bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract P5-05-12: Heparan Sulfatase 2 Regulates Breast Cancer Tumorigenesis and Promotes Invasive Ductal Lesions
Authors: Khurana A , McKean H , Mcguire J , Roberts L , Goetz MP , Shridhar V. 
Published: April 2011

Abstract
Ductal carcinoma in situ (DCIS) of the breast is a heterogenous group of proliferative cellular lesions that have the potential to become invasive. Very little is known about the molecular alterations involved in the progression from DCIS to invasive ductal carcinoma (IDC). Heparan endosulfatases HSulf-1 and -2 catalyze or edit sulfate moieties on heparan sulfate proteoglycans and have been implicated in modulating heparin binding growth factor signaling. Several studies have suggested that while expression of HSulf-1 is down-regulated and is considered a tumor suppressor gene, expression of HSulf-2 is over-expressed in primary breast tumors and cell lines. However, the role of HSulf-2 in breast tumorigenesis is poorly understood. Here we have evaluated the effect of HSulf-2 in an in vivo model of breast cancer progression (MCF10DCIS.com). These cells form comedo type DCIS and progress to IDC when transplanted in immune-deficient mice and therefore are an ideal model to study breast cancer progression.
Methods: Stable clones depleted of HSulf-2 in MCF10DCIS.com cells were generated and injected into mouse mammary fat pads with non-targeted control shRNA clones (NTC) as controls. HSulf-1, HSulf-2, smooth muscle actin immunostaining and H&E staining was perfromed at weeks 3, 5 and 7. Matrixmetallo Protease (MMP) expression by real time PCR was evaluated at Weeks 3 and 5 in both groups.
Results: A significant reduction in tumor growth was observed in HSulf-2 depleted clones compared to NTC clones (p=&lt;0.01). HSulf-2 immunohistochemical staining showed distinct pattern of staining localized to ductal lesions as well as stromal (myoepithelial and myofibroblasts cells). In contrast, the closely related family member, HSulf-1 expression was restricted largely to stromal and myoepithelial layer in ductal lesions. While HSulf-2 staining remained intense from weeks 3 to 7, HSulf-1 expression was diminished during the same time period. In NTC clones, ductal like lesions were observed by week 3 with disruption of the basement membrane and appearance of invasive phenotype by weeks 5 and 7. In contrast, HSulf2 knockdown xenografts showed retention of ductal like structures even at week 5 and retention of basement membrane (p=&lt;0.05). Additionally marked necrotic areas were also observed in HSulf-2 silenced clones. Previous studies have shown that the malignant epithelium induces the development of the stroma necessary for the development of invasive phenotype. Consistent with this observation, we demonstrated decreased MMP expression in xenografts derived from HSulf-2 depleted clones compared with NTC xenografts (p=&lt;0.05). Conclusions: Our data suggest that HSulf-2 may play an important role in the transition from DCIS to an invasive phenotype (IDC) potentially by up regulating MMP expression and activity.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-12.
Abstract P5-05-13: HER2-Induced MMP-1 Expression Is Mediated by HER1 Though AR, EGF, and TGF Independent of HER3 through HRG in Breast Cancer Cells
Authors: Jung HH , Park YH , Ahn JS , Im Y-H. 
Published: April 2011

Abstract
introduction. However, one of plausible explanation for resistance to trastuzumab is associated with HER dimerization. Furthermore, HER3 seems to be the preferred dimerization partner when signaling occurs through the PI3K pathway and, as such, is emerging as a key target for inhibition of HER signaling. Recently, we reported that HER2 could induce enhanced MMP-1 expression. In addition, this up-regulation of MMP1 expression was shown to be mediated MAPK pathway synergized with ER. Heregulin (HRG) acts as a preferential ligand for HER3 and/or HER4 receptors. Upon binding, HRG activates the EGFR/HER2 receptors via dimerization with HER3 and/or HER4. In the other words, HRG-β was reported to promote MMP-7 expression via HER2-mediated AP-1 activation in MCF-7 cells. In addition, MMP-9 is associated with HRG-≥1/HER2 aggressive phenotype of breast cancer.
Based on these backgrounds, we conducted this study to test the role of HER3 in breast cancer cells in relation to the expression of HER2 and MMP-1.
Our result showed that MMP-1 and MMP-13 expressions were up-regulated via EGF and amphiregulin (AR) independent of HRG in HER2 transfected MCF-7 and SK-BR-3 breast cancer cells through Western immunoblotting and quantitative RT-PCR. Enhanced MMP-1 and MMP-13 expressions were markedly decreased by treating HER2 targeting agents in AR, EGF, and TGF-β stimulating cell lines, while these findings were not observed in HRG1-β and HRG1-β stimulating cell lines in quantitative RT-PCR. In addition, these findings were reversed by treating siEGFR and siHER2, not by siHER3. Consequently, we could confirm the downstream pathways were mediated by MMP-1 AP-1 not by HRG1-β, nor HRG1-β with EMSA.
This study showed that HER2-induced MMP-1 expression was mediated by HER1 though AR, EGF, and TGF independent of HER3 through HRG in breast cancer cells. This result implies that the dimerization responsible for MMP-1 up-regulation by HER2 is HER1/HER2, not HER2/HER3. Further implications of MMP-1 in HER2 overexpressing breast cancer through HER1 are going to following.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-13.
Abstract P5-06-13: Reduced NIMA Related Kinase 2 (Nek2) Expression Level Enhances Anti-Cancer Drug Sensitivity in the Breast Cancer Cells
Authors: Lee J , Lee K , Gollahon L. 
Published: April 2011

Abstract
Although the anticancer drugs paclitaxel and doxorubicin are commonly used to treat many types of solid tumors, their effectiveness is highly variable due to tumor cell resistance. Therefore, it is important to find mechanisms that can be targeted to increase the sensitivity of cancer cells to antitumor agents such as paclitaxel and doxorubicin. NIMA related kinase 2 (Nek2), a serine/threonine kinase is emerging as an important oncogene because of its regulatory role in mammalian cell mitosis. Thus, regulation of the Nek2 expression levels may prove important as a target for cancer treatment. In this study, we investigated whether drug sensitivity was increased in the triple negative breast cancer cell line MDA-MB-468 by using antisense oligonucleotides (ASOs) and siRNA against Nek2. MDA-MB-468 breast cancer cells transfected with Nek2 siRNA or ASO were exposed to various concentrations of paclitaxel and doxorubicin. Cell viability, cell cycle distribution, and apoptosis were evaluated. We observed that drug susceptibility in these transfected cells was dramatically increased compared with either agent alone. Interestingly, the cell proliferation of the Nek2 ASO transfected breast cancer cells was much lower than the Nek2 siRNA transfected breast cancer cells. Our FACS data showed paclitaxel and doxorubicin treated cells transfected with ASOs arrested in G2/M phase as expected due to Nek2 regulatory function in centrosome duplication. We observed that ASOs and siRNA against Nek2 work with paclitaxel and doxorubicin by arresting cells at G2/M and progressing the cells to apoptosis. Our results suggest that these drugs in combination with Nek2 siRNA or Nek2 ASO treatment greatly improve the sensitivity of cancer cells. Thus, these results provide a possible new strategy for the treatment of breast cancer.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-06-13.
Abstract P5-06-14: Ezrin Binding Protein Ebp50 Negatively Regulates HER2 and Alters Tamoxifen Sensitivity
Authors: Gu G , Covington KR , Fernandez NM , Ando S , Fuqua SAW. 
Published: April 2011

Abstract
Background: Targeted therapy with antiestrogens and aromatase inhibitors are the mainstay of treatment for estrogen receptor (ER) a-positive breast cancer; however, resistance to these therapies ultimately occurs and patients recur with resistant disease.
Materials and Methods: Stable overexpression of a shRNA to Ebp50 or full-length Ebp50 was performed in MCF-7 breast cancer cells. Cell responsiveness to tamoxifen (Tam) was evaluated using soft agar and MTT growth assays. The molecular signaling pathways affected by Ebp50 knockdown were explored using immunoblot analysis, and immunoprecipitation assays. Cellular localization was determined using confocal microscopy.
Results: Modulation of Ebp50 levels altered cellular response to Tam treatment. Overexpression enhanced Tam sensitivity, whereas knockdown decreased Tam sensitivity. Thus reduction in Ebp50 levels analygous to that discovered in resistant patients, conferred resistance to the growth inhibitory effects of Tam. Since it is known that Ebp50 can bind to a number of cell surface receptors, we performed binding assays, and found that Ebp50 was able to bind to HER-2 in MCF-7 cells. In addition, this interaction was enhanced with Tam treatment. Enhanced activation of phospho-HER2 and total-HER2 levels, as well as pAKT were seen in the Ebp50 knockdown cells. We expressed deletion mutants of Ebp50 we saw increased levels of cytoplasmic ERa when the ERM domain was deleted. The ERM domain, which is know to be important for the binding cytoplasmic proteins, may thus be a determinant of ERa localization. Ebp50 was also shown to affect ERa transcriptional activity. Discussion: Our data suggest that Ebp50 may be a negative regulator of HER2 signaling, and its loss confered homrone resistance. Ebp50 loss might function to stabilize HER2 levels.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-06-14.
Abstract P5-06-15: JNK2 Regulates Mammary Epithelial Cell Differentiation Through Inhibition of p53 and Notch-1 Expression
Authors: Cantrell M , Ebelt ND , Van Den Berg CL 
Published: April 2011

Abstract
The classification of patient tumors by clinical subtype has gained great interest due to the implications for prognosis and treatment. For example, it is known that the five-year survival rate of patients diagnosed with the basal subtype of breast cancer is considerably lower than those with luminal subtypes because they lack expression of common drug targets. For this reason, it is important to elucidate the molecular mechanisms responsible for differentiation of mammary stem cell populations into basal and luminal cell lineages. Data presented in this poster outline a novel pathway whereby a ubiquitously expressed protein kinase, JNK2, influences the differentiation of basal, myoepithelial cells (MEps) while suppressing luminal cell (LEp) populations. Gross anatomical observation of whole-mounted mammary glands from wildtype (jnk2wt) and jnk2-/- (jnk2ko) mice revealed that JNK2 increases the rate of pubertal development. Loss of jnk2 increased the rate of ductal invasion, branching, and terminal end bud (TEB) count. Although a slight increase in proliferation was seen in jnk2ko glands, data suggested that it was not due to growth factor signaling. Instead, it was noticed that jnk2ko glands possess 35% fewer MEps (p=0.0078) with a corresponding increase in LEp populations (p=0.100). LEps are known to proliferate at a higher rate than MEps. The differentiation phenotype was corroborated through 3D culture of primarymammary epithelial cells (MECs). The rate of growth of 3D MEC acini was 31.5% higher in jnk2ko cultures than in jnk2wt (P&lt;0.0001). Notch-1, a well-known regulator of MEC difFerentiation, is present in its active form in a greater proportion of cells within jnk2ko TEBs than jnk2wt (p=0.0118, 0.0209). Further, jnk2ko glands were found to possess greater amounts of Notch-1 protein as well as 10.5 times more expression of the Notch target gene, Hes-1 (p=0.005). These results were corroborated with 3D culture of primary MECs as well as in mammary tumor cells. Inhibition of Notch signaling with GSI, in 3D cultures of primary MECs, normalized growth (p=0.8992) and differentiation to jnk2wt control levels. QPCR results showed that jnk2ko glands possess 6.4 times more notch-1 transcript in vivo (P&lt;0.0001). Luciferase assays comparing activity of the notch-1 wildtype promoter to a notch-1 promoter with mutated p53 response elements revealed a dependence of increased notch-1 promoter activity in jnk2ko cells on the p53 response element. QPCR showed 2.78 times more p53 transcript in jnk2ko mammary glands as compared to jnk2wt. These data suggest that JNK2 regulates MEC differentiation through p53 and its target, Notch-1. These findings are significant because they reveal JNK2 as a possible drug target for novel differentiation therapy of basal type breast tumors.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-06-15.
Abstract P5-10-06: TACT2 Randomised Adjuvant Trial in Early Breast Cancer (EBC): Tolerability and Toxicity of Standard 3 Weekly Epirubicin (E) Versus Accelerated Epirubicin (aE) in 129 UK Hospitals (4391 Patients) (CRUK/05/019)
Authors: Cameron D , Barrett-Lee P , Velikova G , Canney P , Moyses H , McDermaid M , Banerji J , Gaunt C , Reynolds C , Wardley A , Bowman A , Bertelli G , Murray N , Bliss JM 
Published: April 2011

Abstract
Introduction: TACT2, a multicentre phase III trial with E-CMF as control (NEAT Poole NEJM 2006), tests 2 hypotheses in a 2x2 factorial design: A) accelerating chemotherapy (CT) offers superior benefits (CALGB9741 Citron JCO 2003); B) the oral 5FU prodrug capecitabine (X) gives similar efficacy but preferential side-effect profile to CMF. Here, focus is on hypothesis A with results for compliance, QL and acute toxicities (physician & patient (pt) reported) during E vs aE.
Materials &
Methods: Dec 2005-08 4391 pts (4371 women, 20 men) with node +ve or high risk node -ve invasive EBC were recruited. Pts were randomised to receive either E (100mg/m2 x 4) q3wk or q2wk aE (100mg/m2 x 4 plus pegfilgrastim, 6mg d2) followed by classical CMF q4wk x 4 or X (2500mg/m2/day x 14) q3wk x 4. Detailed CTCAE toxicity was to be assessed in subset (38 centres, 2137 pts). Of these, 2127 pts received ≥1 cycle CT. 1279 of these pts completed pt-reported outcomes (EORTC QLQ-C30 and pre-defined pt-reported TACT2 toxicities) with 910 completing baseline on time and having end cycle 4 data. P&lt;0.01 classed as significant. Toxicities/which differ between groups are shown where p-values are trend tests across all grades & % grade 3+ are reported. QL assessed via linear regression models.
Results: 33/4391 pts did not start CT. 4259 (97%) completed the initial 4 cycles (E 2143 (96%), aE 2116 (98%) with RDI &gt;85% for E 2058 (93%) and aE 1983 (91%). During cycles 1-4 higher rates of hand-foot syndrome & anaemia were seen with aE vs E (P&lt;0.001). Few were grade 3+ (E 0%, aE 0.9% & E 0%, aE 0.1%) respectively. Lower grade 3+ rates (all P&lt;0.001) were seen with aE vs E for: ANY toxicity (E 27%, aE 18%), leucopenia (E 4%, aE 1%), neutropenia (E 17%, aE 2%) & febrile neutropenia (E 4%, aE 2%). Change from baseline in global QL, role functioning & fatigue was worse with aE vs E (P&lt;0.001); no differences were reported for aE vs E for impact of individual pre-defined TACT2 toxicities on daily activities (Nausea, vomiting, tingling hands and/or feet, diarrhoea, constipation, sore mouth, mouth ulcers).
Conclusion: In TACT2, the largest adjuvant trial reporting acute toxicities for standard vs accelerated E in EBC, serious CTCAE toxicity was less common with aE compared with E, however pts report poorer global QL, fatigue & role function which does not appear to be explained by impact on daily activities due to individual side-effects.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-10-06.
Abstract P5-10-07: TACT2 Randomised Adjuvant Trial in Early Breast Cancer (EBC): Tolerability and Toxicity of Standard 3 Weekly Epirubicin (E) Versus Accelerated Epirubicin (aE) Followed by Capecitabine (X) or CMF in 129 UK Hospitals (CRUK/05/019)
Authors: Bliss JM , Canney P , Velikova G , Barrett-Lee P , Moyses H , McDermaid M , Banerji J , Gaunt C , Reynolds C , Agrawal R , Murray P , Clark P , Goodman A , Cameron D 
Published: April 2011

Abstract
Introduction: TACT2, a multicentre phase III trial with E-CMF as control (NEAT Poole NEJM 2006), tests 2 hypotheses in a 2x2 factorial design: A) accelerating chemotherapy (CT) offers superior benefits (CALGB9741 Citron JCO 2003); B) the oral 5FU prodrug X gives similar efficacy but preferential side-effect profile to CMF. Here, focus is on hypothesis B with results for compliance, QL & acute toxicities (physician & patient (pt) reported) during CMF vs. X
Materials &
Methods: 4391 pts (4371 women, 20 men) with node+ve/high risk node-ve invasive EBC were recruited between Dec 2005-08. Treatment was E(100mg/m2 x 4) q3wk vs q2wk aE(100mg/m2 x 4 + pegfilgrastim 6mg d2) followed by classical CMF q4wk x 4 vs X(2500mg/m2/day x 14) q3wk x 4. Detailed CTCAE toxicity was assessed in a subset (38 centres, 2086 pts receiving ≥1 cycle CMF or X). 1279/2086 also participated in substudy of pt-reported outcomes (EORTC QLQ-C30 and TACT2-specific toxicities). 826 had complete dataset (baseline, cycles 4&8). P-values are trend tests across all grades & %grade 3+ are reported. QL assessed via linear regression models adjusted for baseline, end cycle 4 score, & aE vs E. P&lt;0.01 classed as significant
QL in cycles 5-8 was better with X than CMF for global QL& fatigue (P&lt;0.001). Pts reporting clinically meaningful deterioration (&gt;10 points): global QL CMF 106/398 (27%), X 79/428 (18%); fatigue CMF 164/398 (41%), X 122/428 (29%)).
Conclusion: TACT2, the largest adjuvant EBC trial with X, confirms that X has preferential side-effect profile and global QL compared to CMF, with no evidence that prior aE compromised treatment delivery. Dose delivery data are consistent with advanced disease observations that for some pts, 2000mg/m2/day may be correct dose.
Results: 4264 (97%) pts continued on CT beyond cycle 4. 3726 completed all 8 cycles (E-CMF 951 (85%), aE-CMF 938 (86%), E-X 932 (84%), aE-X 905 (83%). For cycles 5-8, %RDI &gt; 85% was 69% after E and 68% after aE. Cycles delivered on time CMF 59%, X 63%; cycles without dose reduction CMF 75%, X 62%. 15 deaths in total within 30 days of CT: 9 on CMF, 6 on X. Worst grade toxicities which differed between CMF & X during cycles 5-8:

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-10-07.
Abstract P5-10-09: Comparison of the (Non-)Hematological Toxicity and Compliance of Different Anthracycline Containing Regimen with the Standard AC/EC in Elderly Patients with Primary Breast Cancer
Authors: Linder M , von Minckwitz G , Harbeck N , Janni W , Kümmel S , Kaufmann M , Elling D , Nekljudova V , Loibl S. 
Published: April 2011

Abstract
Background:
There is a paucity on toxicity (tox) data in elderly patients (pts) above 65 years (yrs) treated with anthracycline containing standard chemotherapy (ct) for primary breast cancer. We investigated the tox of standard anthracycline based regimen (AC/EC) and compared those with alternatives like dose-dense monotherapies and combinations (Edd; ADdd), intensified combinations (Canadian FEC) and taxane/anthracycline combinations (TAC) in various age groups. Patients and methods:
In this pooled analysis, individual pts data from 4 German randomized trials (n= 4775) were evaluated regarding tox and compliance. Docetaxel/doxorubicin/cyclophosphamide (75/500/500mg/m2) 3qw (A=TAC), 5-fluorouracil/epirubicin (500/60mg/m2) iv d1+8 followed by cyclophosphamide (75mg/m2) po d1-14 q4w (B=cFEC), doxorubicin (epirubicin)/cyclophosphamide (60(90)/600mg/m2) q3w followed by docetaxel (100mg/m2) or paclitaxel (175mg/m2) q3w (C= AC/EC-D/p), doxorubicin/docetaxel (50/75mg/m2) q2w (D= AD dose dense(dd)) and epirubicin-paclitaxel q2w (120/175 mg/m2) (E= sequence dd) were given. Only the anthracycline containing cycles were investigated. A descriptive analysis was performed on compliance and (non-) hematological tox. Pts were grouped according to their age: &lt; 60yrs [1], 60-64 yrs [2] and &gt;64yrs [3]. Primary G-CSF support was given in A, D, E.
Results:
73.6% were &lt;60 yrs, 15.8% were 60-64 and 10.6% were &gt; 64. A total of 22,306 anthracycline containing cycles were administered, of those 74.6% were given to [1], 15.2% to [2] and 10.0 % to [3]. 41.6% of the pts received TAC; 11.5% cFEC; 29.2% AC/EC-D/P; 8.2% ADoc dd and 9.5% Edd-Pdd.
The incidence of febrile neutropenia (FN) did not significantly differ between the 3 age groups (overall A:10.0%; B:2.9%; C:1.0%; D:4.7%) apart from E with 0.9% ([1]0%; [2]6.4%; [3]0% P&lt;0.001). FN in pts &gt;64 was lower in B (2.4%) than in A (14.4%) and D (5.1%). Diarrhea grade 3+4 increased with age to 8.2% for [3] under TAC (p= 0.01) and mucositis grade 3+4 under E to 12% for &gt;64 (P&lt;0.001). Conclusion:
In total, range and intensity of tox increased with age. Neutropenia did significantly increase with age in standard regimen but not in the dd groups
Rate of dose reduction increased with age in A ([1] 6.1%; [2]6.5%; [3] 11.5; p=0.02) and B ([1]15.7%; [2]26.9%; [3]39.3%; p&lt; 0.001). Similar significant results were observed for discontinuations and hospitalizations in A, B, D. Neutropenia grade 3+4 in [3] was 77% with cFEC, vs 55%with TAC (with primary G-CSF).
Leucopenia Grade 3+4 in %
(D, E). The severity of the non-hematotox differed between age groups and regimen. Monotherapies and sequential regimen seem more favorable in elderly pts. cFEC without G-CSF is not an option in pts &gt;64.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-10-09.
Abstract P5-10-15: First Safety Data from a Randomised Phase III (CIBOMA 2004- 01/GEICAM 2003-11) Trial Assessing Adjuvant Capecitabine Maintenance Therapy after Standard Chemotherapy for Triple-Negative Early Breast Cancer
Authors: Lluch A , Gomez H , Ruiz-Borrego M , Bines J , Llombart A , Ramos M , Torres R , Martins-Segalla JG , Torrecillas L , Barrios C. 
Published: April 2011

Abstract
Background: A large adjuvant trial programme is exploring the role of capecitabine (C) in early breast cancer (EBC). CIBOMA 2004-01/GEICAM 2003-11 is a multicenter, international randomised phase III trial that focuses on adjuvant C maintenance therapy after conventional induction chemotherapy in triple-negative EBC. Here we report interim safety data from the trial.
Materials and methods: Patients (pts) with operable, node-positive (or node-negative with tumour diameter ≥1 cm), hormone receptor-negative, HER2-negative EBC (central laboratory confirmation) who have received standard anthracycline-and/or taxane-containing chemotherapy in the (neo)adjuvant setting are eligible. Pts are randomised to receive 8 cycles of C (1000 mg/m2 bid, d1-14 q21d) (Arm A) or observation (Arm B). The primary endpoint is disease-free survival (DFS). Assuming 30% risk reduction in DFS rate at 5 years (from 64.7% to 73.7%, HR of 0.701) with a power of 80% and a two-tailed log-rank test at 0.05, 834 evaluable patients will be needed. With an expected drop-out rate of 5%, 876 pts will be included. We anticipate completing recruitment by the end of 2010. Efficacy analysis will be triggered after 255 events.
Results: To date, 724 pts have been recruited; here we report safety data from the first 278 pts (136 in Arm A and 142 in Arm B). Baseline characteristics are well balanced between the treatment arms.
Baseline Characteristics
In total, 920 cycles of C were administered (median 8.0, range 0-8). All 8 cycles were completed by 74.3% of pts. Median relative dose-intensity of C was 91.1%. The most common grade 3/4 clinical AEs related to C were: hand-foot syndrome (Grade 3 19.1%), diarrhoea (3.0%), vomiting (1.5%), fatigue (1.5%), nail changes (1.5%) and bilirubin elevation (1.5%).
Conclusions: The safety profile of adjuvant C as maintenance therapy is consistent with its known toxicity profile. Safety data from the first 400 randomised pts will be reviewed by an Independent Data Monitoring Committee and used to update this analysis at the meeting.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-10-15.