David Balli

Detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next generation sequencing of cell-free circulating tumor DNA.

Purpose: The expanding number of targeted therapeutics for non–small cell lung cancer (NSCLC) necessitates real-time tumor genotyping, yet tissue biopsies are difficult to perform serially and often yield inadequate DNA for next-generation sequencing (NGS). We evaluated the feasibility of using cell-free circulating tumor DNA (ctDNA) NGS as a complement or alternative to tissue NGS. Experimental Design: A total of 112 plasma samples obtained from a consecutive study of 102 prospectively enrolled patients with advanced NSCLC were subjected to ultra-deep sequencing of up to 70 genes and matched with tissue samples, when possible. Results: We detected 275 alterations in 45 genes, and at least one alteration in the ctDNA for 86 of 102 patients (84%), with EGFR variants being most common. ctDNA NGS detected 50 driver and 12 resistance mutations, and mutations in 22 additional genes for which experimental therapies, including clinical trials, are available. Although ctDNA NGS was completed for 102 consecutive patients, tissue sequencing was only successful for 50 patients (49%). Actionable EGFR mutations were detected in 24 tissue and 19 ctDNA samples, yielding concordance of 79%, with a shorter time interval between tissue and blood collection associated with increased concordance (P = 0.038). ctDNA sequencing identified eight patients harboring a resistance mutation who developed progressive disease while on targeted therapy, and for whom tissue sequencing was not possible. Conclusions: Therapeutically targetable driver and resistance mutations can be detected by ctDNA NGS, even when tissue is unavailable, thus allowing more accurate diagnosis, improved patient management, and serial sampling to monitor disease progression and clonal evolution. Clin Cancer Res; 22(23); 5772–82. ©2016 AACR.

Foxm1 transcription factor is required for macrophage migration during lung inflammation and tumor formation

Macrophages have a key role in tumor-associated pulmonary inflammation that supports the proliferation of tumor cells and promotes lung tumor growth. Although increased numbers of tumor-associated macrophages are linked to poor prognosis in lung cancer patients, little is known regarding the transcriptional mechanisms controlling recruitment of macrophages during lung tumorigenesis. Forkhead Box m1 (Foxm1) transcription factor is induced in multiple cell types within tumor lesions and its increased expression is associated with poor prognosis in patients with lung adenocarcinomas. To determine the role of Foxm1 in recruitment of tumor-associated macrophages, a mouse line with macrophage-specific Foxm1 deletion was generated (macFoxm1−/−). Lung tumorigenesis was induced using a 3-methylcholanthrene/butylated hydroxytoluene (BHT; 3,5-di-t-butyl-4-hydroxytoluene) tumor initiation/promotion protocol. Ablation of Foxm1 in macrophages reduced the number and size of lung tumors in macFoxm1−/− mice. Decreased tumorigenesis was associated with diminished proliferation of tumor cells and decreased recruitment of macrophages during the early stages of tumor formation. The expression levels of the pro-inflammatory genes iNOS, Cox-2, interleukin-1b (IL-1b) and IL-6, as well as the migration-related genes macrophage inflammatory protein-1 (MIP-1α), MIP-2 and MMP-12, were decreased in macrophages isolated from macFoxm1−/− mice. Migration of Foxm1-deficient macrophages was reduced in vitro. The chemokine receptors responsible for monocyte recruitment to the lung, CX3CR1 and CXCR4, were decreased in Foxm1-deficient monocytes. In co-transfection experiments, Foxm1 directly bound to and transcriptionally activated the CX3CR1 promoter. Adoptive transfer of wild-type monocytes to macFoxm1−/− mice restored BHT-induced pulmonary inflammation to the levels observed in control mice. Expression of Foxm1 in macrophages is required for pulmonary inflammation, recruitment of macrophages into tumor sites and lung tumor growth.

Endothelial Cell-specific Deletion of Transcription Factor FOXM1 Increases Urethane-induced Lung Carcinogenesis

Vascular endothelial cells provide essential support to the tumor microenvironment, but little is known about the transcriptional control of endothelial functions during tumorigenesis. Here we define a critical role for the Forkhead transcription factor FoxM1 in modulating the development of tumor-associated endothelial cells. Pulmonary tumorigenesis induced by urethane administration was compared in mice genetically deleted for FoxM1 in endothelial cells (enFoxm1(-/-) mice). Notably, lung tumor number and size were increased in enFoxm1(-/-) mice. Increased tumorigenesis was associated with increased proliferation of tumor cells and increased expression of c-Myc and cyclin D1. Furthermore, perivascular infiltration by inflammatory cells was elevated and inflammatory cells in BAL fluid were increased. Expression of Flk-1 (vascular endothelial growth factor receptor 2) and FoxF1, known regulators of pulmonary inflammation, was decreased in enFoxm1(-/-) mice. siRNA-mediated knockdown of FoxM1 in endothelial cells reduced Flk-1 and FoxF1 expression, which was driven by direct transcriptional induction by FoxM1 as target genes. Endothelial specific deletion of FoxM1 in vivo or in vitro also decreased expression of Sfrp1 (secreted frizzled-related protein 1), a known inhibitor of canonical Wnt signaling, in a manner that was associated with increased Wnt signaling. Taken together, our results suggest that endothelial-specific expression of FoxM1 limits lung inflammation and canonical Wnt signaling in lung epithelial cells, thereby restricting lung tumorigenesis.

Foxm1 Expression in Prostate Epithelial Cells is Essential for Prostate Carcinogenesis.

Background: Foxm1 is up-regulated in prostate adenocarcinomas and its expression correlates with the poor prognosis. Results: Conditional depletion of Foxm1 in prostate epithelial cells inhibits tumor cell proliferation, angiogenesis, and metastasis. Conclusion: Foxm1 expression in prostate epithelial cells is essential for prostate carcinogenesis in mouse models. Significance: Foxm1 may play a key role in the pathogenesis of prostate cancer in human patients. The treatment of advanced prostate cancer (PCa) remains a challenge. Identification of new molecular mechanisms that regulate PCa initiation and progression would provide targets for the development of new cancer treatments. The Foxm1 transcription factor is highly up-regulated in tumor cells, inflammatory cells, and cells of tumor microenvironment. However, its functions in different cell populations of PCa lesions are unknown. To determine the role of Foxm1 in tumor cells during PCa development, we generated two novel transgenic mouse models, one exhibiting Foxm1 gain-of-function and one exhibiting Foxm1 loss-of-function under control of the prostate epithelial-specific Probasin promoter. In the transgenic adenocarcinoma mouse prostate (TRAMP) model of PCa that uses SV40 large T antigen to induce PCa, loss of Foxm1 decreased tumor growth and metastasis. Decreased prostate tumorigenesis was associated with a decrease in tumor cell proliferation and the down-regulation of genes critical for cell proliferation and tumor metastasis, including Cdc25b, Cyclin B1, Plk-1, Lox, and Versican. In addition, tumor-associated angiogenesis was decreased, coinciding with reduced Vegf-A expression. The mRNA and protein levels of 11β-Hsd2, an enzyme playing an important role in tumor cell proliferation, were down-regulated in Foxm1-deficient PCa tumors in vivo and in Foxm1-depleted TRAMP C2 cells in vitro. Foxm1 bound to, and increased transcriptional activity of, the mouse 11β-Hsd2 promoter through the −892/−879 region, indicating that 11β-Hsd2 was a direct transcriptional target of Foxm1. Without TRAMP, overexpression of Foxm1 either alone or in combination with inhibition of a p19ARF tumor suppressor caused a robust epithelial hyperplasia, but was insufficient to induce progression from hyperplasia to PCa. Foxm1 expression in prostate epithelial cells is critical for prostate carcinogenesis, suggesting that inhibition of Foxm1 is a promising therapeutic approach for prostate cancer chemotherapy.

Immune cytolytic activity stratifies molecular subsets of human pancreatic cancer

Purpose: Immunotherapy has the potential to improve the dismal prognosis in pancreatic ductal adenocarcinoma (PDA), but clinical trials, including those with single-agent PD-1 or PD-L1 inhibition, have been disappointing. Our aim was to examine the immune landscape of PDA as it relates to aspects of tumor biology, including neoepitope burden. Experimental Design: We used publicly available expression data from 134 primary resection PDA samples from The Cancer Genome Atlas to stratify patients according to a cytolytic T-cell activity expression index. We correlated cytolytic immune activity with mutational, structural, and neoepitope features of the tumor. Results: Human PDA displays a range of intratumoral cytolytic T-cell activity. PDA tumors with low cytolytic activity exhibited significantly increased copy number alterations, including recurrent amplifications of MYC and NOTCH2 and recurrent deletions and mutations of CDKN2A/B. In sharp contrast to other tumor types, high cytolytic activity in PDA did not correlate with increased mutational burden or neoepitope load (MHC class I and class II). Cytolytic-high tumors exhibited increased expression of multiple immune checkpoint genes compared to cytolytic-low tumors, except for PD-L1 expression, which was uniformly low. Conclusions: These data identify a subset of human PDA with high cytolytic T-cell activity. Rather than being linked to mutation burden or neoepitope load, immune activation indices in PDA were inversely linked to genomic alterations, suggesting that intrinsic oncogenic processes drive immune inactivity in human PDA. Furthermore, these data highlight the potential importance of immune checkpoints other than PD-L1/PD-1 as therapeutic targets in this lethal disease. Clin Cancer Res; 23(12); 3129–38. ©2016 AACR.

Tumor Cell-Intrinsic Factors Underlie Heterogeneity of Immune Cell Infiltration and Response to Immunotherapy

Summary The biological and functional heterogeneity between tumors—both across and within cancer types—poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell‐inflamed and non‐T‐cell‐inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co‐injecting tumor cell clones revealed the non‐T‐cell‐inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8+ T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor‐cell‐intrinsic production of the chemokine CXCL1 as a determinant of the non‐T‐cell‐inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell‐intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy. Graphical Abstract Figure. No caption available. HighlightsGenerated a library of congenic pancreatic cancer cell clones derived from KPC miceEach tumor elicited unique immune infiltration correlating with therapeutic responseTumors lacking T cells exhibit different epigenetic and transcriptomic statusCXCL1 was increased in therapy‐resistant tumors that lacked T cell infiltration &NA; Using a library of pancreatic cancer cell clones, Li et al. identify heterogeneous and multifactorial pathways regulating tumor‐cell‐intrinsic mechanisms that dictate the immune microenvironment and thereby responses to immunotherapy. This tumor clone library provides a tool for identifying new targets responsible for thwarting responses to immunotherapy in resistant tumors.

Tumor Immunity and Survival as a Function of Alternative Neopeptides in Human Cancer

Analysis of neopeptides across multiple tumor types revealed that “alternatively defined neopeptides,” derived from tumor-associated mutations, exhibited higher peptide affinity for MHC compared with nonmutant counterparts. These neopeptides were strong predictors of immune phenotype and patient survival. The immune system exerts antitumor activity via T cell–dependent recognition of tumor-specific antigens. Although the number of tumor neopeptides—peptides derived from somatic mutations—often correlates with immune activity and survival, most classically defined high-affinity neopeptides (CDNs) are not immunogenic, and only rare CDNs have been linked to tumor rejection. Thus, the rules of tumor antigen recognition remain incompletely understood. Here, we analyzed neopeptides, immune activity, and clinical outcome from 6,324 patients across 27 tumor types. We characterized a class of “alternatively defined neopeptides” (ADNs), which are mutant peptides predicted to bind MHC (class I or II) with improved affinity relative to their nonmutated counterpart. ADNs are abundant and molecularly distinct from CDNs. The load of ADNs correlated with intratumoral T-cell responses and immune suppression, and ADNs were also strong predictors of patient survival across tumor types. These results expand the spectrum of mutation-derived tumor antigens with potential clinical relevance. Cancer Immunol Res; 6(3); 276–87. ©2018 AACR.

FOXM1 activates AGR2 and causes progression of lung adenomas into invasive mucinous adenocarcinomas

Lung cancer remains one of the most prominent public health challenges, accounting for the highest incidence and mortality among all human cancers. While pulmonary invasive mucinous adenocarcinoma (PIMA) is one of the most aggressive types of non-small cell lung cancer, transcriptional drivers of PIMA remain poorly understood. In the present study, we found that Forkhead box M1 transcription factor (FOXM1) is highly expressed in human PIMAs and associated with increased extracellular mucin deposition and the loss of NKX2.1. To examine consequences of FOXM1 expression in tumor cells in vivo, we employed an inducible, transgenic mouse model to express an activated FOXM1 transcript in urethane-induced benign lung adenomas. FOXM1 accelerated tumor growth, induced progression from benign adenomas to invasive, metastatic adenocarcinomas, and induced SOX2, a marker of poorly differentiated tumor cells. Adenocarcinomas in FOXM1 transgenic mice expressed increased MUC5B and MUC5AC, and reduced NKX2.1, which are characteristics of mucinous adenocarcinomas. Expression of FOXM1 in KrasG12D transgenic mice increased the mucinous phenotype in KrasG12D-driven lung tumors. Anterior Gradient 2 (AGR2), an oncogene critical for intracellular processing and packaging of mucins, was increased in mouse and human PIMAs and was associated with FOXM1. FOXM1 directly bound to and transcriptionally activated human AGR2 gene promoter via the -257/-247 bp region. Finally, using orthotopic xenografts we demonstrated that inhibition of either FOXM1 or AGR2 in human PIMAs inhibited mucinous characteristics, and reduced tumor growth and invasion. Altogether, FOXM1 is necessary and sufficient to induce mucinous phenotypes in lung tumor cells in vivo.

An integrated flow cytometry-based platform for isolation and molecular characterization of circulating tumor single cells and clusters

Comprehensive molecular analysis of rare circulating tumor cells (CTCs) and cell clusters is often hampered by low throughput and purity, as well as cell loss. To address this, we developed a fully integrated platform for flow cytometry-based isolation of CTCs and clusters from blood that can be combined with whole transcriptome analysis or targeted RNA transcript quantification. Downstream molecular signature can be linked to cell phenotype through index sorting. This newly developed platform utilizes in-line magnetic particle-based leukocyte depletion, and acoustic cell focusing and washing to achieve >98% reduction of blood cells and non-cellular debris, along with >1.5 log-fold enrichment of spiked tumor cells. We could also detect 1 spiked-in tumor cell in 1 million WBCs in 4/7 replicates. Importantly, the use of a large 200μm nozzle and low sheath pressure (3.5 psi) minimized shear forces, thereby maintaining cell viability and integrity while allowing for simultaneous recovery of single cells and clusters from blood. As proof of principle, we isolated and transcriptionally characterized 63 single CTCs from a genetically engineered pancreatic cancer mouse model (n = 12 mice) and, using index sorting, were able to identify distinct epithelial and mesenchymal sub-populations based on linked single cell protein and gene expression.

Abstract 3801: A novel sensitive flow-cytometry based platform for isolation and molecular characterization of circulating tumor single cells and clusters

Circulating tumor cells (CTCs) are shed from solid tumors and found at extremely low frequencies in the blood of patients in most cancers. A subset of these cells can seed and give rise to metastases, which is the primary cause of cancer-related mortality. Isolation and characterization of these cells from blood as a ‘liquid biopsy’ can be a sensitive, non-invasive method for early detection, disease monitoring and therapy selection. CTCs can be found even at early disease stages in preclinical models and patients. There is increasing evidence that clusters of CTCs in blood are associated with higher metastatic potential; however, efficient isolation and interrogation of these rare clusters is challenging. In this study, we utilize an in-line rare cell enrichment platform developed by Becton Dickinson (BD), coupled with the BD FACS TM Influx cell sorter to rapidly isolate both single cells and clusters from blood. This platform utilizes magnetic particle-based depletion of unwanted leukocytes and combines acoustic focusing to remove red blood cells and debris while enriching for rare cells of interest. We achieved 30-fold enrichment of tumor cells spiked into blood along with >6-fold improvement in sort efficiency with the enrichment process as compared to without. Importantly, the large 200μm nozzle and low sheath pressure (3.5 psi) on the Influx minimizes shear forces and maintains cell viability and integrity of clusters during sorting, thereby enabling discrimination of single cells and clusters based on side scatter. Using this strategy, we could sort enriched populations of viable cell clusters (range 2-10 cells). Finally, this workflow can be seamlessly integrated with downstream molecular analysis such as next-generation sequencing. We successfully performed whole-transcriptome single-cell sequencing (sc-RNAseq) on cells isolated with this streamlined platform where pooled cells and bulk RNA were used as comparisons. As proof of principle, we used this optimized workflow to isolate and characterize CTCs from a pancreatic cancer mouse model (KPCY) in which all tumor cells are labeled with Yellow Fluorescent Protein (YFP). We sorted YFP+ CTCs and CD45+ white blood cells (WBCs) from the blood of 7 KPCY mice along with YFP+ cells from the matched tumor. We recovered an average of 88 CTCs/ml of blood (range 5–258). RNA sequencing was completed on pooled and single cells from all samples. Epithelial genes including Krt18 and Krt19 as well as lineage specific genes such as Sox9 were significantly overexpressed in CTCs and tumors compared to WBCs, suggesting the utility of this approach. This integrated CTC workflow provides a sensitive approach to identify pancreatic cancer-specific markers, which will be evaluated for their ability to improve sensitivity of positive-selection of CTCs in patient blood and for development of a clinically relevant diagnostic assay. Citation Format: Neha Bhagwat, Charles H. Pletcher, Ling Wang, William DeMuth, Keely Dulmage, David Balli, Stephanie S. Yee, Liping Yu, Jonni S. Moore, Ben Z. Stanger, Eric P. Dixon, Erica L. Carpenter. A novel sensitive flow-cytometry based platform for isolation and molecular characterization of circulating tumor single cells and clusters [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 3801. doi:10.1158/1538-7445.AM2017-3801