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Close proximity between cytotoxic T lymphocytes and tumour cells is required for effective immunotherapy. However, what controls the spatial distribution of T cells in the tumour microenvironment is not well understood. Here we couple digital pathology and transcriptome analysis on a large ovarian tumour cohort and develop a machine learning approach to molecularly classify and characterize tumour-immune phenotypes. Our study identifies two important hallmarks characterizing T cell excluded tumours: 1) loss of antigen presentation on tumour cells and 2) upregulation of TGFβ and activated stroma. Furthermore, we identify TGFβ as an important mediator of T cell exclusion. TGFβ reduces MHC-I expression in ovarian cancer cells in vitro. TGFβ also activates fibroblasts and induces extracellular matrix production as a potential physical barrier to hinder T cell infiltration. Our findings indicate that targeting TGFβ might be a promising strategy to overcome T cell exclusion and improve clinical benefits of cancer immunotherapy.

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Purpose: Up to one third of ovarian cancer patients are intrinsically resistant to platinum-based treatment. However, predictive and therapeutic strategies are lacking due to a poor understanding of the underlying molecular mechanisms. This study aimed to identify key molecular characteristics that are associated with primary chemoresistance in epithelial ovarian cancers.Experimental Design: Gene expression profiling was performed on a discovery set of 85 ovarian tumors with clinically well-defined response to chemotherapies as well as on an independent validation dataset containing 138 ovarian patients from the chemotreatment arm of the ICON7 trial.Results: We identified a distinct "reactive stroma" gene signature that is specifically associated with primary chemoresistant tumors and was further upregulated in posttreatment recurrent tumors. Immunohistochemistry (IHC) and RNA in situ hybridization (RNA ISH) analyses on three of the highest-ranked signature genes (POSTN, LOX, and FAP) confirmed that modulation of the reactive stroma signature genes within the peritumoral stromal compartments was specifically associated with the clinical chemoresistance. Consistent with these findings, chemosensitive ovarian cells grown in the presence of recombinant POSTN promoted resistance to carboplatin and paclitaxel treatment in vitro. Finally, we validated the reactive stroma signature in an independent dataset and demonstrated that a high POSTN expression level predicts shorter progression-free survival following first-line chemotherapy.Conclusions: Our findings highlight the important interplay between cancer and the tumor microenvironment in ovarian cancer biology and treatment. The identified reactive stromal components in this study provide a molecular basis to the further development of novel diagnostic and therapeutic strategies for overcoming chemoresistance in ovarian cancer.

Purpose: Tailoring cancer treatment to tumor molecular characteristics promises to make personalized medicine a reality. However, reliable genetic profiling of archived clinical specimens has been hindered by limited sensitivity and high false-positive rates. Here, we describe a novel methodology, MMP-seq, which enables sensitive and specific high-throughput, high-content genetic profiling in archived clinical samples.Experimental Design: We first validated the technical performance of MMP-seq in 66 cancer cell lines and a Latin square cross-dilution of known somatic mutations. We next characterized the performance of MMP-seq in 17 formalin-fixed paraffin-embedded (FFPE) clinical samples using matched fresh-frozen tissue from the same tumors as benchmarks. To demonstrate the potential clinical utility of our methodology, we profiled FFPE tumor samples from 73 patients with endometrial cancer.Results: We demonstrated that MMP-seq enabled rapid and simultaneous profiling of a panel of 88 cancer genes in 48 samples, and detected variants at frequencies as low as 0.4%. We identified DNA degradation and deamination as the main error sources and developed practical and robust strategies for mitigating these issues, and dramatically reduced the false-positive rate. Applying MMP-seq to a cohort of endometrial tumor samples identified extensive, potentially actionable alterations in the PI3K (phosphoinositide 3-kinase) and RAS pathways, including novel PIK3R1 hotspot mutations that may disrupt negative regulation of PIK3CA.Conclusions: MMP-seq provides a robust solution for comprehensive, reliable, and high-throughput genetic profiling of clinical tumor samples, paving the way for the incorporation of genomic-based testing into clinical investigation and practice. Clin Cancer Res; 20(8); 2080-91. Ó2014 AACR.

In our recent studies exploring the biophysical characteristics of resistant cell lipids, and the role they play in drug transport, we demonstrated the difference of drug-resistant breast cancer cells from drug-sensitive cells in lipid composition and biophysical properties, suggesting that cancer cells acquire a drug-resistant phenotype through the alteration of lipid synthesis to inhibit intracellular drug transport to protect from cytotoxic effect. In cancer cells, epigenetic changes (e.g., DNA hypermethylation) are essential to maintain this drug-resistant phenotype. Thus, altered lipid synthesis may be linked to epigenetic mechanisms of drug resistance. We hypothesize that reversing DNA hypermethylation in resistant cells with an epigenetic drug could alter lipid synthesis, changing the cell membrane’s biophysical properties to facilitate drug delivery to overcome drug resistance. Herein we show that treating drug-resistant breast cancer cells (MCF-7/ADR) with the epigenetic drug, 5-aza-2′-deoxycytidine (decitabine), significantly alters cell lipid composition and biophysical properties, causing the resistant cells to acquire biophysical characteristics similar to those of sensitive cell (MCF-7) lipids. Following decitabine treatment, resistant cells demonstrated increased sphingomyelinase activity, resulting in a decreased sphingomyelin level that influenced lipid domain structures, increased membrane fluidity, and reduced P-glycoprotein expression. Changes in the biophysical characteristics of resistant cell lipids facilitated doxorubicin transport and restored endocytic function for drug delivery with a lipid-encapsulated form of doxorubicin, enhancing the drug efficacy. In conclusion, we have established a new mechanism for efficacy of an epigenetic drug, mediated through changes in lipid composition and biophysical properties, in reversing cancer drug resistance.

To the best of our knowledge, this study is the first to compare dual inhibition of PI3K/mammalian target of rapamycin (mTOR) by apitolisib (GDC-0980) against single inhibition of mTORC1 by everolimus in metastatic renal cell carcinoma (mRCC). Patients and MethodsPatients with clear-cell mRCC who progressed on or after vascular endothelial growth factortargeted therapy were randomly assigned to apitolisib 40 mg once per day or to everolimus 10 mg once per day. End points included progression-free survival, safety, overall survival, and objective response rate. Biomarker assessments were conducted. ResultsEighty-five patients were randomly assigned. After 67 events, stratified analysis revealed that median progression-free survival was significantly shorter for apitolisib than for everolimus (3.7 v 6.1 months; hazard ratio, 2.12 [95% CI, 1.23 to 3.63; P , .01]); apitolisib was not favored in any stratification subgroup. Median overall survival was not significantly different but trended in favor of everolimus (16.5 v 22.8 months; hazard ratio, 1.77 [95% CI, 0.97 to 3.24; P = .06]). The objective response rate was 7.1% for apitolisib and 11.6% for everolimus. Patients administered apitolisib with a greater incidence of grade 3 to 4 adverse events were more likely to discontinue treatment (31% v 12% for everolimus). No drug-related deaths were observed. Apitolisib in comparison with everolimus was associated with substantially more high-grade hyperglycemia (40% v 9%) and rash (24% v 2%). Apitolisib pharmacokinetics suggested a relationship between exposure, and rash and hyperglycemia. Retrospective biomarker analyses revealed a relationship between VHL mutation status and outcome with everolimus but not with apitolisib. High hypoxia-inducible factor 1a protein expression was associated with better outcome in both arms. ConclusionThis study demonstrated that dual PI3K/mTOR inhibition by apitolisib was less effective than was everolimus in mRCC, likely because full blockade of PI3K/mTOR signaling resulted in multiple ontarget adverse events. VHL mutation and hypoxia-inducible factor 1a expression may be predictive of an mTOR inhibitor benefit, although prospective validation is required.

BACKGROUND This single arm, open-label trial was designed to evaluate the activity of apitolisib (GDC-0980), a dual PI3K/mTOR inhibitor, in patients with advanced endometrial cancer (EC). METHODS Patients with recurrent or persistent EC treated with 1–2 prior lines of chemotherapy but no prior PI3K/mTOR inhibitor received oral apitolisib 40 mg daily during 28-day cycles until progression or intolerable toxicity. Type I/II diabetic patients requiring insulin were excluded. The primary endpoints were progression-free survival (PFS) at 6 months and objective response rate (ORR). RESULTS A total of 56 women were enrolled including 13 (23%) with well-controlled diabetes. Discontinuation reasons were disease progression (24, 43%), adverse events (13, 23%), and withdrawal by subject (12, 21%). Grade 3/4 apitolisib-related adverse events were hyperglycemia (46%), rash (30%), colitis (5%), and pneumonitis (4%). PFS at 6 months was 20% (KM estimate 95% CI: 7%–33%). ORR was 6% (confirmed). Median PFS was 3.5 months (95% CI: 2.7–3.7 months); median OS was 15.7 months (95% CI: 9.2–17.0 months). Nineteen patients discontinued prior to first tumor assessment. Dose reductions were required for 4 (31%) diabetic and 18 (42%) non-diabetic patients. Comprehensive molecular profiling of 46 evaluable archival tumor samples showed 57% of patients had at least one alteration in PIK3CA, PTEN, or AKT1. All three patients with a confirmed response had at least one alteration in a PI3K pathway gene. CONCLUSION Anti-tumor activity of 40 mg apitolisib daily was limited by tolerability, especially in diabetic patients. Patients with PI3K pathway mutation may have derived enhanced benefit from apitolisib.