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Bone metastases are a frequent complication of cancer that are associated with considerable morbidity. Current treatments may temporarily palliate the symptoms of bone metastases but often fail to delay their progression. Bones provide a permissive environment because they are characterized by dynamic turnover, secreting factors required for bone maintenance but also stimulating the establishment and growth of metastases. Insulin-like growth factors (IGF) are the most abundant growth factors in bone and are required for normal skeletal development and function. Via activation of the IGF-1 receptors (IGF-1R) and variant insulin receptors, IGFs promote cancer progression, aggressiveness, and treatment resistance. Of specific relevance to bone biology, IGFs contribute to the homing, dormancy, colonization, and expansion of bone metastases. Furthermore, preclinical evidence suggests that tumor cells can be primed to metastasize to bone by a high IGF-1 environment in the primary tumor, suggesting that bone metastases may reflect IGF dependency. Therapeutic targeting of the IGF axis may therefore provide an effective method for treating bone metastases. Indeed, anti–IGF-1R antibodies, IGF-1R tyrosine kinase inhibitors, and anti–IGF-1/2 antibodies have demonstrated antitumor activity in preclinical models of prostate and breast cancer metastases, either alone or in combination with other agents. Several studies suggest that such treatments can inhibit bone metastases without affecting growth of the primary tumor. Although previous trials of anti–IGF-1R drugs have generated negative results in unselected patients, these considerations suggest that future clinical trials of IGF-targeted agents may be warranted in patients with bone metastases.

Purpose: Insulin-like growth factor 1 (IGF1) signaling regulates breast cancer initiation and progression and associated cancer phenotypes. We previously identified E-cadherin (CDH1) as a repressor of IGF1 signaling and in this study examined how loss of E-cadherin affects IGF1R signaling and response to anti-IGF1R/insulin receptor (InsR) therapies in breast cancer. Experimental Design: Breast cancer cell lines were used to assess how altered E-cadherin levels regulate IGF1R signaling and response to two anti-IGF1R/InsR therapies. In situ proximity ligation assay (PLA) was used to define interaction between IGF1R and E-cadherin. TCGA RNA-seq and RPPA data were used to compare IGF1R/InsR activation in estrogen receptor-positive (ER+) invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) tumors. ER+ ILC cell lines and xenograft tumor explant cultures were used to evaluate efficacy to IGF1R pathway inhibition in combination with endocrine therapy. Results: Diminished functional E-cadherin increased both activation of IGF1R signaling and efficacy to anti-IGF1R/InsR therapies. PLA demonstrated a direct endogenous interaction between IGF1R and E-cadherin at points of cell–cell contact. Increased expression of IGF1 ligand and levels of IGF1R/InsR phosphorylation were observed in E-cadherin–deficient ER+ ILC compared with IDC tumors. IGF1R pathway inhibitors were effective in inhibiting growth in ER+ ILC cell lines and synergized with endocrine therapy and similarly IGF1R/InsR inhibition reduced proliferation in ILC tumor explant culture. Conclusions: We provide evidence that loss of E-cadherin hyperactivates the IGF1R pathway and increases sensitivity to IGF1R/InsR targeted therapy, thus identifying the IGF1R pathway as a potential novel target in E-cadherin–deficient breast cancers. Clin Cancer Res; 24(20); 5165–77. ©2018 AACR.

Although major advances in our understanding of the molecular underpinnings of hormone receptor–positive (HR+) breast cancer have led to new therapies that have substantially improved patient outcomes, endocrine-resistant disease still remains a leading cause of breast cancer mortality. Comprehensive molecular profiling of breast cancers has highlighted tremendous tumor heterogeneity, and analysis of paired primary and metastatic tumors has shown the evolution that can occur during acquired resistance to systemic therapies. Novel techniques for monitoring tumor load under treatment pressure, including “liquid biopsy” techniques, such as circulating free tumor DNA (cfDNA) and circulating tumor cells, have shown promise as biomarkers to direct treatment without invasive tumor biopsies. However, more research is needed to deepen our understanding of breast cancer alterations under treatment pressure to reveal mechanisms of drug resistance and apply precision medicine in biomarker-driven clinical trials. Clin Cancer Res; 23(5); 1126–31. ©2016 AACR.

Background: Estrogen receptor-positive (ER+) breast cancer is often intractable due to endocrine therapy resistance. Although ER basepair hotspot mutations have been well characterized in ~20-30% of endocrine-resistant disease, accumulating evidence from our group and others suggests that other genetic alterations such as ER fusions (e.g., ESR1-DAB2) could also play a role. As an important component of liquid biopsies, exosomes have recently gained much attention in the field of clinical oncology. It is well established that some species of RNA, typically microRNA and mRNA, are packaged into these small membrane-enclosed structures, which could potentially serve as a noninvasive biomarker to constantly and comprehensively monitor tumor progression and tumor cell evolution. We tested whether ESR1 fusions could be detected in exosomal RNA (exoRNA) produced by breast cancer cell lines. Methods: Multiple methods were used to characterize exosomes isolated from cell culture media: 1) Nanoparticle tracking analysis (NTA) confirmed concentration and size of exosomes, 2) immunoblotting identified protein markers that are enriched or lacking in exosomes, 3) transmission electron microscopy validated the size and shape of exosomes. During exoRNA extraction, DNA contamination was removed by DNase treatment. Fusion-specific regions in exoRNA were amplified by standard PCR. Results: We used breast cancer cell lines stably infected with cDNA encoding ESR1 fusions ESR1-DAB2 and ESR1-SOX9. NTA shows that our isolated exosomes had a size ranging from 30-150 nm, with the peak at 80 nm. Immunoblotting showed TSG101 and CD81 as positive markers of our exosomes, while Grp94 was negative. By RT-PCR, we identified a fusion-specific region amplified for both ESR1-SOX9 and ESR1-LPP from exoRNA for the fusion cell lines. Conclusions: Collectively, these data indicate that multiple ESR1 fusions are detectable in breast cancer cell line-derived exoRNA. Citation Format: Tiantong Liu, Jagmohan Hooda, Nils Ludwig, Theresa Whiteside, Jennifer M. Atkinson, Steffi Oesterreich, Adrian Lee. Detection of ESR1 gene fusions in breast cancer cell-derived exosomal RNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B58.

Purpose: We previously reported an insulin-like growth factor (IGF) gene expression signature, based on genes induced or repressed by IGF-I, which correlated with poor prognosis in breast cancer. We tested whether the IGF signature was affected by anti–IGF-I receptor (IGF-IR) inhibitors and whether the IGF signature correlated with response to a dual anti–IGF-IR/insulin receptor (InsR) inhibitor, BMS-754807. Experimental Design: An IGF gene expression signature was examined in human breast tumors and cell lines and changes were noted following treatment of cell lines or xenografts with anti–IGF-IR antibodies or tyrosine kinase inhibitors. Sensitivity of cells to BMS-754807 was correlated with levels of the IGF signature. Human primary tumorgrafts were analyzed for the IGF signature and IGF-IR levels and activity, and MC1 tumorgrafts were treated with BMS-754807 and chemotherapy. Results: The IGF gene expression signature was reversed in three different models (cancer cell lines or xenografts) treated with three different anti–IGF-IR therapies. The IGF signature was present in triple-negative breast cancers (TNBC) and TNBC cell lines, which were especially sensitive to BMS-754807, and sensitivity was significantly correlated to the expression of the IGF gene signature. The TNBC primary human tumorgraft MC1 showed high levels of both expression and activity of IGF-IR and IGF gene signature score. Treatment of MC1 with BMS-754807 showed growth inhibition and, in combination with docetaxel, tumor regression occurred until no tumor was palpable. Regression was associated with reduced proliferation, increased apoptosis, and mitotic catastrophe. Conclusions: These studies provide a clear biological rationale to test anti–IGF-IR/InsR therapy in combination with chemotherapy in patients with TNBC. Clin Cancer Res; 17(8); 2314–27. ©2011 AACR.

Purpose: This study aimed to identify molecular determinants of sensitivity of non–small cell lung cancer (NSCLC) to anti–insulin-like growth factor receptor (IGF-IR) therapy. Experimental Design: A total of 216 tumor samples were investigated, of which 165 consisted of retrospective analyses of banked tissue and an additional 51 were from patients enrolled in a phase II study of figitumumab, a monoclonal antibody against IGF-IR, in stage IIIb/IV NSCLC. Biomarkers assessed included IGF-IR, epidermal growth factor receptor, IGF-II, IGF-IIR, insulin receptor substrate 1 (IRS-1), IRS-2, vimentin, and E-cadherin. Subcellular localization of IRS-1 and phosphorylation levels of mitogen-activated protein kinase and Akt1 were also analyzed. Results: IGF-IR was differentially expressed across histologic subtypes (P = 0.04), with highest levels observed in squamous cell tumors. Elevated IGF-IR expression was also observed in a small number of squamous cell tumors responding to chemotherapy combined with figitumumab (P = 0.008). Because no other biomarker/response interaction was observed using classical histologic subtyping, a molecular approach was undertaken to segment NSCLC into mechanism-based subpopulations. Principal component analysis and unsupervised Bayesian clustering identified three NSCLC subsets that resembled the steps of the epithelial to mesenchymal transition: E-cadherin high/IRS-1 low (epithelial-like), E-cadherin intermediate/IRS-1 high (transitional), and E-cadherin low/IRS-1 low (mesenchymal-like). Several markers of the IGF-IR pathway were overexpressed in the transitional subset. Furthermore, a higher response rate to the combination of chemotherapy and figitumumab was observed in transitional tumors (71%) compared with those in the mesenchymal-like subset (32%; P = 0.03). Only one epithelial-like tumor was identified in the phase II study, suggesting that advanced NSCLC has undergone significant dedifferentiation at diagnosis. Conclusion: NSCLC comprises molecular subsets with differential sensitivity to IGF-IR inhibition. Clin Cancer Res; 16(18); 4654–65. ©2010 AACR.

Objective: Therapeutic success currently seen with immune checkpoint blockade (ICB) occurs primarily in immunogenic tumors and can be partly explained by the high rates of non-synonymous genetic mutations (single nucleotide variants, SNV) in tumor cells, leading to neoepitope presentations and robust T-cell infiltration. Despite showing mutations in fewer genes (most frequently TP53), high-grade serous ovarian cancer (HGSOC) displays signs of T-cell inflammation and responds, albeit modestly, to ICB. We focus on immune therapy tailored to the genomic characteristics of the tumor, especially to structural aberrations (SAs), like gene fusions. Our hypothesis is that SAs that are expressed as proteins are antigenic and immunogenic, and represent a source of neoantigens for combination immunotherapies that can overcome locoregional immune suppression. DNA structural aberrations can result in intergenic fusions and intragenic rearrangements, such as exon duplications (dup) and deletions (del). Here we focus on identifying gene fusion and exon dup/del-derived neoepitopes based on RNA-seq data for a combination of retrospective and prospective sample collections. Our hypothesis is that these gene rearrangements resulting from SVs may contribute to the immunogenicity and antigenicity of HGSOC. Methods: FusionCatcher was used to evaluate RNA sequencing data for in-frame gene fusions events in banked human HGSOC. HLAminer was used to determine individual haplotypes. Upon detection of fusion events (validated via PCR) we identified the MHC-I and MHC-II restricted peptides with predicted high or intermediate (IC50 Results: Our results demonstrate neoepitopes-specific human T-cell responses in vitro. RNA-seq data were available for nine patients; eight MHC-I and five MHC-II neoantigens were synthesized from gene fusions from three different patients represented by six sets of haplotype specific PBMCs. In vitro, neoantigen-treated PBMCs showed increased cell clumping. An objective IFN gamma response on ELISA was seen for six peptides at two different concentrations and five peptides at one concentration. Analysis of RNA-seq of murine tumors shows that the metastatic MKP-Lung cells (which carry molecular signatures associated with recurrent ovarian cancer), have an increased number of mutations (SNVs, indels) including gene fusions, in line with findings from human tumors. Using in silico prediction tools, we identified 16 potential peptide binders (IC50< 500), two with strong and 14 with moderate binding. The transcript variants were validated via PCR. The vaccine comprised the top seven MHC-I restricted peptide candidates with IC50 Conclusion: Our results demonstrate that PBMC pulsed with a combination of short and large peptides release increased IFNγ, supporting the proposed vaccine design. Structural variants contribute to tumor immunogenic potential. Immunogenicity data from this project will be further used to refine the target selection for individualized, mutanome-based vaccines. Citation Format: Malcolm S. Ross, Ma Tianzhou, Lixin Zhang, Nolan Priedigkeit, George Tseng, Adrian V. Lee, Robert P. Edwards, Anda M. Vlad. Neoepitope peptide vaccines and immune checkpoint blockade in a new preclinical ovarian cancer model. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B33.

Objective: Primary treatment for ovarian low malignant potential (LMP) tumors is surgical resection with little proven effect of cytotoxic chemotherapy. New therapeutic strategies are needed to prevent and treat recurrence. The objective of this study is to investigate the role of hormone receptors in ovarian low malignant potential tumors. Methods: We obtained formalin-fixed, paraffin embedded (FFPE) tissue samples from patients with ovarian LMP tumors. Clinicopathologic data were collected for all available specimens. Immunohistochemistry (IHC) was performed to determine protein expression. Gene expression was measured using the NanoString nCounter platform. Differences in expression between histologic subtypes were by t-test, followed by a Bonferroni adjustment for multiple comparisons (p Results: Thirty-eight patients were included in this study. The histologic subtypes were divided between 20 serous and 18 mucinous tumors. All serous tumors were ERα and PR positive by IHC and 6 of 20 (30%) were ERβ positive. Only one of the 18 mucinous tumors (5.6%) was positive for both ERα and PR, and all the samples were negative for ERβ. ERβ mRNA levels were elevated in the mucinous LMP tumors and ERα mRNA was higher in the serous samples. ERα and PR protein expression correlated with mRNA expression of ESR1 (r2=0.5606) and PR (r2=0.42), respectively. ERβ expression did not correlate with mRNA expression of ESR2 (r2=0.038). Measurement of estrogen-regulated genes and genes involved in ER signaling (n=236) revealed a clear separation of serous from mucinous subtypes, with 44 genes showing a statistically significant difference (p Conclusion: High levels of ERα, PR, and downstream ERα targets in serous LMP tumors suggest hormone responsiveness and that these tumors are potentially treatable with endocrine therapy. Mucinous tumors largely did not express hormone receptors. In light of this, differences in gene expression observed are more likely attributable to underlying lineage differences than differences in estrogen signaling. Additional large-scale analyses are warranted to better understand the differences between subtypes and to identify avenues for targeted therapy in mucinous LMPs. Citation Format: Sarah E. Taylor, Courtney L. Andersen, Rohit Bhargava, George Tseng, Steffi Oesterreich, Robert P. Edwards, Adrian V. Lee. Hormone receptor expression and response in ovarian tumors of low malignant potential. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B25.

Background: The majority of ovarian cancers (OvCa) are exquisitely sensitive to primary cytoreductive surgery and platinum-based agents, yet up to 80% of late-stage disease will relapse and develop deadly resistance to subsequent therapies. Identifying molecular mediators driving this acquired resistance is essential to improve the tragic 12- to 18-month prognosis for patients with recurrent disease. We hypothesize that relapsed ovarian cancers—which are largely uncharacterized—are molecularly distinct from primary disease and acquire druggable vulnerabilities throughout their life histories. To test this hypothesis, we undertook a transcriptome-wide analysis of 19 longitudinally collected patient-matched pairs of chemotherapy-naïve and recurrent cancers. Materials and Methods: Ilumina TruSeq Total RNA-sequencing was performed on 19 flash-frozen patient-matched pairs of primary and recurrent OvCa. Time to recurrence was up to 64 months with a median of 25 months—a shared variant analysis confirmed all paired samples were patient-matched. Adapter-trimmed RNA-sequencing reads were quantified with k-mer based lightweight-alignment (Salmon v0.8.2) and transcript-abundance estimates were collapsed to gene-level with tximport. Differentially expressed genes were determined with DESeq2 using a paired model to account for patient-matched samples. To identify gains and losses in clinically actionable genes (DGIdb 2.0), pair-specific, outlier fold-change thresholds were defined as Q1/Q3 -/+ [1.5 X IQR], using each pairs’ expression fold-change values (recurrence vs. primary) as the distribution. These discrete, longitudinal transcriptional remodeling events (LTREs) in relapsed OvCa were then assessed for recurrence across all cases. Given that OvCa is thought to be driven largely by genomic structural variation, fusion RNAs were then called with FusionCatcher v0.99.7b. Identified fusions were filtered for cancer specificity by discarding fusions detected in normal tissue (Human Protein Atlas and BodyMap). The same fusion analysis was performed on CCLE OvCa cell line RNA-seq and selected fusions were validated with RT-PCR and Sanger sequencing. Results: A suite of genes were consistently upregulated in OvCa recurrences, the most significant being NTRK2 (adjusted p-value < 0.001)—a targetable tyrosine kinase. LTREs were also common with the most shared LTRE gains in recurrences being INHBA (44%) and IGF1 (39%). 18 of 19 (95%) recurrent cancers acquired cancer-specific fusion RNAs that were undetectable in the primary lesion. An in-frame, recurrence-acquired fusion between TOP2A, a target of doxorubicin and known chemoresistance mediator, and STAU1 was confirmed with RT-PCR. Lastly, we discovered recurrent (2 of 19 cases), in-frame CCDC6-ANK3 fusions that persisted throughout therapy in both the primary and relapsed lesions, each with distinct breakpoints. A CCDC6-ANK3 fusion was also validated in the chemoresistant OVCAR3 cell line. 15 of 19 cases (79%) harbored additional preserved fusions, albeit none were shared between cases. Conclusions: Collectively, these results define multimodal transcriptomic mechanisms of ovarian cancer evolution in late disease. Considering that some acquisitions are highly recurrent and readily druggable (NTRK2, IHBA, IGF1), further preclinical studies are demanded and currently ongoing. Lastly, we establish acquired fusions involving known chemoresistance modulators and preserved fusion transcripts—which are maintained throughout therapy—as common somatic events in OvCa. Because fusion breakpoints are cancer specific, they may serve as promising patient-specific nucleotide targets and biomarkers. Citation Format: Nolan Priedigkeit, Sarah Taylor, Shannon Grabosch, Jahnik Kurukulasuriya, Peter C. Lucas, Silvia Liu, Ester Elishaev, Amit Lugade, Kevin Eng, Anda Vlad, George C. Tseng, Kunle Odunsi, Robert P. Edwards, Adrian V. Lee. Recurrent transcriptional remodeling events and acquired fusion RNAs in relapsed ovarian cancers. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B55.

Purpose: This study aimed to test the ability of a new insulin-like growth factor receptor (IGF-IR) tyrosine kinase inhibitor, BMS-536924, to reverse the ability of constitutively active IGF-IR (CD8-IGF-IR) to transform MCF10A cells, and to examine the effect of the inhibitor on a range of human breast cancer cell lines. Experimental Design: CD8-IGF-IR-MCF10A cells were grown in monolayer culture, three-dimensional (3D) culture, and as xenografts, and treated with BMS-536924. Proliferation, cell cycle, polarity, and apoptosis were measured. Twenty-three human breast cancer cell lines were treated in monolayer culture with BMS-536924, and cell viability was measured. MCF7, MDA-MB-231, and MDA-MB-435 were treated with BMS-536924 in monolayer and 3D culture, and proliferation, migration, polarity, and apoptosis were measured. Results: Treatment of CD8-IGF-IR-MCF10A cells grown in 3D culture with BMS-536924 caused a blockade of proliferation, restoration of apical-basal polarity, and enhanced apoptosis, resulting in a partial phenotypic reversion to normal acini. In monolayer culture, BMS-536924 induced a dose-dependent inhibition of proliferation, with an accumulation of cells in G0/G1,, and completely blocked CD8-IGF-IR–induced migration, invasion, and anchorage-independent growth. CD8-IGF-IR-MCF10A xenografts treated with BMS-536924 (100 mg/kg/day) showed a 76% reduction in xenograft volume. In a series of 23 human breast cancer cell lines, BMS-536924 inhibited monolayer proliferation of 16 cell lines. Most strikingly, treatment of MCF7 cells grown in 3D culture with BMS-536924 caused blockade of proliferation, and resulted in the formation of hollow polarized lumen. Conclusions: These results show that the new small molecule BMS-536924 is an effective inhibitor of IGF-IR, causing a reversion of an IGF-IR–mediated transformed phenotype.

The Fifth International Conference on Recent Advances and Future Directions in Endocrine Manipulation of Breast Cancer, co-chaired by Steven Come, M.D., and Aman Buzdar, M.D., was held in Cambridge, Massachusetts, June 13-14, 2005. The conference was organized with the objective of analyzing recent

Blockade of the estrogen receptor (ER) with antiestrogens and aromatase inhibitors is effective in the treatment of breast cancer. Why ER plays such a dominant role in breast cancer and represents such an excellent target remains to be defined. The ability of ER to respond to multiple inputs and to control expression of multiple downstream genes may be one of the reasons why ER is such a powerful target for breast cancer treatment. The recent modest performance of a number of targeted therapies in breast cancer has raised the question whether we will ever develop therapies that have such success as antiestrogens. Targeted therapies tend to inhibit a single pathway that is probably altered in only a subset of patients. Even within this subset, only a limited number of patients respond. The evidence that virtually all pathways can cross-talk and that they exhibit several layers of redundancy reveals a complexity of signaling networks that may defy the generation of targeted therapies with efficacy similar to antiestrogens. However, there are clearly regulatory nodes that can integrate multiple upstream inputs and elicit diverse downstream outputs. We provide evidence and rationales for integrins, insulin receptor substrates (IRSs), and cyclin D1 as potential therapeutic targets. These proteins, similar to ER, can integrate and coordinate multiple signals in breast cancer cells and thus mediate diverse aspects of breast cancer progression. New treatment targets will emerge in light of more global models of signal transduction that fully integrate all aspects of cell biology such as the role of the extracellular matrix and will hopefully result in the development of targeted therapies that show efficacy similar to antiestrogens.

The Third International Conference on Recent Advances and Future Directions in Endocrine Manipulation of Breast Cancer, cochaired by Steven Come, MD, and Aman Buzdar, MD, was held in Cambridge, Massachusetts, June 21–22, 2003. The conference was organized with the objective of analyzing recent