Yasmin A. Lyons

Immune cell profiling in cancer: molecular approaches to cell-specific identification

The immune system has many important regulatory roles in cancer development and progression. Given the emergence of effective immune therapies against many cancers, reliable predictors of response are needed. One method of determining response is by evaluating immune cell populations from treated and untreated tumor samples. The amount of material obtained from tumor biopsies can be limited; therefore, gene-based or protein-based analyses may be attractive because they require minimal tissue. Cell-specific signatures are being analyzed with use of the latest technologies, including NanoString’s nCounter technology, intracellular staining flow cytometry, cytometry by time-of-flight, RNA-Seq, and barcoding antibody-based protein arrays. These signatures provide information about the contributions of specific types of immune cells to bulk tumor samples. To date, both tumor tissue and immune cells have been analyzed for molecular expression profiles that can assess genes and proteins that are specific to immune cells, yielding results of varying specificity. Here, we discuss the importance of profiling tumor tissue and immune cells to identify immune-cell-associated genes and proteins and specific gene profiles of immune cells. We also discuss the use of these signatures in cancer treatment and the challenges faced in molecular expression profiling of immune cell populations.

Macrophages Facilitate Resistance to Anti-VEGF Therapy by Altered VEGFR Expression

Purpose: VEGF-targeted therapies have modest efficacy in cancer patients, but acquired resistance is common. The mechanisms underlying such resistance are poorly understood. Experimental Design: To evaluate the potential role of immune cells in the development of resistance to VEGF blockade, we first established a preclinical model of adaptive resistance to anti-VEGF therapy. Additional in vitro and in vivo studies were carried out to characterize the role of macrophages in such resistance. Results: Using murine cancer models of adaptive resistance to anti-VEGF antibody (AVA), we found a previously unrecognized role of macrophages in such resistance. Macrophages were actively recruited to the tumor microenvironment and were responsible for the emergence of AVA resistance. Depletion of macrophages following emergence of resistance halted tumor growth and prolonged survival of tumor-bearing mice. In a macrophage-deficient mouse model, resistance to AVA failed to develop, but could be induced by injection of macrophages. Downregulation of macrophage VEGFR-1 and VEGFR-3 expression accompanied upregulation of alternative angiogenic pathways, facilitating escape from anti-VEGF therapy. Conclusions: These findings provide a new understanding of the mechanisms underlying the modest efficacy of current antiangiogenesis therapies and identify new opportunities for combination approaches for ovarian and other cancers. Clin Cancer Res; 23(22); 7034–46. ©2017 AACR.

Antitumor and antiangiogenic effects of aspirin-PC in ovarian cancer

To determine the efficacy of a novel and safer (for gastrointestinal tract) aspirin (aspirin-PC) in preclinical models of ovarian cancer, in vitro dose–response studies were performed to compare the growth-inhibitory effect of aspirin-PC versus aspirin on three human (A2780, SKOV3ip1, and HeyA8) and a mouse (ID8) ovarian cancer cell line over an 8-day culture period. In the in vivo studies, the aspirin test drugs were studied alone and in the presence of a VEGF-A inhibitor (bevacizumab or B20), due to an emerging role for platelets in tumor growth following antiangiogenic therapy, and we examined their underlying mechanisms. Aspirin-PC was more potent (vs. aspirin) in blocking the growth of both human and mouse ovarian cancer cells in monolayer culture. Using in vivo model systems of ovarian cancer, we found that aspirin-PC significantly reduced ovarian cancer growth by 50% to 90% (depending on the ovarian cell line). The efficacy was further enhanced in combination with Bevacizumab or B20. The growth-inhibitory effect on ovarian tumor mass and number of tumor nodules was evident, but less pronounced for aspirin and the VEGF inhibitors alone. There was no detectable gastrointestinal toxicity. Both aspirin and aspirin-PC also inhibited cell proliferation, angiogenesis, and increased apoptosis of ovarian cancer cells. In conclusion, PC-associated aspirin markedly inhibits the growth of ovarian cancer cells, which exceeds that of the parent drug, in both cell culture and in mouse model systems. We also found that both aspirin-PC and aspirin have robust antineoplastic action in the presence of VEGF-blocking drugs. Mol Cancer Ther; 15(12); 2894–904. ©2016 AACR.

Sequencing of mutational hotspots in cancer-related genes in small cell neuroendocrine cervical cancer

OBJECTIVES Small cell cervical cancer is a rare malignancy with limited treatment options for recurrent disease. We sought to determine if tumor specimens of small cell cervical cancer harbor common somatic mutations and if any of these are actionable. METHODS Using a registry of patients with neuroendocrine cervical cancer, we identified 44 patients with pure or mixed small cell cervical cancer who had undergone mutational analysis. Mutations had been detected using next generation sequencing of mutational hotspots in 50 cancer-related genes. RESULTS Thirty-five mutations were identified in 24 patients (55%). Fifteen of these 24 patients (63%) had 1 mutation, 7 patients (29%) had 2 mutations, and 2 patients (8%) had 3 mutations. In all 44 patients, the most commonly seen mutations were mutations in PIK3CA (8 patients; 18%), KRAS (6 patients; 14%), and TP53 (5 patients; 11%). No other mutation was found in >7% of specimens. Of the 24 patients who had a mutation, 21 (88%) had at least 1 alteration for which there currently exists a class of biological agents targeting that mutation. In the entire cohort of 44 patients, 48% had at least 1 actionable mutation. CONCLUSION Although no single mutation was found in the majority of patients with small cell cervical cancer, almost half had at least 1 actionable mutation. As treatment options for patients with recurrent small cell cervical cancer are currently very limited, molecular testing for targetable mutations, which may suggest potential therapeutic strategies, may be useful for clinicians and patients.

Response to MEK inhibitor in small cell neuroendocrine carcinoma of the cervix with a KRAS mutation

Highlights • Molecular testing may play a role in the determination of targeted therapy treatment options.• Targeted therapy provides treatment options for recurrent cervical cancer.• MEK inhibitor is a treatment option for recurrent cervical cancer in a patient with KRAS mutation.

Macrophage depletion through colony stimulating factor 1 receptor pathway blockade overcomes adaptive resistance to anti-VEGF therapy

Anti-angiogenesis therapy has shown clinical benefit in patients with high-grade serous ovarian cancer (HGSC), but adaptive resistance rapidly emerges. Thus, approaches to overcome such resistance are needed. We developed the setting of adaptive resistance to anti-VEGF therapy, and performed a series of in vivo experiments in both immune competent and nude mouse models. Given the pro-angiogenic properties of tumor-associated macrophages (TAMs) and the dominant role of CSF1R in macrophage function, we added CSF1R inhibitors following emergence of adaptive resistance to anti-VEGF antibody. Mice treated with a CSF1R inhibitor (AC708) after anti-VEGF antibody resistance had little to no measurable tumor burden upon completion of the experiment while those that did not receive a CSF1R inhibitor still had abundant tumor. To mimic clinically used regimens, mice were also treated with anti-VEGF antibody and paclitaxel until resistance emerged, and then a CSF1R inhibitor was added. The addition of a CSF1R inhibitor restored response to anti-angiogenesis therapy, resulting in 83% lower tumor burden compared to treatment with anti-VEGF antibody and paclitaxel alone. Collectively, our data demonstrate that the addition of a CSF1R inhibitor to anti-VEGF therapy and taxane chemotherapy results in robust anti-tumor effects.

Non-16/18 high-risk HPV infection predicts disease persistence and progression in women with an initial interpretation of LSIL

The current management strategy for women with low‐grade squamous intraepithelial lesions (LSILs) is inefficient and expensive because of the inability to identify patients at high risk for disease progression. The current study was designed to determine the genotypic patterns of human papillomavirus (HPV) associated with the persistence and progression of cervical lesions in women with an initial interpretation of LSIL.

Induction of anti-VEGF therapy resistance by upregulated expression of microseminoprotein (MSMP)

Anti-vascular endothelial growth factor (VEGF) therapy has demonstrated efficacy in treating human metastatic cancers, but therapeutic resistance is a practical limitation and most tumors eventually become unresponsive. To identify microenvironmental factors underlying the resistance of cancer to antiangiogenesis therapy, we conducted genomic analyses of intraperitoneal ovarian tumors in which adaptive resistance to anti-VEGF therapy (B20 antibody) developed. We found that expression of the microseminoprotein, prostate-associated (MSMP) gene was substantially upregulated in resistant compared with control tumors. MSMP secretion from cancer cells was induced by hypoxia, triggering MAPK signaling in endothelial cells to promote tube formation in vitro. Recruitment of the transcriptional repressor CCCTC-binding factor (CTCF) to the MSMP enhancer region was decreased by histone acetylation under hypoxic conditions in cancer cells. MSMP siRNA, delivered in vivo using the DOPC nanoliposomes, restored tumor sensitivity to anti-VEGF therapy. In ovarian cancer patients treated with bevacizumab, serum MSMP concentration increased significantly only in non-responders. These findings imply that MSMP inhibition combined with the use of antiangiogenesis drugs may be a new strategy to overcome resistance to antiangiogenesis therapy.

Perioperative inhibition of β-adrenergic and COX2 signaling in a clinical trial in breast cancer patients improves tumor Ki-67 expression, serum cytokine levels, and PBMCs transcriptome

Catecholamines and prostaglandins are secreted abundantly during the perioperative period in response to stress and surgery, and were shown by translational studies to promote tumor metastasis. Here, in a phase-II biomarker clinical trial in breast cancer patients (n = 38), we tested the combined perioperative use of the β-blocker, propranolol, and the COX2-inhibitor, etodolac, scheduled for 11 consecutive perioperative days, starting 5 days before surgery. Blood samples were taken before treatment (T1), on the mornings before and after surgery (T2&T3), and after treatment cessation (T4). Drugs were well tolerated. Results based on a-priori hypotheses indicated that already before surgery (T2), serum levels of pro-inflammatory IL-6, CRP, and IFNγ, and anti-inflammatory, cortisol and IL-10, increased. At T2 and/or T3, drug treatment reduced serum levels of the above pro-inflammatory cytokines and of TRAIL, as well as activity of multiple inflammation-related transcription factors (including NFκB, STAT3, ISRE), but not serum levels of cortisol, IL-10, IL-18, IL-8, VEGF and TNFα. In the excised tumor, treatment reduced the expression of the proliferation marker Ki-67, and positively affected its transcription factors SP1 and AhR. Exploratory analyses of transcriptome modulation in PBMCs revealed treatment-induced improvement at T2/T3 in several transcription factors that in primary tumors indicate poor prognosis (CUX1, THRa, EVI1, RORa, PBX1, and T3R), angiogenesis (YY1), EMT (GATA1 and deltaEF1/ZEB1), proliferation (GATA2), and glucocorticoids response (GRE), while increasing the activity of the oncogenes c-MYB and N-MYC. Overall, the drug treatment may benefit breast cancer patients through reducing systemic inflammation and pro-metastatic/pro-growth biomarkers in the excised tumor and PBMCs.

Adrenergic-mediated increases in INHBA drive CAF phenotype and collagens

Adrenergic signaling is known to promote tumor growth and metastasis, but the effects on tumor stroma are not well understood. An unbiased bioinformatics approach analyzing tumor samples from patients with known biobehavioral profiles identified a prominent stromal signature associated with cancer-associated fibroblasts (CAFs) in those with a high biobehavioral risk profile (high Center for Epidemiologic Studies Depression Scale [CES-D] score and low social support). In several models of epithelial ovarian cancer, daily restraint stress resulted in significantly increased CAF activation and was abrogated by a nonspecific β-blocker. Adrenergic signaling-induced CAFs had significantly higher levels of collagen and extracellular matrix components than control tumors. Using a systems-based approach, we found INHBA production by cancer cells to induce CAFs. Ablating inhibin β A decreased CAF phenotype both in vitro and in vivo. In preclinical models of breast and colon cancers, there were increased CAFs and collagens following daily restraint stress. In an independent data set of renal cell carcinoma patients, there was an association between high depression (CES-D) scores and elevated expression of ACTA2, collagens, and inhibin β A. Collectively, our findings implicate adrenergic influences on tumor stroma as important drivers of CAFs and establish inhibin β A as an important regulator of the CAF phenotype in ovarian cancer.