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Introduction: Homologous recombination (HR) is essential for repairing double-stranded DNA breaks. Tumors deficient in DNA HR display high levels of copy number aberrations including amplifications, deletions, and breakpoints. Studies in BRCAmut cancers have shown that HR deficiency (HRD) renders triple negative breast carcinoma (TNBC) sensitive to carboplatin or Poly(ADP-ribose) polymerase (PARP) inhibitors via a synthetic lethal interaction, as previously described. Clinical observations in breast and other solid tumors suggest that the HRD phenotype may arise independently of BRCA1/2 mutation. The implications of HRD enrichment in PD-L1, PD-L2, Jak2 (PDJ) 9p24.1 amplified TNBC are not known. Our goal was to determine the HRD status in PDJ enriched tumors, and the potential association of the PDJ amplicon with a BRCA like HRD phenotype. Methods: Archival fresh frozen and formalin-fixed paraffin embedded (FFPE) tumor samples from 41 patients with TNBC were obtained from 1998-2015. Tumor populations (diploid, tetraploid, and aneuploidy) were sorted using DNA content flow cytometry and interrogated with oligonucleotide array comparative genomic hybridization (aCGH). Tumor homologous recombination deficiency (HRD) scores were calculated by number of interstitial aberrations (IA) in their genomes (HRD-IA). A cut-off of < 40 (low), > 40 (high) or > 50 (very high) was assigned based on our studies of known BRCAmut tumors. Data were compared between HRD groups using chi-square, ANOVA F-test or log-rank test. Results: 14/41 (34%) TNBC patients had high HRD-IA score and 6/41 (14.6%) had very high HRD-IA score. The median age was 61.8 years (SD = 12.8), the average tumor size was 3.1cm (SD = 2.6), 42% were node positive, 24% were pre-menopausal, 19% were Black, Hispanic, or Asian/Pacific Islander. Genetic testing was negative for BRCA1/2 mutations in 9/41 patients, including 3 high and very high HRD-IA score patients. There was no difference in the OS at 5-years [74% versus 59% (p = 0.907)] in the high HRD-AI group. 12/41 (29%) patients were PDJ+. In the PDJ+ patients, 4/12 (33.3%) had HRD > 40 and 1/12 (8.3%) had HRD > 50 compared to the PDJ- patients where 10/29 (34%) had high HRD-IA score and 5/29 (17%) had a very high HRD-IA score, p=0.463. Patients with inflammatory TNBC were significantly more likely to have high HRD-IA score 4/6 (66%) compared to non-inflammatory TNBC 8/31 (26%), p=0.05. Conclusions: We describe a unique group of TNBC patients with similar patient characteristics, tumor size, nodal status, and stage when compared by low, high and very high HRD-IA score. For inflammatory TNBC, there was statistically significant percentage of patients with a high HRD-IA score. We did not see a statistically significant correlation with 9p24.1 amplification based on HRD-IA score which has been suggested in other data sets. The HRD-IA score did not statistically impact OS but there was a suggestion that those with a higher score may have an improved OS potentially due to chemotherapy sensitivity. Further study with a greater number of patients to adequately power the study is needed to determine whether our initial findings can be confirmed and whether the HRD-IA score can be used to define a specific subset of TNBC patients. Citation Format: Gawryletz CD, Anderson KS, Northfelt DW, Kosiorek HE, Linnaus ME, Ocal IT, McCullough AE, Pockaj BA, Barrett MT. Homologous recombination deficiency in PD-L1, PD-L2, Jak2 (PDJ) amplified triple negative breast carcinoma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-19.

Metaplastic breast carcinoma (MBC) is a rare subtype of triple negative breast carcinoma with frequent metastasis and poor response to chemotherapy. MBCs are defined by the presence of “metaplastic” components of spindle, squamous, or sarcomatoid histology (e.g. chondroid, osseous). To elucidate the molecular determinants of MBC, our lab analyzed the proteomics landscape of MBC which revealed that squamous MBC are significantly enriched for proteins that mediate inflammation and leukocyte activation, compared to spindle and sarcomatoid MBC. The presence and the significance of inflammation in MBC has not been investigated. To evaluate this, we retrieved 36 MBCs resected from 1988-2015 at our institution. Tumor sections were reviewed by three pathologists, and used to develop a tissue microarray (TMA) in duplicate. H&E whole sections were reviewed for MBC subtype and inflammation. The inflammatory infiltrate was assessed for type (lymphocytes, plasma cells, neutrophils, macrophages), localization (peripheral and/or intratumoral), and degree (0, 1, 2, 3). When the combined degree of inflammation at the periphery and intratumoral was ≥ 4, the degree was considered high. TMAs were immunostained for CD3/CD20 and CD4/CD8 double stains for further characterization. Of the 36 MBCs, 9 (25%) were chondroid, 7 (20%) were spindled, and 20 (55%) were squamous. When present, inflammation was predominantly lymphoid or lymphoplasmacytic (94%). Seventy-five % (15/20) of squamous MBC demonstrated significantly higher intratumoral inflammation (score ≥2) compared to 43% (3/7) and 11% (1/9) of spindle and chondroid, respectively (p=0.005). Squamous MBCs had a significantly higher degree (peripheral and intratumoral) of inflammation (15/20, 75%) compared to spindled (3/7, 43%) and chondroid (1/9, 11%) MBC's (p=0.004). The inflammatory infiltrate consisted of a mixture of B and T lymphocytes. Ninety-five % of cases with a T-cell infiltrate showed a predominance of CD4+ over CD8+ T-cells. Lymph node metastasis was seen in 11/30 MBC, and 9/11 (81%) demonstrated squamous morphology and a high degree of inflammation (p=0.017). Our results demonstrate that squamous MBC have a higher degree of inflammation within and at the periphery of the tumor compared to other MBC subtypes. A high degree of inflammation consisting of B and T lymphocytes in squamous MBC correlates with lymph node metastasis. The inflammatory infiltrate may contribute to the aggressive clinical behavior of squamous MBC, which warrants further investigation. Citation Format: Emily McMullen, Stephanie L. Skala, Sabra Djomehri, May P. Chan, Celina G. Kleer. Lymphocytic inflammation is a distinct feature of squamous metaplastic breast carcinomas and is associated with metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 981.

Introduction: A Disintegrin and Metalloproteinase 9 (ADAM9) promotes cancer progression via its metalloproteinase and disintegrin domains, and upregulated ADAM9 is found in pancreatic, prostate and breast carcinomas. The expression of ADAM9 in triple negative breast carcinoma (TNBC), however, is still unclear. We investigated the expression of ADAM9 in TNBC, its role in tumor progression and prognostic implication in TNBC patients. Materials and Methods: The expression of ADAM9 was investigated in TNBC and non-TNBC cell lines as well as TNBC and non-TNBC tumor tissues. ADAM9 mRNA expression data in TNBC cell lines (n = 14) and non-TNBC cell lines (n = 11) were obtained from Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/) database. A total of 24 TNBC and 20 non-TNBC breast tumor specimens were also collected. The Kaplan-Meier (KM) plotter was used to investigate the association between ADAM9 expression level and survival in TNBC patients. Cell proliferation, migration and invasion assays were performed to investigate the role of ADAM9 in TNBC cell line (MDA-MB-231). Results: ADAM9 mRNA expression in TNBC cell lines was significantly higher than that of non-TNBC cell lines (p = 0.038). Strong staining of ADAM9 was detected in primary tumor tissues from TNBC patients, but only weak staining was found in primary non-TNBC tumors. Statistical analysis showed that the ADAM9 staining intensity (p < 0.05) and expression level (p < 0.05) were markedly increased in TNBC group compared with the non-TNBC group. High level of ADAM9 mRNA expression was associated with poorer overall survival (p < 0.01), more distant metastasis-free survival (p < 0.01) and shorter relapse-free survival (RFS) (p < 0.001). Furthermore, high ADAM9 mRNA level was also significantly associated with poor RFS in TNBC (p < 0.01). ADAM9 mRNA expression level was associated with RFS in grade II and III tumors, but not in grade I tumors (p = 0.21). An increased hazard ratio was also found with grade II tumors (p = 0.2) and grade III tumors (p = 0.003). Scrambled siRNA with green fluorescent protein was transfected in MDA-MB-231 cells by lipofectamine 2000. ADAM9 protein and mRNA expression were successfully inhibited in these transfected MDA-MB-231 cells, which lead to suppression in cell proliferation, migration and invasion on functional assays. Conclusion: Our results showed the ADAM9 was overexpressed in TNBC and was associated with poor prognosis in TNBC patients. ADAM9 was found to play a key role and may represent a potential treatment target because inhibition of ADAM9 mRNA and protein expression was associated with suppression of cell proliferation, migration and invasion in TNBC cell line. Keywords: ADAM9, Prognosis, Triple negative breast cancer Citation Format: Rui Zhou, William CS Cho, Victor WS Ma, Wah Cheuk, Grace Yik-ka So, SC Cesar Wong, Lawrence WC Chan. ADAM9 plays a key role in the aggressive behavior of triple negative breast carcinoma and represents a potential treatment target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4301.

Background Cancer surgery remains the most effective single treatment modality for curing patients with solid tumors. However, only half of all cancer patients undergo curative resection. Of those, a significant number will eventually relapse from occult metastases. Administration of therapy prior to surgery, known as neoadjuvant therapy, has revolutionized cancer surgery. It not only decreases overall tumor burden, rendering previously inoperable disease operable, but also improves survival by providing systemic disease control in patients at high risk of having microscopic distant metastases at the time of surgery. While traditional neoadjuvant therapy using conventional chemotherapy and radiation therapy has improved patient outcomes across a spectrum of solid tumors, the potential of neoadjuvant immunotherapy is currently unknown. Various immune modulating agents have achieved significant clinical success in the management of metastatic and treatment resistant cancers, yet their utility in the neoadjuvant setting remains unclear. In theory, neoadjuvant immunotherapy is particularly interesting because it utilizes the primary tumor as a rich source of antigens for T cell priming. As a result, it may be successful in eliciting an immune response where adjuvant immunotherapy treatment has failed. Here we designed a preclinical study utilizing neoadjuvant immunotherapy and demonstrate the efficacy of employing immune stimulating agents synergistically with surgical resection. Methods We used two different murine models of solid tumors – a metastatic triple negative breast carcinoma and a colorectal carcinoma to compare neoadjuvant immunotherapy, resection alone, or immunotherapy alone treatments on local and systemic disease control, and survival. We found that neoadjuvant immunotherapy offered the best survival as well as the best locoregional and metastatic disease control when compared to the other groups. In fact, in the highly aggressive and spontaneously metastatic breast carcinoma model the neoadjuvant treatment group was the only group in which a long-term surviving cohort of mice was cured. When these long-term survivors were rechallenged with the same tumor, they were immune, confirming that the treatment successfully conferred immunological memory. When exposed to a new tumor type, however, they remained susceptible, confirming the specificity of this immunologic memory. Disease control was found to be T cell dependent as depletion of either both CD4+ and CD8+, or CD8+ only T cells resulted in loss of therapeutic benefit in mice treated with neoadjuvant immunotherapy. Conclusion We found that neoadjuvant immunotherapy successfully initiated cell mediated antitumor activity both locally and systemically at an anatomically distant disease site. This resulted in improved survival in the neoadjuvant group when compared to groups receiving resection alone or immunotherapy alone. As increased options to incorporate immunotherapy in the clinic become available to clinicians, this study explores the timely and clinically important question of how immunotherapy may optimally be combined with surgical resection. The results of the study are readily translatable and may be utilized to inform establishment of neoadjuvant immunotherapy regimens for patients with early stage and locally advanced solid tumors. Citation Format: Wan Xing Hong, Idit Sagiv-Barfi, Debra Czerwinski, Ronald Levy. Neoadjuvant immunotherapy for solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5568.

Backgroud: Brain metastasis (BM) is a fatal event that nontheless alter seriously survival of patients suffering from breast cancer but also reduce their quality of life. Selection of an enriched patients population at high risk for BM is warranted to develope preventive strategies and/or to evaluate the impact early treatment of BM in prospective trials. Methods: Electronic medical records of patients, treated in the Institut Claudius Regaud, with metastatic breast cancer and without BM at stade IV diagnosis or in the first month, were retrospectively reviewed for the period between January 2005 and December 2012. We first study the Graesslin’s nomogram (J Clin Oncol., 2010; 28(12): 2032-37) characteristics in our patients. The discrimination prediction of subsequent BM was evaluated by the area under the receiver operating characteristic curve (AUC) and we performed the calibration. Moreover, we have evaluated average and maximal errors between predictions and observations obtained from the calibration curve. Then, competing risk analysis was used to identify prognostic factors with time to BM appearance and death before BM. Results: We identified 446 patients without BM at stage IV diagnosis or in the first month, 70 of them developed subsequent BM. Young age (≤ 50 years) at the diagnosis of breast cancer (p=0.01), ductal carcinoma (p=0.02), negative status of hormone receptor (p 50 years (p=0.015) and triple negative status (p Conclusion : We have validate the robustness of Graesslin’s nomogram to predict subsequent BM in patients with metastatic breast cancer and suggest the best utility in patients with HER2+ breast cancer, looking forward new systemic therapies to control non BM in triple negative breast carcinoma. Citation Format: Ludivine Genre, Henri Roché, Monia Ouali, Thomas Filleron, Florence Dalenc. Predict subsequent brain metastasis in patients with metastatic breast cancer: External validation of a published nomogram and competing risk regression analysis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-16-07.

The androgen receptor (AR) and its pathway have been implicated in tumorigenesis and progression of breast cancer. Anti-androgen therapy has shown efficacy in the metastatic breast cancer and numerous clinical trials are underway to study efficacy in various clinical settings. 15 splicing variants of AR (AR-Vs) have been described in prostate cancer. Structurally, AR-Vs have insertions of cryptic exons downstream of the exons that encode the DNA-binding domain or deletions of the exons encoding the ligand-binding domain, resulting in a disrupted AR open reading frame and expression of ligand-binding-domain-truncated AR proteins. In prostate cancer, some of the AR-Vs especially AR-V7 are associated with aggressive disease and resistance to anti-AR therapy. The AR-V prevalence in human breast cancer specimens has hitherto not been studied. We aimed at studying the expression of AR-Vs in breast cancer specimens and present the data on AR-V1, AR-V7, AR8, and ARV567, in AR-positive triple negative (TNBC) and ER+/Her2- breast cancer. Design: 98 cases of TNBC, 40 cases of ER+/Her2 breast cancer and 17 cases with reduction mammoplasty were abstracted from NYULMC pathology database and screened for AR expression by immunohistochemistry (IHC). IHC for AR was performed using antibody clone N-20 (Santa Cruz) at 1:100 dilution. Normal breast tissue was used as internal control and 10% nuclear staining was used for categorizing a tumor as AR positive. A subset of cases which over-expressed AR were macrodissected from formalin fixed paraffin embedded sections with total RNA extracted by using the PureLink® FFPE RNA Isolation Kit (Invitrogen). Reverse-transcription was performed by using the SuperScript® III Reverse Transcriptase Kit (Invitrogen). AR-V expression was presented as cycle number difference to housekeeping gene (delta CT) for real-time PCR or as absolute copy number for digital PCR. Results: AR+ TNBC and AR+/ER+ cases ranged from stage 1A to IIIA. IHC for AR showed > 10% staining in 27 of 98 TNBC cases and in 39 of 40 ER+/Her2- cases. AR Positive Prevalence in TNBC and ER+ Breast Carcinoma AR+AR-TNBC2771ER+/Her2-391 AR-V7 was expressed in 11 of 13 AR+/TNBC cases (p < 0.05); AR-V1 and AR-V4 were expressed in 4; ARV8 and ARv567es were expressed in 3 and 2 cases respectively. In 25 AR+/ER+/Her2- cases AR-V7 was expressed in 14; AR-V4 in 9; ARV567 in 5, AR-V1 in 3 cases. AR Spliced Variant Incidence in TNBC, ER+ and Benign breast tissue Benign (N=17)ER+/AR+ (N=25)TNBC+/AR+(N=13)P ValueAR-V113(12%)4(25%)0.145AR-V419(36%)4(25%)0.35AR-V78(47%)14(56%)11(85%)0.021AR-V801(4%)3(19%)0.474AR-V56715(20%)2(12.5%)0.092 Conclusion: We report expression of various spliced variants in TNBC, ER+/Her2- breast cancer. A statistically significant expression of AR-V7 is seen in TNBC. Since AR-V7 predicts for poor prognosis and lack of response to anti-AR therapy in prostate cancer, AR-V7 expression maybe a useful biomarker to analyze response data in on going breast cancer clinical trials. Citation Format: Singh B, Deng F-M, Kane Y, Zhan Y, Qi Y, Liu X, Zhang H, Dong Y, Brody R, Wieczorek R, Lee P. High prevalence of splicing variant AR-V7 in triple negative breast carcinoma. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-08.

Based on the mechanism of action of PARP inhibitors, BRCAness breast and ovarian cancers could be defined as the optimal group of tumours to target with such drugs. As the BRCAness phenotype is not fully defined at present, a surrogate phenotype of triple negative breast cancer (TNBC) has been proposed. Results of recent clinical trials involving PARP inhibitors in the context of TNBC do not demonstrate a significant improvement in overall survival or progression free survival, partly due to the inaccurate selection of certain tumours. Data from cell culture studies suggests that loss of PTEN expression could be a marker of PARP inhibitor sensitivity. Materials and Methods: A retrospective study of 160 TNBC resected at Institut Bergonie between 1989 and 2010 was undertaken. Inclusion criteria were: ER & PR immunohistochemcial (IHC) negativity ( Results: Twenty percent of sporadic and familial TNBC demonstrated reduced BRCA1 expression with a greater than 7 fold reduction in expression. Almost half of tumours demonstrated a complete loss of PTEN expression as assessed by IHC, with a deleterious PTEN point mutation identified in 5% of cases. Although a slight correlation is observed between the complete loss of PTEN and a reduction in BRCA1 expression, a complete overlap of these two alterations was not observed; tumours with reduced BRCA1 expression do not always demonstrate complete negativity on PTEN IHC and certain tumours displaying complete negativity on PTEN IHC do not show a reduction in BRCA1 expression. Full immunohistochemical characterization of the tumour series (ER, PR, HER2, AR, CK5/6 & EGFR) together with the results of PTEN, BRCA1 and 53BP1 analyses will be presented. Conclusion These results suggest that at present, loss of PTEN expression is not a suitable surrogate marker for tumours with reduced BRCA1 expression. This should be borne in mind for ancillary studies of clinical trials involving PARP inhibitors in the treatment of breast tumours and further investigation is warranted. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD10-04.

Purpose: Triple negative breast carcinoma (TNBC) is currently being required to be classified pluralistically in order to provide the most appropriate therapy to the patients. We attempted to subclassify TNBC cases into subgroups based on clinical outcome or prognosis of the patients with TNBC using archival specimens. Materials and Methods: We analyzed 102 Japanese cases of invasive TNBC who underwent surgery between January 1998 and December 2007. The clinicopathological factors and clinical information of these patients were retrospectively retrieved from charts of the patients. Immunohistochemical staining was performed for estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor 1 (EGFR1), CK5/6, Ki-67 and CD31 for microvessel density (MVD). Results: Median follow-up time of the patients was 68.5 months. Multivariable analysis demonstrated that the pathologic node status was the most significantly associated with relapse-free survival (RFS) and breast cancer-specific survival (BCSS) of the patients. Pathological tumor size, basal-like type, Ki-67 labeling index (LI) and MVD were also independently associated with RFS and BCSS. Based on these results, we devised the Risk Score system reflecting Hazard ratios (HRs) of these prognostic factors above. Multivariate analysis and the Risk Score for TNBC patients. With this system, TNBC patients in this study were classified into three subgroups; patients with score 0-3 are in low risk group, score 4-7 in intermediate risk group and score 8- in high risk group. These three groups had statistically significant differences for risk of relapse and breast cancer-specific death, respectively. Conclusions: We propose the Risk Score system, which incorporated pathologic nodal status, size of the primary tumor, the presence or absence of basal-like features, Ki67 LI and MVD of the patients in order to predict postoperative clinical course of the Japanese patients with TNBC. Such a classification which can be performed in diagnostic pathology laboratory can be useful as a decision-making tool for triple negative patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-38.

Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) possesses the ability to induce apoptosis in cancer cells independent of their p53 status while exhibiting minimal toxicity to normal, non-transformed cells, and thus, it is a promising anti-cancer therapeutic. However, rhTRAIL-induced apoptosis is not as effective in a majority of breast cancers due to the up-regulation of anti-apoptotic proteins, down-regulation of pro-apoptotic proteins, and/or down-regulation of death receptors (DRs) 4 and 5. A combinatorial approach of rhTRAIL with the “mother nature”-derived compound ursolic acid (UA) has been applied to sensitize rhTRAIL-resistant triple negative breast carcinoma. UA is derived from the leaves and berries of various plants and found in the coatings of fruits and does not exhibit toxicity to normal, non-transformed cells. UA has been revealed to possess the ability to up-regulate DR5 and diminish the expression of anti-apoptotic proteins survivin and FLIP in cancer cells and thereby, making UA an encouraging choice to be utilized as a sensitizing agent. The aim of this study was to determine the capacity of UA to sensitize rhTRAIL-resistant triple negative breast cancer (TNBC) BT-20 and HCC1937 cells to rhTRAIL-induced apoptosis and elucidate the underlying mechanisms for UA's sensitization. The combinatorial treatment of UA and rhTRAIL augmented the induction of apoptosis when compared to single agent UA and rhTRAIL treatments as detected by Annexin V/PI assays and through by the execution of the extrinsic pathway as marked by the activation of caspase 8, activation of the executioner caspases 3 and 7, and eventual PARP cleavage (a hallmark of apoptosis). The underlying mechanisms for UA's sensitization of rhTRAIL-resistant TNBCs were established to be through the down-regulation of the anti-apoptotic protein FLIP and through the up-regulation of DR4 and DR5. Overall, these findings reveal that UA is an efficacious sensitizing agent for rhTRAIL-resistant TNBCs. Citation Format: Jasmine Manouchehri, Michael Kalafatis. Ursolic acid sensitizes rhTRAIL-resistant triple negative breast carcinoma to rhTRAIL-induced apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1863.

Background. Breast cancer negative for ER, PR, and HER2, so called triple negative breast cancer (TNBC) occurs at a younger age and has a worse prognosis. It is considered hard to treat because it tends to rapidly develop resistance to conventional therapy and targeted therapy is not available. Therefore more efficient therapy is warranted to improve patient survival. In the development of new therapies, the availability of preclinical in vivo models is essential. Ideally, these in vivo models would recapitulate the heterogeneity in histogenetic characteristics, expression profiles, signaling pathways, and response to therapy found in human breast cancers. Xenograft models, in which human tumor tissue is engrafted directly into recipient mice, may be very useful for this purpose (Marangoni et al, Clin Can Res 2007; 13:3989). Methods. We have set up a panel of xenografts in which human TNBC can be studied in a dynamic and reproducible manner by implanting fresh breast cancer fragments in immunocompromised mice. Tumor outgrowth occurs 3 to 10 months after implantation. After outgrowth, tumors are characterized based on histology, immunohistochemistry, aCGH and gene expression profile and serially transplanted. Subsequently, responses to chemotherapeutic agents are being tested. Results. We generated five TNBC xenografts, which were all classified as basal-like based on expression profiling, and BRCA1-like based on aCGH analysis (Joosse et al., Breast Cancer Res Treat (2009) 116: 479). One model was shown to be BRCA2 mutated, whereas no BRCA1 or -2 mutations were found in the other models. We treated the TNBC xenograft models with cisplatin, since recent clinical studies have shown good response of TNBC to cisplatin (Silver et al, J Clin Oncol 2010; 28:1145, Byrski et al, J Clin Oncol 2010; 28:375). When treated with cisplatin, the BRCA2 mutated model showed only limited response, with reduced tumor growth, but no tumor eradication. In contrast, the other four TNBC xenograft models responded very well to treatment with cisplatin, resulting in complete disappearance of a palpable lesion in most cases after 21 days of treatment. However, tumors did relapse and had to be treated again. Interestingly, all tumors remained responsive to cisplatin treatments even after several relapses, but could not be completely eradicated. We have recently started treating TNBC xenograft models with the bifunctional alkylators melphalan and nimustine, because these drugs were shown to cause tumor eradication in genetically engineered mouse models of BRCA2-associated breast cancer (Evers et al, Clin Can Res 2010; 16:99). The BRCA2 mutated tumor again showed only partial response, whereas the other TNBC models showed very good initial responses, although relapses did occur. Conclusion. Triple negative breast cancer xenograft models resemble their human counterpart very well and are useful to study response to cytotoxic drugs and targeted agents in a preclinical setting, which could speed up introduction of promising treatments into clinical practice. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-03-03.

Background. Triple Negative Breast Cancers (TNBCs) still represent a question mark in breast cancer biology and a primary issue in clinics. A promising approach for an efficient targeted treatment of TNBCs seems to be represented by antiangiogenic therapies. Methods. Effects of different compounds on proliferation of TNBC was evaluated in vivo and in vitro respectively by xenograft tumor volume analysis and SRB assay. Vasculogenic Mimicry (VM) properties were evaluated in vitro and in vivo respectively by tube formation assay and Transmission Electron Microscopy (TEM). Results. Treatment of triple negative (TN) MDA-MB-231 xenografts with Sunitinib induced tumor regression, versus only a slight growth inhibition in tumors derived from MCF7 luminal cell line, whereas Bevacizumab determined only a modest decrease in tumor growth in both models. Accordingly, the efficacy of Sunitinib in blocking in vitro growth of breast cancer cell lines was higher in MDA-MB-231 cells in comparison with MCF7 cells (IC50 at 72h in MDA-MB-231 was 5 uM vs 25 uM in MCF7, as evaluated by SRB), and sensitivity to Bevacizumab was comparable between the two different cell lines. Investigating the undifferentiated nature of TNBCs, we observed that these tumors present an endothelial like phenotype and behavior, as supported respectively by the expression of endothelial markers, and the formation of vascular-like channels in vitro. In fact, all six TN cell lines evaluated (MDA-MB-231, MDA-MB-157, MDA-MB-468, BT-549, BT-20 and HCC1937) were able to form vascular channels when seeded on the murine tumor-derived basement membrane (Matrigel), whereas luminal (MCF7, T47D and ZR-75-1) and HER2- positive (MDA-MB-361, BT474 and SKBr3) breast carcinoma cell lines did not exhibit vascular structures. Then, we evaluated the VM in xenograft tumors derived from MDA-MB-231, MCF7 and MDA-MB-361 using transmission electron microscopy (TEM). MDA-MB-231 xenografts displayed channel-like structures formed by tumor cells encompassing erytrocytes, whereas in MCF7 and MDA-MB-361 xenografts the endothelial lining delimiting blood vessels was clearly visible. Notably, blood vessels surrounded by tumor cells were also identified in human TN specimens processed for TEM, and these structures were significantly more frequent in TN compared to non-TN tumors. 60% reduction in TN vascular channel formation in vitro by an anti-bFGF monoclonal antibody along with no effect using anti-VEGF antibody indicated that TN breast carcinoma cells can generate vascular channels through bFGF-mediated pathway. Silencing of different receptors involved in bFGF signal (i.e. FGFR2 and PDGFR β) abrogated VM in TN cells. Finally, TNBC cells able to perform vascular-like channels were found to express significantly higher (p= 0.003) levels of FGFR related genes, described associated with basal-like BC aggressiveness, compared to all the other tested cell lines. Conclusions. Our findings point to the possibility that TNBC cells are maintained by proangiogenic signals and that increased sensitivity to Sunitinib probably relies on the specific impairment of PDGFRβ and FGFR2-mediated pathways, which might represent a possible specific therapeutic target. Partially supported by AIRC and Italian Bureau of Health. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-08-01.

Introduction: Triple negative breast carcinoma (TNBC) is a subtype of breast cancer with a paucity of therapeutic targets and a poor prognostic phenotype. In a retrospective cohort, we sought to determine the prevalence of the amplicon targeting the 9p24.1 locus, resulting in over-expression of PD-L1, PD-L2, and JAK2 (PDJ), and potentially actionable therapeutic targets. We then probed the genomic landscape of these PDJ positive tumors and identified co-occurring copy number aberrations, including focal amplifications and homozygous deletions. The presence of the PDJ amplicon and selected co-occurring aberrations provide a unique description of a clinically relevant subtype of TNBC. Methods: We evaluated fresh frozen and formalin-fixed paraffin embedded tumor samples from 64 patients with triple negative breast cancer whom underwent definitive surgical resection. Clinical annotation was available in 60 of the samples. Tumor populations (diploid, tetraploid, and aneuploidy) were sorted from each biopsy using DNA content flow cytometry. Each sorted sample was interrogated with oligonucleotide array comparative genomic hybridization (aCGH). All microarray slides were scanned using an Agilent 2565C DNA scanner and the images were analyzed with Agilent Feature Extraction version 10.7. The aCGH data was assessed with a series of QC metrics then analyzed using an aberration detection algorithm. Results: We detected a high level (log2ratio greater than or equal to 2) amplicon targeting 9p24.1 in 18 of 64 patients (28%) genomic profiles with triple negative breast carcinoma. In the PDJ positive population, we detected 8 of 18 patients (44%) with co-amplification targeting myc at 8q24, 3 of 18 patients (17%) with co-amplifications targeting EGFR at 7p11, 3 of 18 patients (17%) with co-amplifications targeting PIK3CA. These co-occurring genomic events in PDJ positive tumors may provide clinically actionable targets. Other selected amplifications detected included NOTCH3, KRAS, RUNX1, TUBAL3, FGFR2, AKT1, AKT2, YPEL2, PBXL7, KIT. We detected PTEN homozygous deletion in 2 of 18 patients (11%) PDJ positive tumors. Other homozygous deletions identified in the genomic landscape included FAT1, SOX3, Park2, TNFAIP3, GPC3, RB1, and CREBBP. Conclusions: In our retrospective analysis, the amplification of chromosome 9p24.1 involving PD-L1, PD-L2, and JAK2 is present in approximately 28% of triple negative breast cancer patients. The genomic landscape of these PDJ positive TNBCs include recurring high-level focal amplifications and targeted homozygous deletions of clinically relevant genes. The clinical implications of these data are under current investigation using model systems and are in early phase clinical trials. The efficacy of immune checkpoint inhibitors including nivolumab, pembrolizumab, and JAK2 inhibitors including ruxolitinib, in PDJ positive triple negative breast carcinoma is intriguing and remains to be elucidated. Citation Format: Gawryletz CD, Anderson KS, Cunliffe HE, Northfelt DW, McCullough AE, Lenkiewicz E, Malasi S, Pockaj BA, Barrett MT. The genomic landscape of PD-L1, PD-L2, Jak2 (PDJ) amplified triple negative breast carcinoma. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-03-13.

Several studies including our own have made it increasingly clear that genetic alterations of tumor cells alone are not the sole driving force behind tumor development but that tumor initiation, growth, progression and response to combinatorial treatments are rather intimately controlled by the microenvironment. Improved knowledge of cancer biology and investigation of the complex functional interrelation between the cellular and noncellular compartments of the tumor microenvironment have provided an ideal platform for the evolution of novel cancer nanotherapies. In the present study we have developed an in-vitro method to co-culture color coded triple negative breast carcinoma cells and cell types that exist in the tumor/tumor microenvironment in a “hanging drop” of medium to the size of an avascular microtissue (∼800 µm). Changes in the fluorescence intensity of different cell types and spheroid/microtissue sizing is being utilized as a method for high throughput therapeutic evaluation of the combination therapy using nanoparticles. When implanted orthotopically and in the dorsal skin fold window chambers in nude mice they facilitate the imaging of neovascularization and understanding response to therapy. We have also observed that radiation (3Gy) can significantly enhances expression of Galectin-1 in tumor vasculature associated endothelial cells which can serve as a specific ligand for the 33 amino acid antiangiogenic, anginex peptide. Thus Galectin-1 is a promising candidate for actively targeting irradiated tumors with anginex conjugated nanoparticles. The objective of this study is to establish a novel strategy for radiation therapy-amplified delivery of dual drugs for targeting the microenvironment-responsive endothelial component of the tumor vasculature. Use of radiation-guided anginex bound nanoparticles for delivery and controlled release of dual drugs (Arsenic-cisplatin) are expected to simulate metronomic therapy and preferentially target the tumor endothelial cells at the irradiated tumor site via tumor stroma enriched Galectin-1 protein. Our molecular studies indicate differential vulnerability for arsenic and cisplatin in cell types constituting the tumor/tumor microenvironment. This is the first study to understand a novel combinatorial nanotherapeutic system in an in-vitro/ in-vivo breast tumor model which incorporates characteristics of the tumor microenvironment and is thus expected to better predict treatment response in patients. Supported by NCI grant CA173609 and (CNPP) U01CA151461 Citation Format: Pallavi Sethi, Amar Jyoti, Elden Swindell, Ulrich W. Langner, William H. St. Clair, Ronald C. McGarry, Thomas V. O'Halloran, Meenakshi Upreti. Impact of tumor microenvironment on tumor growth, metastasis and response to combination therapy via microenvironment-responsive dual drug-loaded nanoparticles and radiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4589. doi:10.1158/1538-7445.AM2014-4589

Breast cancer is the most common malignancy among women in developed countries, affecting more than a million women per year worldwide. Of these, triple negative breast carcinoma represents 10-17 %. Triple negative breast carcinomas, characterized by estrogen, progesterone and HER2 receptor negativity are very aggressive tumors with poor prognosis. Breast Cancer Stem derived from triple negative parental Breast Cancer tumors, are a subpopulation of cells within the parental breast cancer population within the individual which are positive for the following markers: CD133+CD44+CD24+ESA+SSEA-1+TRA-1-61+& TRA1-81+ and Oct ¾ these Breast Cancer Stem cells are highly tumorigenic and possess the stem cell-like properties of self-renewal and the ability to produce differentiated progeny. Breast Cancer Stem cells also demonstrate up regulation of SSEA3+, SSEA4+ upon differentiation into parental cancer phenotype. Individualized treatment (tailored therapy) based on molecular biology markers of tumor and patient is the trend in clinical practice these days. However, molecular targets and predictors for the treatment of triple negative breast carcinoma do not currently exist. With the identification and characterization of Breast Cancer Stem Cells from parental triple negative tumors, enables one to screen novel drug candidates for potential development of therapeutics for triple negative Breast Cancer Patients. In this study we have utilized Breast Cancer Stem Cells from triple negative Tumors to screen potential drug candidates. The Breast Cancer Stem Cell based assay system may provide novel therapeutic approaches into treatment of triple negative breast cancer patients, which are resistant to standard chemotherapy and radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3319.

Triple negative (TN) breast cancers account for about 15-20% of breast cancers and are associated with aggressiveness, poor prognosis and shorter survival. Because of the lack of specific therapy guidelines for this subgroup there is an urgent need to define specific markers for therapeutic targeting in these tumors. We observed that TN breast cancer cells lines are able to perform “Vasculogenic Mimicry” (VM) in vitro. In fact, all four TN cell lines evaluated (MDA-MB-231, MDA-MB-157, MDA-MB-468 and HCC1937) were able to form vascular channels when seeded on the murine tumor-derived basement membrane (Matrigel), whereas luminal (MCF7 and ZR-75-1) and HER2-positive (MDA-MB-361 and SKBr3) breast carcinoma cell lines did not exhibit vascular structures. Then, we evaluated the VM in xenograft tumors derived from MDA-MB-231, MCF7 and MDA-MB-361 using transmission electron microscopy (TEM). In MDA-MB-231 xenografts it has been revealed channel-like structures formed by tumor cells encompassing erytrocytes, whereas in MCF7 and MDA-MB-361 xenografts the endothelial lining delimiting blood vessels was clearly visible. Notably, blood vessels surrounded by tumor cells were also identified in human TN specimens processed for TEM, and these structures were significantly more frequent in TN compared to non-TN tumors. 60% reduction in TN vascular channel formation in vitro by an anti-bFGF monoclonal antibody together with no effect using anti-VEGF antibody indicated that TN breast carcinoma cells can generate vascular channels through bFGF-mediated pathway. Silencing different receptors involved in bFGF signal (i.e. FGFR2 and PDFGR) abrogated VM in TN cells. Treatment of MDA-MB-231 xenografts with a TKI able to block both FGFR and PDGFR induced tumor regression, versus only slight growth inhibition in tumors derived from MCF7 luminal cell line. In conclusion our findings point to the possibility that TN tumors generate vascular channels independent of tumor angiogenesis and that targeting this VM capability may represent a valid therapeutic tool. (Partially supported by AIRC). Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1598.

Abstract #3044 Introduction: Obesity is an independent risk factor for breast cancer and obese breast cancer patients exhibit a higher risk for larger tumor burden, increased metastasis and poor response to endocrine therapy. Obesity affects breast carcinogenesis by autocrine and paracrine effects of adipocytokine leptin and IGF-1. We have previously shown that leptin induces growth stimulation of breast cancer cells by recruiting histone acetyltransferases and mediator complex to cyclin D1 promoter via activation of Stat3. In the present study, we found a novel bidirectional crosstalk between IGF-1 and leptin signaling that promotes invasion and migration of triple-negative (ER, PR, HER2 negative) breast cancer cells; MDA-MB-231, MDA-MB-468, MDA-MB-435 and HCC-1806. Methods and Results: IGF-1 induced significant tyrosine phosphorylation of leptin receptor (Ob-Rb) and leptin induced tyrosine phosphorylation of IGF-1 Receptor (IGF-1R). Combined treatment of leptin and IGF-1 induced synergistic activation of both Ob-Rb and IGF-1R along with activation of Akt and ERK. Furthermore, IGF-1 and leptin synergistically transactivated epidermal growth factor receptor (EGFR) and induced proliferation of triple negative breast cancer cells. We also found that tyrosine phosphorylation of EGFR induced by combined treatment of leptin and IGF-1 was significantly inhibited not only by EGFR tyrosine kinase inhibitor, AG1478 but also with a broad-spectrum matrix metalloproteinase inhibitor, GM6001 indicating that leptin and IGF-1 induced EGFR transactivation is dependent on proteolytic release of EGFR ligands. Intriguingly, we also found that combined treatment of leptin and IGF-1 potently induced invasion of triple negative breast cancer cells in Matrigel invasion and quantitative Electric Cell-Substrate Impedance Sensing invasion assays. Inhibition of leptin and IGF-1 mediated EGFR activation using a highly potent, reversible inhibitor of HER1/EGFR tyrosine kinase, erlotinib or dual epidermal growth factor receptor (EGFR)/human EGFR-2 (HER2) kinase inhibitor, lapatinib effectively inhibited leptin and IGF-1 induced invasion. Also, inhibition of EGFR activation using erlotinib and lapatinib reduced leptin- and IGF-1-induced migration of triple negative breast cancer cells. Discussion: Taken together these data indicate a novel bidirectional crosstalk between leptin and IGF-1 signaling that transactivates EGFR and promotes metastatic properties, invasion and migration of triple negative breast carcinoma cells. Our novel findings indicate the possibility of using EGFR inhibitors erlotinib and lapatinib to counter the pro-cancerous effects of leptin and IGF-1 signaling in triple negative breast cancers. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3044.

Introduction: Obesity is an independent risk factor for breast cancer and obese breast cancer patients exhibit a higher risk for larger tumor burden, increased metastasis, and poor response to endocrine therapy. Obesity affects breast carcinogenesis by autocrine and paracrine effects of adipocytokine leptin and IGF-1. We have previously shown that leptin induces growth stimulation of breast cancer cells by recruiting histone acetyltransferases and mediator complex to cyclin D1 promoter via activation of Stat3. In the present study, we found a novel bidirectional crosstalk between IGF-1 and leptin signaling that promotes invasion and migration of triple-negative (ER, PR, HER2 negative) breast cancer cells MDA-MB-231, MDA-MB-468, MDA-MB-435, and HCC-1806. Methods and Results: IGF-1 induced significant tyrosine phosphorylation of leptin receptor (Ob-Rb) and leptin induced tyrosine phosphorylation of IGF-1 receptor (IGF-1R). Combined treatment of leptin and IGF-1 induced synergistic activation of both Ob-Rb and IGF-1R along with activation of Akt and ERK. Furthermore, IGF-1 and leptin synergistically transactivated epidermal growth factor receptor (EGFR) and induced proliferation of triple negative breast cancer cells. We also found that tyrosine phosphorylation of EGFR induced by combined treatment of leptin and IGF-1 was significantly inhibited not only by EGFR tyrosine kinase inhibitor AG1478 but also with a broad-spectrum matrix metalloproteinase inhibitor, GM6001, indicating that leptin and IGF-1 induced EGFR transactivation is dependent on proteolytic release of EGFR ligands. Intriguingly, we also found that combined treatment of leptin and IGF-1 potently induced invasion of triple negative breast cancer cells in Matrigel invasion and quantitative Electric Cell-Substrate Impedance Sensing invasion assays. Inhibition of leptin and IGF-1 mediated EGFR activation using a highly potent, reversible inhibitor of HER1/EGFR tyrosine kinase (erlotinib) or dual EGFR/HER2 kinase inhibitor (lapatinib) effectively inhibited leptin and IGF-1 induced invasion. Also, inhibition of EGFR activation using erlotinib and lapatinib reduced leptin- and IGF-1 induced migration of triple negative breast cancer cells. Conclusions: Taken together, these data indicate a novel bidirectional crosstalk between leptin and IGF-1 signaling that transactivates EGFR and promotes metastatic properties, invasion, and migration of triple negative breast carcinoma cells. Our novel findings indicate the possibility of using EGFR inhibitors erlotinib and lapatinib to counter the pro-cancerous effects of leptin and IGF-1 signaling in triple negative breast cancers.