Rachel score: a nomogram model for predicting the prognosis of lung neuroendocrine tumors.

BACKGROUND: Lung NET, classified in typical carcinoids (TC) and atypical carcinoids (AC), are highly heterogeneous in their biology and prognosis. The histological subtype and TNM stage are well-established prognostic factors for lung NET. In a previous work by our group, we demonstrated a significant impact of laterality on lung NET survival outcomes. MATERIALS AND METHODS: We developed a nomogram that integrates relevant prognostic factors to predict lung NET outcomes. By adding the scores for each of the variables included in the model, it was possible to obtain a prognostic score (Rachel score). Wilcoxon non-parametric statistical test was applied among parameters and Harrell's concordance index was used to measure the models' predictive power. To test the discriminatory power and the predictive accuracy of the model, we calculated Gonen and Heller concordance index. Time-dependent ROC curves and their area under the curve (AUC) were used to evaluate the models' predictive performance. RESULTS: By applying Rachel score, we were able to identify three prognostic groups (specifically, high, medium and low risk). These three groups were associate to well-defined ranges of points according to the obtained nomogram (I: 0-90, II: 91-130; III: > 130 points), providing a useful tool for prognostic stratification. The overall survival (OS) and progression free survival (PFS) Kaplan-Meier curves confirmed significant differences (p < 0.0001) among the three groups identified by Rachel score. CONCLUSIONS: A prognostic nomogram was developed, incorporating variables with significant impact on lung NET survival. The nomogram showed a satisfactory and stable ability to predict OS and PFS in this population, confirming the heterogeneity beyond the histopathological diagnosis of TC vs AC.

KRAS G12C mutation and risk of disease recurrence in stage I surgically resected lung adenocarcinoma.

KRAS G12C mutations are found in about 12-13% of LUAD samples and it is unclear whether they are associated with worse survival outcomes in resected, stage I LUAD. We assessed whether KRAS-G12C mutated tumours had worse DFS when compared to KRAS-nonG12C mutated tumours and to KRAS wild-type tumours in a cohort of resected, stage I LUAD (IRE cohort). We then leveraged on publicly available datasets (TCGA-LUAD, MSK-LUAD604) to further test the hypothesis in external cohorts. In the stage I IRE cohort we found a significant association between the KRAS-G12C mutation and worse DFS in multivariable analysis (HR: 2.47). In the TCGA-LUAD stage I cohort we did not find statistically significant associations between the KRAS-G12C mutation and DFS. In the MSK-LUAD604 stage I cohort we found that KRAS-G12C mutated tumours had worse RFS when compared to KRAS-nonG12C mutated tumours in univariable analysis (HR 3.5). In the pooled stage I cohort we found that KRAS-G12C mutated tumours had worse DFS when compared to KRAS-nonG12C mutated tumours (HR 2.6), to KRAS wild-type tumours (HR 1.6) and to any other tumours (HR 1.8); in multivariable analysis, the KRAS-G12C mutation was associated with worse DFS (HR 1.61). Our results suggest that patients with resected, stage I LUAD with a KRAS-G12C mutation may have inferior survival outcomes..

Clonal KEAP1 mutations with loss of heterozygosity share reduced immunotherapy efficacy and low immune cell infiltration in lung adenocarcinoma.

BACKGROUND: KEAP1 mutations have been associated with reduced survival in lung adenocarcinoma (LUAD) patients treated with immune checkpoint inhibitors (ICIs), particularly in the presence of STK11/KRAS alterations. We hypothesized that, beyond co-occurring genomic events, clonality prediction may help identify deleterious KEAP1 mutations and their counterparts with retained sensitivity to ICIs. PATIENTS AND METHODS: Beta-binomial modelling of sequencing read counts was used to infer KEAP1 clonal inactivation by combined somatic mutation and loss of heterozygosity (KEAP1 C-LOH) versus partial inactivation [KEAP1 clonal diploid-subclonal (KEAP1 CD-SC)] in the Memorial Sloan Kettering Cancer Center (MSK) MetTropism cohort (N = 2550). Clonality/LOH prediction was compared to a streamlined clinical classifier that relies on variant allele frequencies (VAFs) and tumor purity (TP) (VAF/TP ratio). The impact of this classification on survival outcomes was tested in two independent cohorts of LUAD patients treated with immunotherapy (MSK/Rome N = 237; DFCI N = 461). Immune-related features were studied by exploiting RNA-sequencing data (TCGA) and multiplexed immunofluorescence (DFCI mIF cohort). RESULTS: Clonality/LOH inference in the MSK MetTropism cohort overlapped with a clinical classification model defined by the VAF/TP ratio. In the ICI-treated MSK/Rome discovery cohort, predicted KEAP1 C-LOH mutations were associated with shorter progression-free survival (PFS) and overall survival (OS) compared to KEAP1 wild-type cases (PFS log-rank P = 0.001; OS log-rank P < 0.001). Similar results were obtained in the DFCI validation cohort (PFS log-rank P = 0.006; OS log-rank P = 0.014). In both cohorts, we did not observe any significant difference in survival outcomes when comparing KEAP1 CD-SC and wild-type tumors. Immune deconvolution and multiplexed immunofluorescence revealed that KEAP1 C-LOH and KEAP1 CD-SC differed for immune-related features. CONCLUSIONS: KEAP1 C-LOH mutations are associated with an immune-excluded phenotype and worse clinical outcomes among advanced LUAD patients treated with ICIs. By contrast, survival outcomes of patients whose tumors harbored KEAP1 CD-SC mutations were similar to those with KEAP1 wild-type LUADs.

MicroRNA-based signatures impacting clinical course and biology of ovarian cancer: a miRNOmics study.

BACKGROUND: In Western countries, ovarian cancer (OC) still represents the leading cause of gynecological cancer-related deaths, despite the remarkable gains in therapeutical options. Novel biomarkers of early diagnosis, prognosis definition and prediction of treatment outcomes are of pivotal importance. Prior studies have shown the potentials of micro-ribonucleic acids (miRNAs) as biomarkers for OC and other cancers. METHODS: We focused on the prognostic and/or predictive potential of miRNAs in OC by conducting a comprehensive array profiling of miRNA expression levels in ovarian tissue samples from 17 non-neoplastic controls, and 60 tumor samples from OC patients treated at the Regina Elena National Cancer Institute (IRE). A set of 54 miRNAs with differential expression in tumor versus normal samples (T/N-deregulated) was identified in the IRE cohort and validated against data from the Cancer Genoma Atlas (TCGA) related to 563 OC patients and 8 non-neoplastic controls. The prognostic/predictive role of the selected 54 biomarkers was tested in reference to survival endpoints and platinum resistance (P-res). RESULTS: In the IRE cohort, downregulation of the 2 miRNA-signature including miR-99a-5p and miR-320a held a negative prognostic relevance, while upregulation of miR-224-5p was predictive of less favorable event free survival (EFS) and P-res. Data from the TCGA showed that downregulation of 5 miRNAs, i.e., miR-150, miR-30d, miR-342, miR-424, and miR-502, was associated with more favorable EFS and overall survival outcomes, while miR-200a upregulation was predictive of P-res. The 9 miRNAs globally identified were all included into a single biologic signature, which was tested in enrichment analysis using predicted/validated miRNA target genes, followed by network representation of the miRNA-mRNA interactions. CONCLUSIONS: Specific dysregulated microRNA sets in tumor tissue showed predictive/prognostic value in OC, and resulted in a promising biological signature for this disease.

KEAP1-driven co-mutations in lung adenocarcinoma unresponsive to immunotherapy despite high tumor mutational burden.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have demonstrated significant overall survival (OS) benefit in lung adenocarcinoma (LUAD). Nevertheless, a remarkable interpatient heterogeneity characterizes immunotherapy efficacy, regardless of programmed death-ligand 1 (PD-L1) expression and tumor mutational burden (TMB). KEAP1 mutations are associated with shorter survival in LUAD patients receiving chemotherapy. We hypothesized that the pattern of KEAP1 co-mutations and mutual exclusivity may identify LUAD patients unresponsive to immunotherapy. PATIENTS AND METHODS: KEAP1 mutational co-occurrences and somatic interactions were studied in the whole MSKCC LUAD dataset. The impact of coexisting alterations on survival outcomes in ICI-treated LUAD patients was verified in the randomized phase II/III POPLAR/OAK trials (blood-based sequencing, bNGS cohort, N = 253). Three tissue-based sequencing studies (Rome, MSKCC and DFCI) were used for independent validation (tNGS cohort, N = 289). Immunogenomic features were analyzed using The Cancer Genome Atlas (TCGA) LUAD study. RESULTS: On the basis of KEAP1 mutational co-occurrences, we identified four genes potentially associated with reduced efficacy of immunotherapy (KEAP1, PBRM1, SMARCA4 and STK11). Independent of the nature of co-occurring alterations, tumors with coexisting mutations (CoMut) had inferior survival as compared with single-mutant (SM) and wild-type (WT) tumors (bNGS cohort: CoMut versus SM log-rank P = 0.048, CoMut versus WT log-rank P < 0.001; tNGS cohort: CoMut versus SM log-rank P = 0.037, CoMut versus WT log-rank P = 0.006). The CoMut subset harbored higher TMB than the WT disease and the adverse significance of coexisting alterations was maintained in LUAD with high TMB. Significant immunogenomic differences were observed between the CoMut and WT groups in terms of core immune signatures, T-cell receptor repertoire, T helper cell signatures and immunomodulatory genes. CONCLUSIONS: This study indicates that coexisting alterations in a limited set of genes characterize a subset of LUAD unresponsive to immunotherapy and with high TMB. An immune-cold microenvironment may account for the clinical course of the disease.

Immunotherapy in HER2-positive breast cancer: state of the art and future perspectives.

Breast cancer (BC) is a complex disease with primary or acquired incurability characteristics in a significant part of patients. Immunotherapeutical agents represent an emerging option for breast cancer treatment, including the human epidermal growth factor 2 positive (HER2+) subtype. The immune system holds the ability to spontaneously implement a defensive response against HER2+ BC cells through complex mechanisms which can be exploited to modulate this response for obtaining a clinical benefit. Initial immune system modulating strategies consisted mostly in vaccine therapies, which are still being investigated and improved. However, the entrance of trastuzumab into the scenery of HER2+ BC treatment was the real game changing event, which embodied a dominant immune-mediated mechanism. More recently, the advent of the immune checkpoint inhibitors has caused a new paradigm shift for immuno-oncology, with promising initial results also for HER2+ BC. Breast cancer has been traditionally considered poorly immunogenic, being characterized by relatively low tumor mutation burden (TMB). Nevertheless, recent evidence has revealed high tumor infiltrating lymphocytes (TILs) and programmed cell death-ligand 1 (PD-L1) expression in a considerable proportion of HER2+ BC patients. This may translate into a higher potential to elicit anti-cancer response and, therefore, wider possibilities for the use and implementation of immunotherapy in this subset of BC patients. We are herein presenting and critically discussing the most representative evidence concerning immunotherapy in HER2+ BC cancer, both singularly and in combination with therapeutic agents acting throughout HER2-block, immune checkpoint inhibition and anti-cancer vaccines. The reader will be also provided with hints concerning potential future projection of the most promising immutherapeutic agents and approaches for the disease of interest.

Epigenetic Changes Induced by Green Tea Catechins a re Associated with Prostate Cancer.

Prostate cancer is one of the most difficult cancers to treat especially when it becomes hormone resistant such as castrate resistant prostate cancer (CRPC) and subsequent metastatic CRPC. Apart from the genetic alterations in prostate cancer, epigenetic modifications also play an important role in the development and neoplastic progression of this disease. These include DNA methylation, histone modifications, and non-coding microRNAs. miRNAs are a novel class of small endogenous single-stranded non-coding RNAs of 19-25 nucleotides in length that typically silence gene expression. Considering the reversibility of epigenetic alterations in early carcinogenesis process, reversion (correction) of these modifications by green tea catechins could be a promising strategy for cancer chemoprevention and therapy. Recent evidence suggests that green tea catechins such as epigallocatechin gallate (EGCG) not only act as epigenetic modulators but can also modify miRNA expression and their target mRNAs, consistently contributing to the inhibition of prostate carcinogenesis. Various studies also indicate that several green tea polyphenols (GTPs) exert synergistic effects with other cancer chemotherapeutic agents. Therefore, the use of appropriate combinations of green tea catechins with the existing chemotherapeutics will lead to a reduction in side effects without decreasing the chemotherapeutic effects. This review will summarize the key results from recent studies detailing the effects of green tea catechins such as EGCG on epigenetic alterations and miRNA expression in prostate cancer.

Stearoyl-CoA-desaturase 1 regulates lung cancer stemness via stabilization and nuclear localization of YAP/TAZ.

This corrects the article DOI: 10.1038/onc.2017.75.

Stearoyl-CoA-desaturase 1 regulates lung cancer stemness via stabilization and nuclear localization of YAP/TAZ.

Recent evidences suggest that stearoyl-CoA-desaturase 1 (SCD1), the enzyme involved in monounsaturated fatty acids synthesis, has a role in several cancers. We previously demonstrated that SCD1 is important in lung cancer stem cells survival and propagation. In this article, we first show, using primary cell cultures from human lung adenocarcinoma, that the effectors of the Hippo pathway, Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), are required for the generation of lung cancer three-dimensional cultures and that SCD1 knock down and pharmacological inhibition both decrease expression, nuclear localization and transcriptional activity of YAP and TAZ. Regulation of YAP/TAZ by SCD1 is at least in part dependent upon ß-catenin pathway activity, as YAP/TAZ downregulation induced by SCD1 blockade can be rescued by the addition of exogenous wnt3a ligand. In addition, SCD1 activation of nuclear YAP/TAZ requires inactivation of the ß-catenin destruction complex. In line with the in vitro findings, immunohistochemistry analysis of lung adenocarcinoma samples showed that expression levels of SCD1 co-vary with those of ß-catenin and YAP/TAZ. Mining available gene expression data sets allowed to observe that high co-expression levels of SCD1, ß-catenin, YAP/TAZ and downstream targets have a strong negative prognostic value in lung adenocarcinoma. Finally, bioinformatics analyses directed to identify which gene combinations had synergistic effects on clinical outcome in lung cancer showed that poor survival is associated with high co-expression of SCD1, ß-catenin and the YAP/TAZ downstream target birc5. In summary, our data demonstrate for the first time the involvement of SCD1 in the regulation of the Hippo pathway in lung cancer, and point to fatty acids metabolism as a key regulator of lung cancer stem cells.

Risk Differences Between Prediabetes And Diabetes According To Breast Cancer Molecular Subtypes.

Hyperglycemia and hyperinsulinemia may play a role in breast carcinogenesis and prediabetes and diabetes have been associated with increased breast cancer (BC) risk. However, whether BC molecular subtypes may modify these associations is less clear. We therefore investigated these associations in all cases and by BC molecular subtypes among women living in Southern Italy. Cases were 557 patients with non-metastatic incident BC and controls were 592 outpatients enrolled during the same period as cases and in the same hospital for skin-related non-malignant conditions. Adjusted multivariate logistic regression models were built to assess the risks of developing BC in the presence of prediabetes or diabetes. The analyses were repeated by strata of BC molecular subtypes: Luminal A, Luminal B, HER2+, and Triple Negative (TN). Prediabetes and diabetes were significantly associated with higher BC incidence after controlling for known risk factors (OR = 1.94, 95% CI 1.32-2.87 and OR = 2.46, 95% CI 1.38-4.37, respectively). Similar results were seen in Luminal A and B while in the TN subtype only prediabetes was associated with BC (OR = 2.43, 95% CI 1.11-5.32). Among HER2+ patients, only diabetes was significantly associated with BC risk (OR = 3.04, 95% CI 1.24-7.47). Furthermore, when postmenopausal HER2+ was split into hormone receptor positive versus negative, the association with diabetes remained significant only in the former (OR = 5.13, 95% CI 1.53-17.22). These results suggest that prediabetes and diabetes are strongly associated with BC incidence and that these metabolic conditions may be more relevant in the presence of breast cancer molecular subtypes with positive hormone receptors. J. Cell. Physiol. 232: 1144-1150, 2017. © 2016 Wiley Periodicals, Inc.

Immunoscore and Immunoprofiling in cancer: an update from the melanoma and immunotherapy bridge 2015.

The fifth "Melanoma Bridge Meeting" took place in Naples, December 1-5th, 2015. The main topics discussed at this meeting were: Molecular and Immuno advances, Immunotherapies and Combination Therapies, Tumor Microenvironment and Biomarkers and Immunoscore. The natural history of cancer involves interactions between the tumor and the immune system of the host. The immune infiltration at the tumor site may be indicative of host response. Significant correlations were shown between the levels of immune cell infiltration in tumors and patient's clinical outcome. Moreover, incredible progress comes from the discovery of mutation-encoded tumor neoantigens. In fact, as tumors grow, they acquire mutations that are able to influence the response of patients to immune checkpoint inhibitors. It has been demonstrated that sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. The road ahead is still very long, but the knowledge of the mechanisms of immune escape, the study of tumor neo-antigens as well as of tumor microenvironment and the development of new immunotherapy strategies, will make cancer a more and more treatable disease.

Body weight and risk of molecular breast cancer subtypes among postmenopausal Mediterranean women.

Breast cancer (BC) is the most common malignant tumor in women, obesity is associated with increased BC incidence and mortality and high levels of circulating insulin may negatively impact on cancer incidence. In the present study, we investigated whether the strength of several anthropometric and metabolic parameters varies between BC molecular subtypes. Eligible cases were 991 non-metastatic BC patients recruited between January 2009 and December 2013. Anthropometric, clinical and immunohistochemical features were measured. Multivariate logistic regression models were built to assess HER2 positive BC risk, comparing (a) triple positive (TP) with luminal A, luminal B and triple negative (TN) and (b) HER2-enriched group with luminal A, luminal B and TN. We stratified patients in pre- and post-menopause: significant differences emerged for luminal A in relation to age: they were more likely to be older compared to other groups. Among postmenopausal patients, the adjusted multivariate analysis showed that high BMI and high waist circumference were inversely correlated to TP subtype when compared to luminal B (OR=0.48 and OR=0.49, respectively). Conversely, HOMA-IR was a risk factor for TP when compared to luminal A and TN (OR=2.47 and OR=3.15, respectively). Our findings suggest a potential role of higher abdominal fat in the development of specific BC molecular subtypes in postmenopausal women. Moreover, they support a potential role of insulin resistance in the development of HER2 positive BC, although this role appears to be stronger when hormone receptors are co-expressed, suggesting a difference in the etiology of these two BC subtypes.

Decreased LRIG1 in fulvestrant-treated luminal breast cancer cells permits ErbB3 upregulation and increased growth.

ErbB3, a member of the ErbB family of receptor tyrosine kinases, is a potent activator of phosphatidyl inositol-3 kinase (PI3K) and mammalian target of rapamycin (mTOR) signaling, driving tumor cell survival and therapeutic resistance in breast cancers. In luminal breast cancers, ErbB3 upregulation following treatment with the antiestrogen fulvestrant enhances PI3K/mTOR-mediated cell survival. However, the mechanism by which ErbB3 is upregulated in fulvestrant-treated cells is unknown. We found that ErbB3 protein levels and cell surface presentation were increased following fulvestrant treatment, focusing our attention on proteins that regulate ErbB3 at the cell surface, including Nrdp1, NEDD4 and LRIG1. Among these, only LRIG1 correlated positively with ERα, but inversely with ErbB3 in clinical breast cancer data sets. LRIG1, an estrogen-inducible ErbB downregulator, was decreased in a panel of fulvestrant-treated luminal breast cancer cells. Ectopic LRIG1 expression from an estrogen-independent promoter uncoupled LRIG1 from estrogen regulation, thus sustaining LRIG1 and maintaining low ErbB3 levels in fulvestrant-treated cells. An LRIG1 mutant lacking the ErbB3 interaction motif was insufficient to downregulate ErbB3. Importantly, LRIG1 overexpression improved fulvestrant-mediated growth inhibition, whereas cells expressing the LRIG1 mutant were poorly sensitive to fulvestrant, despite effective ERα downregulation. Consistent with these results, LRIG1 expression correlated positively with increased disease-free survival in antiestrogen-treated breast cancer patients. These data suggest that ERα-dependent expression of LRIG1 dampens ErbB3 signaling in luminal breast cancer cells, and by blocking ERα activity with fulvestrant, LRIG1 is decreased thus permitting ErbB3 accumulation, enhanced ErbB3 signaling to cell survival pathways and blunting therapeutic response to fulvestrant.

Electro-gene-transfer as a new tool for cancer immunotherapy in animals.

The concept of vaccines based on the direct inoculation of plasmid DNA gained initial proof-of-concept in small rodent species. Further development was hampered by the difficulty to confirm immunogenicity and efficacy in large animal species and, most importantly, in human clinical trials. These negative findings led to the search of complementary technologies which, in combination with intradermal or intramuscular plasmid DNA injection would result in more robust delivery, decreased interindividual variability, clear evidence of clinical efficacy and which would eventually lead to market approval of new vaccine products. The use of high-pressure, needleless devices as an enhancing tool for plasmid DNA delivery led to recent approval by USDA of Oncept™, a therapeutic cancer vaccine directed against tyrosinase for the therapy of melanoma in dogs. An alternative approach to improve plasmid DNA delivery is electro-gene-transfer (EGT). In this article, we briefly review the principles of DNA-EGT and the evidences for efficacy of a telomerase reverse transcriptase vaccine in a dog clinical trial, and provide perspectives for the use of this technology for broader applications in pet animals.

Stearoyl-CoA desaturase-1 is a key factor for lung cancer-initiating cells.

In recent years, studies of cancer development and recurrence have been influenced by the cancer stem cells (CSCs)/cancer-initiating cells (CICs) hypothesis. According to this, cancer is sustained by highly positioned, chemoresistant cells with extensive capacity of self renewal, which are responsible for disease relapse after chemotherapy. Growth of cancer cells as three-dimensional non-adherent spheroids is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from malignant pleural effusions (MPEs) of patients affected by adenocarcinoma of the lung are able to efficiently form spheroids in non-adherent conditions supplemented with growth factors. By expression profiling, we were able to identify a set of genes whose expression is significantly upregulated in lung tumor spheroids versus adherent cultures. One of the most strongly upregulated gene was stearoyl-CoA desaturase (SCD1), the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study, we show both by RNA interference and through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in MPE-derived primary tumor cultures. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker, most of them positive also for the stemness marker ALDH1A1, thus suggesting that the SCD1 inhibitor is selectively killing cells with stem-like properties. Furthermore, SCD1-inhibited lung cancer cells were strongly impaired in their in vivo tumorigenicity and ALDH1A1 expression. These results suggest that SCD1 is a critical target in lung cancer tumor-initiating cells.

Intratumor RNA interference of cell cycle genes slows down tumor progression.

Small interfering RNAs (siRNAs) are emerging as promising therapeutic tools. However, the widespread clinical application of such molecules as modulators of gene expression is still dependent on several aspects that limit their bioavailability. One of the most promising strategies to overcome the barriers faced by gene silencing molecules involves the use of lipid-based nanoparticles (LNPs) and viral vectors, such as adenoviruses (Ads). The primary obstacle for translating gene silencing technology from an effective research tool into a feasible therapeutic strategy remains its efficient delivery to the targeted cell type in vivo. In this study, we tested the capability of LNPs and Ad to transduce and treat locally tumors in vivo. Efficient knockdown of a surrogate reporter (luciferase) and therapeutic target genes such as the kinesin spindle protein (KIF11) and polo-like kinase 1 were observed. Most importantly, this activity led to a cell cycle block as a consequence and slowed down tumor progression in tumor-bearing animals. Our data indicate that it is possible to achieve tumor transduction with si/short hairpin RNAs and further improve the delivery strategy that likely in the future will lead to the ideal non-viral particle for targeted cancer gene silencing.

An oral TLR7 agonist is a potent adjuvant of DNA vaccination in transgenic mouse tumor models.

In vivo electroporation of plasmid DNA (DNA-EP) is an efficient and safe method for vaccines resulting in increased DNA uptake, enhanced protein expression and increased immune responses to the target antigen in a variety of species. To further enhance the efficacy of DNA-EP, we have evaluated the toll-like receptor7 (TLR7) agonist-2, 9, substituted 8-hydroxyadenosine derivative or SM360320--as an adjuvant to vaccines against HER2/neu and CEA in BALB-neuT and CEA transgenic mice (CEA.Tg), respectively. SM360320 induced in vivo secretion of interferon alpha (IFNalpha) and exerted a significant antitumor effect in CEA.Tg mice challenged with a syngenic tumor cell line expressing CEA and an additive effect with a CEA vaccine. Additionally, combination of SM360320 with plasmid encoding the extracellular and transmembrane domain of ratHER2/neu affected the spontaneous tumor progression in BALB-neuT mice treated in an advanced disease setting. The antitumor effect in mice treated with DNA-EP and SM360320 was associated with an anti-CEA and anti-p185(neu) antibody isotype switch from IgG1 to IgG2a. These data demonstrate that SM360320 exerts significant antitumor effects and can act in association with DNA-EP for CEA-positive colon cancer and HER2-positive mammary carcinoma. These observations therefore emphasize the potential of SM360320 as immunological adjuvant for therapeutic DNA vaccines.

CD4+CD25+ regulatory T-cell-inactivation in combination with adenovirus vaccines enhances T-cell responses and protects mice from tumor challenge.

Cancer vaccines are a promising approach to treating tumors or preventing tumor relapse through induction of an immune response against tumor-associated antigens (TAA). One major obstacle to successful therapy is the immunological tolerance against self-antigens which limits an effective antitumor immune response. As a transient reduction of immunological tolerance may enable more effective vaccination against self-tumor antigens, we explored this hypothesis in a CEA tolerant animal model with an adenovirus expressing CEA vaccine in conjunction with inactivation of CD4(+)CD25(+) regulatory T cells. This vaccination modality resulted in increased CEA-specific CD8(+), CD4(+) T cells and antibody response. The appearance of a CD4(+) T-cell response correlated with a stronger memory response. The combined CD25(+) inactivation and genetic vaccination resulted in significant tumor protection in a metastatic tumor model. Non-invasive tumor visualization showed that not only primary tumors were reduced, but also hepatic metastases. Our results support the viability of this cancer vaccine strategy as an adjuvant treatment to prevent tumor relapse in cancer patients.

CD8+ T-cell tolerance can be broken by an adenoviral vaccine while CD4+ T-cell tolerance is broken by additional co-administration of a Toll-like receptor ligand.

T-cell tolerance to tumor antigens is a considerable challenge to cancer immunotherapy. The existence of a murine model transgenic for human carcinoembryonic antigen (CEA) allows CEA vaccination efficacy to be studied in a physiologically tolerant context. Immunization of CEA-transgenic mice with an adenoviral vector coding for CEA induced a significant CD8+ T-cell response specific to CEA but failed to induce CEA-specific CD4+ T cells and antibodies. To overcome CD4+ T-cell tolerance, we explored the effect of adjuvants inducing in vivo dendritic cell maturation. Two different Toll-like receptor ligands, monophosphoryl lipid A (MPL) and CpG motif-containing oligodeoxynucleotides (CpG-ODN), were tested. CD4+-mediated IFN-gamma production was induced in the CEA-transgenic mice only when the genetic immunization was performed in the presence of these adjuvants. Moreover, CpG-ODN had a greater effect than MPL in inducing CD4+ T-cell response and enabling anti-CEA antibody production.