bluuebunny/crossref_metadata_2025 huggingface dataset:
Mammary Tumors in STAT1-Deficient Mice Recapitulate Characteristics of Human Luminal Breast Cancers.
Authors: Chan S. , Schreiber R.
Published: December 2010

Human breast cancer of the luminal subtype represents approximately 70% of all newly diagnosed breast cancers. This particular subtype of tumors are estrogen receptor α (ERα)-positive and/or progesterone receptor (PR)-positive. However, 50% of these patients do not derive benefit from endocrine therapy. One obstacle in the study of hormone receptor-positive breast cancer is that there is currently a deficit in murine models that can fully recapitulate the biology of this highly prevalent breast cancer subtype. Here we show that mice lacking the transcription factor signal transducers and activators of transcription 1 (STAT1) spontaneously developed mammary gland adenocarcinomas that showed remarkable similarities to human luminal breast cancer at histopathological, biological and molecular levels. More than 90% of the tumor cells in both early and advanced lesions were ERα+ and/or PR+. STAT1-/- mammary tumor cells failed to grow in ovariectomized mice, demonstrating that these tumors are addicted to ovarian hormones for proliferation. Supplementing with exogenous estrogen was sufficient to rescue tumor growth in ovariectomized mice. STAT1 functions, at least in part, as an intrinsic tumor suppressor since reconstitution of STAT1 expression in STAT1-/- mammary tumor cells induced spontaneous apoptosis. Induction of cell death required the critical tyrosine residue at position 701, suggesting that the transcriptional function of STAT1 is involved in tumor suppression.In contrast to the anti-oncogenic effect of STAT1, STAT3 was constitutively phosphorylated by JAK2 in the STAT1-/- mammary tumor cells. Inhibition of JAK2 or STAT3 activation resulted in tumor cell death, indicating that STAT3 promotes tumor cell survival. Gene expression profiles of the STAT1-/- mammary tumors showed significant overlaps with that of the human luminal breast cancers, strongly supporting that the STAT1-/- mammary tumors recapitulate the molecular signatures of human luminal breast cancers. Consistent with the results that STAT1 can act as an intrinsic tumor suppressor, approximately 10% of the human ERα+ breast cancer cases exhibit either undetectable or very low level of STAT1 in the mammary epithelial compartment. This study uncovers a unique role of STAT1 in suppressing mammary gland tumorigenesis. Importantly, this physiologically relevant model of human luminal breast cancer will provide an exceptional tool for the development and evaluation of novel therapeutics.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 65.
Targeting Aldose Reductase: A Novel Strategy in Treating Endocrine Resistance Using Combination Therapy.
Authors: Tekmal R. , Nair H. , Huffman S. , Kirma N. , Rao P. , Evans D. , Ramana K. , Srivastava S.
Published: December 2010

Breast cancer is the most commonly diagnosed form of cancer in women. Among breast cancer patients about 2/3 are initially hormone sensitive or estrogen receptor (ER) positive and respond to endocrine therapy. Aromatase inhibitors (AI's) are superior class of hormonal therapeutic agents effectively control ER positive breast cancer in postmenopausal women. Acquired resistance to AI's is expected to be an emerging serious problem in clinics and recent studies have shown that tumors use adaptive signaling mechanisms to overcome AI sensitivity. Thus there is an urgent need for newer treatment modalities. Combination of endocrine and non endocrine agents that block these signaling pathways may prevent or delay the adaptive mechanism and thereby onset of resistance to hormonal therapy. In our study we have found that Fidarestat, an aldose reductase (enzyme which catalyzes the rate limiting step of glucose to fructose or sorbitol formation in polyol pathway) inhibitor effectively re-sensitize letrozole resistant LTLT-Ca breast cancer cells to letrozole. 1µM of fidarestat + 1uM letrozole was found very effective in inducing maximum cell death in LTLT-Ca cells when compared to fidarestat alone. The combination treatment not only restored ER-α levels but also down regulated HER2/MAPK signaling proteins. Aldose reductase siRNA (100nM)- treated MCF-7/Aro and MCF-7 cells upregulated ER-α in western blot and ER-functionality assays. On the other hand in aldose reductase-siRNA- treated LTLT-Ca cells, ER-α levels were down- regulated as in fidarestat treatment. Pretreatment of LTLT-Ca with fidarestat for one week showed reduced proliferation of cells and the effect was maintained until four passages with 1µM letrozole alone. Fidarestat treatment up-regulated E2-mediated transcription in LTLT-Ca cells. In order to enhance the efficacy and targeted delivery of fidarestat in LTLT–Ca cells we have used a nanoparticle-based therapeutic formulation. Folate receptor, highly expressed on epithelial carcinomas, could be a potential molecular target for tumor selective drug delivery. Physcio-chemically well characterized Fidarestat–folate nanoparticles (FFNP's) were prepared to increase the tumor selective intracellular delivery. FFNP's were found superior in exerting cytotoxicity when compared to fidarestat alone. Combination therapy was equally effective in controlling LTLT-Ca cell growth using xenograft model. Taken together, the increased glucose metabolism in LTLT-Ca cells may be critically contributing to chemotherapeutic resistance by increasing drug metabolism and decreasing uptake. Hence targeting aldose reductase in endocrine resistance may be attractive alternative to increase the sensitivity of hormonal therapy.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 67.
Central Review of ER, PgR and HER2 in BIG 1-98 Evaluating Letrozole vs. Letrozole Followed by Tamoxifen vs. Tamoxifen Followed by Letrozole as Adjuvant Endocrine Therapy for Postmenopausal Women with Hormone Receptor-Positive Breast Cancer.
Authors: Viale G. , Regan M. , Dell'Orto P. , Mastropasqua M. , Rasmussen B. , MacGrogan G. , Braye S. , Orosz Z. , Giobbie-Hurder A. , Neven P. , Knox F. , Oehlschlegel C. , Thuerlimann B. , Coates A. , Goldhirsch A.
Published: December 2010

Background: The BIG 1-98 trial, a large international Phase III study, evaluated letrozole (Let) for 5 years (n=1546) vs. Let for 2 years followed by tamoxifen (Tam) for 3 years (L→T; n=1540) and vs. Tam for 2 years followed by Let for 3 years (T→L; n=1548) as initial adjuvant endocrine therapy for postmenopausal women with hormone receptor-positive breast cancer. Patients with HER2-positive disease did not receive trastuzumab. Efficacy results were presented in SABCS 2008 and showed that neither of the sequential therapy arms significantly improved disease-free survival (DFS) compared with letrozole alone. Centrally-assessed ER, PgR and HER2 have now been evaluated as potential predictive factors for treatment selection.Methods: Tumor blocks for 3975 of the 4634 (86%) patients on these three treatment arms were centrally collected and assessed by the IBCSG Central Pathology Laboratory. The tumors were evaluated for ER and PgR content by immunohistochemistry (IHC), and for HER2 immunoreactivity by IHC and by FISH for IHC 1+ or 2+ tumors. DFS according to centrally-assessed ER, PgR and HER2 status was analyzed using multivariable Cox modeling. STEPP methodology was used to explore patterns of DFS differences according to quantitative values of receptor levels (% staining cells).Results: Upon central review, 3885 tumors were confirmed to express ER and are the focus of this analysis. PgR expression did not predict differential treatment effects among the 3 treatment arms. With only 240 (6.2%) HER2-positive tumors, there was a suggestion of differential treatment benefit relative to HER2 status, in particular of a benefit of Let for 5 yrs over Tam→Let in HER2-positive tumors.Discussion: Given the caveat of a low prevalence of HER2-positive tumors and absence of HER2-directed therapy in the BIG 1-98 trial population, there is a suggestion that Let for 5 years may be a better option than a sequence of Tam and Let among women with HER2-positive tumors. Thus it may be better to initiate and continue treatment of endocrine-responsive, HER2+ tumors with Let rather than a sequence of Tam and Let.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 76.
Joint Disorders Frequency and Structural Changes during Anastrozole Adjuvant Treatment in Early Breast Cancer: A Prospective Trial (D5392L00013).
Authors: Delozier T. , Daban A. , Dieras V. , Mauriac L. , Gligorov J. , Tubiana-Hulin M. , Goldwasser F. , Briot K. , Roux C. , Amrate A. , Guastalla J.
Published: December 2010

Background:Aromatase inhibitors (AIs) are standard adjuvant endocrine-therapy in postmenopausal women with hormone-sensitive breast cancer. However, main side effects reported are musculoskeletal symptoms. The pathogenic and anatomic features of AI-induced arthralgia have not been clearly defined. The objective of the study was to describe the joint symptoms and structural joint changes in women received adjuvant anastrozoleMethods: Postmenopausal women with hormone receptor positive early breast cancer were enrolled in this open, multicentre trial. Anastrozole was administrated 1mg/day p.o. All of them were naïve of AI and tamoxifen treatment. Patients were followed one year with clinical examination every 3 months, ultrasound (US) examination every 6 months, measurements of inflammation biomarkers and cartilage biomarkers of degradation U-CTX-II (urinary C-terminal crosslinked telopeptide of type II collagen) every 6 months and X-rays at baseline and 12 monthsResults: From June 2006 to December 2007, one hundred and fourteen patients (114) were included and 106, with a median age of 61.5 (49-79) years, were followed-up for 1 year.Overall, 33% of patients were previously treated by chemotherapy, (anthracyclines 97%, taxanes 63 %) and 40.6% were receiving hormone replacement therapy (HRT) at tumour diagnosis. The prevalences of arthralgia were 40.6, 59.1 and 60.6 % at baseline, 6 months and 12 months respectively.Among the 63 (59.4%) patients without arthralgia at baseline, 37 patients presented with arthralgia during treatment (26 between baseline and M6, 11 between M6 and M12) with median time to onset of 4 months.In univariate logistic analysis, significant determinant factors of joint disorders at 12 months were previous osteoarthritis and personal history of arthralgia but not the previous HRT or previous adjuvant chemotherapy with taxanes. In multivariate analysis, none of those factors were significant.Clinical examination by rheumatologist and US examination did not show any significant changes of the number of synovitis and tenosynovitis over 12 months.There were no significant changes of the U-CTX-II levels and of joint damages assessed by radiographs. Inflammation biomarkers did not significantly change over 12 months.Conclusion: This prospective study with a systematic rheumatologist follow up of the patients showed that arthralgia occurs early after the beginning of anastrozole therapy (first 6 months); and suggest that joint disorders were not associated with any cartilage degradation after 1 year of treatment.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 801.
Protective Effect of Estrogen (E2) and Increased Risk of E2 Plus Progestin (Prog) on Breast Cancer (BrCa). The 2009 Review of the Women's Health Initiative (WHI) Hormone Replacement Therapy (HRT) Published Trials.
Authors: Ragaz J. , Le N. , Budlovsky J. , Spinelli J.
Published: December 2010

INTRODUCTION & METHODOLOGY.The objective of this review is to identify from the two WHI published randomized HRT trials the BrCa risk or benefit, with a special focus on different impact of E2-alone vs E2+Prog on human breast carcinogenesis (BrCa-genesis).In the 1st trial (JAMA 2002, 2007), healthy women age 50 - 79 were randomized to E2 plus Prog versus placebo (Plac). Reviewed were Hazard ratios (HRs) for all pts and also for those with or without prior Hormone use. Mean follow up was 5.2 years.In the 2nd trial (JAMA 2004, 2006), women with hysterectomy age 50 - 79 were randomized to E2 alone (without Prog) versus Plac. Mean follow up was 7.1 years. Reviewed were all pts, and also subsets with or without prior E2 or Prog; with or without prior history of Benign Breast Disease (PHBrD); and with or without prior history of a 1st degree relative with BrCa (PH1stRelBrCa).RESULTS.Invasive BrCa incidence:N (%)HR95% C.I.1st TrialAll participants:16,608 (100%)1.260.83 - 1.97No prior Hormone use:12,304 (74.1%)1.060.81 - 1.38Prior Hormone use <5 yrs:3,005 (18.1%)2.131.15 - 3.94*Prior Hormone use 5 - 10 yrs:783 (4.7%)4.611.01 - 21.02*2nd TrialAll participants:10,739 (100%)0.800.62 - 1.04No prior hormone use:5,763 (53.7%)0.650.46 - 0.92*Prior E2 use:4,976 (46.3%)0.980.67 - 1.44Prior E2 + PROG use:468 (4.4%)2.350.60 - 9.14No PHBrD:7,681 (71.5%)0.570.41 - 0.78*No PH1stRelBrCa8,554 (80.0%)0.680.50 - 0.92*CONCLUSION:1. Results from the 1st trial show that concomitant use of E2 with Prog, particularly in women with prior Hormone use, is associated with increased BrCa hazards.2. The results of the 2nd HRT trial indicate that use of HRT with E2 alone is associated with a reduction in BrCa incidence, significant in the majority of women with no prior BrCa risk factors. Also the results show that prior Prog but not prior E2 elevate the risk.3. These data indicate that HRT with E2 alone has a BrCa protective effect, while adding Prog increases the risk.4. These results are also compatible with a dual E2 effect on BrCa-genesis, where exogenous E2 are protective, but endogenous E2 [endE2] are carcinogenic, as shown from decades of anti-end E2 research with Tamoxifen, oophorectomy or Aromatase inhibitors.5. Based on these data we propose that in women with hysterectomy and no prior history of risk factors, the use of HRT with E2 alone as chemoprevention is plausible, and needs urgent verification.* significance, "p" <0.05.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 908.
p16 Expression and Breast Cancer Risk in Women with Atypical Hyperplasia.
Authors: Santisteban M. , Berman H. , Reynolds C. , Gauthier M. , Frost M. , Vierkant R. , Pankratz S. , Anderson S. , Tlsty T. , Hartmann L.
Published: December 2010

Background: p16 is a nuclear protein encoded by the p16INK4a gene that regulates the G1-S cell cycle checkpoint. Its overexpression could reflect normal proliferative arrest, but also the evolution of carcinogenic processes that have overcome the p16 block, with progression from hyperplasic lesions to invasive breast cancer. Our aim with this study was to correlate p16 expression, both individually and in combination with expression of COX-2 and Ki67, with subsequent risk of breast cancer among women with atypical hyperplasia. Methods: p16 expression was assessed by immunohistochemical assays in archival paraffin-embedded, formalin-fixed sections from 233 women with atypia whose biopsy specimens were obtained via surgical excision at the Mayo Clinic from January 1, 1967, through December 31, 1991. p16 expression was scored as percent of cells positive and intensity of staining. Risk factor information and follow-up for breast cancer events were obtained via study questionnaire and the medical records. Standardized incidence ratios (SIRs) were used to compare observed numbers of breast cancer events to population-based expected counts, both at 10 years and over the entire course of follow-up. Results: Forty-seven patients (20%) developed breast cancer with a mean follow-up of 14.3 (SD=6.96) years. The overall risk of developing breast cancer was not modified by increasing p16 overexpression (low percent of staining with RR=3.89, 95% CI=2.72 to 5.56; high percent of staining with RR=3.91, 95% CI=2.43 to 6.28; similar results for intensity of staining). However, overexpression of both p16 and COX2 conveyed a stronger risk of breast cancer (SIR=4.01, 95% CI 2.28 to 7.07) as compared to dual low expression (SIR 1.78, 95% CI 0.67-4.74). Moreover, overexpression of both Ki67 and p16 was related to a higher 10-year breast cancer risk (SIR 6.25, 95% CI 2.35-16.6) as compared to low expression of both markers (SIR 0.87, 95% CI 0.22-3.49). Conclusions: although single expression of p16 does not stratify breast cancer risk, its overexpression in concert with biomarkers that reflect other carcinogenic processes (e.g. Ki67 and COX2) could help to define risk of tumor development.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 910.
Results of Breast Conservation Therapy of DCIS without Radiation: Comparison to Mastectomy in a Prospective Study.
Authors: Decker T. , Kettritz U. , Obenaus R. , Morack G. , Jacob R.
Published: December 2010

Aim:To assess the long-term outcome for women with DCIS of the breast treated in clinical practice by breast conservation surgery (BCS) without definitive breast irradiation in comparison to those treated by mastectomy (ME).Patients and MethodsWe studied 534 (of 599) patients with DCIS treated between 1992 and 2003 in a German regional cancer center. 287 patients underwent BCS without adjuvant radiotherapy whereas 247 patients were treated by ME. The decision was made after radiologic-pathological correlation and based on results of standardized pathology work-up including radiograms of whole specimen and slices. The decision was based on margins in 6 directions and maximum size of DCIS. In all cases with margins <10 mm a re-resection was performed, all patients with lesions >40 mm with margins <10 mm were advised to ME. All patients who decided for ME although their lesions did not show these criteria as all patients who could not followed were excluded. The median follow-up for the study population was 82 months.ResultsDuring the follow-up period 8/287 (2.8 %) patients of the BCS group and 10/247 (4.0 %) patients of the ME showed local recurrences. 2/8 (25 %) and 5/10 (50 %) of the recurrences were invasive. Neither regional nor systemic metastases were found, no patient died. Regarding these events there was no statistical significant difference between both groups.The BCS and ME group differed significantly in maximum diameters of DCIS (median 23 mm vs. 64 mm; p<0.001). There was no significant difference in nuclear grade distribution (BCS: 52 (18 %) grade 1, 115 (40 %) grade 2, 120 (42 %) grade 3; ME: 54 (22 %) grade 1, 99 (40 %) grade 2, 94 (38 %) grade 3).ConclusionsOur results support that under the precondition of margins >10mm, patients treated with BCS without radiotherapy can expect very low local recurrence risks similar to those treated with ME. This study continues to support the incremental benefit of margin width on recurrence under the precondition of standardised readilogic and pathological work-up of the specimens.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 953.
Outcome of Active Surveillance and Endocrine Therapy Alone for DCIS.
Authors: Meyerson A. , Joe B. , Wolverton D. , Lessing J. , Hwang E.
Published: December 2010

Objective: There are currently no nonsurgical options offered to patients with DCIS. The purpose of this study was to assess clinical outcome in a cohort of women declining surgery for pathologically proven ductal carcinoma in situ (DCIS) who have opted for endocrine therapy and clinical/radiographic follow-up.Methods: Between 2003 and 2008, 52 women with ER-positive DCIS enrolled in an IRB-approved single-arm study of 3 months of neoadjuvant enodocrine therapy. The study design included definitive surgical excision. However, 9 women withdrew from the parent study at completion of 3 months and declined surgery, opting instead for a plan of active surveillance, with the intent to proceed with conventional therapy in the event of disease progression or radiographic suspicion for invasive disease. All women were amenable for follow up every 6 months with diagnostic mammography and breast MRI, and wished to continue endocrine therapy, despite unproven benefit in this setting. Endocrine treatment consisted of tamoxifen for premenopausal women or an aromatase inhibitor for postmenopausal women. Each patient was informed of the current treatment guidelines for DCIS, as well as the increased future probability for invasive cancer in the absence of standard treatment for DCIS.Results: The median age of the cohort was 48 years (41-60 years) at enrollment. Of these, 5 women were premenopausal on Tamoxifen, and 4 were post menopausal (3 on letrozole and 1 on arimidex). At the time of analysis, median follow up was 23 months (range 9-66 months). During follow up, 4 patients proceeded with surgery either electively (1 patient) or due to changes at imaging (3 patients). Two of these patients had node-negative invasive breast carcinoma identified at surgery, measuring 6 mm and 4 mm. Five patients have elected to continue surveillance (median follow up 34 months; range 10-66 months), and have had either stable disease or continued regression based on both mammographic and MRI criteria. Four patients remain on endocrine therapy. The patient with longest follow up has had almost complete resolution of radiographic abnormalities at 6 years of surveillance.Conclusion: In this pilot cohort, two-thirds of women who continued endocrine therapy and surveillance for ER-positive DCIS had radiographically stable disease or regression with short-term follow up. Women who initially declined surgery elected to undergo excision in the face of radiographic progression. Active surveillance with endocrine therapy for ER-positive DCIS is feasible, and a well-informed patient cohort is essential to minimize risk. The impact of such an approach on long-term morbidity is unknown, but requires further investigation. Ongoing efforts to improve the detection of invasive cancer in women with DCIS are expected to facilitate such an approach.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 954.
Outcomes Following Accelerated Partial Breast Irradiation in ASTRO Consensus Statement Unsuitable Patients.
Authors: McHaffie D. , Patel R. , Das R. , Cannon G.
Published: December 2010

Purpose/Objective: There are limited data to guide patient selection for accelerated partial breast irradiation (APBI). In the recent ASTRO consensus statement, an unsuitable group was defined for whom APBI should only be offered in the context of a clinical trial. In this analysis, we reviewed the outcomes in this cohort of potentially higher risk patients.Materials/Methods: Between 2/2001 and 6/2006, a total of 322 consecutive patients were treated with HDR APBI at the University of Wisconsin. A total of 107 “unsuitable” patients were identified: 80 pts (74.7%) age < 50, 19 pts (17.8%) node positive, 20 pts (18.7%) with LVSI, 3 pts (2.8%) with tumors > 3 cm, and 2 pts (1.9%) with multifocal disease. 17 pts (15.9%) had multiple unsuitable features. In addition, 53 pts (49.5%) possessed “cautionary” features, including 19 pts (17.8%) ER negative and 10 pts (9.3%) with pure DCIS. The median age at diagnosis was 47 (range 25 – 84). All patients received 32 – 34 Gy in 8 - 10 bid fractions using multicatheter (93.5%) or Mammosite balloon (6.5%) HDR brachytherapy.Results: With a median follow-up of 60 months (range 1.1 - 97.8 months), there were 8 loco-regional recurrences (3 local only, 2 regional and 3 loco-regional and distant). Four of 5 patients without distant disease were successfully salvaged and had no evidence of disease at last follow-up. The 6 yr actuarial loco-regional control rate is 91.3% +/- 5.9%, with a latency to recurrence ranging from 10 – 41 months. The 6 yr disease free survival for the entire cohort is 89%, with a cause-specific survival and overall survival of 92.5% and 89%, respectively. There were no loco-regional recurrences observed among those with age < 50 without other unsuitable (LVSI, node +, > 3 cm, multifocality) or cautionary (ER -, EIC, tumor 2 – 3 cm, lobular histology, DCIS, < 2 mm margins) features (n = 31). In comparison, the loco-regional recurrence rate is 12.3% for those deemed unsuitable by virtue of surgical and pathologic factors (p = .064). Loco-regional recurrences were observed in 2/19 node positive pts, 3/20 pts with LVSI, 1/2 pts with multifocal disease, 2/34 with age < 50 alone and cautionary features, and 0/3 pts with tumors > 3 cm.Conclusions: Currently, uncertainty exists regarding patient selection for APBI. Based upon the observed local recurrence rates in the current analysis and the well-established benefits of whole breast radiotherapy, we continue to encourage clinical trial participation among this cohort of patients, specifically RTOG 0412/NSABP B-39. Although not statistically significant, the absence of local failures among women < 50 yrs without adverse histopathologic features suggests they may be appropriate candidates for APBI.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 955.
Breast Cancer Stem Cells and the Epithelial-Mesenchymal Transition.
Authors: Weinberg R.
Published: December 2010

The acquisition of highly malignant traits by carcinoma cells is often associated with the activation of a transdifferentiation program termed the epithelial-mesenchymal transition (EMT). The EMT program is deployed in a variety of steps of embryonic morphogenesis, enabling the interconversion of epithelial and mesenchymal cell types. When both normal and neoplastic epithelial cells undergo an EMT, they acquire addition changes in addition to expressing characteristic mesenchymal cell markers. In particular, they acquire motility, an increased resistance to apoptosis and, in the case of carcinoma cells, invasiveness. The motility and invasiveness components associated with the EMT would appear to empower many types of cancer cells to metastasize. An interesting question is how the resulting disseminated cancer cells are able to spawn a macroscopic tumor mass at distant anatomical sites, since this clonal expansion would appear to require an ability to self-renew. Recently, we have examined the cellular products of an EMT. Contrary to our expectations, the resulting cells acquired many of the attributes of stem cells, including the expression of stem cell-associated cell-surface markers. Conversely, mammary epithelial cells that naturally express these markers also express greatly elevated levels of at least four pleiotropically acting transcription factors that have been found able to induce an EMT. More recent studies have confirmed that the product of an EMT in mouse mammary epithelial cells is a cell that has a greatly increased ability to generate an entire mammary ductal epithelial tree. Moreover, we find that cells with the attributes of stem cells exist naturally as minor subpopulations in cultured human mammary epithelial cells. These cells can differentiate spontaneously into non-stem cells and, conversely, the non-stem cells appear capable of dedifferentiating spontaneously into cells with progenitor or stem-cell phenotypes. Hence, the study of certain aspects of mammary epithelial cell biology, including the biology of mammary carcinoma stem cells, may be feasible with cultured cells.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr A1-1.
Membrane ER in Endocrine Resistance in the Lab and the Clinic: Should We Be Convinced?
Authors: Dowsett M.
Published: December 2010

There is clear evidence, including from our own lab, that in vitro some ER-alpha localises to the plasma membrane in MCF7 cells that acquire resistance to tamoxifen (TamR cells) in contrast to the nuclear localisation in wild-type cells. Immunoprecipitation shows co-localisation of ER and HER2 with immunohistochemical studies unequivocally demonstrating the latter being exclusively membrane bound. Similar membrane localisation of ER-alpha is demonstrable in BT474 cells which are highly HER2-amplified. Clinical samples prepared after formalin-fixation and paraffin-embedding however do not show specific membrane staining with a variety of anti-ER-alpha antibodies, irrespective of whether these tumours are HER2 positive or negative or whether they have acquired tamoxifen resitance, including a small number of samples where this resistance is associated with a gain of HER2 expression. We have found that formalin-fixation of TamR cells leads to loss of ER-alpha membrane staining and that this might be considered a potential explanation for the discrepancy. However, a more compelling explanation is seen in the absence of membrane staining when these cells are grown in 3D culture, despite their identical fixation to those in 2D culture. The clinical and biological response of breast cancer to estrogen deprivation correlates well with nuclear levels of ER. We are currently assessing whether unique gene expression patterns seen with estrogen dendramer conjugates (kindly provided by John and Benita Katzenellenbogen) that cannot enter the nucleus of MCF7 cells correlate with gene expression changes seen in ER-alpha positive breast carcinomas after estrogen deprivation. While there is strong evidence for membrane localisation of ER-alpha of some breast cancer cells in some culture conditions no convincing evidence has yet been gathered for its occurrence in human breast carcinomas.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr BS2-2.
Cancer Immunoediting: Immunologic Control and Sculpting of Cancer.
Authors: Schreiber R.
Published: December 2010

Cancer Immunoediting is the process by which the immune system controls and shapes cancer. We originally envisaged that cancer immunoediting would occur in three phases: Elimination (also known as cancer immunosurveillance, the host protective phase of the process), Equilibrium (the phase in which tumor cells that survive immune elimination remain under immunologic growth control resulting in a state of functional tumor dormancy) and Escape (the phase where clinically apparent tumors emerge because immune sculpting of the tumor cells has produced variants that display either reduced immunogenicity or enhanced immunosuppressive activity). Strong experimental data has now been obtained using mouse models of cancer to demonstrate the existence of each phase of the cancer immunoediting process and compelling clinical data suggests that a similar process may also occur during the evolution of certain types of human cancer. Our efforts now focus on elucidating the molecular and cellular mechanisms that underlie each phase of cancer immunoediting and identifying the critical checkpoints that regulate progression from one phase of the process to the next. This approach has led to the discovery of a new mouse cancer model that shows remarkable developmental, molecular and functional similarity to human estrogen receptor alpha positive (ERα+) luminal breast cancer. We have found that the cancers in our mice arise as a result of dysfunctional JAK-STAT pathway signaling and, interestingly, this observation has led to the discovery of a heretofore-unrecognized similar phenotype in a subset of human ERα+ luminal breast cancers. The therapeutic implications of our work will be discussed.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr BS3-1.
Epigenetic Therapy.
Authors: Issa J.
Published: December 2010

Epigenetic changes such as alterations in DNA methylation and histone modifications have been well described in various malignancies and are now recognized as targets of therapy (epigenetic therapy). The aim of epigenetic therapy is to reverse epigenetic changes and reactivate important genes thereby modifying the malignant phenotype and inducing clearance of the malignant clone by various mechanisms. Epigenetic modifying agents may also have mechanisms of anticancer action unrelated to gene reactivation. The hypomethylating drugs azacitidine and decitabine induce clinically meaningful remissions or improvements in 30-60% of patients with myeloid leukemias and prolong survival compared to standard approaches including chemotherapy. Both drugs been approved in the USA for the treatment of advanced and/or symptomatic MDS. Their clinical activity in other malignancies including breast cancer is poorly defined because published studies used drug doses and schedules of administration that are not optimal for epigenetic activation. New clinical trials are addressing their potential role in solid tumors. Histone deacetylase inhibitors belong to another class of epigenetic modifying agents. Clinically, they have impressive single agent activity in lymphoid malignancies, but have been disappointing so far in other cancers. However, they are synergistic with many cytotoxic or targeted agents, and their ultimate clinical role may well lay in combination studies. Current studies are focusing on identifying drugs that target additional epigenetic pathways such as the polycomb group proteins, as well as combinations of drugs to affect multiple epigenetic pathways simultaneously.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr ES4-3.
Clinical Trials 102: New Strategies in Targeted Therapy Clinical Trials.
Authors: Hilsenbeck S. , Piccart M.
Published: December 2010

The practice of clinical oncology is guided by randomized phase III clinical trials that are powered to detect average benefit of a therapeutic intervention when compared to standard treatment in large group of breast cancer patients. Although such “evidence-based” practice has moved clinical oncology from empiricism to scientific rigor, it is difficult for oncologists to apply these standards and predict whether a particular treatment will be effective for an individual patient. Recent advances in molecular profiling have begun to unravel the biological variations that underlie individual susceptibility, disease behavior, and response to therapy. Our improved understanding of the biological basis of breast cancer has coincided with the development of rationally designed “targeted therapies” that modulate specific oncogenic signaling pathways. The clinical development of trastuzumab, a monoclonal antibody against the HER2 receptor, illustrates how a targeted therapy that is associated with a well-defined predictive marker can produce dramatic gains in clinical effectiveness. In contrast, bevacizumab, a monoclonal antibody against the vascular endothelial growth factor, has shown promising activity in advanced breast cancer, although its clinical development has been hindered by the lack of a clear predictive marker that can be used to guide therapy. In the future, the clinical testing of targeted therapies must be linked with biomarker discovery and validation at earlier stages of clinical development so that patients likely to benefit can be identified. In addition, the traditional approach of conducting early development trials in the second-line metastatic setting where relevant tissue for biomarker development is often not available, and where resistance may have developed, also hampers targeted therapy development. Neoadjuvant and "window of opportunity" studies coupled with biopsy of the primary tumor, offer possible solutions, and are being used more oftern. The Breast International Group (BIG) will launch a program of randomized phase II neo-adjuvant clinical trials (NeoBIG) to accelerate drug and biomarker development. These studies will integrate emerging technologies, such as genomics, proteomics, metabolomics, pharmacogenetics and functional imaging, to identify predictive markers of efficacy that can be validated in subsequent adjuvant registration trials.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr ES5-1.
Obesity, Insulin Resistance and Insulin.
Authors: Goodwin P.
Published: December 2010

There is growing acceptance that overweight and obesity are associated with poor outcomes in early stage breast cancer. This effect has been reported in pre-and postmenopausal women, regardless of hormone receptor status. Postulated mechanisms have included presentation of cancer at a more advanced stage, underdosing of chemotherapy and higher levels of endogenous estrogens in overweight and obese women. Recent research has provided strong evidence that insulin may play a key role in mediating these prognostic effects. Overweight and obesity are associated with insulin resistance (and resulting hyperinsulinemia) in both the general population and in breast cancer patients; both insulin resistance and high insulin levels have been associated with increased risk of breast cancer recurrence or death, independent of traditional prognostic factors. Breast cancer cells commonly overexpress insulin receptors (frequently a fetal form of the receptor that may hybridize with the IGF-1 receptor), providing a biologic basis for a prognostic effect of insulin. These receptors are not downregulated by circulating insulin; as a result, cancer cell growth may be stimulated by high circulating insulin levels, leading to poor prognosis. Preclinical work provides strong evidence for a mitogenic role of insulin in breast cancer. Taken together, these observations have led to the development of lifestyle interventions targeting insulin and/or insulin resistance in breast cancer patients. Interventions include weight loss (which reduces insulin levels in the general population) and physical activity (which has been shown to reduce insulin in breast cancer patients). Randomized trials of weight loss are ongoing and planned. One completed trial (Women's Intervention Nutrition Study, J Natl Cancer Inst 2006;98:1767-1776) reported improved relapse free survival in breast cancer patients randomized to a reduced fat diet (that was associated with significant weight loss). Effects were greatest in hormone receptor negative breast cancer. Recent interest has focused on the use of metformin, a biguanide commonly used to treat type 2 diabetes. Metformin reduces insulin resistance and lowers insulin levels in many populations, including early stage breast cancer patients. In addition to this indirect (insulin lowering) effect, metformin may also exert direct anti-tumor effects via mTOR inhibition. Studies using metformin in the neoadjuvant setting in breast cancer are ongoing. The effect of metformin on breast cancer outcomes (with embedded correlative studies that will examine direct and indirect effects of the drug) will be examined in NCIC MA.32, a randomized trial of metformin versus placebo involving 3582 women with early breast cancer that will be activated in early 2010. Together, these lifestyle and pharmacologic studies will provide important information on the effect of modification of a key host factor (obesity with associated hyperinsulinemia) on breast cancer outcomes.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr MS2-1.
The Development of PI3K Pathway Modulators for Cancer.
Authors: Sellers W.
Published: December 2010

The phosphoinositide-3 kinase pathway is a key oncogenic pathway in breast cancer where direct mutation of PIK3CA, loss-of-function of PTEN and amplification of HER2 all result in constitutive pathway activation. The pathway, under physiologic control, integrates extracellular growth factors signals with nutrient and other environmental conditions such as hypoxia to enact directed changes in cell proliferation, survival, migration and the regulation of key metabolic pathways such as glucose homeostasis. In cancer the constitutive activation of the pathway leads to the untoward induction of proliferation and cell survival. In addition, pathway function is likely required for hypoxia-mediated induction of angiogenesis. These observations/discoveries have motivated and set the stage for the development of therapeutic molecules targeting key pathway components. The progression of allosteric mTOR inhibitors, catalytic mTOR inhibitors, dual mTOR/PI3K inhibitors and selective PI3K inhibitors in the clinic heralds the opportunity to answer critical questions regarding the role of this pathway in breast cancer. The data emerging from the pre-clinical and clinical development of a series of distinct pathway inhibitors targeting mTOR and PI3K will be discussed with a focus on understanding pathway dependence pre-clinically and in humans.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr P2-1.
Quantitative intra-operative RT-PCR assay predicts size of sentinel node metastases.
Authors: Blumencranz P , Pieretti M , Asad H , Zanchi A
Published: December 2010

Abstract #1002

The GeneSearch™ Breast Lymph Node (BLN) Assay (Veridex, LLC) has been FDA approved to detect metastases > 0.2 mm in the sentinel lymph nodes (SLNs) of breast cancer patients and has been in clinical use in our institute for 11 months. This assay gives a qualitative (negative/positive) result by applying cutoff values to the cycle times (Ct) of the two mRNA markers analyzed: mammaglobin (MG) (cutoff value = 31 Cts) and cytokeratin 19 (CK19) (cutoff value = 30 Cts). This is the first study to evaluate if the quantitative Ct values of MG and CK19 can be used to estimate probable size of nodal metastases.  To date 184 patients have been tested using the BLN Assay during its clinical use at Morton Plant Hospital. An additional 172 patients were tested with the BLN Assay at our institute during investigational studies. In both cases, the SLNs of breast cancer patients were divided into ∼2 mm pieces. Alternating pieces were homogenized and were processed intra-operatively. The other pieces of the SLN were used for permanent section H&E analysis. The patient was considered H&E positive when a metastasis > 0.2 mm was identified. During clinical use, same-surgery complete axillary dissection was performed when the assay was positive (n = 34).  Performance of the assay's qualitative result against permanent section H&E on adjacent node pieces was similar for the two groups (total N = 356), for a total sensitivity of 96% (81/84), specificity of 93% (254/272), and overall agreement of 94%. The marker Ct values correlated with metastases size as determined by H&E in adjacent node pieces (r = 0.74 for MG and 0.72 for CK19, p < .0001). The lower the Ct values, the greater the likelihood of macrometastases reported by H&E, as described in Table 1. For example if CK19 is <21.0 and MG is < 25.0, there is 100% chance for H&E positivity in the SLNs, with the majority being macrometastases.    Additionally, BLN Assay Ct values in the SLN were predictive of metastases in the non-SLN nodes. For example, of the 12 patients who had a SLN with CK19 < 24 Cts, 9 (75%) had H&E positive non-SLNs. For the same 12 patients, SLNs were H&E positive (5 macrometastases, 5 micrometastases, and 2 reported as > 0.2 mm).  The BLN Assay's high intra-operative qualitative performance minimizes the need for second surgeries for complete axillary dissections. Results from this investigational study examining the marker Ct values suggest that the assay may provide valuable individual tumor volume data intra-operatively or post-operatively.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1002.
Intraoperative molecular sentinel lymph node analysis with OSNA: multicentre prospective UK evaluation.
Authors: Kissin MW , Snook KL , Layer GT , Jackson P , de Vries CS , Shousha S , Sinnett HD , Singhal H , Nigar E , Cunnick G , Chia Y
Published: December 2010

Abstract #1003

Introduction: Rapid intraoperative molecular analysis of sentinel lymph nodes (SLNs) in breast cancer (BC) patients has the advantage of providing the surgeon with an immediate automated result, without the opinion of a consultant histopathologist, allowing axillary dissection to proceed in SLN positive patients. We undertook a multicentre, prospective evaluation of the OSNA system (One Step Nucleic Acid Amplification) for molecular analysis, to evaluate this new diagnostic technique's accuracy and feasibility for use in our hospital system. This abstract reports our 'Phase 2 - Intraoperative' experience.  Method: Four study centres, both district general and major teaching hospitals took part in the evaluation. SLNs from breast cancer patients were excised using standard surgical techniques. Fresh SLNs were defatted and cut into 4 x 1 or 2mm slices depending on size and weight. Alternate slices underwent lysate preparation and immediate OSNA molecular analysis (detection and amplification of CK19 mRNA); remaining slices were processed for permanent section - intensive haematoxylin & eosin and immunohistochemistry (CK19 and AE1/AE3) examination (25μm multistep sections, 5 levels). OSNA results were correlated with final histopathology findings. Processing times were also recorded. No clinical decision was made based on the intraoperative OSNA result.  Results: 396 lymph node samples were included in the analysis. Concordance with histopathology was 96.2% overall with all sites reaching concordance >95%. Overall sensitivity was 91.5%, specificity 97.2%, positive predictive value 87.7%, negative predictive value 98.1%. Concordance of OSNA with histopathology for lobular carcinoma (34 SLN samples) was >95%. There were 14 cases of tissue allocation bias confirmed on repeated molecular and histology testing that were excluded from overall results. Minimum time to reach a result on a single node was 22 minutes.  Conclusion: The OSNA molecular system for SLN diagnosis provides an accurate and feasible intraoperative assessment of SLN status, achieving consistent results across multiple centres. This system may replace current histopathology-based techniques for intraoperative SLN analysis due to high concordance with histology and that it does not require input from a consultant pathologist. OSNA could allow more breast cancer patients in the UK access to one step axillary surgery.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1003.
Intra-operative sentinel lymph node metastasis detection by “one-step nucleic acid amplification (OSNA)”: results of the french multicentric study.
Authors: LeFrere Belda M , Bats A , Charon-Barra C , Crouet H , Houvenaeghel G , Clough K , Khaddage A , Leroux A , Lucas N , Nos C , Penault-Llorca F , Poulet B , Lecuru F
Published: December 2010

Abstract #1004

Background: Sentinel node (SLN) biopsy is widely used as a staging procedure in early breast cancer. usual procedures for intra-operative assessment have a low sensitivity and lead to reoperations for axillary dissection when the SLN is metastatic. The semi-automated OSNA test is based on a short sample preparation step and subsequent rapid amplification of CK19 mRNA. Results are available within 30 - 40 minutes. The OSNA method was developed to accurately detect metastases (≥ 0.2mm) in an intra-operative setting. The objective of this study was to evaluate diagnostic performance of OSNA in comparison to intensive histological examination.  Methods: A total of 509 fresh SLN (234 patients) were cut into four slices. Two alternate slices were analysed by OSNA, the remaining ones were cut at 200µm intervals. On each level, Haematoxylin & Eosin and immunohistochemical staining (CK19 and AE1/AE3) were performed. In case of discordant results, the lysates of samples were subjected to QRT-PCR and Western Blot (Discordant Case Investigation, DCI) in order to detect whether the discordances were caused by tissue allocation bias (TAB): localisation of tumour deposits in only one slice due to the study design.  Results: results were concordant for 202/234 patients (33 positive and 169 negative). 17/32 discordant cases were due to TAB. 2 cases were ininterpretable due to procedure errors. For remaining discordant cases, 2 were histology + / OSNA - and 11 were histology - / OSNA + (8 of them had CK 19 expression levels close to the cutoff). The sensitivity was 94.30% and specificity 93.90%. The overall concordance rate was 93.96%.  Conclusion: OSNA is a rapid and accurate tool for intra-operative assessment of SLN and could reduce the reoperation rate for secondary axillary dissection when the SLN is metastatic.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1004.