Ve Breast Cancer Research Articles

Abstract CS1-1: Adjuvant endocrine therapy for premenopausal ER+ breast cancer

Abstract Adjuvant therapy decisions for premenopausal ER+ve breast cancer are challenging. An analysis from TEXT found that therapy varied by geography.1 Approximately half the women with node negative tumors enrolled from North America received optional adjuvant chemotherapy, in addition to protocol ovarian suppression plus oral endocrine therapy, while only one quarter of women with node negative tumors enrolled from Italy received chemotherapy. For women with node positive tumors, the use of chemotherapy was almost universal (93%) in North America, while 27% enrolled from Italy did not receive chemotherapy. Use of multigene assays may reduce future use of chemotherapy in ER+ve breast cancer, however limited information exists for outcomes of young women with low risk assay results, treated with tamoxifen alone. Among 6,693 women enrolled in MINDACT, only 44 women were under age 35 with low genomic risk.2 Only 58 women aged 40 or under were included in the Tailor-X Recurrence Score 0-10 cohort and not all received tamoxifen alone.3 Chemotherapy may provide an indirect endocrine effect in premenopausal ER+ve breast cancer, due to suppression of ovarian estrogen production. The EBCTCG meta-analysis published in 1998 reported that 5 years of adjuvant tamoxifen was effective, regardless of age, menopausal status or chemotherapy.4 Adjuvant ovarian ablation is effective for women under 50 as a sole adjuvant therapy,5 but the value of ovarian suppression/ablation in premenopausal women with ER+ve tumors who also receive tamoxifen, with or without adjuvant chemotherapy, has been uncertain.6 SOFT randomized premenopausal women with hormone receptor-positive breast cancer to receive 5 years of tamoxifen or tamoxifen plus ovarian suppression or exemestane plus ovarian suppression.7 The initial results, after median 5.6 years follow-up, did not show a significant benefit from addition of ovarian suppression to tamoxifen in the overall premenopausal population. SOFT was stratified by use of prior chemotherapy and in the cohort who did not receive chemotherapy, women allocated tamoxifen alone had few recurrences and virtually no distant recurrences during the first 5 years. However, for patients in SOFT deemed at sufficient risk to receive chemotherapy and who remained premenopausal, the addition of ovarian suppression to tamoxifen did reduce risk of invasive recurrence during the first 5 years, and the use of exemestane with ovarian suppression further reduced recurrence. The joint analysis of TEXT and SOFT found that exemestane plus ovarian suppression significantly improved DFS compared with tamoxifen plus ovarian suppression.8 The absolute benefit of endocrine therapies that included ovarian suppression in SOFT and TEXT, as compared with tamoxifen alone, was most striking in women under age 35, although women with a high risk of recurrence from a combination of clinical-pathologic factors (composite risk) could also derive benefit.9

Abstract P4-04-05: Primary endocrine therapy (PETx) induces PAM50 intrinsic subtype migration with prognostic implications

Abstract Backgorund: ER activation is a major ruler of cell biology in ER+ve breast cancer. Hence, ER dependent gene expression at diagnosis, may unveil most of the oncogenic mechanisms responsible of potential tumour relapse and metastasis. Thus, we hypothesized that oestrogen deprivation through PETx may unveil underlying tumour biology with deeper prognostic implications. To explore this, we studied changes in PAM50 intrinsic subtyping and Risk of Recurrence score throughout PETx and their correlation with known prognostic factors. Methods: Clinical-pathological data were evaluated in a series of patients with stage I-III ER-positive/HER2-negative breast cancer treated in 6 centers in Spain with PETx during more than two months with available baseline and surgical samples. The expressions of 50 genes were measured in baseline samples and surgical specimens using the nCounter platform. Intrinsic subtypes and Risk of Recurrence score (ROR) were determined by the research-based PAM50 predictor. Response by ultrasonography (US) and magnetic resonance (RMI) between diagnosis and before surgery and PEPI score in surgical samples were used as the endpoints. Association between two variables was evaluated using χ2 test or Pearson correlation. All statistical tests were two-sided and considered significant when P≤0.05. Results: Gene expression profile was feasible in 58 pre/post sample pairs with a median of 7.8 months (range 2.5-40.6) of PETx with AIs (98.3%) or tamoxifen (1.7%). At baseline, 68.9%(n=40) were classified as Luminal A, followed by Luminal B (24.1%; n=14), HER2 enriched (HER2-E) (5.2%; n=3) and Normal-like (1.8%; n=1). Radiologic response did not change significantly according to intrinsic subtype either by MRI or US (P>0.05). Instead, PEPI score varied according to intrinsic subtype (P=0.024).Thirteen (32%) of LumA, while neither of LumB or HER2-E tumours showed a PEPI score Group 1. PETx resulted in changes in the intrinsic subtype in 29 (50%) of tumours Table 1 Her2-E postLumA postLumB postNormal postHer2 pre2 (66.7%)1 (33.33%)00LumA pre1 (2.5%)21 (52.5%)3 (7.5%)15 (37.5%)LumB pre06 (42.86%)5 (35.71%)3 (21.43%)Normal pre0001 (100%) . Of note, 2 of 3 (66.7%) HER2 tumors, and 5 of 14 (35.7%) LumB tumors did not change their profile. Forty-eight (83%) tumours showed a decrease in ROR score after PETx (P<0.001). ROR was over 30 in 29 tumours (49.1%) at baseline and 16 (27%) at surgery. Change in ROR was moderately correlated with percentage of change in Ki-67 [correlation: 0.417 (P<0.01)]. Correlation of surgical tumour subtype with IHC markers of cell-viability/apoptosis will be presented. Conclusion: Oestrogen deprivation of luminal tumours through PETx results in profound changes in tumour biology including a migration in intrinsic subtype in 50% of tumours. Correlation of the largely decreased ROR with changes in Ki67 reveals the potential prognostic additional information generated by profiling tumours after PETx. ER-blockade may unveil underlying tumour oncogenic capabilities for relapse, survival and metastasis. Hence, the post-PETx gene expression profile, molecular subtype and ROR may bear incremental prognostic and predictive information generating a novel scenario for optimal clinical decision making. Citation Format: Larburu L, Paré L, Rezola R, Carrera M, Buch E, Gimenez J, Quiroga V, Fernandez M, Aragón S, Pascual T, Prat A, Urruticoechea A. Primary endocrine therapy (PETx) induces PAM50 intrinsic subtype migration with prognostic implications [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 P4-04-05.

Abstract P1-09-09: Efficacy of estrogen receptor β agonists in the prevention of breast cancer progression to therapy resistance

Abstract Estrogen plays an important role in the initiation and progression of breast cancer (BCa). Approximately, 70% of breast tumors are estrogen receptor (ER) positive at the time of presentation. Endocrine therapy using aromatase inhibitors (AI), or anti-estrogen (AE) molecules are widely used for treating ER+ve BCa. However, their efficacy is limited by intrinsic and acquired therapy resistance and most patients develop resistance to these drugs. The transcriptional effects of estrogen are mediated by two ERs (ERα and ERβ) and both are expressed in normal breast tissue. Unlike ERα, ERβ functions as tumor suppressor. However, role of ERβ specific agonists in the prevention of BCa progression remains elusive. In this study, we investigated the effectiveness of two ERβ agonists (S-Equol and LY500307) in the prevention of BCa progression using endocrine therapy sensitive (MCF7-aro) and letrozole resistant (MCF7-aro-LTLT) cells. Our results demonstrated that treatment with ERβ agonists inhibit short-and long-term growth of both endocrine therapy sensitive and resistant BCa cells. In addition, ERβ agonists treatment inhibited invasion and migration of both MCF7-aro and MCF7-aro-LTLT cells. Importantly, cell cycle analysis revealed that ERβ agonists induced cell cycle arrest. Our gene microarray analysis demonstrated that both ERβ agonists significantly modulated genes involved in the cell cycle progression, DNA replication and cell death pathways. Further, gene enrichment analysis of differentially expressed genes revealed that genes involved in the cell cycle checkpoints emerged as significant pathway modulated by ERβ agonists treatment in MCF7-aro cells. Interestingly, in letrozole-resistant MCF7-aro cells, DNA replication was significantly affected by ERβ agonists treatment. Pathway analysis also identified enrichment for chemokine signaling pathways. We confirmed pathway analysis by qRT-PCR and western blot analysis. Accordingly, treatment of in vivo syngeneic xenografts with ERβ agonists significantly inhibited BCa progression. Collectively, these results from this study suggest that ERβ agonists have potential to prevent the progression of BCa progression. Citation Format: Ramasamy K, Samayoa C, Krishnegowda NK, Vadlamudi RK, Tekmal RR. Efficacy of estrogen receptor β agonists in the prevention of breast cancer progression to therapy resistance [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 P1-09-09.

Association of damaging nsSNPs of XRCC1 with breast cancer

BER is a pivotal repair system that works against endogenous DNA damages arising due to ROS produced from cellular metabolism. XRCC1 protein is a crucial protein of BER pathway that interacts with many proteins of this pathway and helps in maintaining genomic integrity. Single-nucleotide polymorphisms (SNPs) of this gene are reported to be associated with cancer risks. In our study, genotyping of SNPs was done for age matched cases (120) and controls (130) using PCR-RLFP method, which was further, validated using sequencing method. The association was assessed by Odds ratio and confidence interval calculated by Fisher's exact test. We found that AA genotype of rs25487 is significantly associated with increased risk of breast cancer means positive correlation of SNP rs25487 with breast cancer in West Indian population. Heterozygous (GA) genotype of SNP rs25487 increases the susceptibility to ER −ve breast cancer. Women with AA genotype (rs25487) were only postmenopausal and women with GG genotype were more at postmenopausal phase.

The use of trastuzumab in New Zealand women with breast cancer.

Trastuzumab was first funded in New Zealand for use in HER2+ve stage I-III breast cancer in 2007. This observational study aims to ascertain the patterns of use of trastuzumab in women with invasive HER2+ve breast cancer, and assess the effectiveness of adjuvant trastuzumab in women with stage I-III HER2+ve breast cancer. The Waikato and Auckland Breast Cancer Registries have clinical details of 12372 women diagnosed with invasive breast cancer between June 2000 and May 2013. The proportion of women with HER2+ve breast cancer treated with trastuzumab was examined by age, ethnicity, stage and year of diagnosis. Differences in outcomes including the development of metastases and death were assessed for women with stage I-III HER2+ve breast cancer treated with both chemotherapy and trastuzumab, compared to women treated with chemotherapy alone. Among the 1587 HER2+ve breast cancer patients, 888 (56.0%) women received trastuzumab. The probability of having trastuzumab decreased with higher age and comorbidity score and increased with year of diagnosis, tumor size and cancer stage. Māori and Pacific women were less likely to be treated with trastuzumab. After adjustment for potential confounding factors, the treatment with trastuzumab improved breast cancer-specific mortality (adjusted hazard ratio 0.57, 95% CI: 0.35-0.93). Overall, this observational study has shown a substantial improvement in survival for women with HER2+ve stage I-III breast cancer, and much of this improvement can be attributed to the introduction of trastuzumab. Changes in chemotherapy also appear to have led to improved outcomes.

Abstract 4156: Molecular and phenotypic characterization of breast and colorectal cancer cells selected for resistance to antibody-dependent cell-mediated cytotoxicity (ADCC)

Abstract Monoclonal antibody (mAb) therapy is limited by clinical resistance. For example, alterations in PI3K/Akt /Ras-MAPK signaling, over activation/expression of alternate receptor kinases (c-Met/ IGF-1R), or proteolysis of extracellular domains harboring target epitopes are postulated mechanisms of clinical resistance to trastuzumab. ADCC is a major mechanism of action for mAbs. Most studies have focused on cell models of resistance through in vitro selection, in presence of mAbs, in the absence of immune effector cells. We developed a unique model of immune selection, where target cells (HER2 +ve breast cancer cells SKBR3, BT474; and EGFR +ve colon cancer cells HT29, DLD1) were subjected to ADCC (>90% cell death) with saturating concentrations of trastuzumab or cetuximab (100 µg/ml), respectively, and human peripheral blood mononuclear cells (PBMCs). Selection (in triplicate) was continued for 10 consecutive passages and surviving cells were allowed to grow to confluence over a period of 8-10 weeks. Mock-treated parent cells, IgG1 isotype control, mAb-alone and PBMCs only were utilized as controls. Compared to parent controls, immune-selected DLD1 and SKBR3 cells demonstrated statistically significant increase in proliferation (WST viability assays, P<0.01), and significantly higher colony forming efficiency (soft agar assays, P<0.001); immune-selected HT29 and SKBR3 cells showed significantly higher motility (trans-well migration assays, P<0.05, and P<0.001 respectively). In vitro ADCC assays (calcein labeling) suggested significant reduction (max 20%, p<0.005) in cell lysis in all the immune-selected cell lines compared to parent. Immune-selected HT29 cells demonstrated a 5-fold reduction in gene copy number of EGFR by fluorescence in situ hybridization (FISH, p<0.0001), 2-fold reduction in mRNA levels (qPCR, p< 0.01) and 1.8-fold reduction in protein levels of EGFR (densitometry scans, western blot analyses, p<0.01). Transcriptome-wide next-generation RNA sequencing (Illumina NextSeq 500, 2 x 75 bp paired-end, with over 100 million paired-end reads/sample), coupled with bioinformatic analyses (Reactome pathway database) revealed gene expression changes in ADCC-selected cells. These involve changes in key immune signaling pathways such as class I/II MHC antigen processing, cell chemotaxis, inflammatory and immune regulatory pathways among others. Our data indicate immune-selection by effector cells contributes to ADCC resistance in vitro. Further investigation will determine whether mAb combination with an agonist antibody against CD137 (co-stimulatory NK cell receptor) or Fc-engineered MAbs (e.g. afucosylated) will sensitize the resistant cells. It is hoped that this investigation will better inform mAb therapy by elucidating potentially targetable pathways/ markers that emerge from immune-selection with therapeutic mAbs. Citation Format: Tanuka Biswas, Rebecca Fritzemeier, Adam Mark, Tobias Meißner, Brandon Young, Brian-Leyland Jones, Mark Pegram. Molecular and phenotypic characterization of breast and colorectal cancer cells selected for resistance to antibody-dependent cell-mediated cytotoxicity (ADCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4156. doi:10.1158/1538-7445.AM2017-4156

Adjuvant endocrine therapy for premenopausal women: Type and duration

The Early Breast Cancer Trialists' Collaborative Group (EBCTCG) meta-analysis of randomized tamoxifen trials, found that women age <45 years with estrogen receptor-positive (ER+ve) breast cancer, allocated 5 years of adjuvant tamoxifen, have substantial long-term reduction of breast cancer recurrence. Breast cancer mortality was reduced by about one-third through the first 15 years. Increasing the duration of tamoxifen to 10 years can further reduce the risk of recurrence. For women age <45 years allocated 5 years of tamoxifen, the risk of contralateral breast cancer is halved over 15 years.The initial results from the Suppression of Ovarian Function Trial (SOFT) indicate tamoxifen is a suitable therapy for premenopausal women with low risk clinical-pathologic features. For women at sufficient risk to receive chemotherapy who have premenopausal E2 levels within 8 months of completion, the addition of ovarian suppression to tamoxifen for 5 years resulted in some reduction of recurrence. The use of ovarian suppression combined with an aromatase inhibitor exemestane for 5 years, resulted in further reduction of recurrence.The joint analysis of SOFT and Tamoxifen and Exemestane Trial (TEXT) found the combination of ovarian suppression plus exemestane significantly reduced recurrence, compared with ovarian suppression plus tamoxifen. Premenopausal women with ER+ve HER2-negative breast cancer with high-risk features can derive a meaningful improvement in 5-year invasive breast cancer-free interval with exemestane plus ovarian suppression, as an alternative to tamoxifen. Very young women under age 35 with ER+ve breast cancer have higher risks of recurrence, and the use of ovarian suppression with oral endocrine therapy should be considered.

Cardiac glycosides use and the risk and mortality of cancer; systematic review and meta-analysis of observational studies.

BackgroundCardiac glycosides (CGs) including digitalis, digoxin and digitoxin are used in the treatment of congestive heart failure and atrial fibrillation.Pre-clinical studies have investigated the anti-neoplastic properties of CGs since 1960s. Epidemiological studies concerning the association between CGs use and cancer risk yielded inconsistent results. We have performed a systematic review and meta-analysis to summarize the effects of CGs on cancer risk and mortality.MethodsPubMed, Scopus, Cochrane library, Medline and Web of Knowledge were searched for identifying relevant studies. Summary relative risks (RR) and 95% confidence intervals (CI) were calculated using random-effects model.ResultsWe included 14 case-control studies and 15 cohort studies published between 1976 and 2016 including 13 cancer types. Twenty-four studies reported the association between CGs and cancer risk and six reported the association between CGs and mortality of cancer patients.Using CGs was associated with a higher risk of breast cancer (RR = 1.330, 95% CI: 1.247–1.419). Subgroup analysis showed that using CGs increased the risk of ER+ve breast cancer but not ER-ve. Using CGs wasn’t associated with prostate cancer risk (RR = 1.015, 95% CI: 0.868–1.87). However, CGs decreased the risk in long term users and showed a protective role in decreasing the risk of advanced stages. CGs use was associated with increased all-cause mortality (HR = 1.35, 95% CI: 1.248–1.46) but not cancer-specific mortality (HR = 1.075, 95% CI: 0.968–1.194).ConclusionThe anti-tumor activity of CGs observed in pre-clinical studies requires high concentrations which can’t be normally tolerated in humans. However, the estrogen-like activity of CGs could be responsible for increasing the risk of certain types of tumors.

Patient/oncologist decisions about adjuvant chemotherapy in ER+ve, HER2-ve early breast cancer following endopredict testing.

e12002 Background: Endopredict is a multigene test including tumor size and nodal status; it predicts low or high risk of distant recurrence in patients (pts) with ER+ve, HER2–ve breast cancer treated with adjuvant endocrine therapy alone. We compared adj chemotherapy decisions pre and post Endopredict test results, pts’ anxiety, decisional conflict and oncologists’ (oncs) confidence about decisions made. Methods: 14 oncs in 8 UK hospitals saw 149 pts judged by clinical teams to have equivocal indications for chemotherapy. Pts and oncs discussed provisional treatment decisions based on conventional prognostic factors. Initial decisions were reconsidered when Endopredict results were available. Pre and post-test pts completed Spielberger’s State/Trait Anxiety inventory (STAI) and a decision conflict scale (DCS). Oncs answered questionnaires probing:- basic demographic,/clinical details, agreement with, and confidence about treatment decisions (endocrine (E) therapy +/- chemotherapy(C)) Results: 66.7% pts with an initial E alone decision and a high risk result upgraded to E+C. 9.4% pts with initial E+C decisions and high risk results down-graded to E. None of 46 pts initially favouring E alone who were low risk changed decisions. 82.8% who initially wanted E+C and had low risk scores downgraded to E alone. Endopredict results increased oncs’ confidence (8% ‘strongly agreed’ pre-test, 50% post-test). Oncs neither agreeing nor disagreeing with decisions fell (24% to 5%). Anxiety was stable in pts with unchanged decisions. Pts whose therapy was downgraded had significantly lower anxiety scores (p&lt;0.01); those whose treatment was upgraded had increased scores (p&lt;0.001). Likewise overall uncertainty on DCS fell post-test (p&lt;0.023) Conclusions: Endopredict results increased oncs’ and pts’ decision-making confidence, improved matching of risk with therapy decisions and thus a potential for improved outcomes. Clinical trial information: ISRCTN69220108. [Table: see text]

Targeted sequencing in a phase III trial of luminal breast cancer: Identification of novel targets.

505 Background: The International Cancer Genome Consortium and The Cancer Genome Atlas have had a global transformative impact on our understanding of cancer. These programs have mapped the genomic landscape of common and rare tumors setting the scene for a comprehensive change in the approach to cancer diagnosis and treatment. However, the task remains incomplete until these mutational events are linked to clinical outcomes in the context of current therapeutic intervention to drive future stratified medicine approaches. Methods: We performed targeted sequencing in patients from the Tamoxifen Exemestane Adjuvant Multicentre trial. DNA was extracted and a 101 gene panel analysed using a novel mutation calling pipeline. Both a priori and machine learning analyses were performed using distant recurrence free survival as the primary endpoint. Results: In 1,491 successfully analyzed samples 1,070 (71.76%) samples exhibited at least one single nucleotide mutation (range 0-94, 1.828+/-0.133, mean+/-s.e.). 98/101 genes were mutated in at least one patient. Only variants in PIK3CA, TP53, MLL3, CDH1 were detected in 5% or more of samples. Twenty genes were associated with increased risk of recurrence in multivariate analyses corrected for clinic-pathological variables, 50% of these genes were involved in transcriptional regulation or RNA/protein processing. In a multivariate analysis, two combined signalling modules were independently prognostic for residual risk following hormone therapy (HRvalidation 3.10 95%CI 1.78-5.40 and HRvalidation 2.70 95%CI 1.57-4.64). Conclusions: We successfully performed a signalling pathway-based targeted sequencing analysis within predefined signalling modules. In supervised and unsupervised analyses we identified multiple signalling cassettes linked to poor outcome in patients with ER+ve breast cancers treated with modern endocrine therapy in the context of a phase III clinical trial. These results identify novel candidates as targets to treat endocrine refractory breast cancers.

Danazol has potential to cause PKC translocation, cell cycle dysregulation, and apoptosis in breast cancer cells.

Danazol, the established clinical drug, has given promising therapeutic results in a series of clinical trials with breast cancer patients. Danazol shares structural similarities with several known PKC agonists and fits well into the C1 domain. Danazol binds to the C1b domain of PKC with Kd of 5.64±1.27μm. MD simulation studies further support that the PKC-danazol molecular model is stable and showing minimum distortion to the structure during the simulation period. Immunofluorescence and Western blotting studies indicate that MDAMB-231 cells stimulated with danazol exhibit translocation of PKCα to the plasma membrane. Cells stimulated with danazol causes appearance of several phosphorylated proteins in lysate and plasma membrane. In addition, danazol affects carcinogenic molecule (PMA)-induced intracellular signaling in cancer cells. It halted the cancer cells in the G1 phase of the cell cycle and reduced the viability of ER+ve and triple-negative breast cancer cells with an IC50 of 31±2.63 and 65±4.27μg/ml, respectively. DNA fragmentation and flow cytometry experiments revealed that the cell death follows the apoptotic pathway. It affects mitochondrial membrane potentials and releases cytochrome-C from mitochondria to induce downstream apoptosis in breast cancer cells. Hence, the current study may help clinicians to re-design their treatment strategy to optimize therapeutic potentials of the molecule.

Pilot Study of Combined Type I FLT3 Tyrosine Kinase Inhibitor, Crenolanib with Sorafenib in Pediatric Patients with Relapsed/Refractory FLT3+Ve AML

Background: Crenolanib is a novel type I pan-FLT3 inhibitor, which inhibits both FLT3-ITD, as well as FLT3 tyrosine kinase domain (TKD) activating mutations. Combination of crenolanib (Type I) and sorafenib (Type II) has been shown to have synergistic apoptotic effects in FLT3+ve AML blasts derived from patients (pts) who have relapsed after prior sorafenib treatment. We hypothesized that the combination of crenolanib and sorafenib may provide enhanced FLT3 inhibition by targeting both inactive as well as active FLT3 kinase conformations. We here report the results of a dose finding study of sorafenib given with fixed doses of crenolanib in pediatric pts with FLT3+ve AML.Design: This pilot study was designed to assess the tolerability of escalating doses of sorafenib with full doses of crenolanib (66.7 mg/m2 TID) in pediatric pts with relapsed or refractory FLT3+ve AML. Crenolanib was administered continuously at 66.7 mg/m2 TID starting on d1. Sorafenib was administered continuously starting from d8 at 150 mg/m2/d (dose level 1). The rolling-6 design was used and if no DLTs were seen, sorafenib was escalated to 200 mg/m2/d (dose level 2). Additionally, triple intrathecal therapy with methotrexate, hydrocortisone, and cytarabine was given on d8 and continued weekly in pts with CNS leukemia until CSF was free of blasts.Results: From April 2015 to March 2016, 7 children (median age, 9yrs; range, 5-14yrs) have been enrolled. All pts had progressed after a median of 3 prior AML therapies (range, 1-7). Four pts had progressed after frontline treatment, which included sorafenib maintenance; the other 3 pts had progressed after standard chemotherapy. Two pts had undergone prior allo SCT. Median WBC count prior to therapy was 1.7 x 109/L (range 0.7-7.5 x 109/L) with median blast percentage of 15% (range 0-60%). All 7 pts had a FLT3-ITD (mutant to wild-type FLT3 ratio ranged from 0.048 to 22, with one pt not having any wild type FLT3 allele). At the time of study entry, 2 pts had active CNS leukemia and required additional intrathecal therapy.Crenolanib 66.7 mg/m2 TID was well tolerated with no pt requiring crenolanib dose reduction. Sorafenib was administered to the first 3 pts at 150 mg/m2 QD from d8 onward. As no DLTs were observed at 150 mg/m2 QD dose, sorafenib was dose escalated to 200 mg/m2 QD for the next 4 pts. No DLTs were observed at either of the sorafenib doses. The combination of crenolanib/sorafenib was well tolerated with most AEs being grade 1 and 2 in severity. Rash was frequently seen in pts following the addition of sorafenib and one pt developed grade 2 hand-foot syndrome which resolved within 5d on the same dosing regimen. No grade 4 LFT elevations were seen in any pt.During cycle 1 of therapy, crenolanib/sorafenib combination resulted in clearance of peripheral blood blasts in 5/6 pts and 1 pt without blasts at baseline had resolution of extramedullary neck mass. Reduction of bone marrow blasts was seen in 4/7 pts. By Cheson criteria, one patient achieved a CR (with FLT3 negativity), two had a PR, and one had a marrow response of CRi but with persistent CNS disease. Three pts had NR although 2 had transient benefit from crenolanib on day 8 as determined by BM blast percentage. Peripheral blood and bone marrow blast percentages are summarized in Figure 1 and Table 1, respectively. Median overall survival was 221d (Figure 2) with 2 pts currently still alive.Conclusions: Crenolanib 66.7 mg/m2 TID can be safely combined with sorafenib at 200 mg/m2 QD. Even in pts with prior exposure to sorafenib, this combination showed clinical benefits, represented by rapid reduction of bone marrow or peripheral blasts, and was able to have improved quality of life. Although dual TKI combinations targeting the same receptor have not been tried in AML, such strategies have been shown to have efficacy in other diseases like HER2+ve breast cancer. Pharmacokinetics of crenolanib and sorafenib and FLT3-TKD mutation analysis will be presented. Accrual to this trial continues. [Display omitted] [Display omitted] [Display omitted] DisclosuresInaba:Arog: Research Funding. Eckardt:Arog: Employment, Equity Ownership.

F44 - Subcutaneous Trastuzumab (scT) and metronomic oral Vinorelbine (mVRL) combination in HER2 + ve advanced breast cancer (ABC) patients (pts): a pilot evaluation of toxicity. Preliminary results of the VICTOR-4 study

Background: The combination of Trastuzumab (T) and Vinorelbina (VRL) is wildly administered in metastatic HER2 + ve breast cancer. This combination is associated to a high incidence of grade 3-4 neutropenia compared to taxane-based treatment. Recent data demonstrated that scT is not-inferior to ivT in terms of efficacy, with a similar pharmacokinetic and safety profile. In a previous study (VICTOR-1), we tested the metronomic administration (low-dose, continuing schedule) of VRL, demonstrating an incidence of grade 3-4 toxicities below 5%. The easier schedule of mVRL, together with the preference for scT by pts, suggest the possibility to combine these two schedules in HER2+ pts. Here we present the preliminary results of toxicity in an unselected group of HER2+ pts, as a pivotal experience for a Phase II study. Materials and methods: HER2+ ABC pts were treated with VRL 40 mg thrice a week continuously + scT 600 mg fixed dose every 3 weeks, until progression or unacceptable toxicity. Drug-related toxicity was collected at the beginning of each scT administration. Tumor radiological assessment has been planned every 3 cycles (1 cycle = 3 weeks), according to the clinical practice. Results: From August 2014 to March 2016, 10 pts entered this pivotalstudy. At the moment of the present analysis, a total of 93 cycles are evaluable for toxicity. Median age of the pts was 69 years; 9 pts have been previously treated with taxanes; 2 patients received mVRL + scT as 1stline,4 pts as 2nd, 2 pts as 3rd and 2 pts as 4th line of treatment. Table 1 summarizes the incidence of side effects. Grade 3 toxicity was observed in 3.2% of the cycles; no Grade 4 toxicity has been reported. Results regarding efficacy are too early at the moment to be presented. Conclusion: In this pilot evaluation, scT and mVRL combination seems to be feasible with a low incidence of toxicity, in comparison to what observed in the TRAVIOTA Trial. Further evaluation of a greater number of cycles is needed to confirm these preliminary results.Table: F44G1G2G3G4Nausea1310Diarrhoea2000Vomiting0100Abdominal pain1000Myalgia1100Neuropathy2000Neutropenia0310Asthenia2010Total9830 Open table in a new tab

Abstract 4518: 4CSeq analysis of a breast cancer susceptibility locus on 6q25.1

Abstract Single nucleotide polymorphisms within the genomic region immediately upstream of ESR1, a gene on 6q25.1 encoding the estrogen receptor α (ER), have been found to be associated with increased risk of breast cancer1,2. Three genes within this region, CCDC170, RMND1 and ARMT1 are co-expressed with ESR1 in ER+ve breast cancers and CCDC170 expression is inversely associated with proliferation and recurrence free survival in a tamoxifen-treated patient cohort3. In addition, gene fusions between ESR1 and CCDC170 have been associated with aggressive and endocrine-resistant luminal B tumours4. This study aimed to investigate chromatin structure within the 6q25.1 region using targeted chromatin confirmation capture (3C) and circularised chromatin confirmation capture analysis sequencing (4Cseq) to explore whether the spatial organisation of the chromatin might be associated with the regulation of genes and mutational patterns at this locus. To carry out 4Cseq analysis, chromatin from MCF7 cells was cross-linked, enzymatically digested, ligated, re-digested, circularised and then amplified before the purified PCR products were sequenced on an Illumina HiSeq2500. After the sequence reads were trimmed and processed, FourSig software was used to identify cis and trans interactions from the nine anchor points used in the assay. For 3C analysis, chromatin was cross-linked, digested, ligated, and analysed by quantitative real-time PCR. Data was normalised to a synthetically generated fragment. Analyses revealed that the ESR1 promoter region contacted a number of different upstream regions including the promoters of RMND1 and CCDC170. A high frequency of interactions was observed in the intergenic region between CCDC170 and ESR1 where a number of SNPs associated with breast cancer risk have been found. In addition, contact between the 3’ end of the ESR1 gene and the 3’ end of CCDC170 suggests that there may be a regulatory interaction in this region. Contacts were also observed within CCDC170, immediately adjacent to the point at which gene fusions between ESR1 and CCDC170 have previously been observed4. Overall, these data support the hypothesis that chromatin structure may drive the co-expression of the genes at this locus and suggests that some interactions within the region occur in areas where genomic rearrangements in metastatic breast cancers have been observed.

Abstract 1332: Dual antibody gold nanoconjugate significantly enhances cytotoxicity in metastatic breast cancer

Abstract Background: Metastatic breast cancer is the second leading cause of cancer-related deaths of women in the United States. First line treatment for Her2-positive metastatic breast cancer patient is combination therapy involving pertuzumab, trastuzumab, and docetaxel. Both pertuzumab and trastuzumab targets Her2, but works in complementary ways in increasing the cytotoxicity. Indeed, combination of these two antibody drugs for treatment has shown excellent survival benefits. We hypothesized that by covalently attaching these two antibodies to a single nanoparticle would accelerate the cancer cell death in lower concentrations of antibodies. In this study, we covalently conjugated trastuzumab and pertuzumab to gold nanoparticle (AuNP) and investigated the cytotoxicity in metastatic breast cancer cells. Methods: Gold nanoparticles of core size 16 nm were prepared and modified using heterofunctional end terminal carboxylic polyethylene glycol (PEG) linker. The carboxyl terminals were activated by conventional EDC/NHS procedure followed by simultaneous addition of antibodies. The dual antibody gold nanoconjugates (AuNP-Dual Ab) was purified by HPLC using size exclusion column and characterized in detail by routine analytical techniques. Detailed proteomic analysis was performed on the dual antibody gold nanoconjugate. The binding affinity of AuNP-Dual Ab was investigated on HER2+ve SkBR3 cells using dark field microscopy. In vitro cytotoxicity evaluations of AuNP-Dual Ab were performed on HER2 expressing SKBR3 and BT474 cell lines. The effect of AuNP-Dual Ab on signal transduction and phosphorylation status mediated by HER family proteins were evaluated by monitoring the HER2, HER3, AKT, ERK, MEK protein expression through western blot analysis. In vivo studies have been performed in metastatic breast tumor mice models to evaluate the therapeutic efficacy of the nanoconjugate. Results: AuNP-Dual Ab was isolated from the free antibodies by HPLC. We confirmed both the presence and the structural identity of antibodies covalently attached to AuNP using proteomics. TEM immunohistochemistry and dark field microscopy showed AuNP-Dual-Ab possess high receptor binding affinity with Her2+ve breast cancer cells. We investigated the cytotoxicity of AuNP-Dual-Ab in SKBR-3 and BT474 human breast cancer cells. Our studies reveal that AuNP-Dual-Ab is 60 fold higher cytotoxic than non-antibody coated AuNPs, antibodies, and combination antibodies. Western blot analysis showed down regulation of AkT and phospho-AkT proteins while expression of MEK and phospho-MEK proteins was un-altered. We believe the increase in cytotoxicity is possibly due to effective transport of antibodies to cancer cells. Conclusions: The in vitro studies demonstrate that AuNP-Dual Ab is promising and can potentially overcome the problem of resistance. In vivo studies are ongoing on metastatic breast tumor mice models for therapy evaluation. Citation Format: Ajit Zambre, Anandhi Upendran, Matthew Leevy, Sarah Chapman, Raghuraman Kannan. Dual antibody gold nanoconjugate significantly enhances cytotoxicity in metastatic breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1332.

Rab25 acts as an oncogene in luminal B breast cancer and is causally associated with Snail driven EMT.

The Rab GTPases regulate vesicular trafficking machinery that transports and delivers a diverse pool of cargo, including growth factor receptors, integrins, nutrient receptors and junction proteins to specific intracellular sites. The trafficking machinery is indeed a major posttranslational modifier and is critical for cellular homeostasis. Deregulation of this stringently controlled system leads to a wide spectrum of disorders including cancer. Herein we demonstrate that Rab25, a key GTPase, mostly decorating the apical recycling endosome, is a dichotomous variable in breast cancer cell lines with higher mRNA and protein expression in Estrogen Receptor positive (ER+ve) lines. Rab25 and its effector, Rab Coupling Protein (RCP) are frequently coamplified and coordinately elevated in ER+ve breast cancers. In contrast, Rab25 levels are decreased in basal-like and almost completely lost in claudin-low tumors. This dichotomy exists despite the presence of the 1q amplicon that hosts Rab25 across breast cancer subtypes and is likely due to differential methylation of the Rab25 promoter. Functionally, elevated levels of Rab25 drive major hallmarks of cancer including indefinite growth and metastasis but in case of luminal B breast cancer only. Importantly, in such ER+ve tumors, coexpression of Rab25 and its effector, RCP is significantly associated with a markedly worsened clinical outcome. Importantly, in claudin-low cell lines, exogenous Rab25 markedly inhibits cell migration. Similarly, during Snail-induced epithelial to mesenchymal transition (EMT) exogenous Rab25 potently reverses Snail-driven invasion. Overall, this study substantiates a striking context dependent role of Rab25 in breast cancer where Rab25 is amplified and enhances aggressiveness in luminal B cancers while in claudin-low tumors, Rab25 is lost indicating possible anti-tumor functions.

The clinical impact of early immunological responses in human HER2-positive breast cancers on responsiveness to trastuzumab-based therapy.

587Background: ‘TCHL’ an ICORG phase-II neo-adjuvant study in HER2+ve Breast cancer (BC) assessed the impact of TCH (Docetaxel, Carboplatin, Trastuzumab) versus TCHL (Docetaxel, Carboplatin, Trastu...

OPTIMA (Optimal Personalised Treatment of early breast cancer usIng Multi-parameter Analysis): A prospective trial to validate the predictive utility and cost-effectiveness of gene expression test-directed chemotherapy decisions.

TPS623Background: Multi-parameter gene expression assays (MPAs) are widely used to estimate individual patient residual risk and guide chemotherapy use in hormone sensitive HER2-ve early breast cancer, but prospective validation data are not available. OPTIMA aims to validate the use of MPA testing to predict chemotherapy sensitivity in a largely node-positive breast cancer population. Methods: OPTIMA is a partially blinded multi-center, phase 3 RCT with an adaptive two-stage design. The preliminary phase (OPTIMA prelim) evaluated the performance of MPAs to identify a suitable test to be used in the main efficacy trial, and demonstrated the feasibility and acceptability of a large UK trial. Eligible patients (pts) are men or women aged ≥ 40 years who have surgically resected early stage ER+ve and HER2-ve breast cancer, and have either 1-9 involved axillary lymph nodes or tumors of ≥ 30mm diameter. Randomization is to standard management (chemotherapy followed by endocrine therapy) or to MPA-directed thera...

Analysis of treatment options and outcomes for operable ER+ve breast cancer in women aged 70 years and older

Analysis of treatment options and outcomes for operable ER+ve breast cancer in women aged 70 years and older

Is Interleukin 10 (IL10) Expression in Breast Cancer a Marker of Poor Prognosis?

Interleukin 10 (IL10) is a poor prognostic marker in several cancers. Its role in breast cancer is not well elucidated. The present study is designed to see the expression of IL10 in breast cancer tissue and to evaluate its correlation with the established markers of prognosis. Sixty female patients who underwent surgery for breast cancer were enrolled for the study. Immediately after surgery, 2-5g of tumour tissue and similar volume of peritumoural normal breast tissue were collected for IL10 assay. IL10 expression was assayed by immunohistochemistry. IL10 expressing tumours and IL10 non expressing tumours were compared. Chi square/Fisher exact test and student's t test were used to compare the data. p- valueless than 0.05 was considered as statistically significant. Thirty six patients (60%) of carcinoma breast showed IL 10 expression in tumour tissue as compared to no IL 10 expression in any peritumouralnormal breast tissue (p<0.01). IL10 expression had statistically significant correlation with locally advanced disease, tumour grade, HER2+ve tumours and ER-ve, PR-ve, HER2+ve breast cancer subtypes (p=0.001, 0.001, 0.001 and 0.01 respectively). No correlation could be found with patient's age, tumour size, tumour histology and ER and PR status. Correlation of IL10 expressing tumours with several established poor prognostic markers of breast cancer may indicate the possible association of IL10 expression with poor prognosis. Large studies with long term follow up are needed to substantiate the association of IL10 with poor prognosis.