breast cancer patients to detect possible association to correlation with immune system antitumor response [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-07-01.

Evaluation of the prognostic value of RANK, RANKL, OPG and OPN mRNA expression in high-risk early breast cancer patients treated with anthracycline-containing adjuvant chemotherapy.

e23244Background: Prevention of bone metastases is a major issue for breast cancer patients, as it would improve quality of life in a population where long survival with metastatic disease is antic...

Abstract P5-15-06: 10-year follow-up of adjuvant ovarian suppression in high risk premenopausal breast cancer

Abstract Background: A large randomized study has shown the value of temporary ovarian suppression during the administration of chemotherapy in the treatment of high risk estrogen receptor positive (ER+) breast cancer (BC) (Pagani et al. N Engl J Med 2014). In another study ovarian suppression added to chemotherapy appeared to protect ovarian function (Moore et al. N Engl J Med 2014) and improve the expected outcome of estrogen receptor negative (ER-) BC. Nevertheless, both these studies have a median follow-up time limited to 68 months and 49 months, respectively. Here we present the data of a large non randomized phase II study of adjuvant ovarian suppression in ER+ and ER- breast cancer with a median follow-up of 120 months (range 90-220 months). The primary end point was the ovarian function preservation rate, secondary end points were disease-free survival (DFS) and overall survival (OS). Methods: Between 06-1997 and 06-2007, 200 premenopausal, high risk early BC patients entered the study. All patients received the LH-RH analogue before starting chemotherapy. Breast conserving and radical surgery were performed in 74% and 26% of patients, respectively. Systemic therapy was tailored to the biological characteristics of each patient, and followed by radiation therapy and hormonal therapy in ER+ tumors. Results: The median patient's age was 43 years (range 26-45). The mean number of positive axillary nodes was 3.2 (range 1-25). Seventy-one % of patients were ER+ and/or progesterone receptor positive (PGR+), 29% were ER - and PGR-. The median KI-67 was 30% (range 15% -100%). Twenty-one % of patients were c-ErbB-2 positive. After a median follow up of 120 months (range 90-220), normal menses returned in 90% of patients younger than 40 years and in 56% of patients older than 40 years. The 10 and 15-year DFS rate were 85.5% and 71%, respectively, while the 10 and 15-year OS rate were 91%, and 71%, respectively. The standard pattern of toxicity of chemotherapy was observed. Hot flashes and G1 osteopenia occurring after LH-RH analogue administration were temporary and subsided after the cessation of therapy. Conclusions: LH-RH administration, concurrent with chemotherapy is tolerable and effective. Five full term pregnancies were documented. A favourable impact on the expected DFS and OS was observed. ER- patients had late new primaries, but no recurrence after 5 years, while ER+ patients had disease recurrence even after 13 years. Citation Format: Recchia F, Candeloro G, Rosselli M, Bratta M, Rea S. 10-year follow-up of adjuvant ovarian suppression in high risk premenopausal breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-15-06.

Evaluation of the prognostic significance of HER family mRNA expression in high-risk early breast cancer: a Hellenic Cooperative Oncology Group (HeCOG) validation study

BackgroundThe aim of the study was to evaluate the prognostic ability of the transcriptional profiling of the HER family genes in early breast cancer, as a validation analysis of another previously published HeCOG study.MethodsRNA was extracted from 663 formalin-fixed paraffin-embedded (FFPE) tumor tissue samples of high-risk early breast cancer patients enrolled in the randomized HE10/00 trial. Relative mRNA expression of all four HER family members was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).ResultsIn compliance with our previous study, the overall agreement between qRT-PCR and IHC/FISH for HER2 status determination was good (69%). Likewise, the overall concordance between qRT-PCR and IHC for EGFR status was high (81%). In line with our previously reported data, we demonstrated a positive association between HER2 and HER3 mRNA expression. Similarly, mRNA expression of HER3 and HER4 was positively associated with each other and negatively associated with EGFR. Regarding relationships with clinico-pathological parameters, our findings are also in agreement with our previous results. Generally, increased EGFR and HER2 mRNA expression was related to unfavorable, whereas high HER3 and HER4 mRNA expression was associated with favorable clinico-pathological parameters. In univariate analysis, no significant association between EGFR, HER2 and HER3 mRNA expression and overall survival (OS) or disease-free survival (DFS) was demonstrated. However, high EGFR protein expression was associated with significantly shorter OS (log-rank, p = 0.015). In compliance with our previously published data, increased HER4 mRNA expression had a significantly favorable prognostic value in terms of OS (p = 0.044) and DFS (p = 0.047). In multivariate analysis, among all HER receptors, only EGFR protein expression was found to affect OS (Wald’s p = 0.028) and DFS (p = 0.015) independently. Concerning the combined expression of all four HER family receptors, the combination of high EGFR, high HER2, low HER3 and low HER4 mRNA expression was associated with a trend for shorter OS (log-rank, p = 0.065) and significantly worse DFS (p = 0.033), compared with all other co-expression profiles.ConclusionsThese data indicate that qRT-PCR may represent a valid alternative method for evaluating the expression of HER family members in FFPE breast carcinoma tissue samples.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12609001036202

Abstract CT301: Late disease recurrence in estrogen receptor positive (ER+) premenopausal breast cancer (BC)

Abstract Background: BC is often a slowly growing disease. For this reason, in order to establish the validity of a therapeutic approach, we need long term follow up, especially in the adjuvant setting. In addition, estradiol (E2) plays a key role in premenopausal BC growth through the induction of vascular endothelial growth factor (VEGF) and the expansion of T-Regulatory cells (T-Regs). We hypothesized that, decreasing plasma E2, the most important inducer of BC, we could reduce VEGF and, therefore, decrease the recurrence rate in premenopausal patients with high risk breast cancer. After a median follow-up &amp;gt;100 months here we present the results of an adjuvant approach in high-risk premenopausal early breast cancer: Luteinizing hormone releasing hormone (LHRH), tailored chemotherapy, radiation therapy and an aromatase inhibitor in ER+ patients. Primary endpoint of this study was the evaluation of E2 and VEGF; secondary end points were the 5 and 15-year disease-free survival (DFS) and overall survival (OS) rates. Methods: Between June 1997 and June 2007, 200 premenopausal high risk early BC patients were entered into the study. The LH-RH analogue was administered before starting chemotherapy. Breast conserving and radical surgery were performed in 74% and 26% of patients, respectively. Systemic therapy was tailored to the biological characteristics of each patient and followed by radiation therapy and by an aromatase inhibitor to ER+ patients. Results: Median patient's age was 43 years (range 26-45). Mean number of positive axillary nodes was 3.2 (range 1-25). 71% of patients were ER+ and/or progesterone receptor (PGR+), 29% were ER - and PGR-. Median KI-67 was 30% (range 15% -100%). After a median follow-up of 104 months (range 63-180), a statistically significant decrease of E2 (P&amp;lt;0.0001) and VEGF (P&amp;lt;0.0001) were observed. 5-year DFS and OS rate were 89% and 96%, respectively, while the 15-year DFS and OS rate were 62%, and 73%, respectively. ER+ patients had a better DFS (P&amp;lt;0.05) with respect to ER- patients, while no difference was observed in OS. The standard pattern of toxicity of chemotherapy was observed, while hot flashes and G1 osteopenia occurred after LH-RH analogue administration. Conclusions: E2 deprivation with an LH-RH analogue is able to decrease E2 and VEGF and to improve the expected DFS and OS rate in premenopausal high risk ER+ and ER- BC patients, at the price of a moderate toxicity. No recurrence after 5 years was observed in ER- patients who had late new primaries. ER+ patients had recurrences even after 15 years. Long term therapy and new immunologic approaches are being evaluated in premenopausal patients with ER+ tumors. Citation Format: Francesco Recchia, Giampiero Candeloro, Stefano Necozione, Silvio Rea, William Soo Hoo, Tom Cantor. Late disease recurrence in estrogen receptor positive (ER+) premenopausal breast cancer (BC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT301. doi:10.1158/1538-7445.AM2014-CT301

Abstract P6-09-06: Estrogen (E2) suppression following diagnosis of node positive (N+) breast cancer (BC) in premenopause. Long term follow-up of a phase II study

Abstract Background: E2 plays a key role in human reproduction and in premenopausal BC growth through the induction of vascular endothelial growth factor (VEGF) and the expansion of T-Regulatory cells (T-Regs). In addition, few clinical data exist on long-term outcome of high-risk N+ premenopausal BC treated with E2 deprivation, by luteinizing hormone releasing hormone (LH-RH). We hypothesized that E2 suppression, following diagnosis of N+ BC in young premenopausal patients, could decrease VEGF and T-Regs and, therefore, could reduce the recurrence rate. Primary endpoint of this study was the evaluation of T-Regs and VEGF of premenopausal patients with high-risk N+, estrogen receptor positive (ER+) and negative (ER-) early BC, treated with an LH-RH analogue. Secondary endpoints were the 10 and 15-year disease-free survival (DFS) and overall survival (OS) rates. Methods: Between 06-1997 and 06-2007, 200 premenopausal high risk early BC patients were entered into the study. Before starting chemotherapy, the LH-RH analogue was administered, 3.75 mg every 28 days for 1 year to the first 64 patients. The same monthly dose was given, for 2 years, to 36 patients. Having observed a decrease of 31% in the recurrence rate for the longer administration of LH-RH analogue, we gave the 11.25 mg dose every 84 days, for 5 years, to the remaining 100 patients. Breast conserving and radical surgery were performed in 74% and 26% of patients, respectively. Systemic therapy was tailored to the biological characteristics of each patient and followed by radiation therapy, and hormonal therapy in ER+ tumors. Results: Median patient's age was 43 years (range 26-45). Mean number of positive axillary nodes was 3.2 (range 1-25). 71% of patients were ER+ and/or progesterone receptor (PGR+), 29% were ER - and PGR-. Median KI-67 was 30% (range 15% -100%). 21% of patients were c-ErbB-2 positive and 11 of them were treated with trastuzumab for 1 year. After a median follow up of 102 months (range 60-180), a statistically significant decrease of VEGF and T-Regs was observed, both in ER+ (P&amp;lt;0.0001) and ER- patients (P&amp;lt;0.0001). Ten-year DFS and OS rate were 86% and 90%, respectively, while the 15-year DFS and OS rate were 62%, and 73%, respectively. Two c-ErbB-2 positive patients (4.7%), had disease recurrence. ER+ patients had a better DFS (P&amp;lt;0.05) and OS (P&amp;lt;0.0001) with respect to ER- patients. The standard pattern of toxicity of chemotherapy was observed, while hot flashes and G1 osteopenia occurred after LH-RH analogue administration. Conclusions: E2 deprivation with an LH-RH analogue is able to decrease plasma VEGF and T-Regs in premenopausal high risk ER+ and ER- BC patients, at the price of a mild toxicity. A favourable impact on DFS and OS was observed. ER- patients had late new primaries, but no recurrence after 5 years, while ER+ patients had disease recurrence even after 15 years. Long term therapy should have further evaluation in premenopausal patients with ER+ tumors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-09-06.

Evaluation of the prognostic significance of RACGAP1, Ki67, and TOP2A mRNA expression in high-risk early breast cancer.

582 Background: Proliferation is a major process in carcinogenesis. RACGAP1 is a protein involved in cell growth regulation and metastasis, Ki67 is a known proliferation marker and TOP2A has a key role in DNA replication and remodeling. The aim of the present study was to explore the prognostic significance of a signature of proliferation markers, such as RACGAP1, Ki67 and TOP2A on disease-free survival (DFS) and overall survival (OS) in high-risk early breast cancer patients. Methods: A total of 1681high-risk breast cancer patients, enrolled in two consecutive phase III trials, were treated with anthracycline-based adjuvant chemotherapy. Formalin-fixed paraffin-embedded tumor tissue samples from 963 of these patients were extracted using a standardized fully automated isolation method for total RNA based on silica-coated magnetic beads, followed by multiplex RT-qPCR for assessing RACGAP1, Ki67 and TOP2A mRNA expression. CALM2 was included in the same reaction, as a reference gene. Results: After a median follow-up of 107 months, 289 patients (30.0%) demonstrated disease progression and 261 (27.1%) patients died. Univariate analysis revealed that poor OS was associated with high RACGAP1 mRNA expression (p=0.0185, log-rank), high Ki67 (p=0.0219), as well as high TOP2A (p=0.0019) mRNA expression, while in multivariate analysis only TOP2A retained significance (Wald’s p=0.008). In an effort to improve prognostic significance, combinations of the expression of two or all three genes were tested, with low mRNA expression of the three genes being associated with improved DFS (HR=0.74, CI=0.56-0.98, p=0.035) and OS (HR=0.60, CI=0.42-0.85, p=0.004). However, in multivariate analysis, none of the combinations retained prognostic significance, except the combination of high RACGAP1 and TOP2A mRNA expression, which was found to be associated with decreased DFS (HR=1.26, CI=0.96-1.63, p=0.092) and OS (HR=1.49, CI=1.10-2.03, p=0.009). Conclusions: High RACGAP1 and TOP2A mRNA expression was found, in multivariate analysis, to be of adverse prognostic significance in high-risk early breast cancer patients treated with anthracycline-containing adjuvant chemotherapy.

The prognostic and predictive value of mRNA expression of vascular endothelial growth factor family members in breast cancer: a study in primary tumors of high-risk early breast cancer patients participating in a randomized Hellenic Cooperative Oncology Group trial

IntroductionThe main prognostic variables in early breast cancer are tumor size, histological grade, estrogen receptor/progesterone receptor (ER/PgR) status, number of positive nodes and human epidermal growth factor receptor 2 (HER2) status. The present study evaluated the prognostic and/or predictive value of vascular endothelial growth factor (VEGF) family members in high-risk early breast cancer patients treated with adjuvant chemo-hormonotherapy.MethodsRNA was isolated from 308 formalin-fixed paraffin-embedded primary tumor samples from breast cancer patients enrolled in the HE10/97 trial, evaluating adjuvant dose-dense sequential chemotherapy with epirubicin followed by cyclophosphamide, methotrexate, fluorouracil (CMF) with or without paclitaxel (E-T-CMF versus E-CMF). A fully automated method based on magnetic beads was applied for RNA extraction, followed by one-step quantitative RT-PCR for mRNA analysis of VEGF-A, -B, -C and vascular endothelial growth factor receptor (VEGFR) 1, 2, 3.ResultsWith a median follow-up of 8 years, 109 patients (35%) developed a relapse and 80 patients (26%) died. In high VEGF-C and VEGFR1 mRNA expressing tumors, ER/PgR-negative tumors (Fisher's exact test, P = 0.001 and P = 0.021, respectively) and HER2-positive tumors (P <0.001 and P = 0.028, respectively) were more frequent than in low VEGF-C and VEGFR1 expressing tumors, respectively. From the VEGF family members evaluated, high VEGFR1 mRNA expression (above the 75th percentile) emerged as a significant negative prognostic factor for overall survival (OS; hazard ratio (HR) = 1.60, 95% confidence interval (CI): 1.01 to 2.55, Wald's P = 0.047) and disease-free survival (DFS; HR = 1.67, 95% CI: 1.13 to 2.48, P = 0.010), when adjusting for treatment group. High VEGF-C mRNA expression was predictive for benefit from adjuvant treatment with paclitaxel (E-T-CMF arm) for OS (test for interaction, Wald's P = 0.038), while in multivariate analysis the interaction of VEGF-C with taxane treatment was significant for both OS (Wald's P = 0.019) and DFS (P = 0.041) and continuous VEGF-B mRNA expression values for OS (P = 0.019).ConclusionsThe present study reports, for the first time, that VEGF-C mRNA overexpression, as assessed by qRT-PCR, has a strong predictive value in high-risk early breast cancer patients undergoing adjuvant paclitaxel-containing treatment. Further studies are warranted to validate the prognostic and/or predictive value of VEGF-B, VEGF-C and VEGFR1 in patients treated with adjuvant therapies and to reveal which members of the VEGF family could possibly be useful markers in identifying patients who will benefit most from anti-VEGF strategies.Trial registrationAustralian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12611000506998

Prognostic significance of RACGAP1 mRNA expression in high-risk early breast cancer: a study in primary tumors of breast cancer patients participating in a randomized Hellenic Cooperative Oncology Group trial

RACGAP1 is a Rac GTPase-activating protein involved in cell growth regulation, cell transformation and metastasis. The aim of the present study was to explore the prognostic and/or predictive significance of RACGAP1 mRNA expression on disease-free survival (DFS) and overall survival (OS) in high-risk early breast cancer patients and compare it to that of Ki67 protein expression and to the Nottingham prognostic index (NPI). A total of 595 high-risk breast cancer patients were treated in a two-arm trial evaluating postoperative dose-dense sequential chemotherapy with epirubicin followed by CMF with or without paclitaxel. RNA was extracted from 314 formalin-fixed paraffin-embedded primary tumor tissue samples followed by one-step quantitative RT-PCR for assessing RACGAP1 mRNA expression. High RACGAP1 mRNA expression (above the median) was associated with poor DFS (log-rank, p = 0.002) and OS (p < 0.001). High histological grade, as well as high Ki67 protein expression, was more frequent in the high-expression group of RACGAP1. Results of the Cox multivariate regression analysis revealed that high RACGAP1 mRNA expression independently predicted poor overall survival (Wald's p = 0.008). High Ki67 protein expression was also an adverse prognostic factor for death (p = 0.016), while high NPI score values were not. High RACGAP1 mRNA expression, as assessed by qRT-PCR, was found to be of adverse prognostic significance in high-risk early breast cancer patients treated with dose-dense sequential chemotherapy. The utility of RACGAP1 mRNA expression in patient selection for treatment with aggressive chemotherapy regimens should be further explored and validated in larger cohorts.

A multicenter, open-label phase II trial of liposomal doxorubicin (LD) and docetaxel (DX), followed by concurrent chemotherapy and radiation therapy in locally advanced high-risk, early breast cancer (HRBC).

e11041 Background: Taxanes and anthracyclines (ANs) are among the most active agents in the treatment of breast cancer. However, ANs are associated with dose-related cardiac toxicity, which can lead to progressive myocardial damage. Non-pegylated liposome-encapsulated doxorubicin (Myocet®), has been shown to significantly reduce cardiac toxicity even though providing comparable antitumor efficacy. Primary endpoint of this phase II study was the assessment of early and late cardiac toxicity of young patients with HRBC treated with LD and DX. Secondary end-points were the incidence of hematological toxicity, time to progression and overall survival in this cohort of patients. Methods: From 05-2006 to 10-2009, 60 HRBC patients were entered into the study. They were treated with LD 60 mg/m2 and DX 75 mg/m2 every three weeks for six courses. Hematological growth factors were given when indicated. Cyclophosphamide 600 mg/m2, methotrexate 60 mg/m2 and 5-fluorouracil 600 mg/m2 were given for six courses concomitantly with radiation therapy to the breast, axilla and supraclavicular nodes. After chemo-radiation therapy, a hormonal therapy was given to estrogen receptors positive (ER+) patients and trastuzumab to Herb-2 positive patients. LVEF was evaluated at baseline and after 6 months, thereafter, up to 5 years. Results: Median age was 49 years. Mean number of positive axillary nodes was 4.9 (range 0-20). 70% were ER+, 30% were ER - and progesterone receptor (PGR) negative. Median Ki-67 was 30% (range 20%-100%), 18% of patients were Herb-2 positive and 18% of patients had pre-existing hypertension. Delivered dose was 95.7% of the planned dose. No patient showed neither a decrease of EF &gt; 10%, nor congestive heart failure. Febrile neutropenia was observed in 12% of patients. At 44 months of median follow-up, 86% of patients were disease-free and 94% were alive. Conclusions: Chemotherapy with LD and DX has shown low cardiac toxicity and high activity in this group of HRBC patients.

Docetaxel-containing adjuvant chemotherapy in patients with early stage breast cancer. Consistency of effect independent of nodal and biomarker status: a meta-analysis of 14 randomized clinical trials

The benefit of taxanes in the adjuvant setting for node-negative (N0) early breast cancer (EBC) has not yet been established. We conducted a meta-analysis of randomized adjuvant trials comparing docetaxel-containing versus non-taxane-containing regimens. The purpose of this study was to determine whether the incorporation of docetaxel improves disease-free survival (DFS) and overall survival (OS) in early stage breast cancer. Studies were retrieved by searching major databases and the proceedings of leading breast cancer conferences. We extracted hazard ratios (HRs) and 95% confidence intervals (CIs) for DFS and OS and obtained pooled estimates using an inverse-variance model. Fourteen randomized phase III studies were included (25,067 patients). The pooled HR estimate was 0.84 (95% CI 0.78-0.89; P < 0.001) favoring docetaxel for DFS and 0.86 (0.78-0.94; P < 0.001) for OS. In N0 patients (4,274 patients), the pooled HR estimate for DFS was 0.86 (0.73-1.00; P = 0.05). The HR for OS was equal to 1 (0.75-1.34). The improvement in DFS with docetaxel-containing regimens was observed across all subgroups (age, under or over 50; number of involved nodes; hormone receptor or HER2 status (including triple negative status), or administration schedule (sequential or concomitant). The addition of docetaxel to a non-taxane-containing regimen improves DFS and OS in high risk EBC patients. The benefit in DFS was seen across all subgroups regardless of nodal status, age, hormone receptor or HER2 status (including triple negative status), or administration schedule.

HER2 and TOP2A in high-risk early breast cancer patients treated with adjuvant epirubicin-based dose-dense sequential chemotherapy

BackgroundHER2 and TOP2A parameters (gene status, mRNA and protein expression) have individually been associated with the outcome of patients treated with anthracyclines. The aim of this study was to comprehensively evaluate the prognostic/predictive significance of the above parameters in early, high-risk breast cancer patients treated with epirubicin-based, dose-dense sequential adjuvant chemotherapy.MethodsIn a series of 352 breast carcinoma tissues from patients that had been post-operatively treated with epirubicin-CMF with or without paclitaxel, we assessed HER2 and TOP2A gene status (chromogenic in situ hybridization), mRNA expression (quantitative reverse transcription PCR), as well as HER2 and TopoIIa protein expression (immunohistochemistry).ResultsHER2 and TOP2A amplification did not share the same effects on their downstream molecules, with consistent patterns observed in HER2 mRNA and protein expression according to HER2 amplification (all parameters strongly inter-related, p values < 0.001), but inconsistent patterns in the case of TOP2A. TOP2A gene amplification (7% of all cases) was not related to TOP2A mRNA and TopoIIa protein expression, while TOP2A mRNA and TopoIIa protein were strongly related to each other (p < 0.001). Hence, TOP2A amplified tumors did not correspond to tumors with high TOP2A mRNA or TopoIIa protein expression, while the latter were characterized by high Ki67 scores (p = 0.003 and p < 0.001, respectively). Multivariate analysis adjusted for nodal involvement, hormone receptor status, Ki67 score and HER2/TOP2A parameters revealed HER2/TOP2A co-amplification (21.2% of HER2 amplified tumors) as an independent favorable prognostic factor for DFS (HR = 0.13, 95% CI: 0.02-0.96, p = 0.046); in contrast, increased HER2/TOP2A mRNA co-expression was identified as an independent adverse prognostic factor for both DFS (HR = 2.41, 95% CI: 1.31-4.42, p = 0.005) and OS (HR = 2.83, 95% CI: 1.42-5.63, p = 0.003), while high TOP2A mRNA expression was an independent adverse prognostic factor for OS (HR = 2.06, 95% CI: 1.23-3.46, p = 0.006). None of the parameters tested was associated with response to paclitaxel.ConclusionsThis study confirms the favorable prognostic value of HER2/TOP2A co-amplification and the adverse prognostic value of high TOP2A mRNA expression extending it to the adjuvant treatment setting in early high-risk breast cancer. The strong adverse prognostic impact of high HER2/TOP2A mRNA co-expression needs further validation in studies designed to evaluate markers predictive for anthracyclines.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12611000506998.

Do Anthracyclines Still Have a Role in Adjuvant Chemotherapy of Breast Cancer?

Anthracycline-based regimens became the standard of care for early breast cancer patients based on the survival advantage they provide over nonanthracycline-containing regimens. The addition of taxanes, and subsequently trastuzumab in HER2-overexpressing patients, to anthracyclines further improved their efficacy in several studies involving high-risk early breast cancer patients. Concern over toxicity initially surfaced after anthracyclines were reported to carry an increased risk of cardiotoxicity and secondary leukemia. Trastuzumab has since been shown to compound the risk of cardiotoxicity in patients who have received an anthracycline. This has led to the development of regimens featuring a taxane without an anthracycline; these protocols vary in design and have different toxicity and efficacy profiles. Ongoing investigations are centered on the optimization of nonanthracycline regimens, prospective exploration of molecular markers to identify populations of patients who will derive maximal benefit from anthracycline-based chemotherapy, and the identification of less cardiotoxic formulations of existing anthracycline agents. Perhaps most importantly, a rapidly growing understanding of the biological heterogeneity of breast cancer is likely to lead to an individualized standard of care guided by particular patient and tumor characteristics.