bluuebunny/crossref_metadata_2025 huggingface dataset:
P3-18-05: Triple-Negative Breast Cancer: Stem Cells, Cancer and New Treatment Strategy.
Authors: Yin S , Xu L , Reddy KB
Published: June 2012

Background: Women with triple negative breast cancer (TNBC) have a worse prognosis compared with other breast cancer subtypes. Hormonal or Herceptin-based therapies were found to be ineffective because of the loss of target receptors such as ER, PR and HER-2 amplification. TNBC has increased recurrence and poor survival, despite initial response to neoadjuvant chemotherapy. The aim of this study was to determine whether current drug treatment(s) eliminates bulk of tumor cells, but it has a minimal effect on cancer stem cells (CSCs) leading to tumor recurrence.

Materials and Methods: Cancer stem cells were identified by ALDH-positive population by fluorescence-activated cell sorting. CSCs ability to form mammospheres, are generated in nonadherent and nondifferentiated conditions. Apoptosis was assessed using the Cell Death Detection ELISAPLUS kit. Xenograft tumor formation was evaluated using Nod/Scid mice. Cancer stem cell levels in human TNBC and ER+, PR+ and HER2− tumors were obtained by CD44+/ CD24low/− immunohistochemistry.

Results: TNBC tumors express relatively higher levels of cancer stem cells compared to ER-positive, PR-positive and HER2−negative breast tumors. We studied the effects of PARP inhibitors (AZD2281 and BSI-201), paclitaxel, docetaxel, cisplatin and cisplatin plus TRAIL on CSCs derived from CRL-2335 and MDA-MB-468 TNBC cells in vitro. The in vitro data indicates that cisplatin plus TRIAL treatment was most effective in eliminating CSCs compared to PARP inhibitors, cisplatin, paclitaxel and docetaxel. Treatment with cisplatin plus TRAIL also inhibits Wnt-1 signaling and its downstream target, β-catenin, phospho β-catenin, cyclin D1, increased apoptosis, reduced proliferation and mammosphere formation. Inhibition of Wnt-1 by siRNA significantly reduced the ability of CSCs to form mammospheres compared to control. However, maximum effect was seen in cisplatin plus TRAIL treated cells. The combination of cisplatin/TRAIL also inhibited the growth of CRL-2335 Xenografts. Discussion: We demonstrate for the first time that cisplatin plus TRAIL is highly effective in reducing CSCs compared to PARP inhibitors, docetaxel or paclitaxel in TNBC cells in vitro. Inhibition of Wnt-1 signaling pathways by siRNA or cisplatin plus TRAIL reduces cell proliferation and increases apoptosis in CSCs. Taken together the data suggests that cisplatin plus TRAIL treatment has the potential of providing a new strategy for improving the therapeutic outcome in TNBC patients.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-18-05.
P4-01-02: Endocrine Resistance: Mechanism, Tumorigenic Capacities, and New Therapeutic Strategies.
Authors: Morrison GD , Fu X , Ithimakin S , Rimawi MF , Wicha MS , Osborne CK , Schiff R
Published: June 2012

Background

Endocrine therapy is the most effective therapies for estrogen receptor positive (ER+) breast cancer, but resistance is still a major clinical problem. Tumor initiating cells (TICs) in drug resistant tumors are believed to be part of the contributing factors for tumor relapse. Here we focused on the TICs and how they are related to HER2 expression and its downstream factors in acquired endocrine resistance models.

Materials and Methods: We have established a panel of endocrine resistant cell models through long-term treatment with tamoxifen (Tam, 10−7M, >6 months). Resistance to tamoxifen treatment in cells (TamR) was confirmed by growth curve assays (Celigo, Cyntellect Inc., San Diego, CA). In vitro mammosphere assays were applied to assess the mammosphere forming ability of parental and resistant cells. Aldefluor assays (StemCell Technology) were performed to measure the aldehyde dehydrogenase (ALDH-1) expressing cell population as a marker of TICs. Flow analysis was performed to evaluate HER2 expression in the ALDH subpopulations of both parental and TamR cells. Immunofluorescence staining was further used to detect the expression of ER, HER2, phosphorylated STAT3, and β-catenin in sorted ALDH+/− subpopulations.

Results: We found that endocrine resistant cells have a higher mammosphere forming ability than their parental counterparts. In addition, we showed that the ALDH+ subpopulation was significantly upregulated in all TamR derivative lines.

Further analysis of the ALDH populations indicated that HER2, as well as downstream survival factors such as pSTAT3s727 and β-Catenin, are upregulated in the ALDH+ populations as compared to the ALDH-population, especially in TamR derivatives.

Discussion: Our data suggest that deregulated HER or other growth factor receptor signaling can potentially lead to the enrichment of TICs as a potential contributor to endocrine resistance. We will further investigate the role and molecular signaling of TICs in endocrine resistance of breast cancer, as well as whether and to what extent more potent anti-HER inhibitors can improve endocrine sensitivity and circumvent resistance.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-02.
P4-01-04: Effects of CYP2D6 Phenotype and Drug Adherence on Tamoxifen Metabolite Levels.
Authors: Sousa BA , Nasim S , Cole DE , Wong BY , Hill K , Pritchard KI , Trudeau M , Verma S , Dent R , Petrella T , Gandhi S , Ueng S , Vandenberghe U , Romaschin A , Warner E
Published: June 2012

Background

Although the activity of cytochrome P450 2D6 (CYP2D6), the enzyme responsible for conversion of tamoxifen (TAM) to its most important active metabolite endoxifen, varies significantly with genotype. Routine genotype testing in patients on TAM has recently been discouraged. Conflicting results in publications regarding the prognostic utility of this test remain unexplained. Confounding factors could be lack of predicted correlation between CYP2D6 genotype and TAM active metabolites, or variability of patient compliance

Methods: Consecutive breast cancer patients on TAM were asked to enroll in a study to examine the relationship between CYP 2D6 phenotype, patient-reported treatment adherence, and TAM metabolites levels. Patients were genotyped for CYP2D6 polymorphisms using long-range PCR allele-specific amplification and single-nucleotide primer extension assay. From the genotypes, four phenotype groups were defined: Ultra rapid Metabolizer (UM), Extensive Metabolizer (EM), Intermediate Metabolizer (IM) and Poor Metabolizer (PM). Plasma was collected after at least 6 weeks of TAM (20 mg daily). The parent drug TAM, as well as 4-hydroxy N-desmethyl tamoxifen (endoxifen), 4 hydroxy tamoxifen (4OHtam) and N-desmethyl-tamoxifen (NDtam), were determined by Liquid Chromatography tandem mass-spectrometry (LC-MS/MS). Patients also completed a questionnaire about ethinicity, side effects, concurrent medications and tamoxifen adherence. Correlation between metabolite/TAM ratio and phenotype was tested by Spearman correlation test. Relationship between metabolite levels and adherence was tested by Wilcoxon rank sum test. Chi square test was used to compare proportions.

Results: Of the 100 patients enrolled there were 62 Caucasians, 25 Asians, 4 Africans and 6 Unknown. We found a strong correlation between ratio of endoxifen/TAM and phenotype (p <.0001) (Table 1) Over a 2 week period 68 never missed a TAM dose, 25 missed 1–2 times, 2 missed 3–5 times and 2 > 5times (2 missing data). In EM group we found significantly lower levels of TAM (p <.0001), NDtam (p=.008), 4OHtam (p=.003) in less adherent patients. A trend to decreased levels was also shown for endoxifen (p=.081). No associations were found between adherence or phenotype activity and side effects.

Conclusions: Our data suggests the predicted association between endoxifen levels and genotype. However, non-adherence may have a significant confounding effect. Prospective studies to evaluate the prognostic impact of CYP2D6 variants for patients on adjuvant tamoxifen should be done but results could be confounded by variable drug adherence if this is not measured concurrently.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-04.
P4-01-12: The Unfolded Protein Response (UPR) and Pro-Survival Autophagy Contributes to Antiestrogen Resistance in Breast Cancer.
Authors: Clarke R , Shajahan AN , Cook KL , Hickman FE , Facey CO
Published: June 2012

In 2011, most of the 192,000 newly diagnosed cases of invasive breast cancer will be estrogen receptor alpha positive (ER+). Endocrine therapy, administered as an antiestrogen, e.g., Tamoxifen (TAM) or Faslodex (FAS; Fulvestrant; ICI 182,780) or an aromatase inhibitor (AI), e.g., Letrozole or Exemestane is the least toxic and most effective means to manage the hormone-dependent breast cancer in such patients. However, resistance to endocrine therapy remains a significant clinical problem. Previously, we have shown that antiestrogen resistant breast cancer cells over-express X-Box Binding Protein 1 (XBP1), a transcription factor that belongs to the basic region/leucine zipper (bZIP) family. XBP1(U) and XBP1(S) variants result from an unconventional splicing of the XBP1 mRNA by IRE1alpha. In the unfolded protein response (UPR), endoplasmic reticulum proteins (IRE1alpha, ATF6 and PERK) sense cellular stress to regulate the accumulation of unfolded proteins. Initially a compensatory mechanism allowing cells to recover normal endoplasmic reticulum function, prolonged UPR may induce cell death; this is often dependent upon which arm of the UPR predominates. XBP1 is an obligate component in both the IRE1alpha and ATF6 arms of the UPR. In this study, we show that in MCF7/LCC9 [FAS resistant; TAM cross-resistant] cells, there is an increase in UPR signaling as detected by increased expression of BiP/GRP78. Transient expression of XBP1(S) in MCF7/LCC1 [antiestrogen sensitive] cells show decreased sensitivity to FAS that correlated with increased levels of both basal and FAS-induced autophagy as measured by cleaved LC3BII protein fragment, GFP-LC3 activity, and reduced expression of p62/SQSTM1. Furthermore, we show that MCF7/LCC9 cells survive by activating pro-survival autophagy through UPR that is regulated by the transcription factor c-MYC. An inhibitor of c-MYC, 10058-F4, synergized with FAS to inhibit proliferation in MCF7/LCC9 cells by preventing pro-survival autophagy and increasing apoptosis. Thus, our study shows that antiestrogen resistant breast cancer cells evade cell death by activating XBP1-mediated UPR that results in c-MYC-mediated prosurvival autophagy.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-12.
P4-01-14: Fulvestrant Regulates Epidermal Growth Factor (EGF) Ligands and Induces EGF Receptor Activation in MCF-7 Breast Cancer Cells.
Authors: Zhang X , Diaz MR , Yee D
Published: June 2012

Selective Estrogen Receptor Modulators (SERMs) 4 Hydroxy Tamoxifen (4-HT) and fulvestrant (fulv) inhibit estrogen receptor a (ER) positive breast cancer growth. We have observed that treatment (24 to 48 hours) of fulv in MCF-7 cells induced a 190kDa tyrosine phosphorylated band, which was blocked by EGFR and HER-2 tyrosine kinase inhibitors erlotinib and lapatinib. Immunoprecipitation showed EGFR, HER-2, and HER-3 were all phosphorylated after fulv treatment. 4-HT and estradiol did not cause this phosphorylation. No changes in receptor level after fulv treatment were noted. Downstream MAPK signaling was also blocked by erlotinib and lapatinib. Fulv induced activation of EGFR was ER dependent, since fulv treatment in C4-12, an ER negative cell line derived from MCF-7 cells, did not induce EGFR activation. Co-treatment with estradiol and fulv prevented EGFR activation. To explore the possibility that fulv enhanced ligand expression, we collected conditioned media (CM) from MCF-7 cells after 48 hours of treatment. pEGFR was lost when CM was removed, but recurred within 30 minutes. Cycloheximide abolished the ability of fulv to activate EGFR suggesting autorcine production of EGFR ligands was induced by fulv. To detect specific EGFR ligands, we used qPCR to measure various EGFR ligand mRNA levels in MCF-7 and C4-12 cell at 1, 4, 24 and 48 hours after fulv treatment. TGF-α and HB-EGF mRNAs were upregulated over 48 hours, which correlated well with pEGFR activation. 4-HT did not affect mRNA levels of these ligands. In contrast, amphiregulin (AREG) mRNA levels were substantially reduced 48 hours after fulv treatment. A similar trend was seen for AREG mRNA levels in 4HT treated cells but to a much lesser extent. There was no change in any EGFR ligand mRNA levels in ER negative C4-12 cells. These qPCR data suggested differential regulation of EGFR ligands by fulv treatment contributed to EGFR family member activation and was dependent on ER expression. Upon fulv treatment, levels of ER were diminished with decreased detection of S167 and S118 ER phosphorylation sites. Monolayer cell growth analysis showed that while fulv treatment in SFM reduced MCF-7 cell numbers compared to control treatment, erlotinib plus fulv significantly reduced cell numbers below the level of cells maintained in fulv alone. In conclusion, we show that fulv, but not E2 or 4-HT, activates EGFR family members accompanied by upregulation of ligands. Since SERM resistance has been associated with EGFR family member activation, differential control of EGFR ligand gene expression by ER may contribute to the development of fulvestrant resistance in breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-14.
P4-05-04: Arachidonic Acid-Induced Elevated Expression of 5-Lipoxygenase Is Linked to Metastatic Migration of Breast Cancer Cells.
Authors: Roy D , De Chatterjee A , Pal R , Roychowdhury S , Das S
Published: June 2012

Background: In invasive breast cancers, cancerous cells spread outside the ducts of the breast and metastasize to lung and other tissues. Although the rapid arachidonic acid (AA) metabolism and concomitant increase of eicosanoid molecules are involved in the proliferation and invasion of breast cancer cells, the exact mechanism by which AA metabolites regulate these phenomena are not well understood. Here, we show that leukotriene B4 (LTB4), one of the metabolites of AA, which is produced by the action of 5-lipoxygenase (5-LOX), causes the proliferation and metastatic migration of breast cancer cells.

Material and Methods: MDA-MB-231 (invasive breast cancer cells) and MCF7 (non-invasive breast cancer cells) were used in this study. Briefly, cells were treated with AA (100 μM) and nordihydroguaiarectin acid (NDGA, 10 μM), and the secreted eicosanoids were characterized by HPLC. Immunofluorescence microscopy was performed to elucidate the expression and intracellular localization of 5-LOX. The metastatic migration was analyzed by wound-healing assays.

Results: Our results suggest that while MDA-MB-231 cells produce high levels of PGE2 and PGD2, MCF7 cells synthesize excess HETE compounds (HETE5 and HETE8). Interestingly, MDA-MB-231 cells, when stimulated with AA, show the increased syntheses of LTB4 (∼3 fold) and decreased PGE2 and PGD2 (∼2 fold). In MCF7, on the contrary, AA treatment reduced the syntheses of all eicosanoids. Furthermore, the expression of 5-LOX in MDA-MB-231 cells was also increased by ∼2 fold. We observed that AA promotes cell migration in MDA-MB-231 cells, which could be blocked by NDGA, a generic inhibitor of LOX enzymes. Because LTB4 production and its binding with BLT receptors are linked to IL-8 secretion, we measured the level of IL-8 synthesized by both MCF7 and MDA-MB-231 cells. The results suggest that MDA-MB-231 cells secrete excess IL-8 (∼2 fold) when stimulated with AA, suggesting that LTB4 and IL-8 interactive pathways are important for cancer metastasis.

Discussion: Our results demonstrate that LTB4 synthesis is linked to the metastatic migration of MDA-MB-231 cells, and that it therefore should be considered as a target for developing new drugs for the treatment of invasive breast cancers.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-05-04.
P4-06-04: Detection of HER2 Gene Amplification in Circulating Tumor Cells and Disseminated Tumor Cells by Fluorescence In Situ Hybridization Using OncoCEE™.
Authors: Krishnamurthy S , Bischoff FZ , Mayer JA , Wong K , Pham T , Kuerer HM , Lodhi AK , Bhattacharyya A , Hall CS , Lucci A
Published: June 2012

BACKGROUND: The status of HER2 gene amplification in circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) might provide useful information for monitoring response to trastuzumab therapy, and may provide a basis for consideration of trastuzumab in patients with HER2 negative primary tumors who have HER2 positive CTCs and/or DTCs. The majority of techniques utilized for detection of minimal residual disease are limited in their ability to allow detailed phenotypic and genotypic evaluation of the cells. We report the utility of a microfluidic platform (OncoCEE™,Biocept, San Diego) for detecting HER2 gene amplification in CTCs and DTCs in patients with non-metastatic breast cancer.

METHODS: Peripheral blood (10ml) and bone marrow (BM) (1-2ml) were collected from patients with clinical stage I-III breast cancer in acid citrate dextrose solution (BD, Franklin Lakes, NJ) and anti-clumping reagent (OncoCEE-Sure™). Mononuclear cells were recovered using a Percoll density gradient method, incubated with a mixture of 10 primary capture antibodies (Abs), introduced into CEE™ microchannels, stained with fluorescent anti cytokeratin (CK) and CD45 abs and finally processed for fluorescence in situ hybridization (FISH) using probes specific to centromere 17 (spectrum green) and HER2 (spectrum arrange). The ratio of HER2:CEP17 >2.2 in any CK+/CD45- and CK-/CD45- cell was regarded as positive for HER2 gene amplification.

RESULTS: Peripheral blood and/or BM from 78 patients (65 BM; 70 blood; 57 matched blood and BM) with T1NO (39), T1N1 (8), T2N0 (12), T2N1 (2), T2N2 (1), T2N3 (3), T3N0 (2), T3N1 (2), T3N2 (1), T4N0(5), T4N1 (3), with HER2+ (n=12) and HER2− (n=58) primary invasive breast tumors were studied. The 12 patients with HER2+ primary tumors had HER2+ DTCs in 3/12 (25%) and HER2+ CTCs in 1/9 (11%) cases respectively. HER2+ DTCs and HER2+ CTCs occurred in 12/55 (24%) and in 4/63 (6%) of the patients with HER2− primary breast tumors. HER2+ CTCs and DTCs occurred simultaneously in only 2 patients and in either blood (3) or BM (13) in the remaining patients.

CONCLUSION: 1. The cell enrichment and extraction microfluidic technology (OncoCEE™) provides a sensitive platform for evaluation of HER2 gene amplification of CTCs and DTCs.

2. HER2+ primary tumors were associated with either HER2+ CTCs or DTCs in 25% of the patients.

3. HER2+ CTCs or DTCs occurred in 28% of patients with HER2−primary tumor.

4. Discordant HER2 status was contributed mainly by HER2+ DTCs occurring in HER2 - primary tumors.

5. The clinical significance of evaluating the status of HER2 gene amplification in CTCs and DTCs in the management of patients with breast cancer needs to be evaluated prospectively in larger clinical trials.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-06-04.
P4-07-01: Circulating Tumor Cells, Disease Recurrence and Survival in Newly Diagnosed Breast Cancer.
Authors: Franken B , de Groot MR , Terstappen LWMM , Mastboom WJB , Van der Palen J , Tibbe AGJ
Published: June 2012

Background: The presence of circulating tumor cells (CTC) is an independent prognostic factor for progression-free survival and overall survival (OS) for patients with metastatic breast cancer beginning a new line of systemic therapy. The current study was undertaken to explore whether the presence of CTC at the time of diagnosis was associated with recurrence free survival (RFS) and breast cancer related death.

Materials and Methods: In a prospective single center study, CTC were enumerated with the CellSearch system in 4 aliquots of 7.5 ml of peripheral blood of 504 patients before undergoing surgery for breast cancer between September 2003 and January 2009. Four hundred four had stage I-III disease and were followed for 6–90 months (median 48). One hundred had a benign tumor and served as a control group. Results: In the control group 15 (15%) had ≥1 CTC / 30 mL of blood. In stage I-III patients 76 (19%) had ≥1 CTC in 30 mL of blood of whom 16 (21.1%) developed a recurrence. In 328 patients with 0 CTC 38 (11.6%) developed a recurrence. Four year RFS was 88.4% for favorable CTC and 78.9% for unfavorable CTC (p =0.038). Breast cancer related death was 4,3% (14 of 328 patients) for favorable and 14,5% (11 of 76 patients) for unfavorable CTC (p = 0.001). CTC, PR receptor and stage were independent predictors of breast cancer related death in multivariate analysis. The corrected odds ratio after multivariate analysis for breast cancer related death in patiënts with CTC versus patiënts without CTC was 3,47.

Discussion: Presence of CTC in breast cancer patients before undergoing surgery with curative intent is associated with an increased risk for recurrence and breast cancer related death. This supports the notion that the presence of CTC is also important in the primary disease setting. In this study the sensitivity of the CellSearch assay was increased by a fourfold increase in blood volume and a threshold for unfavorable of ≥1 CTC in 30 mL of blood, which lead to a relatively high background in the control group. Thus an increase in specificity is desired before initiation of prospective multicenter trials as well as a more practical way to process larger blood volumes.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-01.
P4-07-09: Automated Quantitative Assessment of HER2 Expression of Circulating Tumor Cells (CTC) in Metastatic Breast Cancer (IC 2006–04 Study).
Authors: Bidard F-C , Ligthart ST , Decraene C , Bachelot T , Delaloge S , Brain E , Campone M , Pierga J-Y , Terstappen LWMM
Published: June 2012

Background

Beyond quantitative CTC analysis for outcome assessment, their qualitative analysis could predict drug response and resistance. Classification of CTC and quantification of treatment targets on CTC can be subjective as they are morphologically heterogeneous. Current HER2 fluorescence assessment methods rely on a visual comparison between CTC images and external controls (cells lines) and are poorly reproducible. We report an automated classification and quantification of HER2 expression on CTC in a large cohort of metastatic breast cancer (MBC) patients (pts).

Materials and Methods: CTC enumeration and HER2 assessment was performed with the CellSearch® system in pts with MBC included in the prospective IC 2006–04 study (Pierga, Ann Oncol 2011), before first line chemotherapy. The FITC conjugated antibody CB11 was used to identify HER2. For quantitative assessment of HER2 the breast cancer lines SKBR3 and MCF7 with known HER2 status were spiked in blood of normal donors and processed with the CellSearch system. Digital images of Cytokeratin-PE, DAPI, CD45-APC, and HER2−FITC from these samples were stored. An algorithm, previously developed in prostate cancer (Ligthart, AACR 2011), was applied on these images to automate identification of CTC. The fluorescence of the HER2 FITC channel for each CTC as well as the CD45+, DAPI+ leukocytes contained within each sample were also quantified using this algorithm. HER2 fluorescence levels of CTC were compared to HER2 status of status of pts.

Results: Among 267 pts included in this study, 103 had at least 1 CTC/7.5 ml and HER2 fluorescence assessment. In these pts, correlation analysis between automated (aCTC) and manual (mCTC) CTC counts showed an excellent correlation (R2 =0.978). HER2 fluorescence of CTC, varied greatly within and between pts, whereas leukocyte fluorescence was more homogeneous. The vast majority of CTC exhibited less fluorescence than SKBR3 cells, even in HER2+ pts. Primary tumor was HER2+ and HER2− in 36 and 58 pts respectively. Percentage of HER2+ CTC was higher in pts with primary HER2+ status.

Although a rare event, the presence within a sample of at least 1 CTC whose HER2 expression is superior to SKBR3 cells was also strongly associated with HER2+ primary tumor (N=15/36 vs. 3/58; p<0.001).

Conclusion: This study is the first to assess HER2 staining intensity in each CTC isolated from a large number of MBC pts. This was made possible by the aCTC count that was highly correlated with the mCTC count in our cohort and permitted HER2 quantification. Our study shows that HER2 staining is highly heterogeneous among CTC within each pt. These findings demonstrate the feasibility of real time quantitative assessment of treatment targets on CTC and opens a path towards personalized treatment.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-09.
P4-09-03: Clinical Significance of HER2+ and Triple-Negative Status in Patients with Tumor Size ≤ 1 cm and Node Negative Breast Cancer.
Authors: Shao T , Boolbol SK , Boachie-Adjei K , Klein P
Published: June 2012

Background

Data regarding the clinical significance of HER2 and triple negative status in patients with small node negative tumors is limited. Several small case series have suggested an increased risk of recurrence in patients with T 1abN0M0 HER2+ or triple negative (TN) tumors. Conversely, a recent study reported that the recurrence risk for T1abN0M0 HER2+ tumors was too low to justify any adjuvant chemotherapy or trastuzumab. Therefore it remains unclear who among those with small lesions might possibly benefit from more aggressive adjuvant therapy.

Methods: We identified all node negative breast cancer patients with tumor size ≤ 1 cm diagnosed between 1/1/1995 and 12/21/2008 through our institutional breast service database. Patients were classified according to their receptor status into three groups: 1) Hormone receptor (HR)+ (ER or PR positive, HER2 negative); 2) HER2+ (IHC 3+ or FISH ≥2); and 3) TN (ER, PR, and HER2 negative). Disease-free survival (DFS) was calculated using KaplanMeier methods. Cox proportional hazards models were used to estimate the association between breast cancer subtypes with DFS adjusting for patient and disease characteristics.

Results: Among 658 patients with tumor ≤ 1 cm, 494 (75%) of the patients were HR+, 109 (17%) were HER2+, and 55 (8%) were TN. Two hundred thirty-two tumors were classified as T1a, and 426 were classified as T1b. Median age was 58 years (27-92). Median follow-up was 3.5 years. The 5-year DFS rates were 97.9%, 95.6%, 93.5% in patients with HR+, HER2+, and TN tumors, respectively (p=0.026). In multivariate analysis, TN status was associated with worse DFS (HR 3.13, 95% CI 1.26−7.73), while HER2+ was not (HR 1.64, 95% CI 0.73−3.69). In the HER2+ group, 5-year DFS was also not different between patients who received chemotherapy +/− trastuzumab (32%) and those who did not (p=0.588). We also analyzed T1aN0 and T1bN0 patients separately in order to differentiate the sub-populations of patients more likely to benefit from systemic adjuvant chemotherapy. TN status remains an independent risk factor for worse DFS in T1bN0 (HR 3.68, 95% CI 1.17−11.6), but not in T1aN0 patients.

Conclusion: TN, but not HER2+ status was associated with worse DFS in patients with T1abN0M0 tumors, and adjuvant chemotherapy should be considered in TN breast cancer patients who have T1bN0 tumors.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-09-03.
P4-10-01: Tumor Markers Predicting Recurrence Type after a Primary Ductal Carcinoma In Situ.
Authors: Zhou W , Johansson C , Jirström K , Ringberg A , Blomqvist C , Amini R-M , Fjällskog M-L , Wärnberg F
Published: June 2012

Background: The risk for recurrence after a primary ductal carcinoma in situ (DCIS) is approximately 8% after ten years. If a recurrence occurs, the risk of the recurrence being invasive is approximately 50%. However, the knowledge of determinants for type of recurrence is limited. The purpose of this study was to investigate the predictable effect of specific biologic markers on the type recurrence after a primary DCIS.

Material and Methods: All women diagnosed with a primary DCIS between 1986 and 2004 in Uppsala and Västerås, Sweden were included (n=458). We also included all women from the SweDCIS Trial with a known recurrence (n= 166). TMA blocks were constructed and immunohistochemistry (IHC) was used to evaluate the status of estrogen receptor (ER), HER2, epidermal growth factor receptor (EGFR/HER1), cytokeratin 5/6 (CK 5/6), forkhead box A1 (FOXA1), forkhead box C1 (FOXC1), Ki 67, CD 10 and transacting T-cell-specific transcription factor GATA-3 (GATA-3) in the primary DCIS tumors. Logistic regression was employed to calculate the difference of expression status of biological markers between groups. Odds ratios (ORs) and 95% confidence interval (CI) were calculated, with adjustment for age, free margins, surgical type and molecular subtype by IHC.

Results: Of the 624 patients, 358 without recurrence were grouped as controls. Of the 266 with recurrence, 130 developed an in situ recurrence, while the remaining 136 developed invasive recurrence. Mastectomy compared to breast conserving surgery (BCS) and free margins were associated with less recurrences (OR 0.07, 95% CI 0.03 — 0.15) and (OR 0.39, 95% CI 0.26 — 0.58). As compared with the most common molecular subtype Luminal A, the subtype Luminal B had statistically significant higher risk of recurrence (OR 2.01, 95% CI 1.12 — 3.59). Higher risk of all recurrences was also observed for FOXA1 (OR 3.16, 95% CI 1.48 — 4.74) and FOXC1 (OR 2.39, 95% CI 1.39 — 4.09). In the analyses of type of recurrence, ER-negative, HER2−positive and EGFR-positive tumors recurred more often as in situ; (OR 2.34, 95% CI 1.17 — 4.71), (OR 2.08, 95% CI 1.11 — 3.88) and (OR 2.20, 95% CI 1.11 — 4.36). The molecular subtype HER2+ was also associated with a lower risk of invasive recurrence (OR 0.27, 95% CI 0.11 — 0.67).

Conclusion: As expected, mastectomy and free margins were associated with a lower risk of recurrence. Molecular subtype Luminal B and tumor markers FOXA1 and FOXC1 had a higher risk of recurrence. Recurrences after molecular subtype HER2+, ER-negative and EGFR-positive DCIS were more often of the in situ type.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-10-01.
P4-11-11: Thyroid Disease Is Associated with Breast Cancer: A Meta-Analysis.
Authors: Hardefeldt PJ , Eslick GD , Edirimanne S
Published: June 2012

Background: The controversial relationship between benign thyroid diseases and breast cancer has been investigated for over 50 years. Despite extensive population studies, the results as a whole have been inconsistent. The purpose of this study was to collate and analyse available data, calculating a pooled odds ratio of the risk of breast cancer in patients diagnosed with benign thyroid diseases.

Materials and Methods: Studies were obtained from a database search of MEDLINE, EMBASE, PubMed, Current Contents and Google Scholar with additional cross checking of reference lists. Inclusion criteria required a confirmed diagnosis of a benign thyroid disease, reporting of an odds ratio or data to calculate an odds ratio (and 95% confidence interval) and the use of an internal control group as the comparator. Collated data was assessed for heterogeneity and a pooled odds ratio calculated. From 276 citations identified in this search, a total of 28 studies were identified meeting our inclusion criteria. No language restrictions were used in the search or study selection. All data were analysed using a random effects model.

Results: There was significant evidence of an increased risk of breast cancer in patients with auto-immune thyroiditis, evident in a pooled odds ratio (OR) of 2.92 (95% CI: 2.13−4.01). No heterogeneity was present (I2=0, p=0.62) and there was no publication bias (p=0.15). In addition, the results supported an increased risk associated with the presence of anti-thyroid antibodies (OR 2.02, 95% CI: 1.63−2.50) and goitre (OR 2.19, 95% CI: 1.44−3.33). Moderate but non-significant heterogeneity was present in the analysis of goitre (I2=49.2, p=0.08), while the risk of publication bias was not signigicant (p=0.08). In the analysis of antibody presence, moderate and non-significant heterogeneity was evident (I2=58.3, p=0.08) with no publication bias (p=0.07). Subgroup analysis of antibody presence revealed increased risk associated with both anti-TPO (OR 2.64, 95% CI: 1.82−3.83) and anti-Tg antibodies (2.71, 95%CI: 1.58−4.69). No heterogeneity was present in the analysis of either subgroup evident in an I2 index of 29.3 (p=.22) and 18.1 (p=0.30), respectively. Publication bias was not significant (p=0.53 and 0.17). Quantitative analysis of hypothyroidism and hyperthyroidism was not significant, evident in pooled odds ratios of 1.56 (95% CI: 0.78−3.12) and 1.56 (95% CI: 0.84−2.88), respectively. Heterogeneity was high in hypothyroidism (I2=80.9, p=0.001) yet minimal in hyperthyroidism (I2=0, p=0.75). Publication bias was not significant in either hypothyroidism (p=0.06) or hyperthyroidism (p=0.20).

Conclusion: While these results indicate a link between thyroid auto-immunity and breast cancer, further prospective studies are required to definitively prove causality.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-11-11.
P4-11-13: Influence of Two Years of Exemestane on Bone Mineral Density in Postmenopausal Women at Increased Risk of Developing Breast Cancer; a Companion Study to the NCIC CTG MAP.3 Trial.
Authors: Goss PE , Richardson H , Ingle JN , Chlebowski RT , Fabian CJ , Garber JE , Sarto GE , Hiltz A , Tu D , Cheung AM
Published: June 2012

Background

Exemestane significantly reduced invasive and preinvasive breast cancers in postmenopausal women at increased risk for breast cancer in the NCIC CTG MAP3 trial with no serious toxicities, including excess fractures or osteoporosis.

Purpose: To provide additional information on the effect of exemestane on bone loss in women at high risk for breast cancer, within a subset of women participating on the NCIC CTG MAP.3B study. The primary hypothesis is that exemestane does not induce clinically significant bone loss in postmenopausal women at increased risk of developing breast cancer at 2 years. The primary objective of this companion study is to examine the effect of exemestane on lumbar spine and total hip BMD by DEXA at 2 years in women participating in the MAP3 trial.

Methods: The MAP.3B bone sub-study registered women from the main MAP. 3 trial from May 2008 to March 2010. Eligible women had to have an acceptable quality BMD scan by DEXA taken within 12 months prior to randomization to MAP.3. A BMD T-score >-2.0 SD (i.e. better than 2 standard deviations below the average peak BMD of a young adult woman) was established as the study population cutoff. A questionnaire including information on height, falls, fractures, lifestyle information including physical activity, tobacco and alcohol use was completed at baseline, 12 months, 24 months and at last visit. Fasting serum for bone biomarkers was collected at 12 months and total hip and L1-L4 (postero-anterior) spine BMD were measured 2 years after randomization on the same Lunar or Hologic scanner. The primary objective was to determine differences in hip and spine BMD at 2 years. Secondary outcomes include number of skeletal fractures and development of osteoporosis 2 years after randomization and changes in bone biomarkers at 1 year after randomization. For the analysis of the primary endpoints, the upper limit of a one sided 95% confidence interval for the difference in mean percentage changes between placebo and exemestane will be calculated for the BMD by DEXA at each site. We will conclude that exemestane does not induce significant bone loss in postmenopausal women at increased risk of developing breast cancer at 2 years when the upper limit is less than 3% for both sites. Similar confidence interval approach will be used to analyze the secondary endpoints.

Results: Between May 2008 and March 2010, 238 postmenopausal women were recruited. Median age was 61.8 years, and the majority of women were Caucasian (91%), with approximately 20% of the participants reporting a recent fall (within past 12 months) and another 13% reporting a recent fracture prior to randomization. We will report results from the primary as well as the secondary endpoints at the SABCS meeting.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-11-13.
P4-11-15: Increased Propability of Triple Negative Breast Cancer (TNBC) in Premenopausal Patients after Exogenous Hormonal Intake (EHI).
Authors: Pistamaltzian NF , Tzavara C , Papadimitriou C , Gyftaki R , Tryfonopoulos D , Panopoulos C , Tsoukalas N , Koumakis G , Demiri S , Koufopoulos N , Misitzis Y , Apostolikas N , Efremidis A
Published: June 2012

Purpose: HRT is a known risk factor for breast cancer (BC) among post-menopausal women. Our knowledge in relation to EHI (estrogens and/or progestin) and BC among pre-menopausal women is scarce. We have studied whether previous EHI influences BC phenotype, clinical and pathologic characteristics and correlation with other known BC risk factors.

Patients and methods: A prospective analysis (data recorded upon patients first presentation) of an electronic database at a tertiary cancer centre was performed. Patients’ demographics, risk factors for BC (smoking, alcohol use, obesity, family history), clinical profile, EHI parameters (duration, cause) and the tumors’ histopathology (type, grade,ER/PgR and HER2 by IHC and FISH) were analyzed. Premenopausal patients without an EHI history consisted control group.

Results: Out of 938 patients treated for BC between 2006 and 2010, 333(35,5%) were premenopausal and 131(39%) of them reported any use of hormones. Median age was identical (43 years,range:20–57) among premenopausal patients with and without EHI history. Mean duration of use was 28 months (range:1–180). Causes of EHI were contraception (35%), pregnancy(17%), menstrual abnormalities(17%) and medically assisted fertilization(17%). Smoking, alcohol use and obesity didn't differ among two groups. Family history for BC was more common (31, 3% vs. 22,8%, p=0,08)among women with EHI. No correlation was found among duration of use and the time of cancer diagnosis, while the mean time from the cessation of hormones to cancer diagnosis was 13 years(range:1–32). Only 18% of breast cancers were diagnosed within the first 5 years after exogenous hormones cessation.

TNBC was found to be significantly increased among premenopausal women with a history for EHI (23,6% vs. 13,4%, p=0,016). This increase was independent of the existence of positive family history for BC (p=0,61). EHI conferred a twofold increase in the risk for a TNBC (OR=1,99 p=0,019). No other clinical or histopathologic parameter showed any difference among the two groups.

Conclusion: Prior use of exogenous hormones, for any cause and irrespective of the coexistence of other risk factors and family history, increases the probability of a triple negative breast cancer diagnosis by twofold. Whether this represents a trend of a changing epidemiology in the types of BC in prior hormone users vs. nonusers, poses an extremely challenging hypothesis to be verified in large epidemiologic studies — given the young age and the treating difficulties of this patient population.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-11-15.
P4-11-17: Noninferiority (NI) Phase III Trials in Advanced Breast Cancer (ABC) over 12 Years.
Authors: Saad ED , Militao MS
Published: June 2012

Background

NI trials have gained increasing importance in oncology. The contemporary practice regarding the design features and the results of NI trials have not been formally assessed in ABC.

Methods: We searched PubMed for articles reporting the efficacy results of phase III trials on systemic antineoplastic treatments for ABC published between 1/1998 and 12/2009 in 11 leading medical journals (Ann Oncol, BCRT, BJC, Cancer, CCR, EJC, JCO, JNCI, Lancet, Lancet Oncol, and NEJM).

Results: We retrieved 93 reports, 12 of which (13%; 95% CI, 7% — 22%) had a NI primary hypothesis. Of note, there were no equivalence (i.e., two-sided question) studies. There was no significant trend in the proportion of NI trials in the two 6-year periods compared (17% vs 9%; P=0.36). The median (range) sample sizes for NI and superiority trials were 404 (160-1354) and 326 (118-907) patients, respectively (P=0.32). NI trials enrolled exclusively first-line patients more frequently than superiority trials (83% vs 51%; P=0.06). The primary endpoint for NI trials was response rate (RR) and progression-free survival/time to tumor progression in 8 and 4 cases, respectively. Despite their nominally higher number of patients, NI trials were more likely than superiority trials to have RR as their primary endpoints (67% vs 32%; P=0.03). The NI margin was reported in all trials, but justified by authors in only one case. Among trials with RR as primary endpoint, the NI margin was 15% in six cases and 25% in two. In trials with progression-free survival/time to tumor progression as primary endpoints, the lower limit of the confidence interval for the hazard ratio to demonstrate NI was always 0.8. Of 12 NI trials, 9 were positive (i.e., proved NI).

Conclusion: NI trials represent 13% of contemporary phase III trials on ABC. Most such trials achieve their primary endpoint. At present, NI margins seem to be chosen on historical basis rather than on statistical reasoning, and such choice could bear implications on trial positivity.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-11-17.
P4-11-19: Healthcare Resource Utilization among Breast Cancer Patients with Bone Metastases and Skeletal-Related Events: A Population-Based Cohort Study in Denmark (1997 - 2009).
Authors: Yong M , Christiansen CF , Gammelager H , Sværke C , Chia V , Atchison C , Fryzek J
Published: June 2012

Background

The healthcare resource utilization (HRU) of breast cancer patients who develop bone metastases and skeletal-related events (SREs) has not been well-characterized. Our objective was to describe the HRU associated with SREs in a large population-based cohort of Danish breast cancer patients with bone metastases and one or more SRE.

Methods: We identified women diagnosed with incident breast cancer from January 1, 1997 through December 31, 2006 using the Danish Cancer Registry. We followed this cohort of patients for development of subsequent bone metastases and SREs identified through the Danish National Registry of Patients through December 31, 2009. SREs were defined as pathologic fracture, spinal cord compression, and radiation or surgery to bone. Among patients with only one SRE, the HRU period, composed of all HRU occurring within a 90-day period after the SRE and within a two-week diagnostic period prior to the SRE, was assessed. For patients with multiple SREs (each one separated by less than 90 days), the HRU period, composed of all HRU occurring within a two-week diagnostic period prior to the first SRE up until 90 days after the last SRE, was described. Patients may have had multiple HRU periods if SREs were separated by more than 90 days. The HRU summarized included number of inpatient hospitalizations, length of hospitalization stay, outpatient physician visits, emergency room visits, and procedures.

Results: We identified 1,148 patients with bone metastases and SREs among 38,485 breast cancer patients. The mean age at breast cancer diagnosis for those who developed bone metastases and SREs was 59 years (SD, 13 years) and the majority (72%) of patients had multiple SREs during the first HRU period. Approximately 20% to 30% of patients with single and multiple SREs, respectively, died within the first HRU period. Overall, length of hospitalization was longest for patients with spinal cord compression followed by patients with pathologic fracture. In general, patients with multiple SREs had higher HRU compared to those with a single SRE in the first HRU period, particularly in length of hospitalization stay.

Conclusion: SREs secondary to bone metastases are serious events. In Denmark, high HRU was observed in all patients with SREs, but especially in those with multiple SREs, where increased lengths of hospitalization were observed.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-11-19.
P4-12-11: Myelodysplastic Syndrome Post Primary Breast Cancer Treatment: Cases from a Community Cancer Center: 1990–2010.
Authors: Kaplan HG , Malmgren JA , Atwood MK
Published: June 2012

Background: Myelodysplastic syndrome (MDS) has not been well documented or described post breast cancer (BC) treatment. Methods: A cohort of all breast cancer patients from 1989 to 2010 at our institution were followed for incidence of blood disorders of any type by our registry system and by linkage to the Surveillance, Epidemiology and End Results (SEER) registry for our area. All cases of post breast cancer leukemia were reviewed for incidence of MDS from a cohort of 9846 cases diagnosed between 1990 and 2010. Coding of cases was assigned by the SEER registry using ICD-0 codes for type of leukemia from pathology reports. 20 cases of MDS were identified and confirmed using SEER coding for MDS and death certificate review if applicable. Variables of interest included age, race, treatment received, recurrence and treatment for recurrence, time interval from breast cancer diagnosis to leukemia diagnosis and time from leukemia diagnosis to follow up or death. Cytogenetic studies were available on 5 of the 20 patients.

Results: Mean age of the patients was 56 years, range 33–79, with 65% < age 65. 90% of the patients were white and 10% Asian. TNM BC stage distribution was stage 0=2, stage I=8, stage II=8, and stage III=2. MDS was diagnosed at a median time post BC diagnosis of 65 months, range 8–205 months including 5 patients treated for recurrence and median 47 months, range 8–205 months excluding patients with recurrence. Nine patients had initial chemotherapy treatment, two without initial adjuvant therapy received chemotherapy for recurrence, and 9 received radiation only. Eight of eleven patients who received chemotherapy were treated with doxorubicin. All eleven received cyclophosphamide. Five of the 20 cases had MDS, not otherwise specified (NOS) (9989), 4 cases had MDSaAML, 3 MDS treatment related (9987), 3 refractory anemia (RA) (9980), 3 refractory anemia with excess blasts (RAEB) (9983), 1 RA with sideroblasts (9982), and 1 MDS with 5q deletion. Three of the five patients with cytogenetics available had chromosome 7 abnormalities and one had 11q21,23. Eleven of the 20 patients (55%) died of MDS or MDSaAML with a mean survival of 17 months, range 0.5-72 months. Six patients were alive at follow up with mean survival 56 months, range 43–89. Two of the surviving patients had stem cell transplants. The remaining three patients died of other causes with mean survival 66 months, range 7–109 months. Mean survival of all patients was 36.26 months, range .53-109 months with a significant difference in survival time between the patients that died of MDS, those alive at follow up and those that died of other causes (F statistic 6.56, p=.008).

Discussion: MDS is a significant complication of breast cancer treatment with radiation and/or chemotherapy with a high rate of associated mortality.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-12-11.