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Despite being the hallmark of cancer that is responsible for the highest number of deaths, very little is known about the biology of metastasis. Metastatic disease typically manifests after a protracted period of undetectable disease following surgery or systemic therapy, owing to relapse or recurrence. In the case of breast cancer, metastatic relapse can occur months to decades after initial diagnosis and treatment. In this review, we provide an overview of the known key factors that influence metastatic recurrence, with the goal of highlighting the critical unanswered questions that still need to be addressed to make a difference in the mortality of breast cancer patients.

Breast cancers are divided into subtypes with different prognoses and treatment responses based on global differences in gene expression. Luminal breast cancer gene expression and proliferation are driven by estrogen receptor alpha, and targeting this transcription factor is the most effective therapy for this subtype. By contrast, it remains unclear which transcription factors drive the gene expression signature that defines basal-like triple-negative breast cancer, and there are no targeted therapies approved to treat this aggressive subtype. In this study, we utilized integrated genomic analysis of DNA methylation, chromatin accessibility, transcription factor binding, and gene expression in large collections of breast cancer cell lines and patient tumors to identify transcription factors responsible for the basal-like gene expression program. Glucocorticoid receptor (GR) and STAT3 bind to the same genomic regulatory regions, which were specifically open and unmethylated in basal-like breast cancer. These transcription factors cooperated to regulate expression of hundreds of genes in the basal-like gene expression signature, which were associated with poor prognosis. Combination treatment with small-molecule inhibitors of both transcription factors resulted in synergistic decreases in cell growth in cell lines and patient-derived organoid models. This study demonstrates that GR and STAT3 cooperate to regulate the basal-like breast cancer gene expression program and provides the basis for improved therapy for basal-like triple-negative breast cancer through rational combination of STAT3 and GR inhibitors. Significance: This study demonstrates that GR and STAT3 cooperate to activate the canonical gene expression signature of basal-like triple-negative breast cancer and that combination treatment with STAT3 and GR inhibitors could provide synergistic therapeutic efficacy.

Background: Approximately 40% of patients with stage I-III triple-negative breast cancer (TNBC) recur after standard treatment, while the remaining 60% experience long-term disease-free survival (DFS). There are currently no clinical tests to assess the risk of recurrence in TNBC patients. We previously developed and validated an MHCII Immune Activation assay, which measures a gene expression signature that includes the major histocompatibility complex class II (MHCII) pathway genes and markers of activated tumor infiltrating lymphocytes. The MHCII Immune Activation assay was recently shown to accurately assess the risk of recurrence in Basal-like TNBC patients using FFPE tissues from an institutional cohort. A limitation of the previous study is that the patients’ treatment regimens in the institutional cohort were heterogeneous. The goal of this study was to determine if the MHCII Immune Activation assay can accurately assess the DFS in Basal-like TNBC patients who received uniform chemotherapy regimens in the context of a randomized clinical trial. Methods: The MHCII Immune Activation assay was applied to RNA isolated from 60 Basal-like TNBC FFPE specimens from the GEICAM/9906 clinical trial, a multicenter randomized phase III study evaluating adjuvant chemotherapy in node-positive operable BC patients. Thirty nine (65%) of the patients were treated with fluorouracil, epirubicin, and cyclophosphamide (FEC), and 21 (35%) patients received FEC followed by paclitaxel (FEC-P) (median follow-up 9.7 years). The MHCII Immune Activation Score was calculated from the assay using methods described previously, and patients were categorized into a High Risk of Recurrence (High ROR) group using pre-specified thresholds. The association between the MHCII Immune Activation Score and DFS was assessed using Kaplan-Meier analysis and cox proportional hazards models. Results: Across both arms, 32/60 (53.3%) patients were classified into the High ROR group based on their Low MHCII Immune Activation Scores. Patients in the High ROR group had significantly shorter DFS (Hazard Ratio (HR)=2.9 (CI95%:1.27-6.66), p-value=0.0081). Cox proportional hazard regression of the log10 transformed MHCII Immune Activation Score confirmed that MHCII Immune Activation Score is significantly associated with DFS (Wald Test p=0.0127). In the FEC arm, there were 22/39 (56.41%) patients classified into the High ROR group (Low MHCII Immune Activation Score). Patients in the High ROR group in the FEC arm showed a trend toward shorter DFS (HR=1.99 (CI95%:0.74-5.31)), but it was not significant (p-value=0.1595). In the FEC-P arm, there were 10/21 (47.62%) patients classified into the High ROR group (Low MHCII Immune Activation Score). Patients in the High ROR group in the FEC-P arm had significantly shorter DFS (HR=6.28 (CI95%:1.28-30.73), p-value=0.0111). Larger cohorts will be needed to confirm if the association between DFS and MHCII Immune Activation Score is stronger when patients are treated with chemotherapy regimens that include paclitaxel. Conclusions: The MHCII Immune Activation assay can be applied to RNA from FFPE tumor specimens to assess risk of recurrence in TNBC patients. The MHCII Immune Activation Score is associated with risk of recurrence in TNBC patients treated with uniform adjuvant chemotherapy regimens. Citation Format: Miguel Martín, Katherine L Updike, Alvaro Rodríguez-Lescure, Lourdes Calvo, Jesús Herranz, Nuria Martín, Philip S Bernard, Katherine E Varley. The MHCII immune activation assay is prognostic for disease free survival in basal-like TNBC breast cancer patients in the GEICAM/9906 clinical trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-09.

Background In preclinical studies, the expression of vascular endothelial growth factor (VEGF) in hormone receptor-positive breast cancer is associated with estrogen-independent tumor growth and resistance to endocrine therapies. This study investigated whether the addition of bevacizumab, a monoclonal antibody against VEGF, to letrozole enhanced the antitumor activity of the letrozole in the preoperative setting. Methods Postmenopausal women with newly diagnosed stage 2 or 3 estrogen and/or progesterone receptor-positive, HER2-negative breast cancer were randomly assigned (2:1) between letrozole 2.5 mg PO daily plus bevacizumab 15 mg/kg IV every 3 weeks (Let/Bev) and letrozole 2.5 mg PO daily (Let) for 24 weeks prior to definitive surgery. Primary objective was within-arm pathologic complete remission (pCR) rate. Secondary objectives were safety, objective response, and downstaging rate. Results Seventy-five patients were randomized (Let/Bev n = 50, Let n = 25). Of the 45 patients evaluable for pathological response in the Let/Bev arm, 5 (11%; 95% CI, 3.7–24.1%) achieved pCR and 4 (9%; 95% CI, 2.5–21.2%) had microscopic residual disease; no pCRs or microscopic residual disease was seen in the Let arm (0%; 95% CI, 0–14.2%). The rates of downstaging were 44.4% (95% CI, 29.6–60.0%) and 37.5% (95% CI, 18.8–59.4%) in the Let/Bev and Let arms, respectively. Adverse events typically associated with letrozole (hot flashes, arthralgias, fatigue, myalgias) occurred in similar frequencies in the two arms. Hypertension, headache, and proteinuria were seen exclusively in the Let/Bev arm. The rates of grade 3 and 4 adverse events and discontinuation due to adverse events were 18% vs 8% and 16% vs none in the Let/Bev and Let arms, respectively. A small RNA-based classifier predictive of response to preoperative Let/Bev was developed and confirmed on an independent cohort. Conclusion In the preoperative setting, the addition of bevacizumab to letrozole was associated with a pCR rate of 11%; no pCR was seen with letrozole alone. There was additive toxicity with the incorporation of bevacizumab. Responses to Let/Bev can be predicted from the levels of 5 small RNAs in a pretreatment biopsy. Trial registration This trial is registered with ClinicalTrials.gov (Identifier: NCT00161291), first posted on September 12, 2005, and is completed.

Model systems that recapitulate the complexity of human tumors and the reality of variable treatment responses are urgently needed to better understand cancer biology and to develop more effective cancer therapies. Here we report development and characterization of a large bank of patient-derived xenografts (PDX) and matched organoid cultures from tumors that represent some of the greatest unmet needs in breast cancer research and treatment. These include endocrine-resistant, treatment-refractory, and metastatic breast cancers and, in some cases, multiple tumor collections from the same patients. The models can be grown long-term with high fidelity to the original tumors. We show that development of matched PDX and PDX-derived organoid (PDxO) models facilitates high-throughput drug screening that is feasible and cost-effective, while also allowing in vivo validation of results. Our data reveal consistency between drug screening results in organoids and drug responses in breast cancer PDX. Moreover, we demonstrate the feasibility of using these patient-derived models for precision oncology in real time with patient care, using a case of a triple negative breast cancer with early metastatic recurrence as an example. Our results uncovered an FDA-approved drug with high efficacy against the models. Treatment with the PDxO-directed therapy resulted in a complete response for the patient and a progression-free survival period more than three times longer than her previous therapies. This work provides valuable new methods and resources for functional precision medicine and drug development for human breast cancer.Graphical Abstract

Breast cancers can be divided into subtypes with different prognoses and treatment responses based on global gene expression differences. Luminal breast cancer gene expression and proliferation are driven by the transcription factors Estrogen Receptor α (ER), FOXA1 and GATA3. Targeting ER is the most effective therapy for treating luminal breast cancer because ER is the master regulator of the luminal gene expression program. In contrast, it is unclear which transcription factors are responsible for driving the gene expression signature that defines basal-like triple negative breast cancer, and there are no targeted therapies approved to treat this aggressive subtype of the disease. This study utilized integrated analysis of DNA methylation, chromatin accessibility, transcription factor binding, and gene expression in large collections of breast cancer cell lines and patient tumors to identify transcription factors responsible for the basal-like gene expression program. The results of this study indicate that glucocorticoid receptor (GR) and signal transducer and activator of transcription 3 (STAT3) bind to the same genomic regulatory regions that are specifically open and unmethylated in basal-like breast cancer. These transcription factors cooperate to regulate expression of hundreds of genes in the basal-like gene expression signature and these downstream genes are associated with poor prognosis in patients. Furthermore, combination treatment with small molecule drugs that inhibit both transcription factors leads to synergistic decreases in cell proliferation in cell lines and patient-derived organoid models. This study demonstrates that GR and STAT3 cooperate to regulate the basal-like breast cancer gene expression program and provides the basis for improved therapy for basal-like triple negative breast cancer through rational combination of STAT3 and GR inhibitors.

// Joy M. McDaniel 1, 2 , Katherine E. Varley 3 , Jason Gertz 3 , Daniel S. Savic 1 , Brian S. Roberts 1 , Sarah K. Bailey 4 , Lalita A. Shevde 4, 6 , Ryne C. Ramaker 1, 7 , Brittany N. Lasseigne 1 , Marie K. Kirby 1 , Kimberly M. Newberry 1 , E. Christopher Partridge 1 , Angela L. Jones 1 , Braden Boone 1 , Shawn E. Levy 1 , Patsy G. Oliver 5 , Katherine C. Sexton 6 , William E. Grizzle 6 , Andres Forero 6 , Donald J. Buchsbaum 5 , Sara J. Cooper 1 , Richard M. Myers 1 1 HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA 2 The University of Alabama in Huntsville, Huntsville, AL 35899, USA 3 Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, 84112, USA 4 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA 5 Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA 6 University of Alabama at Birmingham Comprehensive Cancer Center, Birmingham, AL 35294, USA 7 Department of Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294, USA Correspondence to: Richard M. Myers, email: rmyers@hudsonalpha.org Keywords: TNBC, STAT3, ChIP-seq, RNA-seq, invasion Received: August 15, 2016 Accepted: November 14, 2016 Published: December 24, 2016 ABSTRACT Breast cancer is a heterogeneous disease comprised of four molecular subtypes defined by whether the tumor-originating cells are luminal or basal epithelial cells. Breast cancers arising from the luminal mammary duct often express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth receptor 2 (HER2). Tumors expressing ER and/or PR are treated with anti-hormonal therapies, while tumors overexpressing HER2 are targeted with monoclonal antibodies. Immunohistochemical detection of ER, PR, and HER2 receptors/proteins is a critical step in breast cancer diagnosis and guided treatment. Breast tumors that do not express these proteins are known as “triple negative breast cancer” (TNBC) and are typically basal-like. TNBCs are the most aggressive subtype, with the highest mortality rates and no targeted therapy, so there is a pressing need to identify important TNBC tumor regulators. The signal transducer and activator of transcription 3 (STAT3) transcription factor has been previously implicated as a constitutively active oncogene in TNBC. However, its direct regulatory gene targets and tumorigenic properties have not been well characterized. By integrating RNA-seq and ChIP-seq data from 2 TNBC tumors and 5 cell lines, we discovered novel gene signatures directly regulated by STAT3 that were enriched for processes involving inflammation, immunity, and invasion in TNBC. Functional analysis revealed that STAT3 has a key role regulating invasion and metastasis, a characteristic often associated with TNBC. Our findings suggest therapies targeting STAT3 may be important for preventing TNBC metastasis.

Approximately 40% of patients with stage I–III triple-negative breast cancer (TNBC) recur after standard treatment, whereas the remaining 60% experience long-term disease-free survival (DFS). There are currently no clinical tests to assess the risk of recurrence in TNBC patients. We previously determined that TNBC patients with MHC class II (MHCII) pathway expression in their tumors experienced significantly longer DFS. To translate this discovery into a clinical test, we developed an MHCII Immune Activation assay, which measures expression of 36 genes using NanoString technology. Preanalytical testing confirmed that the assay is accurate and reproducible in formalin-fixed paraffin-embedded (FFPE) tumor specimens. The assay measurements were concordant with RNA-seq, MHCII protein expression, and tumor-infiltrating lymphocyte counts. In a training set of 44 primary TNBC tumors, the MHCII Immune Activation Score was significantly associated with longer DFS (HR = 0.17; P = 0.015). In an independent validation cohort of 56 primary FFPE TNBC tumors, the Immune Activation Score was significantly associated with longer DFS (HR = 0.19; P = 0.011) independent of clinical stage. An Immune Activation Score threshold for identifying patients with very low risk of relapse in the training set provided 100% specificity in the validation cohort. The assay format enables adoption as a standardized clinical prognostic test for identifying TNBC patients with a low risk of recurrence. Correlative data support future studies to determine if the assay can identify patients in whom chemotherapy can be safely deescalated and patients likely to respond to immunotherapy. Significance: The MHCII Immune Activation assay identifies TNBC patients with a low risk of recurrence, addressing a critical need for prognostic biomarker tests that enable precision medicine for TNBC patients.

Introduction Triple negative breast cancer (TNBC) is clinically to denote women with invasive breast cancer whose tumours lack expression of oestrogen receptor (ER-), progesterone receptor (PR-), or overexpression of HER2/Neu. TNB tumours behave aggressively and are not candidates for ER or HER2/Neu targeted therapy. In previous published study of 47 TNBC patients with RNA-seq data, we discovered that twenty-four genes related MCH pathway exhibited significantly higher expression in tumour tissue from patients who did not relapse, including eleven genes representation of the MHC II pathway. In this study, we further investigated 24 genes in MCH pathway in prognosis of TNBC. This work will lead in future guidance in treatment of TNB based on patient’s’ gene profile and also provides means to assess prognosis in TNBC. Material and methods Forty-seven snap frozen primary TNBC tumour specimens were analysed using RNA-seq. Descriptive analysis and logistic regression model were used to identify genes that can predict TNB good prognosis (no relapsed). Odds ratio and 95% two-sided confidence intervals were presented. The optimal cut point of expression level was determined from the receiver operating characteristic analysis and sensitivity and specificity were estimated. This study has approval of Institutional Review Board. Results and discussions MCH II genes CD74 and CIITA had significantly association with TNBC progression free survival. When TNBC patients had high CIITA, they were 10 times likely to have no relapsed than patients with low expression (odds ratio OR=10.8, 95% CI 2.8–41.9, p=0.006). The sensitivity and specificity is 75% and 78.3% respectively; TNB who had higher expression of CD74 had 3 times more likely to have no relapsed (OR=3.1, 95% CI 0.95–10.28, p=0.0609) with sensitivity and specificity of 62.5% and 65.2% respectively. We used multivariable approach with all 24-gene panel and identified three genes, CIITA, CTSH and KRT14 together can predict no relapsed with 87.5% sensitivity 78.3%. The accuracy is 83% if TNBC had high expression of CIITA, CTSH and KRT14. With a total of 47 patients, we predicted 17 out 22 relapsed, and 21 out 25 no relapsed correctly. The results was validated using the public microarray data from 199 patients with TNBC confirmed. Conclusion Higher expression of genes in MCII pathway, e.g. CIITA, CD74, not only associated with TNBC progression free survival, they also have high accuracy to predict TNBC patient’s no relapse regardless of age, race, and tumour grade and tumour stage.

Introduction: Vascular endothelial growth factor overexpression has been associated with resistance to anti-estrogen therapy (Cancer Res 2008; 68: 6232); our preclinical data showed that anti-VEGF therapy reverse resistance to estrogen therapy. We postulated that anti-VEGF therapy would enhance anti-estrogen therapy and thus designed a pilot study to assess the feasibility and efficacy of neoadjuvant letrozole and bevacizumab in post-menopausal women with stage II/III, ER/PR positive breast cancer. Patients and Methods: Eligible patients were treated with a neo-adjuvant regimen of letrozole, 2.5 mg/day (PO) and bevacizumab 15 mg/kg every 3 weeks (IV) for a total of 24 weeks prior to surgical treatment of their breast cancer. Patients were followed for toxicity at three week intervals and for tumor assessment at 6 week intervals. Research tumor biopsies were taken before and 6 weeks after initiation of therapy. The primary endpoint was pathological complete remission (pCR). Patients with inflammatory breast cancer were excluded. Results: Twenty six patients were enrolled and 25 were treated (one patient had a TIA the day before initiation of therapy). The regimen was well tolerated with 2 patients taken off-study due to uncontrolled hypertension. Objective clinical response occurred in 68% of the patients (17/25), 16% with CR and 52% with partial response (PR). Sixteen percent of the patients (4/25) had clinical stable disease (SD) and 2 patients progressed (PD) while on therapy. Three patients had pCR and 1 patient had microscopic residual tumor cells in the LNs but not in the breast (pCR 16%). Thirty two percent of the patients attained stage 0 or 1 status. None of the pCR patients received adjuvant chemotherapy and none have relapsed after a median follow-up of 6.1 years (range, 5.8+ to 7.5+). Eight of the 13 patients with PR did not receive chemotherapy and only one relapsed with a median follow-up of 6.2 years (range, 3.7 to 7.7+). At a median follow-up of 6.4 years, 88% of the patients have not relapsed and 12% relapsed (1 PD [basal-like], 1 PR [Luminal B], 1 SD [HER2] relapsed at 1.7, 4, and 6.8 years respectively). Of the 17 patients with CR and PR, only 1 has relapsed (6%). Next Generation Sequencing Analysis and evaluation of markers of proliferation/apoptosis are underway. Conclusion: Combination neoadjuvant therapy with letrozole and bevacizumab was well tolerated and resulted in an impressive pCR of 16%. At a median of 6.4 years, the relapse free survival is 88% for all comers and 94% for responding patients (Luminal A and B). Full correlation of clinical and genomic/biomarker analysis will be presented at the time of the meeting. This encouraging data has led The Breast Cancer Translational Research Consortium to complete a randomized phase II trial (TBCRC002) of letrozole ± bevacizumab in this patient population. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-15-02.

Patient-derived xenografts (PDX) are valuable, clinically-relevant models of cancer. Their close genomic, phenotypic, and temporal association with patient tumors makes them well-suited for pre-clinical and co-clinical studies that assess the potential of new therapeutics. However, PDX models are not amenable to large-scale drug sensitivity studies due to their high cost and low throughput capacity. To address this, we established and characterized long-term PDX organoid (PDxO) cultures from breast cancer (BC) PDXs and evaluated their utility in therapeutic studies. To establish PDxO culture conditions, we extensively tested medium supplements, including growth factors, kinase inhibitors, conditioned medium, and antioxidants, along with extracellular matrix composition in 3D gels. Using optimized culture conditions for each BC subtype, we established PDxOs from 16 PDX lines in the HCI series, with a PDX to PDxO success rate of 85%. Around 20% of PDxOs contained aggressive mouse stroma, which had to be eliminated by FACS for long-term culture success. PDxOs were maintained for over 1 year, during which we tracked viability, doubling time, organoid size, genomics, epithelial character, and tumorigenicity. Doubling times stabilized after 60 days of initial culture, with a mean of 6.4±1.7 days for triple negative (TN) lines and 7.2±1 days for ER+ lines. Mean organoid size remained stable, with ER+ PDxOs significantly smaller than TN PDxOs, at 91 and 262 cells/organoid respectively (p=0.0012). PDxOs histologically resembled their PDX counterparts when stained for H&E, Vimentin, and CAM 5.2. RNA-Seq and copy-number variation analyses showed that PDxOs clustered with their PDXs and patient tumors. To assess if culturing affected tumorigenicity, we re-implanted PDxOs into mice following early and late passaging. 5/5 early passage and 5/6 late passage PDxOs engrafted and formed tumors. Resulting tumors showed similar gene expression profiles as their original PDXs by RNA-Seq. These data suggest that PDxOs generally recapitulate the molecular and genomic features of their originating PDXs and patient tumors. Having established PDxO cultures, we evaluated their utility as a therapeutic screening platform. We developed a screen to differentiate compounds with cytotoxic and cytostatic effects. NCI CTEP and clinically-approved BC therapies (n=40) were screened in quadruplicate 8-point dose response curves on all 16 PDxOs across three biological replicates. Drug response profiles were stable across biological replicates, spanning up to 1 year in culture. PDxOs exhibited diverse responses to therapies targeting cIAP1, PI3K, and CHK1/2. Initial work to evaluate concordance between PDxO and in vivo PDX responses returned trending linear correlations between PDxO dose response data and change in PDX growth rate following treatment (R2 = 0.52-0.77; p = 0.045-0.12, across 3 PDXs and 9 compounds). Ongoing work aims to confirm concordance between PDX and PDxO models. Our work demonstrates that PDxO models are cost-efficient, easy to maintain, and grow indefinitely - making them renewable and accessible cancer models. PDxOs are a powerful parallel resource to PDX models, especially useful for efficient determination of PDX drug response. We are currently expanding our PDxO bank to include 100 models which will be deposited with the NCI as part of the PDXNet effort. Citation Format: Katrin P. Guillen, Sandra D. Scherer, Yi Qiao, Satya S. Pathi, James M. Graham, Maihi Fujita, Yoko S. DeRose, Jason Gertz, Gabor T. Marth, Katherine E. Varley, Alana L. Welm, Bryan E. Welm. Predicting breast cancer therapy response using a patient-derived xenograft organoid screening platform [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 LB-038.

Endometrial cancer (EC) is the most common gynecological malignancy among American women. Steadily increasing numbers of newly diagnosed cases and deaths emphasize the necessity of improving current management strategies. Rather than the commonly known two types of EC based on histological classification, the Cancer Genome Atlas (TCGA) Research Network recently reported a reclassification that categorizes EC into four subtypes based on genomic characterization. Molecular similarities between these subtypes and other cancers, including colorectal, ovarian, and breast cancers, were identified. But to date, no prospective validation has delivered an improved understanding on how to translate these findings to the clinic in the form of treatments adapting genomic guidance. Objectives: To evaluate drug response according to TCGA classification using a panel of twenty-seven established endometrial cancer patient-derived xenografts (EC-PDXs). Methods: Twenty-seven EC-PDXs representing various stages of disease and histological subtypes were orthotopically transplanted and propagated over multiple generations. Upon thorough characterization including histology, metastatic status, and molecular features, EC-PDXs were further categorized into the four TCGA molecular subtypes on the basis of somatic copy number variations (CNV), microsatellite instability (MSI), and POLE mutations. A PDX clinical trial (PCT) was performed to re-evaluate current standard treatments in comparison to therapeutic agents adopted from treatment regimens for other cancer types based on their molecular similarities. Results: EC-PDXs established from patients with recurrent disease exhibited minimal activity to first-line chemotherapeutic drugs, as expected, but remained sensitive to other second-line drugs. None of the tested drugs exhibited sufficient activity in POLE EC-PDXs. MSI EC-PDXs were found to be less sensitive to platinum-based treatment and resistant to 5-FU as reported for MSI colorectal cancer. CNV-high EC-PDXs were more sensitive to drugs adopted from current ovarian and breast cancer treatment regimens. Conclusions: This is the first study that demonstrates the utility of TCGA classification for treatment guidance based on a PCT. Patients with recurrent and persistent disease are in desperate need of new therapeutic options. Our findings open up the possibility to utilize drugs which are currently used in the treatment of other cancers for the treatment of EC based on molecular similarities. In addition, this is the first study that successfully demonstrated the use of molecular classification to enable the prediction of drug response, and thus may facilitate the refinement of current EC management in a more precise and personalized way. Citation Format: Chieh-Hsiang Yang, David K. Gaffney, Theresa L. Werner, Elke A. Jarboe, Jason Gertz, Katherine E. Varley, Matthew Peterson, Margit M. Janat-Amsbury. Genomic reclassification of endometrial carcinoma predicts drug response and facilitates refinement of current management based on PDX-guided efficacy outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1756. doi:10.1158/1538-7445.AM2017-1756

Endometrial cancer (EC) is the fourth most frequently diagnosed gynecological cancer among American Women. Steady increased numbers of newly diagnosed cases and deaths necessitates an added attention. Despite survival rates between 60-80% for localized, early stage disease, only marginal advances have been made in the treatment of distant (16%) and recurrent (15% of early stage) EC over the past two decades. New treatment strategies, guided by more predictive preclinical disease models incorporating molecular characteristics are crucial. Recent findings from The Cancer Genome Atlas (TCGA) Research Network suggested that the current histo-pathologically-guided classification of EC may not suffice to direct treatment recommendations, and introduced the potential adequacy of genomic profiling for reclassifying EC. However, these findings are hampered by the lack of appropriate in vivo models, which adequately facilitate obtaining the necessary preclinical evidence before translating findings to genome-guided clinical trials. Objectives: To establish patient derived orthotopic endometrial tumor grafts recapitulating histology, metastasis, and molecular characteristics of the original tumor specimen, and integrating TCGA genomic characterization to facilitate the development of genomic-guided treatment. Methods: Patient derived EC tissues were orthotopically transplanted to murine uteri and propagated over multiple generations. Generated tumor grafts were characterized for metastases, histopathology, and molecular profiles to ensure grafts resemblance of the original tumor samples. Tumor grafts were then further categorized into the four reported TCGA clusters on the basis of mutation frequency, somatic copy number alterations, microsatellite instability, and POLE mutation. Results: Twenty EC tumor grafts were successfully generated. Histological features, including tumor grade and hormone receptor status, as well as genomic profiles were maintained for up to 7 generations. Xenografts were capable to form metastasis closely parallel to clinical relevant sites. Results from a survival study demonstrated that the established tumor grafts reflected clinical stage-, and grade-based disease progression, and recapitulate TCGA cluster-based survival findings. Conclusions: This study reports the first successful generation of orthotopic EC patient-derived tumor grafts, which retain crucial histo-pathological characteristics, the capacity to form distant metastasis following known clinical patterns, as well as recapitulating molecular features of the original human tumor specimen. Additionally, our model can indeed be used as a tool to investigate the need for reclassifying EC into four clusters rather than two simplified disease subtypes and feasibility in directing genomic-guided treatment based on TCGA recommendations. Citation Format: Chieh-Hsiang Yang, Elke A. Jarboe, Jason Gertz, Katherine E. Varley, C. Matthew Peterson, Margit M. Janát-Amsbury. Integrated genomic characterization of endometrial cancer tumor grafts: a step toward genomic-guided treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 648.

Prostate cancer and breast cancer are the most prevalent cancers in men and women, respectively, and pancreatic cancer, while more rare than prostate and breast cancer, is an extremely lethal cancer, with a 5-year survival rate of less than 5%. With the recent advances in next generation sequencing technology, there is an opportunity to identify genomic aberrations that will provide knowledge about the biology driving these cancers and serve as novel diagnostic and prognostic biomarkers for these diseases. We performed genome-wide measurements of mRNA, microRNA, and DNA methylation from tumor and patient-matched non-tumor tissues. We have identified DNA methylation and gene expression signatures associated tumor formation, disease recurrence, and treatment sensitivity. These candidate biomarkers may be useful in predicting clinical outcomes for patients, and may suggest pathways that can be targeted with novel treatment regimens.

Crucial to the adoption of next-generation sequencing within clinical cancer research is the ability to deeply sequence hundreds to thousands of targeted genomic regions across a large number of patient samples. Several targeting (PCR) and capture (hybridization) methods have been developed for next-generation sequencing, however these methods have limited utility with cancer patient specimens due to drawbacks such as large input DNA quantity requirements, wasted off-target sequencing, high cost of reagents, low sample throughput, specialized equipment, and inflexible content design. We developed TargetRich cancer gene panels employing Nested PatchPCR, which enables the targeted sequencing of hundreds of genomic regions in large numbers of patient samples to fully and efficiently utilize next-generation sequencers. Nested PatchPCR required only 250 ng genomic DNA, and was compatible with the fragmented DNA obtained from formalin fixed paraffin embedded samples (FFPE). No special equipment was needed, only a standard PCR machine. Patient samples were processed in parallel in a single 96-well plate. For each patient sample, we simultaneously amplified hundreds of exons from genes that are frequently mutated in cancer. We utilized standard library construction and barcoding methods to sequence targeted genes from 96 patient samples on a single Illumina GAIIx flowcell. The TargetRich cancer gene panels were highly specific; 95% of sequencing reads aligned to the targeted regions. The high percentage of on-target sequence data and focused content, allowed us to achieve high coverage across the loci (average of 400X read depth) for the sensitive detection of cancer mutations. We have created two cancer panels: CR (157 regions covering exons from 10 genes) and CS (760 regions covering exons from 62 genes) for use on the Illumina MiSeq, GAIIx & HiSeq platforms. Additionally, a CR panel for the Ion Torrent PGM has been developed with platform-specific primers added during the Nested PatchPCR reaction, allowing direct sequencing of products without time-consuming library construction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-412. doi:1538-7445.AM2012-LB-412

Read-through fusion transcripts that result from the splicing of two adjacent genes in the same coding orientation are a recently discovered type of chimeric RNA. We sought to determine if read-through fusion transcripts exist in breast cancer. We performed paired-end RNA-seq of 168 breast samples, including 28 breast cancer cell lines, 42 triple negative breast cancer primary tumors, 42 estrogen receptor positive (ER+) breast cancer primary tumors, and 56 non-malignant breast tissue samples. We analyzed the sequencing data to identify breast cancer associated read-through fusion transcripts. We discovered two recurrent read-through fusion transcripts that were identified in breast cancer cell lines, confirmed across breast cancer primary tumors, and were not detected in normal tissues (SCNN1A-TNFRSF1A and CTSD-IFITM10). Both fusion transcripts use canonical splice sites to join the last splice donor of the 5′ gene to the first splice acceptor of the 3′ gene, creating an in-frame fusion transcript. Western blots indicated that the fusion transcripts are translated into fusion proteins in breast cancer cells. Custom small interfering RNAs targeting the CTSD-IFITM10 fusion junction reduced expression of the fusion transcript and reduced breast cancer cell proliferation. Read-through fusion transcripts between adjacent genes with different biochemical functions represent a new type of recurrent molecular defect in breast cancer that warrant further investigation as potential biomarkers and therapeutic targets. Both breast cancer associated fusion transcripts identified in this study involve membrane proteins (SCNN1A-TNFRSF1A and CTSD-IFITM10), which raises the possibility that they could be breast cancer-specific cell surface markers. Electronic supplementary material The online version of this article (doi:10.1007/s10549-014-3019-2) contains supplementary material, which is available to authorized users.