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BackgroundThe aggressive clinical behavior of poorly differentiated and anaplastic thyroid cancers (PDTC and ATC) has proven challenging to treat, and survival beyond a few months from diagnosis is rare. Although 30%–60% of these tumors contain mutations in the BRAF gene, inhibitors designed specifically to target oncogenic BRAF have shown limited and only short-lasting therapeutic benefits as single agents, thus highlighting the need for improved treatment strategies, including novel combinations.MethodsUsing a BRAFV600E-driven mouse model of ATC, we investigated the therapeutic efficacy of the combination of BRAF inhibition and oncolytic herpes simplex virus (oHSV). Analyses of samples from tumor-bearing mice were performed to immunologically characterize the effects of different treatments. These immune data were used to inform the incorporation of immune checkpoint inhibitors into triple combination therapies.ResultsWe characterized the immune landscape in vivo following BRAF inhibitor treatment and detected only modest immune changes. We, therefore, hypothesized that the addition of oncolytic virotherapy to BRAF inhibition in thyroid cancer would create a more favorable tumor immune microenvironment, boost the inflammatory status of tumors and improve BRAF inhibitor therapy. First, we showed that thyroid cancer cells were susceptible to infection with oHSV and that this process was associated with activation of the immune tumor microenvironment in vivo. Next, we showed improved therapeutic responses when combining oHSV and BRAF inhibition in vivo, although no synergistic effects were seen in vitro, further confirming that the dominant effect of oHSV in this context was likely immune-mediated. Importantly, both gene and protein expression data revealed an increase in activation of T cells and natural killer (NK) cells in the tumor in combination-treated samples. The benefit of combination oHSV and BRAF inhibitor therapy was abrogated when T cells or NK cells were depleted in vivo. In addition, we showed upregulation of PD-L1 and CTLA-4 following combined treatment and demonstrated that blockade of the PD-1/PD-L1 axis or CTLA-4 further improved combination therapy.ConclusionsThe combination of oHSV and BRAF inhibition significantly improved survival in a mouse model of ATC by enhancing immune-mediated antitumor effects, and triple combination therapies, including either PD-1 or CTLA-4 blockade, further improved therapy.

Background Several prognostic signatures for early oestrogen receptor-positive (ER+) breast cancer have been established with a 10-year follow-up. We tested the hypothesis that signatures optimised for 0–5-year and 5–10-year follow-up separately are more prognostic than a single signature optimised for 10 years. Methods Genes previously identified as prognostic or associated with endocrine resistance were tested in publicly available microarray data set using Cox regression of 747 ER+/HER2− samples from post-menopausal patients treated with 5 years of endocrine therapy. RNA expression of the selected genes was assayed in primary ER+/HER2− tumours from 948 post-menopausal patients treated with 5 years of anastrozole or tamoxifen in the TransATAC cohort. Prognostic signatures for 0–10, 0–5 and 5–10 years were derived using a penalised Cox regression (elastic net). Signature comparison was performed with likelihood ratio statistics. Validation was done by a case-control (POLAR) study in 422 samples derived from a cohort of 1449. Results Ninety-three genes were selected by the modelling of microarray data; 63 of these were significantly prognostic in TransATAC, most similarly across each time period. Contrary to our hypothesis, the derived early and late signatures were not significantly more prognostic than the 18-gene 10-year signature. The 18-gene 10-year signature was internally validated in the TransATAC validation set, showing prognostic information similar to that of Oncotype DX Recurrence Score, PAM50 risk of recurrence score, Breast Cancer Index and IHC4 (score based on four IHC markers), as well as in the external POLAR case-control set. Conclusions The derived 10-year signature predicts risk of metastasis in patients with ER+/HER2− breast cancer similar to commercial signatures. The hypothesis that early and late prognostic signatures are significantly more informative than a single signature was rejected.

Ovarian clear cell carcinoma (OCCC) is a rare subtype of epithelial ovarian cancer characterised by a high frequency of loss-of-function ARID1A mutations and a poor response to chemotherapy. Despite their generally low mutational burden, an intratumoural T cell response has been reported in a subset of OCCC, with ARID1A purported to be a biomarker for the response to the immune checkpoint blockade independent of micro-satellite instability (MSI). However, assessment of the different immune cell types and spatial distribution specifically within OCCC patients has not been described to date. Here, we characterised the immune landscape of OCCC by profiling a cohort of 33 microsatellite stable OCCCs at the genomic, gene expression and histological level using targeted sequencing, gene expression profiling using the NanoString targeted immune panel, and multiplex immunofluorescence to assess the spatial distribution and abundance of immune cell populations at the protein level. Analysis of these tumours and subsequent independent validation identified an immune-related gene expression signature associated with risk of recurrence of OCCC. Whilst histological quantification of tumour-infiltrating lymphocytes (TIL, Salgado scoring) showed no association with the risk of recurrence or ARID1A mutational status, the characterisation of TILs via multiplexed immunofluorescence identified spatial differences in immunosuppressive cell populations in OCCC. Tumour-associated macrophages (TAM) and regulatory T cells were excluded from the vicinity of tumour cells in low-risk patients, suggesting that high-risk patients have a more immunosuppressive microenvironment. We also found that TAMs and cytotoxic T cells were also excluded from the vicinity of tumour cells in ARID1A-mutated OCCCs compared to ARID1A wild-type tumours, suggesting that the exclusion of these immune effectors could determine the host response of ARID1A-mutant OCCCs to therapy. Overall, our study has provided new insights into the immune landscape and prognostic associations in OCCC and suggest that tailored immunotherapeutic approaches may be warranted for different subgroups of OCCC patients.

The tumor microenvironment (TME) is highly heterogeneous and can dictate the success of therapeutic interventions. Identifying TMEs that are susceptible to specific therapeutic interventions paves the way for more personalized and effective treatments. In this study, using a spontaneous murine model of breast cancer, we characterize a TME that is responsive to inhibitors of the heme degradation pathway mediated by heme oxygenase (HO), resulting in CD8+ T cell- and NK cell-dependent tumor control. A hallmark of this TME is a chronic type I interferon (IFN) signal that is directly involved in orchestrating the antitumor immune response. Importantly, we identify that similar TMEs exist in human breast cancer that are associated with patient prognosis. Leveraging these observations, we demonstrate that combining a STING agonist, which induces type I IFN responses, with an HO inhibitor produces a synergistic effect leading to superior tumor control. This study highlights HO activity as a potential resistance mechanism for type I IFN responses in cancer, supporting a therapeutic rationale for targeting the heme degradation pathway to enhance the efficacy of STING agonists.

Understanding the factors, which control ErbB2 and EGF receptor (EGFR) status in cells is likely to inform future therapeutic approaches directed at these potent oncogenes. ErbB2 is resistant to stimulus-induced degradation and high levels of over-expression can inhibit EGF receptor down-regulation. We now show that for HeLa cells expressing similar numbers of EGFR and ErbB2, EGFR down-regulation is efficient and insensitive to reduction of ErbB2 levels. Deubiquitinating enzymes (DUBs) may extend protein half-lives by rescuing ubiquitinated substrates from proteasomal degradation or from ubiquitin-dependent lysosomal sorting. Using a siRNA library directed at the full complement of human DUBs, we identified POH1 (also known as Rpn11 or PSMD14), a component of the proteasome lid, as a critical DUB controlling the apparent ErbB2 levels. Moreover, the effects on ErbB2 levels can be reproduced by administration of proteasomal inhibitors such as epoxomicin used at maximally tolerated doses. However, the extent of this apparent loss and specificity for ErbB2 versus EGFR could not be accounted for by changes in transcription or degradation rate. Further investigation revealed that cell surface ErbB2 levels are only mildly affected by POH1 knock-down and that the apparent loss can at least partially be explained by the accumulation of higher molecular weight ubiquitinated forms of ErbB2 that are detectable with an extracellular but not intracellular domain directed antibody. We propose that POH1 may deubiquitinate ErbB2 and that this activity is not necessarily coupled to proteasomal degradation.

Multi-gene prognostic signatures including the Oncotype® DX Recurrence Score (RS), EndoPredict® (EP) and Prosigna® (Risk Of Recurrence, ROR) are widely used to predict the likelihood of distant recurrence in patients with oestrogen-receptor-positive (ER+), HER2-negative breast cancer. Here, we describe the development and validation of methods to recapitulate RS, EP and ROR scores from NanoString expression data. RNA was available from 107 tumours from postmenopausal women with early-stage, ER+, HER2− breast cancer from the translational Arimidex, Tamoxifen, Alone or in Combination study (TransATAC) where previously these signatures had been assessed with commercial methodology. Gene expression was measured using NanoString nCounter. For RS and EP, conversion factors to adjust for cross-platform variation were estimated using linear regression. For ROR, the steps to perform subgroup-specific normalisation of the gene expression data and calibration factors to calculate the 46-gene ROR score were assessed and verified. Training with bootstrapping ( n  = 59) was followed by validation ( n  = 48) using adjusted, research use only (RUO) NanoString-based algorithms. In the validation set, there was excellent concordance between the RUO scores and their commercial counterparts ( r c (RS) = 0.96, 95% CI 0.93–0.97 with level of agreement (LoA) of −7.69 to 8.12; r c (EP) = 0.97, 95% CI 0.96–0.98 with LoA of −0.64 to 1.26 and r c (ROR) = 0.97 (95% CI 0.94–0.98) with LoA of −8.65 to 10.54). There was also a strong agreement in risk stratification: (RS: κ  = 0.86, p  < 0.0001; EP: κ  = 0.87, p  < 0.0001; ROR: κ  = 0.92, p  < 0.001). In conclusion, the calibrated algorithms recapitulate the commercial RS and EP scores on individual biopsies and ROR scores on samples based on subgroup-centreing method using NanoString expression data.

Background Metastases from primary breast cancers can involve single or multiple organs at metastatic disease diagnosis. Molecular risk factors for particular patterns of metastastic spread in a clinical population are limited. Methods A case-control design including 1357 primary breast cancers was used to study three distinct clinical patterns of metastasis, which occur within the first six months of metastatic disease: bone and visceral metasynchronous spread, bone-only, and visceral-only metastasis. Whole-genome expression profiles were obtained using whole genome (WG)-DASL assays from formalin-fixed paraffin-embedded (FFPE) samples. A systematic protocol was developed for handling FFPE samples together with stringent data quality controls to identify robust expression profiling data. A panel of published and novel gene sets were tested for association with these specific patterns of metastatic spread and odds ratios (ORs) were calculated. Results Metasynchronous metastasis to bone and viscera was found in all intrinsic breast cancer subtypes, while immunohistochemically (IHC)-defined receptor status and specific IntClust subgroups were risk factors for visceral-only or bone-only first metastases. Among gene modules, those related to proliferation increased the risk of metasynchronous metastasis (OR (95% CI) = 2.3 (1.1–4.8)) and visceral-only first metastasis (OR (95% CI) = 2.5 (1.2–5.1)) but not bone-only metastasis (OR (95% CI) = 0.97 (0.56–1.7)). A 21-gene module ( BV ) was identified in estrogen-receptor-positive breast cancers with metasynchronous metastasis to bone and viscera (area under the curve = 0.77), and its expression increased the risk of bone and visceral metasynchronous spread in this population. BV was further orthogonally validated with NanoString nCounter in primary breast cancers, and was reproducible in their matched lymph nodes metastases and an external cohort. Conclusion This case-control study of WG-DASL global expression profiles from FFPE tumour samples, after careful quality control and RNA selection, revealed that gene modules in the primary tumour have differing risks for clinical patterns of metasynchronous first metastases. Moreover, a novel gene module was identified as a putative risk factor for metasynchronous bone and visceral first metastatic spread, with potential implications for disease monitoring and treatment planning.

Background Risk of recurrence from primary ER+ breast cancer continues for at least 20 years. We aimed to identify clinical and molecular features associated with risk of recurrence after 10 years. Methods ER+ breast cancers from patients with and without recurrence were analysed with the BC360 NanoString Panel and an 87 gene targeted-exome panel. Frequency of clinical, pathologic and molecular characteristics was compared between cases (recurred between 10 and 20 years) and controls (no recurrence by 20 years) in the Very Late Recurrence (VLR) cohort. Analogous data from METABRIC were examined to confirm or refute findings. Results VLR cases had larger tumours and higher node positivity. Both VLR and METABRIC cases had higher clinical treatment score at 5 years (CTS5). There was a trend for fewer GATA3 mutations in cases in both VLR and METABRIC but no statistically significant differences in mutation frequency. Cell cycle and proliferation genes were strongly expressed in VLR cases. Immune-related genes and cell cycle inhibitors were highly expressed in controls. Neither of these changes were significant after correction for multiple testing. Conclusions Clinicopathologic features are prognostic beyond 10 years. Conversely, molecular features, such as copy number alterations, TP53 mutations and intrinsic subtype which have early prognostic significance, have little prognostic value after 10 years.

Purpose EndoPredict (EPclin) is a prognostic test validated to inform decisions on adjuvant chemotherapy to endocrine therapy alone for patients with oestrogen receptor-positive, HER2-negative breast cancer. Here, we determine the performance of EPclin for estimating 10-year distant recurrence-free interval (DRFI) rates for those who received adjuvant endocrine therapy (ET) alone compared to those with chemotherapy plus endocrine therapy (ET + C). Methods A total of 3746 women were included in this joint analysis. 2630 patients received 5 years of ET alone (ABCSG-6/8, TransATAC) and 1116 patients received ET + C (GEICAM 2003-02/9906). The primary objective was to evaluate the ability of EPclin to provide an estimate of the 10-year DR rate as a continuous function of EPclin separately for ET alone and ET + C. Cox proportional hazard models were used for these analyses. Results EPclin was highly prognostic for DR in women who received ET alone (HR 2.79 (2.49–3.13), P  < 0.0001) as well as in those who received ET + C (HR 2.27 (1.99–2.59), P  < 0.0001). Women who received ET + C had significantly smaller increases in 10-year DR rates with the increasing EPclin score than those receiving ET alone (EPclin = 5; 12% ET + C vs. 20% ET alone). We observed a significant positive interaction between EPclin and treatment groups ( P - interaction  = 0.022). Conclusions In this comparative non-randomised analysis, the rate of increase in DR with EPclin score was significantly reduced in women who received ET + C versus ET alone. Our indirect comparisons suggest that a high EPclin score can predict chemotherapy benefit in women with ER-positive, HER2-negative disease.

In triple-negative breast cancer, the kinase PIM1, which is highly expressed, functions through the transcription factor c-MYC to promote tumor cell survival and growth. Triple-negative breast cancers (TNBCs) have poor prognosis and lack targeted therapies. Here we identified increased copy number and expression of the PIM1 proto-oncogene in genomic data sets of patients with TNBC. TNBC cells, but not nonmalignant mammary epithelial cells, were dependent on PIM1 for proliferation and protection from apoptosis. PIM1 knockdown reduced expression of the anti-apoptotic factor BCL2, and dynamic BH3 profiling of apoptotic priming revealed that PIM1 prevents mitochondrial-mediated apoptosis in TNBC cell lines. In TNBC tumors and their cellular models, PIM1 expression was associated with several transcriptional signatures involving the transcription factor MYC, and PIM1 depletion in TNBC cell lines decreased, in a MYC-dependent manner, cell population growth and expression of the MYC target gene MCL1 . Treatment with the pan–PIM kinase inhibitor AZD1208 impaired the growth of both cell line and patient-derived xenografts and sensitized them to standard-of-care chemotherapy. This work identifies PIM1 as a malignant-cell-selective target in TNBC and the potential use of PIM1 inhibitors for sensitizing TNBC to chemotherapy-induced apoptotic cell death.