bluuebunny/crossref_metadata_2025 huggingface dataset:
Five year follow-up of patients with operable breast cancer whose sentinel nodes were completely sectioned at 0.25mm.
Authors: Dowlatshahi K , Alvarado R , Harrison M , Witt T , Gatta J , Bloom K
Published: December 2010

Abstract #1017

Background: 20-30% of patients with operable breast cancer and negative axillary lymph nodes develop recurrence within 10 years. With the advent of sentinel lymph node biopsy (SLNB) as a substitute to axillary lymph node dissection for staging of these patients, detailed examination of sentinel nodes for metastases has become feasible. Our group has previously reported on detection of 25% occult metastases in 200 patients with T1-T2 invasive breast cancer whose sentinel nodes were initially sectioned at 2-3 mm intervals and stained with H&E. The blocks were then sectioned at 0.25 mm intervals in the entirety and stained with immuno-histochemical technique (IHC). Some patients were given conventional chemo-hormonal therapy based upon tumor prognostic factors. We now present the disease recurrence in these patients at median follow-up of five years.  Methods: Prospectively registered records of 209 consecutive patients with T1-T2 invasive breast cancer treated with lumpectomy and sentinel node biopsy at Rush University Medical Center by three surgeons between December 1997 and May 2002 were reviewed. Of the remaining 175, the nodes were sectioned at 0.25 mm throughout and stained with IHC. The remaining 123 cases had no metastases. Adjuvant chemo-hormonal therapy was given to 38 patients with unfavorable prognostic factors and none to 85.  Results: The mean age of the patients and their tumor size were 54.6 years and 1.44 cm respectively. The mean length of follow up was 5.8 years. 34 of 209 patients had metastases in their sentinel nodes based on 2-3 mm sectioning and H&E staining. They were offered adjuvant chemo-hormonal therapy. 27 accepted and7 did not. Of the 209 patients, 175 had negative SLNB by routine examination. Of these, 52 (29.7%) were found to have occult metastases (isolated cells: 31, colonies> 0.2 mm: 21) upon 0.25 mm sectioning and IHC staining. Details of the findings are summarized in the table. The incidence of disease recurrence was noted to be similar in the subsets of patients with and without occult metastases.  Conclusion  Complete sectioning of the sentinel nodes in 209 patients with T1 and T2 breast cancer resulted in detection of occult metastases in 52 patients ranging in size from single cells to colonies of cells measuring up to 1500 microns. However, the incidence of disease recurrence at 5 year appears unaffected by these metastases.    m*: Small metastases: isolated cells  M*: Large metastases: colonies of cells.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1017.
The use of ultrasound in pre-operative assessment of the axilla in breast cancer.
Authors: Hogan BV , Shenoy HG , Peter MB , Langlands FE , Dall BJ , Horgan KM
Published: December 2010

Abstract #1019

Introduction  Sentinel lymph node biopsy is now standard practice in axillary staging in breast cancer. It is associated with less morbidity than an axillary node clearance. A drawback of sentinel node biopsy is the need for a second surgical procedure if the sentinel node shows metastases. Clinical examination of the axilla has been the standard pre-operative assessment but this has been shown to be unreliable. More recently ultrasound has been used to identify axillary metastases pre-operatively. The aim of our study is to assess the role of pre-operative ultrasound and fine needle aspirate cytology in refining the selection of patients for whom sentinel node biopsy is appropriate.  Methods  Three hundred patients with primary operable invasive breast cancer had axillary ultrasound preoperatively. If the ultrasound was normal the patient was offered a sentinel node procedure. If it identified equivocal nodes a fine needle cytology was performed. If the cytology was benign a sentinel node biopsy was performed. If the cytology was malignant then an axillary node clearance was performed. In cases where pathological nodes were identified on imaging, cytology was performed to confirm malignancy. An axillary node clearance was then performed.  Results.  Eighty three percent of our patients (n=249) had a normal axillary ultrasound pre-operatively. Of these 74.5% had a benign sentinel node biopsy. Sixty two percent of patients with equivocal nodes on pre-operative imaging had metastases on final histology (n=19). Of these 63% (n=12) were correctly identified pre-operatively with fine needle aspirate cytology of the equivocal node and had an axillary node clearance performed form the outset. Ultrasound identified pathological nodes in 7% of our patients (n=20). Malignancy was confirmed on cytology and an axillary node clearance was performed.  Using ultrasound and fine needle aspirate cytology to assess the axilla pre-operatively sentinel node biopsy was performed in 265 patients (88%). Of these, 74.8% had a benign final histology. Thirty two patients had a pre-operative diagnosis of nodal metastases and had an axillary node clearance. All 32 had lymph node metastases on final histology. Factors that predicted for a malignant sentinel node were lymphovascular invasion (p<0.001), tumour grade (p<0.008) and size of tumour (p<0.001).  Conclusion  Eighty eight percent of our patients had a sentinel lymph node biopsy to stage the axilla. Pre-operative ultrasound combined with cytology correctly determined the status of the node in 78% of cases. Our rate of second axillary operations was reduced by 32%. Importantly all patients diagnosed with node metastases pre-operatively and advised to have an axillary node clearance did have metastases on final histology.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1019.
Sentinel lymph node biopsy after neoadjuvant chemotherapy is accurate in breast cancer patients with a clinically negative axillary nodal status at presentation.
Authors: Gimbergues P , Abrial C , Le Bouedec G , Cachin F , Penault-Llorca F , Mouret-Reynier M , Kwiatkowski F , Maublant J , Tchirkov A , Dauplat J , Chollet P , Durando X
Published: December 2010

Abstract #1020

Background: In breast cancer, neoadjuvant chemotherapy (NAC) is widely used in order to enable a conservative surgery. In patients treated with NAC, the use of sentinel lymph node (SLN) biopsy, which is a good predictor of the axillary nodal status in previously untreated patients, is still discussed. The aim of our study was to determine clinicopathological factors that may influence the accuracy of SLN biopsy after NAC.  Patients and methods : Between March 2001 and December 2006, 129 patients with infiltrating breast carcinoma were studied prospectively. Preoperatively, all of them underwent NAC. At surgery, SLN biopsy followed by axillary lymph node (ALN) dissection was performed. Lymphatic mapping was done using the isotope method.  Results : The SLN identification rate was 93.8% (121/129). Fifty-six out of the 121 sucessfully mapped patients had positive ALN. Eight out of these 56 patients had tumor-free SLN (false-negative rate of 14.3%). The false-negative rate was correlated with larger tumor size (T1-T2 versus T3; p=0.045) and positive clinical nodal status (N0 versus N1-N2; p=0.003) before NAC. In particular, the false-negative rate was 0% (0/29) in N0 patients and 29.6% (8/27) in N1-N2 patients. Clinical and pathological responses to NAC did not influence the accuracy of SLN biopsy.  Conclusion : Our results show that clinical nodal status is the main clinicopathological factor influencing the false-negative rate of SLN biopsy after NAC for breast cancer. SLN biopsy after NAC can predict the ALN status with a high accuracy in patients who are clinically lymph node negative at presentation.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1020.
Subareolar injection may not always identify the same sentinel lymph nodes as peritumoral injection in breast cancer.
Authors: Inokuchi M , Noguchi M , Zen Y
Published: December 2010

Abstract #1022

Background. Sentinel lymph node (SLN) biopsy is widely accepted as a standard surgical procedure for early breast cancer, but the optimal site for injection of mapping tracers is still controversial. We evaluated whether a combination of peritumoral injection and subareolar injection can improve the identification rate of SLN biopsy and decrease the false-negative rate.  Methods. From August 2006 to April 2008, 155 patients with biopsy-proven operable breast cancer underwent SLN biopsy with peritumoral injection of radioisotope (Tc-99m-phytate) and subareolar injection of blue dye (patent blue dye).  Results. The overall identification rate for blue and/or radioactive (hot) lymph nodes was 99.4% (154/155); the identification rate of blue-dyed lymph nodes was 98.1% (152/155) and of hot lymph nodes was 96.8% (150/155). A total number of the excised SLNs were 379 in these 154 patients, and the average number of SLNs in each patient was 2.4 (range, 1-9). The average number of SLNs was 2.0 (range, 1-8) for blue dye and 1.9 (range, 1-6) for radioactive (hot). Two hundred twenty-six nodes (59.6%) were blue and hot, while 79 were blue-only and 74 were hot-only. Consequently, no concordance between the hot node and the blue node was found in 14 of 154 patients (9.1%). Eight of these 14 patients had separate blue-only nodes and hot-only nodes (so-called sequential mismatch). Four patients had SLNs identified by blue dye only, whereas 2 patients had SLNs localized solely by radioisotope labeling. On the other hand, metastatic disease was identified in SLNs of 40 of 154 patients (26.0%). A number of positive SLNs were 55 in these 40 patients, where 34 nodes (61.8%) were blue and hot, 9 were blue-only and 12 were hot-only. Among these 40 patients, 30 patients (75.0%) had blue and hot positive SLN. Four patients had blue-only positive SLN and 4 patients had hot-only positive SLN. Two patients had separate blue-only positive SLN and hot-only positive SLN. The SLN was the only positive node in 24 of the 41 positive SLN mapping (60%). Consequently, the false-negative rates were 9.8% (4/41) for peritumoral injection and 9.8% (4/41) for subareolar injection. There were no significant differences between peritumoral and subareolar injection. However, both injection methods complemented each other, thereby decreasing the false-negative rate to 0%. It is important to note that metastatic disease was identified by peritumoral injection in 4 patients in which the SLN was hot-only. At a median follow-up of 10.5 months (range, 2-22 months), there were no axillary recurrences in any of the 155 patients.  Conclusions. Subareolar injection may not always identify the same SLN as peritumoral injection. Subareolar injection of blue dye and peritumoral injection of radioisotope improves the identification rate of the SLN and decrease the false-negative rate of SLN biopsy.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1022.
Sentinel lymph node detection in breast cancer patients by real-time virtual sonography constructed with three-dimensional computed tomography-lymphography.
Authors: Yamamoto S , Maeda N , Tamesa M , Nagashima Y , Oka M
Published: December 2010

Abstract #1026

Background: Ultrasonography (US) is one tool for preoperative diagnosis of lymph node metastases in breast cancer. However, US cannot detect true sentinel lymph nodes (SLNs).  Methods: We identified SLNs in 60 clinically node-negative breast cancer patients using a real-time virtual sonography (RVS) system to display in real time a virtual multi-planar reconstruction obtained from computed tomography (CT) volume data corresponding to the same cross-sectional image from US. CT volume data were obtained from our original three-dimensional CT lymphography (3DCT-LG), which accurately detects SLNs in breast cancer. SLN metastases were assessed by shape and visibility of the hilum. All patients underwent SLN biopsy and SLN metastases were examined pathologically.  Results: In all 60 patients, we were able to detect the same SLNs visualized by 3DCT-LG. Suspicious SLN metastases were identified in 7 of the 60 patients, and 4 of 7 patients were pathologically positive. Positive predictive value was 57%. The remaining 53 patients displayed non-suspect SLNs in which absence of metastasis from the SLN was confirmed histologically. Overall accuracy was 95%.  Conclusions: This is a first attempt at preoperatively identifying SLNs using US guided by the RVS system in breast cancer patients. Although evaluation of SLN metastases was unsatisfactory, this method may be useful for preoperative fine-needle aspiration cytology for diagnosis of SLN metastases.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1026.
The effect of raloxifene on vascular endothelial growth factor expression in breast carcinomas of postmenopausal women.
Authors: da Silva BB , dos Santos AR , Lopes-Costa PV , Pires CG , Facina G , Nazário AC
Published: December 2010

Abstract #1031

Background: To evaluate the effect of raloxifene on vascular endothelial growth factor (VEGF) expression in breast carcinomas of postmenopausal women. Materials and Methods: Sixteen postmenopausal patients with operable stage II, estrogen receptor-positive, infiltrating ductal breast carcinoma were treated with raloxifene at the dose of 60 mg/day for a period of 28 days prior to definitive surgery. Tumor size varied from 3 to 5 cm (mean 3.7 cm) and the mean age of patients was 61.8 years (range 49-72 years). Tumor samples were obtained by incisional biopsy at the time of diagnosis and again at the time of definitive surgical treatment. VEGF expression was obtained by immunohistochemistry (primary anti-VEGF monoclonal antibody, IgG, C-1, sc-7269, 200 mg/ml, Santa Cruz Biotechnology, Santa Cruz, USA). Evaluation of VEGF expression was assessed semiquantitatively based on the fraction of stained tumor cells and on the intensity of staining. McNemar's test of symmetry was used to evaluate agreement between the positive or negative classification of VEGF expression prior to and following raloxifene treatment (p<0.05). Results: Fourteen of the 16 patients (88%) were classified as positive for VEGF expression prior to raloxifene treatment, while only 5 (31%) were classified as positive following treatment (p<0.007) (see Figure 1 and Table 1).[figure1]     Discussion: The results of the present study show that raloxifene reduced VEGF expression in breast cancer in postmenopausal women, thus suggesting that the drug may reduce neovascularization and the growth of estrogen receptor-positive breast tumors and reinforcing the argument for its possible use both in chemoprevention and in the treatment of breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1031.
Mechanisms of nab-paclitaxel and bevacizumab cooperation in inhibition of breast tumor growth and metastasis.
Authors: Volk L , Stutzman A , Flister M , Hall K , Chihade D , Desai N , Trieu V , Ran S
Published: December 2010

Abstract #1032

Background: Nab-paclitaxel, also known as Abraxane®, is paclitaxel formulated in nanoparticles that consist of human albumin encapsulating the hydrophobic drug moiety. This drug has shown numerous advantages including linear pharmacokinetics, significantly higher tumor retention, and increased maximal-tolerated dose due to reduced toxicity. We previously showed that combination of Abraxane and anti-VEGF-A antibody, bevacizumab, is significantly more efficacious in suppression of breast tumor xenografts and metastasis as compared to treatments with each of these drugs alone. We hypothesized that the mechanism underlying the therapeutic success of the combined therapy might include bevacizumab-dependent abrogation of the stress response elicited by paclitaxel in the tumor cells surviving chemotherapy.  Materials and Methods: Cultured MDA-MB-231 cells were treated with 0, 2.5, 5, 10, and 30 nM of Abraxane followed by detection of angiogenic (VEGF-A), prosurvival (p42 & p44 kinase, bcl-2) and inflammatory (IL-6, IL-8, and TNF-α) proteins using Western blotting and ELISA. MDA-MB-231 tumors were extracted from mice upon cessation of intravenous (IV) Abraxane therapy (10 to 30 mg/kg, qdx5) followed by Western blot and immunohistochemical analyses.  Results: In vitro, Abraxane treatment increased expression of VEGF-A, p42/44 kinase, bcl-2 as well as total and phosphorylated p65 subunit of NF-κB. Treated cells secreted 25- to 30-fold higher concentrations of inflammatory cytokines IL-6, IL-8, and TNF-α into conditioned media as compared with untreated control cells. Likewise, significant increases in bcl-2 and inflammatory cytokines were observed in tumors extracted immediately after paclitaxel therapy in vivo as confirmed by both Western blotting and immunohistochemical analyses.  Discussion: These findings suggest that paclitaxel elicits VEGF-A dependent prosurvival and proinflammatory stress responses in tumor cells surviving the cytotoxic therapy. Activation of these pathways suggests that concurrent therapy with VEGF-A neutralizing antibody might significantly improve the efficacy of paclitaxel-based therapies by counteracting the stress responses in the therapy-spared tumor cells.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1032.
Selective inhibition of VEGFR2 activity with a human anti-VEGF monoclonal antibody decreases tumor growth and macrophage infiltration in an orthotopic model of breast cancer.
Authors: Dineen SP , Lynn KD , Roland CL , Payton LA , Brekken RA
Published: December 2010

Abstract #1034

In general tumor associated macrophages confer a negative prognosis in patients with breast cancer. This is due in part to the fact that macrophages can promote angiogenesis and mediate the angiogenic switch in tumors. Vascular endothelial growth factor (VEGF) is a primary stimulant of angiogenesis as well as a macrophage chemotactic protein. Inhibition of VEGF is an attractive therapeutic strategy that has shown to be beneficial in combination with chemotherapy for some breast cancer patients. However, the mechanism by which inhibition of VEGF affects tumor growth appears to involve more than its effect on endothelial cells. We have previously shown that 2C3 (PGN-310), a mouse monoclonal antibody that prevents VEGF from binding to VEGF receptor 2, decreases tumor growth, angiogenesis, and macrophage infiltration into pancreatic tumors. In this study, we test a fully human version of 2C3, termed r84 (PGN-311), for its effect on established orthotopic MDA-MB-231 breast tumors in SCID mice. Orthotopic tumors were established by injecting one million MDA-MB-231 cells into the mammary fat pad of SCID mice. Therapy with saline (n=5), bevacizumab (n=8), 2C3 (n=5), or r84 (n=9) at 250 μg/ip injection twice weekly was initiated on day 26 post tumor cell injection (mean initial tumor volume = 150 mm3) and continued for approximately 3 weeks. Upon sacrifice, tumor weight was determined and the tumor and other organs were snap frozen or fixed in formalin and paraffin-embedded for histology. Anti-VEGF therapy inhibited growth of orthotopic breast tumors (on day 48, Mean ± SEM: control, 822 ± 160; bevacizumab, 344 ± 29; 2C3, 309 ± 69; r84, 368 ± 38 mm3;all p < 0.001 vs control). Furthermore, immunohistochemical analysis of tumors from control and anti-VEGF treated animals demonstrated that each anti-VEGF therapy reduced microvessel density and macrophage infiltration. Additionally, we also determined that inhibition of VEGF activity with 2C3 reduces VEGF-induced migration of MDA-MB-231 cells in vitro in a transwell assay. In summary, we demonstrate that r84 a unique fully human anti-VEGF antibody is effective in reducing tumor microvessel density, macrophage infiltration into tumors, and the growth of breast tumors implanted into SCID mice. These data suggest that inhibition of VEGF receptor 2 activity with an anti-VEGF monoclonal antibody is sufficient for effective blockade of the pro-tumorigenic activities of VEGF.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1034.
Effect of anticoagulation therapy on bleeding and thromboembolic events (TEs) in the AVADO phase III study of docetaxel (D) ± bevacizumab (BV) in inoperable locally recurrent (LR) or metastatic breast cancer (mBC).
Authors: Wardley A , Lohrisch C , Joy A , Young S , Yi F , Marme A , Dreosti L , Albanell J , Muñoz M , Miles DW
Published: December 2010

Abstract #1035

Background: In first-line mBC, the anti-VEGF monoclonal antibody BV (Avastin®) has demonstrated PFS improvement when combined with taxanes in two large phase III trials (E2100 and AVADO). Thromboses and bleeding are among the possible side effects of BV. Pts receiving full-dose anticoagulation therapy have historically been excluded from clinical trials of BV due to the hypothetical risk of bleeding and TEs. AVADO enrolled pts receiving full-dose anticoagulation provided that their level of anticoagulation was stable for ≥2 weeks prior to study entry. We investigated the incidence of bleeding and arterial and venous TEs (ATEs; VTEs) in pts receiving concomitant anticoagulation therapy in AVADO.  Methods: Pts with inoperable LR or mBC, with no CTx 6 months prior to randomisation (12 months if taxane-based), ECOG PS 0–1 and adequate LVEF were eligible for enrolment. 736 pts were randomised to D 100mg/m2 + placebo (PL) or D + BV 7.5 or 15mg/kg. D was administered q3w for ≥9 cycles, BV/PL q3w until disease progression or unacceptable toxicity. Anticoagulation therapy was defined as coumarin, heparin or low molecular weight heparins.  Results: Safety data are available from 730 pts. Grade ≥3 TEs and grade ≥3 bleeding were each reported by six pts (1.2%) who received BV, compared with nine pts (3.9%) and two pts (0.9%), respectively in the control arm. Eleven pts received anticoagulants at study entry (PL: 4, BV 7.5mg/kg: 6, BV 15mg/kg: 1) and 73 pts began anticoagulants during the study (PL: 21, BV 7.5mg/kg: 26, BV 15mg/kg: 26). No grade ≥3 ATEs were reported in pts receiving anticoagulation therapy either before or during the study.    Conclusions: The rate of grade ≥3 ATEs, VTEs and bleeding events was relatively low in the entire study and did not appear to increase with BV use. The incidence of TEs and bleeding events was not increased relative to the control arm in the subpopulation of pts receiving anticoagulants concurrently with BV, although patient numbers are small, particularly for bleeding events.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1035.
ALDH1 is a useful marker of basalness in human breast cancer and its stem/progenitor cells.
Authors: Jolicoeur F , Garcia de la Fuente I , Gaboury L , Balicki D
Published: December 2010

Abstract #104

Background: The basal cell subtype is a particularly aggressive form of breast cancer. The molecular mechanisms which underlie the aggressive course of basal cell breast cancer, as well as its corresponding tumor markers are currently under intense study. In addition, there is increasing evidence that cancer stem/progenitor cells are integral to the formation and perpetuation of breast cancer. Together with their progenitors, cancer stem cells may determine the therapeutic outcome and the clinical course of cancer. The first isoform of aldehyde dehydrogenase (ALDH1) has recently been reported to be a marker of breast stem/progenitor cells. Thus, we undertook this study to determine whether the immunohistochemical detection of ALDH1 in fixed breast cancer tissues correlates with selected basal and luminal cell surface markers.  Methods: Formalin-fixed, paraffin-embedded specimens were studied in triplicate using tissue microarrays from 57 breast cancer patients. In addition to ALDH1 staining, each of the following markers were studied using immunohistochemistry (IHC) in each sample: ER, PR, HER-2, the basal cell markers EGFR/HER1, CK5, CK14, CK17, SMA, and the luminal cell markers CK19, and EMA.  Results: ALDH1 staining was performed using IHC and subsequently evaluated using the Quick score. The Quick score takes into account both the percentage of positive cells, as well as the corresponding staining intensity. Our results suggest that breast cancer samples expressing the triple negative phenotype (ER-, PR-, HER-2 negative) together with one or more basal cell markers (EGFR/HER1,CK5, CK14, CK17, SMA) are associated with an elevated Quick score for ALDH1 staining, when compared with samples lacking the expression of these markers.  Conclusions: As it has been demonstrated that IHC detection of ALDH1 detects stem/progenitor cells in fixed breast samples, our results suggest that an elevated Quick score is associated with the enrichment of IHC basal cell markers in these samples. We are currently focusing on a larger number of breast cancer samples so that correlations can be studied as a function of therapeutic outcome and overall survival of breast cancer patients.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 104.
Identification of NPM1 as a potential high-penetrance gene in hereditary breast cancer.
Authors: Ren Z , Teugels E , De Brakeleer S , Sermijn E , Bonduelle M , Fontaine C , De Greve J
Published: December 2010

Abstract #1042

Background: In a major proportion of breast cancer patients with a strong familial cancer history no BRCA1 or BRCA2 mutations are detected which prompted us to explore other proteins on the BRCA1/2 pathway. Previously we have identified BARD1 (De Brakeleer et al, SABC 2006) as a rare high penetrance breast cancer predisposing gene. Nucleophosmin (NPM1) is a multifunctional protein that is ubiquitylated and stabilized by the BRCA1-BARD1 complex and thus is part of the BRCA1 pathway. Somatic protein truncating mutations in NPM1 are frequent events in acute myeloid leukemia (AML).  Aim: This study was aimed at determining whether NPM1 germline mutations may contribute to the breast cancer predisposition in families where no BRCA1 or BRCA2 mutation is found.  Methods: NPM1 mutation analysis was performed on genomic DNA extracted from blood of 198 breast cancer patients with strong indications for a hereditary form of the disease and 110 controls. All the 11 exons of the NPM1 gene were separately amplified by PCR. Since a high number of processed NPM1 pseudogenes are dispersed throughout the human genome, PCR primers had to be designed carefully. The PCR products were subsequently analyzed by denaturing gradient gel electrophoresis (DGGE) and when an abnormal migration pattern occurred, sequencing analysis was performed to determine the exact nucleotide-sequence alteration which was verified against the pseudogene sequences.  Results: Out of the six novel sequence variations, only one occurred in an exon: a duplication of thymidine located in the 3' non-coding sequence of exon 12 (c.1042dupT). The remaining 5 variants were located in intron sequences close to the exon borders. Two variants flanking exon7 (c.460-31G>A and c.524+42G>A) were always linked, and occurred at the same high frequency in breast cancer patients and controls. Two intron sequence variants were only found in one patient. One was a duplication of a sequence in intron 8 (c.582+52dupT) and the other a sequence deletion in intron 3 (c.139-9delT). One sequence variation located close to exon 11 (c.771-46C>A) was found in 7 familial breast cancer patients in 4 high incidence families but not in unaffected family members and in none of the control samples. This mutation leads to an alternative splicing of the NPM1 mRNA and is predicted to modify the carboxyterminal end of the protein, removing a nucleolar locating signal.  Discussion: A germline mutation in NPM1 was found only in familial breast cancer cases. This mutation may result in the synthesis of a C-terminal truncated NPM1 protein conferring high susceptibility to breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1042.
Impact of SNPs in ERα corepressor SMRT on breast cancer risk and response to endocrine therapy.
Authors: Hartmaier RJ , Skaar TC , Rae J , Wang J , Li L , Oesterreich S
Published: December 2010

Abstract #1043

Approximately 1/3 of hormone dependent breast cancers do not respond to tamoxifen treatment. The fact that these tumors are intrinsically resistant suggests that underlying genetic factors are responsible for this phenotype. The two major types of genetic variation are single nucleotide polymorphisms (SNPs) and copy number variations (CNVs). SNPs are the most common form of genetic alteration with an estimated 10 million SNPs across the genome and likely influence both disease susceptibility and response to therapy.  Traditionally, researchers have attempted to identify SNPs associated with a clinical outcome in a “top-down” approach by conducting linkage and SNP association studies. However, here we report on a “bottom-up” approach whereby SNPs are first tested in vitro for a functional effect. This awards two benefits over a “top-down” approach: 1) identification of functional SNPs and 2) detailed characterization of the SNP so that the clinical association studies can be designed accordingly.  Tamoxifen acts by blocking estradiol (E2) from binding to the estrogen receptor (ERα) and by recruiting corepressors, which then actively repress gene transcription. The critical role of corepressors, especially SMRT, in the proper action of tamoxifen makes them likely to be involved in tamoxifen resistance. Thus, we used a “bottom-up” approach to identify SNPs in ERα corepressors which affect the ability of tamoxifen to suppress ERα function.  Here we report the detailed in vitro characterization of a number of SNPs in SMRT (NCoR2) with respect to altered ERα function in the presence of tamoxifen. Further, these SNPs are currently being genotyped so that associations can be made in a population of women receiving tamoxifen as either preventative or adjuvant therapy in which extensive surrogate markers for ERα function were collected during the 1-year follow (hot flash rate, hot flash intensity, lipid profiles, and bone mineral density).  Through the discovery of common polymorphisms affecting Tamoxifen response, we hope to identify new diagnostic criteria to predict which patients will respond to anti-estrogen therapy and which will be better candidates for another line of therapy, such as aromatase inhibitors.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1043.
T cell marker metagene predicts a favourable prognosis in estrogen receptor negative and Her2 positive breast cancers.
Authors: Rody A , Ruckhaeberle E , Holtrich U , Gaetje R , Engels K , Hanker L , Solbach C , Ahr A , Metzler D , Karn T , Kaufmann M
Published: December 2010

Abstract #1048

Background: Lymphocyte infiltration (LI) is often seen in breast cancer and has been suggested as a marker of host antitumor immune response but its importance remains controversial. A positive correlation of Her2 amplification/overexpression and LI has been described which was associated with a more favorable outcome. In rapidly proliferating tumors LI is a good prognostic indicator correlating with lymph node negativity, smaller tumor size, lower grade. However the impact of monocytes, B- and T-lymphocytes on prognosis are still a matter of debate.  Material and Methods: A database of 2110 primary invasive breast cancer samples from 14 microarray datasets was established. Only Affymetrix HG-U133A microarrays were included for full comparability. Feature reduction was achieved by generating metagenes from genes with strong correlation in unsupervised clustering. The relationship of the five major metagenes with different cell types in the sample as well as differentiation programs/pathways associated with specific expression profiles was analyzed.  Results: A large cluster of approximately 600 genes with functions in immune cells was consistently obtained in all datasets. The redundant information from several ProbeSets allowed the construction of robust metagenes which can be used as surrogate markers for the amount of different immune cell types in the breast cancer sample. However, rather complex relationships of these immunological metagenes with standard parameters of the tumors were observed. When different subgroups of tumors were analyzed for disease free survival the IgG metagene as a surrogate marker for B cells had no significant prognostic value. In contrast high expression of the T cell surrogate marker (LCK metagene) was beneficial among all subgroups of ER-negative tumors. Moreover a positive prognostic value of LCK metagene expression was also revealed for those ER-positive tumors with a Her2 overexpression. In addition a trend for a better response to neoadjuvant chemotherapy was detected for those ER negative tumors associated with lymphocyte infiltration as deduced from high expression of both IgG and LCK metagenes.  Conclusions: Tumor associated lymphocytes could represent an anti tumor response but on the other hand they might promote tumor progression by shifting the cytokine milieu toward angiogenic factors, inflammatory cytokines and matrix metallo-proteinases. Thus it is crucial to precisely define the specific subtypes of immune cells which are associated with the tumor. Our results demonstrate that this task can be accomplished by a detailed analysis of the expression of metagenes. These surrogate markers define subgroups of tumors with different prognosis.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1048.
Monocytes/macrophages and natural killer cells characterize the tumor microenvironment of inflammatory breast cancer in Egyptian patients.
Authors: Mohamed MM , El-Shinawi M , Cavallo-Medved D , Sobhy M , Nouh MA , Abdelwahab SF , Sloane BF
Published: December 2010

Abstract #1054

Background: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. IBC incidence appears to be higher in Egypt as compared to USA. Although the role of leukocytes in regulating breast cancer dissemination has been studied extensively, their role in IBC progression is not well understood. Previously, we showed that human monocytes augment the invasiveness and proteolytic activity of IBC cell lines. Aim: To compare the immunophenotype of IBC and non-IBC and to identify IBC tumor-infiltrated immune cell that may be associated with poor prognosis of IBC.  Methods: We enrolled two groups of patients: IBC (n=13) and non-IBC (n=27) being treated at Ain Shams University hospitals. All patients underwent modified radical mastectomy. IBC patients had 7 or more metastatic axillary lymph nodes whereas non-IBC patients had 3 or less metastatic axillary lymph nodes. During surgery, blood draining from the breast tumor microenvironment, through branches of axillary vein, and peripheral blood was collected. Total mononuclear cells were isolated from collected blood and their phenotype was examined using flow cytometry. Monoclonal antibodies specific for cell surface markers for particular cells types were used: namely T-helper cells (CD3+CD4+), T-cytotoxic cells (CD3+CD4-), natural killer (NK) cells (CD56+) and monocytes/macrophages (CD14+). Results: Within each group we compared the leukocytic composition of blood collected from the tumor drainage to that of peripheral blood in IBC and non-IBC patients. 1) In IBC, we detected a significant increase in the mean percentage of NK cells and monocytes collected from the tumor site, but no significant difference in CD8+ and CD4+ cells. 2) In non-IBC, the percentage of CD4+ T cells collected from the tumor site was significantly higher than from peripheral blood. No significant differences were detected in CD8+, CD56+ and CD14+ cells.  We compared the leukocyte composition of blood collected from both IBC and non-IBC patients. 1) In peripheral blood of IBC patients, we detected a significant increase in CD4+ T cells, but no significant differences in CD8+, CD56+ and CD14+ cells when compared to non-IBC patients. 2) When we compared the leukocyte content of blood collected from the tumor site in IBC and non-IBC, we found a significant increase in CD56+, and CD14+ cells and a significant decrease in CD4+ T cells in IBC, but no significant difference in CD8+ T cells. Conclusion: Differences in the immunophenotype of IBC versus non-IBC suggests a role for these immune cells in the metastatic dissemination and progression of IBC.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1054.
β1 integrin mediates tamoxifen resistance in breast cancer cells.
Authors: Pontiggia O , Bal de Kier Joffé E , Simian M
Published: December 2010

Abstract #1055

Even though it is now well established that the microenvironment plays a central role in modulating tumor progression and response to therapy, its function as a regulator of endocrine resistance in breast cancer has not yet been explored. To do so we have established a bicellular in vitro cell line, called LM05-Mix, from the estrogen dependent M05 mouse mammary tumor (BCRT, in press). The epithelial and fibroblastic cell populations of the LM05-Mix cell line were further separated to generate the LM05-E and LM05-F cell lines respectively, both of which are estrogen receptor (ER) and progesterone receptor positive. We have previously shown that treatment with 10-6M 4-OH-tamoxifen (TAM) induces apoptosis of the LM05-E cells, and that this effect is inhibited when the epithelial cells are co-cultured with the fibroblastic cell population. Immunofluorescence staining to the extracellular matrix component fibronectin (FN) was found to be especially associated to the fibroblastic cells in the co-culture. To investigate whether FN conferred TAM resistance, LM05-E cells and human MCF-7 cells were cultured on recombinant FN and treated for 48 hours with TAM. In both cases FN conferred resistance (P≤0.001). To understand what signaling pathways were involved, the experiments were repeated in the presence of specific inhibitors and both PD98059 and LY294002 reverted the protective effect, implicating the participation of both the MAPK/ERK and PI3K/AKT pathways. Next, to corroborate the activation of these pathways by FN, starved LM05-E cells were treated with FN (30 μg/ml). Phosphorylation of FAK, MAPK/ERK1/2 and AKT was detected by western blot between 3 and 15 minutes of treatment. To find out whether the activation of these pathways by FN could lead to ligand independent activation of the ER and thus account for the induction of TAM resistance, immunofluorescence staining was carried out to serine 118-phosphorylated ER. An increase in the phosphorylation of ER at this site was evident at 15 minutes of treatment both in LM05-E and MCF-7 cells. Finally, to confirm that the protective effect was mediated by the binding of β1 integrin to FN, cells were pre-incubated with either a specific β1 blocking antibody (AIIB2) or mouse IgG and seeded on FN. The protective effect of FN over TAM induced cell death was only reverted in the AIIB2 treated group (P≤0.01). Our results show that FN through its binding to β1 integrin confers TAM resistance and suggest that the mechanism involved would be through the modulation of the activity of ER. Targeting the interaction of breast cancer cells with the microenvironment could be a novel and future alternative for patients with breast cancer whose tumors progress to TAM resistance. This work is supported by a grant from the Susan G. Komen for the Cure Foundation to MS (BCTR0600341).

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1055.
The microvasculature of invasive ductal breast carcinoma: a sterological approach.
Authors: Mahon S , Miller N , Curran C , Dockery P , Callagy G , Kerin MJ
Published: December 2010

Abstract #1057

Background:  Microvessel density (MVD) is a commonly used parameter to describe tumour vascularity and has been widely studied as a possible prognosticator in breast cancer. Other descriptors of vascular beds have been developed and have been applied to a variety of systems including the reproductive and nervous systems. These stereological approaches are providing objective unbiased assessment of various vascular beds.  Aim:  The aim of this pilot study was to use stereological methods to describe the vasculature in invasive ductal breast carcinoma, to test the hypothesis that microvasculature anatomy effects Tumor Grade, Size and Nodal Status.  Methods:  A cohort of 51 breast cancer patients with pre-menopausal invasive ductal carcinoma and moderate NPI was identified.Sections were stained with CD34 using the automated Ventana Discovery staining platform. A number of stereological parameters were estimated in three tissue compartments (gland,stroma and adipose):Number per Unit area (Na), Length Density (Lv) and Radial Diffusion Distance. Data was analysed using Analyses of Variance methods using Minitab© Statistical software.  Results:  There was no significant difference in the radial diffusion difference when groups were analysed by Grade Size and Nodal Status. Similar results were found for Na and Lv  Conclusion:  This study suggests that there is no simple link between tumor vasculature and Grade, size and nodal status in Invasive Ductal Breast Carcinoma. This study illustrates the potential usefulness of the stereological approach in the study of vascular beds in breast cancer studies by providing access to potentially physiologically relevant anatomical parameters of microvasculature beds.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1057.
Inhibition of Notch signaling reduces the stem-like cell population in T47D breast cancer cells and prevents mammosphere formation.
Authors: Grudzien P , Strack P , Golde T , Miele L , Foreman KE
Published: December 2010

Abstract #106

Breast cancer has been proposed as a stem cell-derived disorder based on studies identifying cancer stem cells (CSCs) in breast tumors and established cell lines. These self-renewing CSCs are thought to be responsible for breast cancer formation, progression and recurrence; therefore, a deeper understanding of the signaling pathways regulating CSC survival will benefit development of novel therapeutic strategies. Notch signaling, which is known to be dysregulated in breast cancer and has been implicated in mammary stem cell self-renewal, can be effectively blocked by gamma-secretase inhibitors (GSIs). While GSIs are currently in clinical trials for breast cancer, it is unclear how these compounds will affect CSCs or if CSCs from different breast cancer phenotypes (estrogen receptor-alpha (ER)-positive and negative) will be differentially affected. Here, we demonstrate stem-like cells derived from the T47D breast cancer cell line show elevated levels of Notch signaling, with an ER-negative subclone (T47D-C42) having higher intrinsic levels of Notch activity compared to an ER-positive subclone (T47D-A18). Blockade of Notch signaling with three structurally distinct GSIs reduced the number of stem-like cells, and completely abolished secondary mammosphere formation, which is a measure of stem cell self-renewal. Importantly, this effect was irreversible with MRK-003, but not with the two other GSIs. Moreover, our results suggest that the ER-negative cell line was more sensitive to the GSIs compared to the ER-positive line. This is consistent with our recent report that loss of estrogen signals results in Notch activation. These novel findings support a role for Notch signaling in CSC self-renewal and proliferation, and they suggest Notch inhibition may have clinical benefits in targeting CSCs.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 106.
Brain metastases (BM) in patients presenting with HER-2 amplified advanced breast cancer (ABC).
Authors: Sweldens C , Huget P , Weytjens R , Prove A , Vermeulen P , Van Dam P , Dirix L
Published: December 2010

Abstract #1060

Background : The HER-2 amplified breast cancer phenotype is suggested to have an increased propensity for CNS metastasis. The introduction of trastuzumab might be related to this association. In order to elucidate this relationship we analyzed our prospectively collected database of all patients presenting with metastatic HER-2 amplified breast cancer in the time frame between Jan 2002 until Dec 2004.  Material and Methods : The AZ-Augustinus Breast Cancer Tissue Bank was initiated in 1999. It ensures the prospective entry of all clinical and pathological features of patients presenting in our Breast Clinic clinical database. The data entry is performed by dedicated Datamanagers and is linked to the Biological Repository Collection (tissue, serum, plasma, bone marrow). Patients presenting with primary metastatic, HER-2 amplified ( FISH +) between 2002 and 2004 were analyzed for this study. Since Jan 2002, all patients presenting with metastatic disease underwent a routine CT-scan of the brain as initial staging procedure.  Results : A total of 33 patients presented with HER-2 +, unpretreated metastatic breast cancer. Mean age: 53 years (range 30-69). Sites of metastases at diagnosis ( % of patients) : liver: 71 %, lung: 36 %, bone: 86 %, brain (BM): 15 % (5), lymph nodes and others: 20%. In total 5/33 (15%)  presented with brain only metastasis. During the follow-up period 17 more patients developed BM, amounting to 22/33 (67%). The median time between diagnosis of BM after diagnosis of ABC was 22 months (0 – 98). Mean time to developing BM was 18 months. In total 62 lesions in the CNS were counted on MRI in these 22 patients. 23 lesions (38%) had a cerebellar localisation.  Conclusions: Assuming that the presentation with metastasis is a reflection of the unperturbed biology of disease, our data suggest that the CNS is not an exceptionally preferred site for metastasis in untreated HER-2 + advanced breast cancer. A preference for a cerebellar localisation is suggested.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1060.