Changes In Tumor Phenotype Research Articles

Combined Therapeutic Strategies Based on the Inhibition of Non-Oncogene Addiction to Improve Tumor Response in EGFR- and KRAS-Mutant Non-Small-Cell Lung Cancer.

Oncogene-driven NSCLC is usually treated with targeted therapies using tyrosine kinase inhibitors (TKIs) to inhibit oncogene downstream signaling pathways, affecting tumor survival and proliferation. EGFR- and KRAS-mutant NSCLCs are the most represented subtypes, and they are treated in clinical practice with oncogene-targeting drugs in the first and second line, respectively. Unfortunately, the development of oncogene-independent resistant clones limits TKI efficacy. Here, we used non-oncogene addiction (NOA) as an innovative therapeutic strategy to target other essential proteins that support changes in tumor phenotype. Specifically, we tested, for the first time, a combination of inhibitors, namely ATR, involved in DNA damage response, and pyruvate dehydrogenase kinases (PDKs), involved in energy metabolism. Sensitive PC9 and the corresponding EGFR-TKI-resistant PC9/OR, EGFR-mutant H1975, and KRAS-mutant A549 NSCLC cells, were treated with TKIs (osimertinib and selumetinib, respectively). In parallel, cells were exposed to two combination regimens: one using the TKI with an ATR inhibitor and the other one combining the two selected NOA inhibitors (ATR inhibitor, M4344; and PDK inhibitor, DCA). The effect of these two combined approaches, compared to TKI alone, produced similar results in terms of cell proliferation, cell death, and migration. Thus, depending on tumor biology, selecting between the proposed therapeutic strategies will be different, to maximize tumor response. The major translational relevance of this study is to exploit new targets for the development of innovative and improved therapeutic strategies with NOA drugs, over combinations including target genes within the oncogene pathway, to overcome resistance to TKI therapies in patients with NSCLC who are oncogene-addicted.

Image-Guided Precision Medicine in the Diagnosis and Treatment of Pheochromocytomas and Paragangliomas.

In this comprehensive review, we aimed to discuss the current state-of-the-art medical imaging for pheochromocytomas and paragangliomas (PPGLs) diagnosis and treatment. Despite major medical improvements, PPGLs, as with other neuroendocrine tumors (NETs), leave clinicians facing several challenges; their inherent particularities and their diagnosis and treatment pose several challenges for clinicians due to their inherent complexity, and they require management by multidisciplinary teams. The conventional concepts of medical imaging are currently undergoing a paradigm shift, thanks to developments in radiomic and metabolic imaging. However, despite active research, clinical relevance of these new parameters remains unclear, and further multicentric studies are needed in order to validate and increase widespread use and integration in clinical routine. Use of AI in PPGLs may detect changes in tumor phenotype that precede classical medical imaging biomarkers, such as shape, texture, and size. Since PPGLs are rare, slow-growing, and heterogeneous, multicentric collaboration will be necessary to have enough data in order to develop new PPGL biomarkers. In this nonsystematic review, our aim is to present an exhaustive pedagogical tool based on real-world cases, dedicated to physicians dealing with PPGLs, augmented by perspectives of artificial intelligence and big data.

Abstract 1595: Analysis of spatiotemporal phenotypic heterogeneity in chemoresistant triple negative breast cancer using imaging mass cytometry

Abstract Shifts in tumor cell phenotype in response to selective pressures (i.e. changing microenvironments, drug treatments) pose one of the biggest obstacles to successful breast cancer therapies. Phenotypically diverse breast tumor and stroma subpopulations, and interactions between them that alter tumor cell biology, represent unique and spatially distinct niches. We hypothesize that localized neighborhoods of breast tumor cells possess specialized phenotypes that mediate chemoresistance and represent novel therapeutic vulnerabilities. In order to assess these potential phenotypes, we utilized imaging mass cytometry (IMC), a highly multiplexed imaging modality that allows simultaneous measurement of 30-40 antigens while retaining the spatial architecture of the cancer tissue. We constructed an IMC antibody panel that combines markers for tissue architecture, tumor and stromal cell phenotyping, and signaling pathway activation. IMC was applied to patient-derived xenograft (PDX) models of triple negative breast cancer (TNBC).Our TNBC PDX collection was established from tumors obtained before and after neoadjuvant Adriamycin and cyclophosphamide (AC). IMC analysis of 18 PDX models representing eight patients revealed that stromal cell phenotypes were generally shared between all models, but tumor cell phenotypes were largely patient-specific. While every model was comprised primarily of a few major tumor cell phenotypes, we noted that each case also harbored several minor, unique populations, suggesting that specialized neighborhoods may exist within the tumor mass. Comparison of paired PDX models showed a wide range of phenotypic responses to chemotherapy, ranging from stable tumor composition to widespread changes in tumor phenotypes. These phenotypic changes arose despite relatively consistent genomic architecture. Vimentinhi fibroblasts were present more often in post-AC models, while SMAhi fibroblasts were unchanged after treatment. Comparison of pre-/post-AC PDX pairs revealed spatially constrained MAPK activation emerged after treatment. To capture acute changes in tumor phenotype, we treated treatment-naïve PDX models with AC and evaluated tumors by IMC. As tumors regressed and then regrew, we identified novel phenotypic shifts, again including increased MAPK signaling localized to discrete neighborhoods, suggesting this property may be a common feature of chemoresistant TNBC. Analysis of adjacent cells revealed seven distinct neighborhoods, and ongoing work is aimed at determining whether these neighborhoods are altered in response to chemotherapy treatment. Taken together, our findings suggest that distinct tumor phenotypes arise following treatment. Our goal is to determine whether these unique phenotypic niches functionally contribute to chemoresistance and if disruption of these niches enhances chemosensitivity. Citation Format: Amanda L. Rinkenbaugh, Vidya C. Sinha, Pankaj Singh, Yuan Qi, Jiansu Shao, Xiaomei Zhang, Gloria V. Echeverria, W. Fraser Symmans, Stacy L. Moulder, Helen Piwnica-Worms. Analysis of spatiotemporal phenotypic heterogeneity in chemoresistant triple negative breast cancer using imaging mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1595.

Abstract 5540: Tumor phenotypic plasticity and resistance to immune-mediated cytotoxicity in models of castration resistant prostate cancer

Abstract Androgen receptor (AR)-targeted therapies are routinely used to treat prostate cancer, including the use of next-generation hormonal therapies such as abiraterone or enzalutamide. Although these therapies are initially effective, a significant proportion of patients exhibit resistance to treatment, therefore developing castration-resistant prostate cancer (CRPC). While immune checkpoint blockade therapies have led to remarkable clinical responses in patients with several tumor types, CRPC remains resistant to immunotherapy. The characterization of tumor resistance mechanisms has led to the identification of various tumor variants that could emerge along the progression of prostate cancer, including tumors undergoing phenotypic plasticity. Our laboratory has previously shown that phenotypic plasticity is a driver of resistance to immunotherapy. Here we investigated whether changes in tumor phenotype could affect the response of CRPC to immune-based therapies, and ways these mechanisms can be mitigated. Models of resistance to enzalutamide (LNCAP-EnzaR) or abiraterone (LNCAP-AbiR) were derived from the androgen sensitive LNCAP prostate cancer cell line. Resistant and parental LNCAP cells were comparatively evaluated for phenotypic features via RT-PCR, ELISA, western blot, immunofluorescence, and RNAseq analysis. Changes in the susceptibility to NK-cell mediated cytotoxicity were evaluated with NK cells isolated from peripheral blood from healthy donors. LNCAP-EnzaR and LNCAP parental cells were also grown in vivo in NSG MHCI/II-deficient mice, and tumors were characterized for phenotypic markers and potential therapeutic targets. Our results demonstrated that acquisition of resistance to both enzalutamide and abiraterone was associated with a significant increase in mesenchymal tumor features, including increased levels of vimentin and fibronectin, and the loss of epithelial E-cadherin. The susceptibility of LNCAP-Enza-R and LNCAP-AbiR cells to NK-cell mediated cytotoxicity and antibody-dependent cell cytotoxicity (ADCC) were significantly reduced (up to 90%), compared with parental cells. RNAseq analysis identified several upregulated gene and gene pathways, including estrogen receptor 1 (ESR1), which was particularly overexpressed in LNCAP-EnzaR cells. In a xenograft model of LNCAP-EnzaR cells, we corroborated the maintenance of tumor phenotypic plasticity and the expression of actionable targets. Our data indicates that acquisition of resistance to AR inhibition is associated with significant reduction of susceptibility to immune attack, and the acquisition of tumor phenotypic plasticity features. Future studies plan to investigate approaches that revert tumor plasticity, including blockade of increased markers such as ESR1, TGF-beta or IL-8, for potential improvement of tumor susceptibility to immune attack in CRPC. Citation Format: Madeline Dahut, Kristen Fousek, Lucas Horn, Haiyan Qin, Jeffrey Schlom, Claudia Palena. Tumor phenotypic plasticity and resistance to immune-mediated cytotoxicity in models of castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5540.

On optimal temozolomide scheduling for slowly growing glioblastomas.

Temozolomide (TMZ) is an oral alkylating agent active against gliomas with a favorable toxicity profile. It is part of the standard of care in the management of glioblastoma (GBM), and is commonly used in low-grade gliomas (LGG). In-silico mathematical models can potentially be used to personalize treatments and to accelerate the discovery of optimal drug delivery schemes. Agent-based mathematical models fed with either mouse or patient data were developed for the in-silico studies. The experimental test beds used to confirm the results were: mouse glioma models obtained by retroviral expression of EGFR-wt/EGFR-vIII in primary progenitors from p16/p19 ko mice and grown in-vitro and in-vivo in orthotopic allografts, and human GBM U251 cells immobilized in alginate microfibers. The patient data used to parametrize the model were obtained from the TCGA/TCIA databases and the TOG clinical study. Slow-growth "virtual" murine GBMs benefited from increasing TMZ dose separation in-silico. In line with the simulation results, improved survival, reduced toxicity, lower expression of resistance factors, and reduction of the tumor mesenchymal component were observed in experimental models subject to long-cycle treatment, particularly in slowly growing tumors. Tissue analysis after long-cycle TMZ treatments revealed epigenetically driven changes in tumor phenotype, which could explain the reduction in GBM growth speed. In-silico trials provided support for implementation methods in human patients. In-silico simulations, in-vitro and in-vivo studies show that TMZ administration schedules with increased time between doses may reduce toxicity, delay the appearance of resistances and lead to survival benefits mediated by changes in the tumor phenotype in slowly-growing GBMs.

Abstract PR01: Analysis of spatiotemporal phenotypic heterogeneity in chemoresistant triple negative breast cancer using imaging mass cytometry

Abstract Shifts in tumor phenotype in response to selective pressures (i.e. changing microenvironments, drug treatments) pose one of the biggest obstacles to successful cancer therapies. Phenotypically diverse tumor and stroma subpopulations, and interactions between them that alter tumor biology, represent unique signaling niches. We hypothesize that these localized neighborhoods of breast tumor cells possess specialized phenotypes that mediate chemoresistance and represent novel therapeutic vulnerabilities. In order to assess these potential phenotypes, we utilized imaging mass cytometry (IMC), a highly multiplexed imaging modality that allows simultaneous measurement of 30-40 antigens while retaining the spatial architecture of the cancer tissue. We have constructed an IMC antibody panel that combines markers for tissue architecture, tumor and stromal cell phenotyping, and signaling pathway activation. IMC was applied to a collection of patient-derived xenograft (PDX) models of triple negative breast cancer (TNBC). Our PDX collection was established from patient tumors obtained before and after neoadjuvant chemotherapy. IMC analysis of 18 PDX models representing eight patients revealed that stromal cell phenotypes were generally shared between all models, but tumor cell phenotypes were largely patient-specific. While every model was comprised primarily of a few major tumor cell phenotypes, we noted that each case also harbored several minor, unique populations, suggesting that specialized neighborhoods may exist within the tumor mass. Sequential PDX models showed a wide range of phenotypic responses after chemotherapy, ranging from stable tumor composition to widespread changes in tumor phenotypes. Importantly, comparison of multiple pre-/post-chemotherapy PDX pairs revealed spatially constrained MAPK activation emerging after treatment. To capture acute changes in tumor phenotype, we treated animals bearing PDX tumors with chemotherapy and evaluated tumors by IMC. As tumors regressed and then regrew, we identified novel phenotypic shifts, again including increased MAPK signaling localized to discrete neighborhoods, suggesting this phenomenon may be a common feature of chemoresistant TNBC. Taken together, our findings suggest that distinct tumor phenotypes arise following treatment. Ongoing work is examining the spatial arrangement of these cell types to determine their local niche compositions, and then correlating these findings with clinical features. Our goal is to determine whether these unique signaling niches functionally contribute to chemoresistance and if disruption of these niches enhances chemosensitivity. Citation Format: Amanda L. Rinkenbaugh, Vidya C. Sinha, Jiansu Shao, Xiaomei Zhang, Gloria V. Echeverria, W. Fraser Symmans, Stacy L. Moulder, Helen Piwnica-Worms. Analysis of spatiotemporal phenotypic heterogeneity in chemoresistant triple negative breast cancer using imaging mass cytometry [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PR01.

Nuclear-Encoded lncRNA MALAT1 Epigenetically Controls Metabolic Reprogramming in HCC Cells through the Mitophagy Pathway

Mitochondrial dysfunction is a metabolic hallmark of cancer cells. In search of molecular factors involved in this dysregulation in hepatocellular carcinoma (HCC), we found that the nuclear-encoded long noncoding RNA (lncRNA) MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) was aberrantly enriched in the mitochondria of hepatoma cells. Using RNA reverse transcription-associated trap sequencing (RAT-seq), we showed that MALAT1 interacted with multiple loci on mitochondrial DNA (mtDNA), including D-loop, COX2, ND3, and CYTB genes. MALAT1 knockdown induced alterations in the CpG methylation of mtDNA and in mitochondrial transcriptomes. This was associated with multiple abnormalities in mitochondrial function, including altered mitochondrial structure, low oxidative phosphorylation (OXPHOS), decreased ATP production, reduced mitophagy, decreased mtDNA copy number, and activation of mitochondrial apoptosis. These alterations in mitochondrial metabolism were associated with changes in tumor phenotype and in pathways involved in cell mitophagy, mitochondrial apoptosis, and epigenetic regulation. We further showed that the RNA-shuttling protein HuR and the mitochondria transmembrane protein MTCH2 mediated the transport of MALAT1 in this nuclear-mitochondrial crosstalk. This study provides the first evidence that the nuclear genome-encoded lncRNA MALAT1 functions as a critical epigenetic player in the regulation of mitochondrial metabolism of hepatoma cells, laying the foundation for further clarifying the roles of lncRNAs in tumor metabolic reprogramming.

325P - Changes in hormone-receptor status in luminal breast cancers between primary tumour and metastases: Results of the observational cohort GIM-13 AMBRA study

BackgroundTumor phenotype may change during breast cancer (BC) progression and discordance between primary and metastases ranges between 16% and 21% for Hormone Receptor (HR) expression in some series. The impact of this change on patient (pts) management and outcome is not yet clear, even though preliminary data indicate that the molecular characterization of recurrent breast cancer may induce clinicians to modify the treatment choice in ∼14% of the cases and that a change in the receptor status is associated with poorer survival. This analysis evaluates the prognostic impact of receptor discordance between paired primaries and recurrences. MethodsWe used data of 879 pts of the longitudinal cohort AMBRA study, identifying 3 different groups: Group A: HR+ BC remaining HR+ at relapse; Group BHR+ BC becoming TNBC; Group C:TNBC remaining TNBC. Change in HR status was evaluated on the biopsy done at the moment of relapse. Time to event was evaluated by Cox-Mantel Hazard Ratio and Logrank Test; Mean/median by Wilcoxon Rank-Sum Test. ResultsPts who underwent biopsy at the time of relapse were 545/879 (62%), of those 462 (84.4%) were classified as Luminal tumors and 83 (15.2%) TNBC on primary. Change in tumor phenotype occurred in 31 (6.7%) of Luminal BC and 16 (19.3%) TNBC, probably due to a higher genomic instability (p=0.021). Median DFS between Luminal BC was not different: HR+àHR+: 74.56 months (95%CI 66.8-82.1) vs HR+àTNBC: 89.73 months (95%CI:44.73-103.46). Table summarizes main data in time to event in the 3 identified groups. *PFS at 1strelapse; **PFS at 2nd relapse; ***Time to Treatment Change at 1strelapse; °OS at 1strelapse; §OS from 1stPD.Table325PTableHR+/HR+ (range) Group AHR+/TNBC (range) Group Bp value Group A vs CTNBC/TNBC (range) Group CPFS1* (months)13.65 (0,77-162,03)9.45 (3,97-52,73)0.368.13 (6.3-10.9)PFS2** (months)6.83 (0,00-57,13)6.12 (2,67-26,30)0.844.20 (0,33-22,17)TTC1*** (months)12.43 (0,13-163,10)8.88 (0,00-48,40)0.507.88 (0,23-27,80)TTC2**** (months)7.45 (0,00-55,97)6.73 (3,77-26,80)nanaOS1° (months)23.1 (20-26)15 (8.7-26.3)0.0313.60 (0,60-25,38)OS (years)8.52 (0,26-115,92)7.86 (0,19-30,67)0.433.58 (2.91-2.39)OS from PD1§ (months)25.8 (0,00-207,53)17.23 (2,67-104,30)0.8217.90 (2,37-53,57) ConclusionsChange in tumor phenotype has a significant impact on OS; BC pts who become TNBC at relapse should be treated in the same way as TNBC pts at diagnosis. Anyway, OS of Luminal BC who change phenotype seems to be better than TNBC. Legal entity responsible for the studyGIM - Gruppo Italiano Mammella. FundingCelgene. DisclosureAll authors have declared no conflicts of interest.

Expression Signatures of Cisplatin- and Trametinib-Treated Early-Stage Medaka Melanomas.

Small aquarium fish models provide useful systems not only for a better understanding of the molecular basis of many human diseases, but also for first-line screening to identify new drug candidates. For testing new chemical substances, current strategies mostly rely on easy to perform and efficient embryonic screens. Cancer, however, is a disease that develops mainly during juvenile and adult stage. Long-term treatment and the challenge to monitor changes in tumor phenotype make testing of large chemical libraries in juvenile and adult animals cost prohibitive. We hypothesized that changes in the gene expression profile should occur early during anti-tumor treatment, and the disease-associated transcriptional change should provide a reliable readout that can be utilized to evaluate drug-induced effects. For the current study, we used a previously established medaka melanoma model. As proof of principle, we showed that exposure of melanoma developing fish to the drugs cisplatin or trametinib, known cancer therapies, for a period of seven days is sufficient to detect treatment-induced changes in gene expression. By examining whole body transcriptome responses we provide a novel route toward gene panels that recapitulate anti-tumor outcomes thus allowing a screening of thousands of drugs using a whole-body vertebrate model. Our results suggest that using disease-associated transcriptional change to screen therapeutic molecules in small fish model is viable and may be applied to pre-clinical research and development stages in new drug discovery.

Cancer Explant Models.

Overcoming the challenges of understanding and treating cancer requires reliable patient-derived models of cancer (PDMCs). For decades, cancer research and therapeutic development relied primarily on cancer cell lines because of their prevalence, reproducibility, and simplicity to maintain. However, findings from research conducted in cell lines are rarely recapitulated in vivo and seldom directly translatable to patients. The tumor microenvironment (TME), tumor-stromal interactions, and associations with host immune cells produce profound changes in tumor phenotype and complexity not captured in traditional monolayer cell culture. In this chapter, we present various cancer explant models and discuss their applicability based on specific research aims. We discuss the appropriateness of these models for basic science questions, drug screening/development, and for personalized, precision medicine. We also consider logistical factors such as resource cost, technical difficulty, and accessibility. We finish this chapter with a practical guide intended to help the reader select the cancer explant model system(s) that best address their research aims.

Extremely Low-Frequency Magnetic Fields and Redox-Responsive Pathways Linked to Cancer Drug Resistance: Insights from Co-Exposure-Based In Vitro Studies.

Electrical devices currently used in clinical practice and common household equipments generate extremely low-frequency magnetic fields (ELF-MF) that were classified by the International Agency for Research on Cancer as “possible carcinogenic.” Assuming that ELF-MF plays a role in the carcinogenic process without inducing direct genomic alterations, ELF-MF may be involved in the promotion or progression of cancers. In particular, ELF-MF-induced responses are suspected to activate redox-responsive intracellular signaling or detoxification scavenging systems. In fact, improved protection against oxidative stress and redox-active xenobiotics is thought to provide critical proliferative and survival advantage in tumors. On this basis, an ever-growing research activity worldwide is attempting to establish whether tumor cells may develop multidrug resistance through the activation of essential cytoprotective networks in the presence of ELF fields, and how this might trigger relevant changes in tumor phenotype. This review builds a framework around how the activity of redox-responsive mediators may be controlled by co-exposure to ELF-MF and reactive oxygen species-generating agents in tumor and cancer cells, in order to clarify whether and how such potential molecular targets could help to minimize or neutralize the functional interaction between ELF-MF and malignancies.

Abstract B191: Synergistic anticancer activity of eribulin plus palbociclib in patient-derived xenograft (PDX) models of ER+/Her2- human breast cancer

Abstract Eribulin is a synthetic analog of the marine sponge natural product halichondrin B. Its clinical formulation is currently approved in numerous countries for treating certain patients with advanced breast cancer or advanced liposarcoma. Eribulin’s anticancer mechanisms include relatively fast antimitotic effects that lead to cell death by apoptosis, and slower complex effects on the tumor microenvironment that include increased vascular perfusion, reduced hypoxia, and changes in phenotype associated with decreased migration and invasiveness in vitro. Such changes in tumor phenotype led us to ask whether eribulin could be successfully combined with inhibitors of cyclin dependent kinases 4 and 6 (cdk 4/6), which work at the G1/S cell cycle checkpoint where phenotypic changes are typically regulated. It is now recognized that patient-derived xenograft (PDX) tumor models more faithfully recapitulate human tumor biology and drug responsiveness than standard xenograft models derived from established human cancer cell lines. Accordingly, in the current study, we used two ER+/PR+/Her2- PDX models developed from patients with luminal B breast cancers, OD-BRE-0192 and OD-BRE-0745, to test in vivo combinations of eribulin and the cdk 4/6 inhibitor palbociclib. Eribulin and palbociclib dose levels were chosen that led to only minimal effects when given alone, and were administered on Q7Dx3 and Q1Dx5[x3] schedules, respectively, with palbociclib withheld the day before and the day of weekly eribulin dosing to avoid cell cycle-based antagonism that could occur between mitotic and G1/S blockers. In the OD-BRE-0745 model only, a fourth cycle of eribulin followed by 5 days of palbociclib was administered starting 13 days after the third eribulin dose. Under these conditions, combining eribulin and palbociclib led to markedly superior anticancer activity in both models (minimum T/C values of 29% and 41%) compared to either agent alone (T/C: 55-67% and 88-98%, respectively). These preclinical PDX results support clinical exploration of eribulin and palbociclib combinations for appropriate patients with ER+/Her2- breast cancers. Citation Format: Kenichi Nomoto, Marc H. de Boisferon, Bruce A. Littlefield. Synergistic anticancer activity of eribulin plus palbociclib in patient-derived xenograft (PDX) models of ER+/Her2- human breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B191.

The Dark Side of IFN-γ: Its Role in Promoting Cancer Immunoevasion.

Interferon-γ (IFN-γ) is a pleiotropic cytokine that has long been praised as an important effector molecule of anti-tumor immunity, capable of suppressing tumor growth through various mechanisms. On the contrary to such a bright side of IFN-γ, it has also been involved in promoting an outgrowth of tumor cells with immunoevasive phenotype suggesting an existence of a dark “tumor-promoting” side effect of IFN-γ. In this review, we will summarize this multi-functional role of IFN-γ in tumor context, how it promotes changes in tumor phenotype towards increased fitness for growth in immunocompetent host. Furthermore, we summarize how IFN-γ is involved in homeostatic or cancer-triggered mechanisms to establish an immunosuppressive tumor microenvironment.

Alterations in three biomarkers (estrogen receptor, progesterone receptor and human epidermal growth factor 2) and the Ki67 index between primary and metastatic breast cancer lesions.

In recurrent breast cancer, the tumor phenotype, as assessed by estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2) status, occasionally changes. This change, in addition to the Ki67 index were evaluated at sites of recurrence and the correlation between changes in tumor phenotype and survival were assessed in breast cancer patients. Comparisons in pathological parameters between primary and metastatic lesions were drawn between ER, PR, HER2, and the Ki67 index in 70 patients with recurrent breast cancer. The association between changes in tumor phenotype and patient survival was assessed. The hormone receptor status changed from positive, in the primary lesions, to negative, in the metastatic lesions in 19.8% (ER) and 39.5% (PR) of patients, respectively. Conversion from negative to positive status was confirmed in 27.2% (ER) and 31.2% (PR) of patients, respectively. A change in HER2 status from negative (primary lesion) to positive (metastatic lesion) occurred in seven patients (10%). The mean Ki67 index of primary lesions with positive hormone receptor status was significantly lower than at sites of recurrence with any hormone receptor status, from 10.9±9.8 standard deviation (SD) to 22.9±18.6 (P=0.031) and 12.2±10.5 SD to 27.4±20.9 (P=0.023), for ER and PR, respectively. The mean overall survival of patients with ER status conversion from positive to negative was 7.4±1.2 standard error (SE) years, and 14.8±1.4 SE years for patients who retained positive ER status (P=0.005, log-rank), with a hazard ratio of 3.44 (95% confidence interval, 1.36-8.33). This difference in survival based upon change in ER status was similarly observed in patients with PR status conversion in the same direction. Thus, ER and PR status conversion at the time of recurrence strongly impact survival, particularly if the change is from positive (primary lesion) to negative (metastatic lesion). Monitoring the biological behavior of breast cancer may benefit a patient by allowing for a novel personalized treatment strategy.

The use of 18F-fluoroestradiol (FES) and 18F-fluorodeoxyglucose (FDG) PET in the evaluation of breast cancer heterogeneity.

11572 Background: 18F-Fluoroestradiol (FES) is an estrogen analogue that has been shown to be a promising biomarker in ER imaging of breast cancer. FES uptake correlates to ER expression, provides qualitative and quantitative assessment of multiple tumor sites simultaneously, and can predict response to endocrine therapies. Tumor heterogeneity is a known feature of metastatic breast cancer. Our work and others has shown that patients that have tumors with high FDG-PET SUV and low FES-PET SUV uptake have a poorer prognosis. Biopsies of metastatic disease may be done initially for diagnosis of metastatic disease, but are generally only performed in the setting of target identification for clinical trials. A change in ER or HER2 expression, however, can result in a change in therapy. FES-PET imaging offers a virtual biopsy and can reveal heterogeneity of the entire tumor burden. Methods: We reviewed our prior evaluations of tumor heterogeneity with FES-PET from 3 different studies. 46 breast cancer patients with metastatic disease (de novo or recurrent) who had biopsy proven ER+ primary breast cancer underwent FES-PET and FDG-PET imaging and a biopsy of a metastatic lesion prior to therapy initiation. ER and HER-2 expression was reviewed. Results: Of the 46 patients, 5 (11%) had ER- metastatic biopsies. One (2%) biopsy changed from ER+/HER-2 neg to ER-/HER-2+, and one (2%) biopsy changed from ER+/HER-2+ to ER+/HER-2-. All 5 patients (11%) with changes in ER/HER2 expression underwent a change in therapy due to the unexpected findings by metastatic biopsy. FDG findings helped to guide selection of biopsy sites. FES quantitative measures correlated with biopsy findings. Conclusions: Biopsy resulted in a change in therapy for > 10 % of patients enrolled in trials of FES imaging. Imaging can help identify heterogeneous tumor locations to assist identification of evolving tumor targets in breast cancer. In addition, FES imaging may reveal a change in tumor phenotype that can ultimately affect choice of therapy. Research Support: P01CA42045, R01CA72064

Abstract PR16: New tools for mapping genetic modifiers of cancer risk in the tumor microenvironment

Abstract The majority of heritable breast cancer risk is unknown. One potential source of “missing heritability” is genetic modifiers in the tumor microenvironment (TME). Although genetic modifiers in the TME have long been suspected, they have rarely been studied and are largely unknown. Here, we used two new techniques: the Consomic Xenograft Model (CXM) and species-specific RNA-seq (SSRS) to map genetic modifiers in the TME. In CXM, human breast cancer xenografts are implanted in immunodeficient consomic rat strains and tracked for tumor progression. Because the rat strains vary by one chromosome (i.e., consomic), whereas the malignant tumor cells do not differ, any observed changes in tumor phenotypes are due to genetic modifiers in the TME and can be localized to one chromosome. The SSRS method uses probabilistic mapping of RNAseq reads to a joint human and rat transcriptome to assess differential expression (DE) in malignant (human) tumor cells and the nonmalignant (rat) TME. Validation of SSRS revealed >99.4% specificity in calling human or rat reads, which was significantly better than conventional RNA-seq. Using CXM, we found that BN-derived genetic variant(s) on rat chromosome 3 significantly reduced growth of MDA-MB-231-Luc (231Luc+) tumors by 49% (P<0.05) in the SS.BN3IL2Rγ CXM strain compared with parental SSIL2Rγ. This coincided with a 3.1-fold (P<0.001) decrease in blood vascular invasion by 231Luc+ tumor cells and 7.3-fold (P<0.05) lower metastatic burden in the lungs in SS.BN3IL2Rγ compared with SSIL2Rγ, despite a paradoxical 27% (P<0.05) increase in blood vessel density (BVD) in SS.BN3IL2Rγ rats. The tumor-associated blood vessels in SS.BN3IL2Rγ rats appeared collapsed and dysfunctional, possibly explaining the decreased tumor growth and metastasis, despite increased BVD. Lymphatic vasculature and lymphogenous metastasis were completely unaffected by the SS.BN3IL2Rγ background, suggesting that the causative variant(s) on BN rat chromosome 3 are vascular cell-type specific. We used SSRS to begin identifying the TME-specific mediators on rat chromosome 3 (RNO3) that inhibit growth and hematogenous metastasis of human 231Luc+ breast cancer xenografts implanted in the SS.BN3IL2Rγ. Compared with SSIL2Rγ tumors, we identified a network of 539 DE transcripts in the TME of SS.BN3IL2Rγ rats, of which 28% (150 genes) reside on RNO3, which was significantly higher (>4-fold; P<0.001) than any other rat chromosome. Moreover, a two-sample Kolmogorov-Smirnov test revealed that the difference in distributions of adjusted p-values for RN03 versus the rest of the genome was highly significantly higher for DE genes (P=3.152e-08) or DE transcripts (P=3.441e-16). Compared with other rat chromosomes, RNO3 also had by far the highest incidence of alternative isoform usage (91% of all instances). Pathway analysis of DE genes using DAVID revealed that the two most significant GO clusters were extracellular matrix (49 genes; P<10-20) and blood vessel development (43 genes; P<10-17), which recapitulated the vascular defects observed in the SS.BN3IL2Rγ tumors. Collectively, our data demonstrate that CXM and SSRS can be used to detect genetic modifiers in the TME. Citation Format: Michael J. Flister, Alexander Stoddard, Shirng-Wern Tsaih, Angela Lemke, Jozef Lazar, Howard Jacob. New tools for mapping genetic modifiers of cancer risk in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr PR16.

Abstract PR08: New tools for mapping genetic modifiers of cancer risk in the tumor microenvironment

Abstract The majority of heritable breast cancer risk is unknown. One potential source of “missing heritability” is genetic modifiers in the tumor microenvironment (TME). Although genetic modifiers in the TME have long been suspected, they have rarely been studied and are largely unknown. Here, we used two new techniques: the Consomic Xenograft Model (CXM) and species-specific RNA-seq (SSRS) to map genetic modifiers in the TME. In CXM, human breast cancer xenografts are implanted in immunodeficient consomic rat strains and tracked for tumor progression. Because the rat strains vary by one chromosome (i.e., consomic), whereas the malignant tumor cells do not differ, any observed changes in tumor phenotypes are due to genetic modifiers in the TME and can be localized to one chromosome. The SSRS method uses probabilistic mapping of RNAseq reads to a joint human and rat transcriptome to assess differential expression (DE) in malignant (human) tumor cells and the nonmalignant (rat) TME. Validation of SSRS revealed >99.4% specificity in calling human or rat reads, which was significantly better than conventional RNA-seq. Using CXM, we found that BN-derived genetic variant(s) on rat chromosome 3 significantly reduced growth of MDA-MB-231-Luc (231Luc+) tumors by 49% (P<0.05) in the SS.BN3IL2Rγ CXM strain compared with parental SSIL2Rγ. This coincided with a 3.1-fold (P<0.001) decrease in blood vascular invasion by 231Luc+ tumor cells and 7.3-fold (P<0.05) lower metastatic burden in the lungs in SS.BN3IL2Rγ compared with SSIL2Rγ, despite a paradoxical 27% (P<0.05) increase in blood vessel density (BVD) in SS.BN3IL2Rγ rats. The tumor-associated blood vessels in SS.BN3IL2Rγ rats appeared collapsed and dysfunctional, possibly explaining the decreased tumor growth and metastasis, despite increased BVD. Lymphatic vasculature and lymphogenous metastasis were completely unaffected by the SS.BN3IL2Rγ background, suggesting that the causative variant(s) on BN rat chromosome 3 are vascular cell-type specific. We used SSRS to begin identifying the TME-specific mediators on rat chromosome 3 (RNO3) that inhibit growth and hematogenous metastasis of human 231Luc+ breast cancer xenografts implanted in the SS.BN3IL2Rγ. Compared with SSIL2Rγ tumors, we identified a network of 539 DE transcripts in the TME of SS.BN3IL2Rγ rats, of which 28% (150 genes) reside on RNO3, which was significantly higher (>4-fold; P<0.001) than any other rat chromosome. Moreover, a two-sample Kolmogorov-Smirnov test revealed that the difference in distributions of adjusted p-values for RN03 versus the rest of the genome was highly significantly higher for DE genes (P=3.152e-08) or DE transcripts (P=3.441e-16). Compared with other rat chromosomes, RNO3 also had by far the highest incidence of alternative isoform usage (91% of all instances). Pathway analysis of DE genes using DAVID revealed that the two most significant GO clusters were extracellular matrix (49 genes; P<10-20) and blood vessel development (43 genes; P<10-17), which recapitulated the vascular defects observed in the SS.BN3IL2Rγ tumors. Collectively, our data demonstrate that CXM and SSRS can be used to detect genetic modifiers in the TME. Citation Format: Michael J. Flister, Alexander Stoddard, Shirng-Wern Tsaih, Angela Lemke, Jozef Lazar, Howard Jacob. New tools for mapping genetic modifiers of cancer risk in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr PR08.

Abstract 3217: NextGen strategies for mapping genetic modifiers in the tumor microenvironment

Abstract The majority of heritable breast cancer risk is unknown. One potential source of “missing heritability” is genetic modifiers in the tumor microenvironment (TME). Although genetic modifiers in the TME have long been suspected, they have rarely been studied and are largely unknown. Here, we used two new techniques: the Consomic Xenograft Model (CXM) and species-specific RNA-seq (SSRS) to map genetic modifiers in the TME. In CXM, human breast cancer xenografts are implanted in immunodeficient consomic rat strains and tracked for tumor progression. Because the rat strains vary by one chromosome (i.e., consomic), whereas the malignant tumor cells do not differ, any observed changes in tumor phenotypes are due to genetic modifiers in the TME and can be localized to one chromosome. The SSRS method uses probabilistic mapping of RNAseq reads to a joint human and rat transcriptome to assess differential expression (DE) in malignant (human) tumor cells and the nonmalignant (rat) TME. Validation of SSRS revealed >99.4% specificity in calling human or rat reads, which was significantly better than conventional RNA-seq. Using CXM, we found that BN-derived genetic variant(s) on rat chromosome 3 significantly reduced growth of MDA-MB-231-Luc (231Luc+) tumors by 49% (P<0.05) in the SS.BN3IL2Rγ CXM strain compared with parental SSIL2Rγ. This coincided with a 3.1-fold (P<0.001) decrease in blood vascular invasion by 231Luc+ tumor cells and 7.3-fold (P<0.05) lower metastatic burden in the lungs in SS.BN3IL2Rγ compared with SSIL2Rγ, despite a paradoxical 27% (P<0.05) increase in blood vessel density (BVD) in SS.BN3IL2Rγ rats. The tumor-associated blood vessels in SS.BN3IL2Rγ rats appeared collapsed and dysfunctional, possibly explaining the decreased tumor growth and metastasis, despite increased BVD. We used SSRS to begin identifying the TME-specific mediators on rat chromosome 3 (RNO3) that inhibit growth and hematogenous metastasis of human 231Luc+ breast cancer xenografts implanted in the SS.BN3IL2Rγ. Compared with SSIL2Rγ tumors, we identified a network of 539 DE transcripts in the TME of SS.BN3IL2Rγ rats, of which 28% (150 genes) reside on RNO3, which was significantly higher (>4-fold; P<0.001) than any other rat chromosome. Moreover, a two-sample Kolmogorov-Smirnov test revealed that the difference in distributions of adjusted p-values for RN03 versus the rest of the genome was highly significantly higher for DE genes (P = 3.152e-08) or DE transcripts (P = 3.441e-16). Compared with other rat chromosomes, RNO3 also had by far the highest incidence of alternative isoform usage (91% of all instances). Pathway analysis of DE genes using DAVID revealed that the two most significant GO clusters were extracellular matrix (49 genes; P<10−20) and blood vessel development (43 genes; P<10−17), which recapitulated the vascular defects observed in the SS.BN3IL2Rγ tumors. Collectively, our data demonstrate that CXM and SSRS can be used to detect genetic modifiers in the TME. Citation Format: Michael Flister, Angela Lemke, Michael Dwinell, Carmen Bergom, James Shull, Howard Jacob. NextGen strategies for mapping genetic modifiers in the tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3217. doi:10.1158/1538-7445.AM2015-3217

Abstract 1500: Development of a Quantitative Immunofluorescence imaging assay to assess β-catenin translocation and multiplex biomarkers for Epithelial-Mesenchymal Transition (EMT) in tumor tissues.

Abstract Background: Wnt/ß-catenin signaling and cadherin-mediated cell adhesion are two processes that promote cell proliferation and epithelial-mesenchymal transition (EMT) of carcinomas. Therapeutic inhibitors of Wnt-induced tumor activation prevent stabilization and nuclear translocation of β-catenin and putatively lead to inhibition of tumor metastasis by upregulation of E-Cadherin and downregulation of N-Cadherin and Vimentin. Methods: A multiplex quantitative immunofluorescence (qIFA) assay was developed to assess changes in levels of EMT biomarkers (E-Cadherin, N-Cadherin, Vimentin/Pancytokeratin) as well as monitor intracellular localization of β-catenin in tumor tissues. A panel of highly specific antibodies against EMT targets, directly-conjugated to various fluorophores, were optimized for multiplex staining of FFPE tissues using colorectal (CRC) and triple-negative breast (TNBC) cancer cell lines and xenograft tissues as tumor controls for target expression. Rat kidney was used as positive control for endogenous biomarker expression in tissues. Image acquisition was performed with Confocal and Epifluorescence microscopy. Image segmentation and quantitative multiplex analysis were performed using NIS Elements and Definiens Tissue Studio software to obtain total mean integrated signal per biomarker within a tissue region of interest. Results: Fitness for purpose evaluation of the biomarkers was performed in vitro with several cell lines and compounds to demonstrate nuclear translocation of β-catenin as well as EMT-mediated changes in E- to N-Cadherin ratio. A431 squamous epithelial cell lines treated with Lysophosphatidic acid (LPA) induced Adherens Junction (AJ) dissociation of membrane E-Cadherin and β-catenin while co-treatment with specific inhibitors to glycogen synthase kinase-3β (GSK-3β) induced a 5-fold increase in the nuclear to cytoplasmic mean intensity ratio of β-catenin after 24h. TGFβ-treatment of the NMuMG mouse epithelial cell line induced a significant decrease in E- to N-Cadherin membrane intensity ratio after 48 and 72h in vitro. Studies with human TNBC or APC-mutated CRC mouse xenograft models using HDAC or Src/Abl kinase inhibitors to compare pre- and post-treatment effects on EMT biomarker staining will be presented. Conclusion: We have developed a quantitative imaging assay that integrates multiplex changes in EMT biomarkers to assay pharmacodynamic activities of potential targeted agents currently in development at the National Cancer Institute (NCI). This assay could also be applied for retrospective analysis of clinical samples to document drug-induced changes in tumor phenotypes. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Tony Navas, Robert J. Kinders, Karun Mutreja, Scott M. Lawrence, Donna Butcher, Melinda Hollingshead, Ralph E. Parchment, Joseph E. Tomaszewski, James H. Doroshow. Development of a Quantitative Immunofluorescence imaging assay to assess β-catenin translocation and multiplex biomarkers for Epithelial-Mesenchymal Transition (EMT) in tumor tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1500. doi:10.1158/1538-7445.AM2013-1500

PI3K/AKT pathway regulates phosphorylation of steroid receptors, hormone independence and tumor differentiation in breast cancer

Using a model of medroxyprogesterone acetate (MPA)-induced mouse mammary tumors that transit through different stages of hormone dependence, we previously reported that the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT (protein kinase B) pathway is critical for the growth of hormone-independent (HI) mammary carcinomas but not for the growth of hormone-dependent (HD) mammary carcinomas. The objective of this work was to explore whether the activation of the PI3K/AKT pathway is responsible for the changes in tumor phenotype and for the transition to autonomous growth. We found that the inhibition of the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway suppresses HI tumor growth. In addition, we were able to induce mammary tumors in mice in the absence of MPA by inoculating HD tumor cells expressing a constitutively active form of AKT1, myristoylated AKT1 (myrAKT1). These tumors were highly differentiated and displayed a ductal phenotype with laminin-1 and cytokeratin 8 expression patterns typical of HI tumors. Furthermore, myrAKT1 increased the tumor growth of IBH-6 and IBH-7 human breast cancer cell lines. In the estrogen-dependent IBH-7 cell line, myrAKT1 induced estrogen-independent growth accompanied by the expression of the adhesion markers focal adhesion kinase and E-cadherin. Finally, we found that cells expressing myrAKT1 exhibited increased phosphorylation of the progesterone receptor at Ser190 and Ser294 and of the estrogen receptor α at Ser118 and Ser167, independently of exogenous MPA or estrogen supply. Our results indicate that the activation of the PI3K/AKT/mTOR pathway promotes tissue architecture remodeling and the activation of steroid receptors.