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1.  The Modulation of Hepatitis C Virus 1a Replication by PKR is Dependent on NF-kB Mediated Interferon Beta Response in Huh7.5.1 Cells 
Virology  2013;438(1):28-36.
Protein kinase R (PKR), a sensor of double-stranded RNA, plays an important role in the host response to viral infection. Hepatitis C genotype 2a virus (HCV 2a) has been shown to induce PKR activation to suppress the translation of antiviral interferon stimulated genes (ISGs), suggesting that PKR inhibitor can be beneficial for treating chronically HCV-infected patients in conjunction with interferon alpha and ribavirin. However, in this study, we found that PKR inhibition using siRNA PKR, shRNA PKR or PKR inhibitor enhanced HCV 1a replication and rendered Huh7.5.1 cells more susceptible to HCV1a infection. Additionally, PKR silencing suppressed NF-kB activation and NF-kB mediated STAT1 phosphorylation in Huh7.5.1 cells and HCV1a persistently-infected Huh7.5.1 cells (2HDD4). These effects were accompanied by a reduction of interferon beta response and thereby enhanced HCV1a replication in Huh7.5.1 cells. We conclude that host cells can employ PKR activation to restrict HCV1a replication through regulation of NF-kB expression.
doi:10.1016/j.virol.2013.01.015
PMCID: PMC3594529  PMID: 23399035
PKR silencing; NF-kB activation; IFN-β; HCV1a replication
2.  Bach2 represses effector programmes to stabilize Treg-mediated immune homeostasis 
Nature  2013;498(7455):10.1038/nature12199.
Through their functional diversification, distinct lineages of CD4+ T cells play key roles in either driving or constraining immune-mediated pathology. Transcription factors are critical in the generation of cellular diversity, and negative regulators antagonistic to alternate fates often act in conjunction with positive regulators to stabilize lineage commitment1. Genetic polymorphisms within a single locus encoding the transcription factor BACH2 are associated with numerous autoimmune and allergic diseases including asthma2, Crohn’s disease3–4, coeliac disease5, vitiligo6, multiple sclerosis7 and type 1 diabetes8. While these associations point to a shared mechanism underlying susceptibility to diverse immune-mediated diseases, a function for Bach2 in the maintenance of immune homeostasis has not been established. Here, we define Bach2 as a broad regulator of immune activation that stabilizes immunoregulatory capacity while repressing the differentiation programmes of multiple effector lineages in CD4+ T cells. Bach2 was required for efficient formation of regulatory (Treg) cells and consequently for suppression of lethal inflammation in a manner that was Treg cell dependent. Assessment of the genome-wide function of Bach2, however, revealed that it represses genes associated with effector cell differentiation. Consequently, its absence during Treg polarization resulted in inappropriate diversion to effector lineages. In addition, Bach2 constrained full effector differentiation within Th1, Th2 and Th17 cell lineages. These findings identify Bach2 as a key regulator of CD4+ T-cell differentiation that prevents inflammatory disease by controlling the balance between tolerance and immunity.
doi:10.1038/nature12199
PMCID: PMC3710737  PMID: 23728300
3.  Baseline Levels and Temporal Stability of 27 Multiplexed Serum Cytokine Concentrations in Healthy Subjects 
PLoS ONE  2013;8(12):e76091.
Background
Cytokines are humoral molecules that elicit regulatory function in immunologic pathways. The level and type of cytokine production has become critical in distinguishing physiologic from pathologic immune conditions. Cytokine profiling has become an important biomarker discovery tool in monitoring of the immune system. However, the variations in cytokine levels in individual subjects over time in healthy individuals have not been extensively studied. In this study, we use multiplex bead arrays to evaluate 27 analytes in paired serum samples taken seven days apart from 144 healthy individuals in order to assess variations over a short time period.
Methods
Fluorescent bead-based immunoassay (Luminex) was used to measure 27 analytes in serum samples. Measurements were performed on matched samples from 144 healthy donors. To assess inter-plate variability, one arbitrarily selected serum sample was analyzed on each of the first ten plates as bridge sample.
Results
Using the bridge sample, we showed minimal inter-plate variations in the measurement of most analytes. In measurement of cytokines from the 144 patients at two time points, we found that three cytokines (IL-2, IL-15 and GM-CSF) were undetectable and five analytes (RANTES, MCP-1, VEGF, MIP-1β and PDGF-BB) showed significant difference in concentrations at Day 0 compared to Day 7.
Conclusions
The current study demonstrated higher variations in cytokine levels among individuals than were observed for samples obtained one week apart from identical donors. These data suggest that a serum sample from each subject for use as a baseline measurement is a better control for clinical trials rather than sera from a paired cohort.
doi:10.1371/journal.pone.0076091
PMCID: PMC3861126  PMID: 24348989
4.  Influence of quercetin-rich food intake on microRNA expression in lung cancer tissues 
Background
Epidemiologic studies have reported that frequent consumption of quercetin-rich foods is inversely associated with lung cancer incidence. A quercetin-rich diet might modulate microRNA (miR) expression; however, this mechanism has not been fully examined.
Methods
miR expression data were measured by a custom-made array in formalin-fixed paraffin-embedded tissue samples from 264 lung cancer cases (144 adenocarcinomas and 120 squamous cell carcinomas). Intake of quercetin-rich foods was derived from a food-frequency questionnaire. In individual-miR-based analyses, we compared the expression of miRs (n=198) between lung cancer cases consuming high-versus-low quercetin-rich food intake using multivariate ANOVA tests. In family-miR-based analyses, we used Functional Class Scoring (FCS) to assess differential effect on biologically functional miRs families. We accounted for multiple testing using 10,000 global permutations (significance at p-valueglobal <0.10). All multivariate analyses were conducted separately by histology and by smoking status (former and current smokers).
Results
Family-based analyses showed that a quercetin-rich diet differentiated miR expression profiles of the tumor suppressor let-7 family among adenocarcinomas (p-valueFCS<0.001). Other significantly differentiated miR families included carcinogenesis-related miR-146, miR-26, and miR-17 (p-valuesFCS<0.05). In individual-based analyses, we found that among former and current smokers with adenocarcinoma, 33 miRs were observed to be differentiated between highest-and-lowest quercetin-rich food consumers (23 expected by chance; p-valueglobal = 0.047).
Conclusions
We observed differential expression of key biologically functional miRNAs between high-versus-low consumers of quercetin-rich foods in adenocarcinoma cases.
Impact
Our findings provide preliminary evidence on the mechanism underlying quercetin-related lung carcinogenesis.
doi:10.1158/1055-9965.EPI-12-0745
PMCID: PMC3538163  PMID: 23035181
5.  HCV RNA Levels in a Multi-Ethnic Cohort of Injection Drug Users: Human Genetic, Viral and Demographic Associations 
Hepatology (Baltimore, Md.)  2012;56(1):86-94.
In patients with chronic hepatitis C, the hepatitis C virus (HCV) RNA level is an important predictor of treatment response. To explore the relationship of HCV RNA with viral and demographic factors, as well as IL28B genotype, we examined viral levels in an ethnically diverse group of injection drug users (IDUs). Between 1998 and 2000, the Urban Health Study (UHS) recruited IDUs from street settings in San Francisco Bay area neighborhoods. Participants who were positive by HCV EIA were tested for HCV viremia by a bDNA assay. HCV genotype was determined by sequencing the HCV NS5B region. For a subset of participants, IL28B rs12979860 genotype was determined by Taqman. Among 1701 participants with HCV viremia, median age was 46 years and median duration of injection drug use was 26 years; 56.0% were African American and 34.0% were of European ancestry (non-Hispanic). HIV-1 prevalence was 13.9%. The overall median HCV RNA level was 6.45 log10 copies/ml. In unadjusted analyses, higher levels were found with older age, male gender, African American ancestry, HBV infection, HIV-1 infection and IL28B rs12979860-CC genotype; compared to participants infected with HCV genotype 1, HCV RNA was lower in participants with genotype 3 or genotype 4. In an adjusted analysis, age, gender, racial ancestry, HIV-1 infection, HCV genotype and IL28B rs12979860 genotype were all independently associated with HCV RNA. Conclusion: The level of HCV viremia is influenced by a large number of demographic, viral and human genetic factors.
doi:10.1002/hep.25652
PMCID: PMC3369001  PMID: 22331649
epidemiology; genetics; HCV; IL28B; viremia
6.  Cancer cell secretion of the DAMP protein HMGB1 supports progression in malignant mesothelioma 
Cancer Research  2012;72(13):3290-3301.
Human malignant mesothelioma (MM) is an aggressive and highly lethal cancer that is believed to be caused by chronic exposure to asbestos and erionite. Prognosis for this cancer is generally poor due to late-stage diagnosis and resistance to current conventional therapies. The damage-associated molecular pattern (DAMP) protein HMGB1 has been implicated previously in transformation of mesothelial cells. Here we show that HMGB1 establishes an autocrine circuit in MM cells that influences their proliferation and survival. MM cells strongly expressed HMGB1 and secreted it at high levels in vitro. Accordingly, HMGB1 levels in MM patient sera were higher than that found in healthy individuals. The motility, survival and anchorage-independent growth of HMGB1-secreting MM cells was inhibited in vitro by treatment with monoclonal antibodies directed against HMGB1 or against the receptor for advanced glycation end products (RAGE), a putative HMGB1 receptor. HMGB1 inhibition in vivo reduced the growth of MM xenografts in SCID mice and extended host survival. Taken together, our findings indicate that MM cells rely on HMGB1 and they offer a preclinical proof of principle that antibody-mediated ablation of HMBG1 is sufficient to elicit therapeutic activity, suggesting a novel therapeutic approach for MM treatment.
doi:10.1158/0008-5472.CAN-11-3481
PMCID: PMC3389268  PMID: 22552293
HMGB1; mesothelioma; inflammation; biomarker; therapy
7.  Molecular signatures mostly associated with NK cells are predictive of relapse free survival in breast cancer patients 
Background
Recent observations suggest that immune-mediated tissue destruction is dependent upon coordinate activation of immune genes expressed by cells of the innate and adaptive immune systems.
Methods
Here, we performed a retrospective pilot study to investigate whether the coordinate expression of molecular signature mostly associated with NK cells could be used to segregate breast cancer patients into relapse and relapse-free outcomes.
Results
By analyzing primary breast cancer specimens derived from patients who experienced either 58–116 months (~5-9 years) relapse-free survival or developed tumor relapse within 9–76 months (~1-6 years) we found that the expression of molecules involved in activating signaling of NK cells and in NK cells: target interaction is increased in patients with favorable prognosis.
Conclusions
The parameters identified in this study, together with the prognostic signature previously reported by our group, highlight the cooperation between the innate and adaptive immune components within the tumor microenvironment.
doi:10.1186/1479-5876-11-145
PMCID: PMC3694475  PMID: 23758773
Breast cancer prognosis; Molecular markers; Innate immunity; NK cells; Tumour relapse; Tumour microenvironment
8.  Future perspectives in melanoma research. Meeting report from the “Melanoma Bridge. Napoli, December 2nd-4th 2012” 
Recent insights into the genetic and somatic aberrations have initiated a new era of rapidly evolving targeted and immune-based treatments for melanoma. After decades of unsuccessful attempts to finding a more effective cure in the treatment of melanoma now we have several drugs active in melanoma. The possibility to use these drugs in combination to improve responses to overcome the resistance, to potentiate the action of immune system with the new immunomodulating antibodies, and identification of biomarkers that can predict the response to a particular therapy represent new concepts and approaches in the clinical management of melanoma. The third “Melanoma Research: “A bridge from Naples to the World” meeting, shortened as “Bridge Melanoma Meeting” took place in Naples, December 2 to 4th, 2012. The four topics of discussion at this meeting were: advances in molecular profiling and novel biomarkers, combination therapies, novel concepts toward integrating biomarkers and therapies into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage, and the knowledge gained from the biology of tumor microenvironment across different tumors as a bridge to impact on prognosis and response to therapy in melanoma. This international congress gathered more than 30 international faculty members who in an interactive atmosphere which stimulated discussion and exchange of their experience regarding the most recent advances in research and clinical management of melanoma patients.
doi:10.1186/1479-5876-11-137
PMCID: PMC3681569  PMID: 23731854
9.  Molecular Signatures Associated with HCV-Induced Hepatocellular Carcinoma and Liver Metastasis 
PLoS ONE  2013;8(2):e56153.
Hepatocellular carcinomas (HCCs) are a heterogeneous group of tumors that differ in risk factors and genetic alterations. In Italy, particularly Southern Italy, chronic hepatitis C virus (HCV) infection represents the main cause of HCC. Using high-density oligoarrays, we identified consistent differences in gene-expression between HCC and normal liver tissue. Expression patterns in HCC were also readily distinguishable from those associated with liver metastases. To characterize molecular events relevant to hepatocarcinogenesis and identify biomarkers for early HCC detection, gene expression profiling of 71 liver biopsies from HCV-related primary HCC and corresponding HCV-positive non-HCC hepatic tissue, as well as gastrointestinal liver metastases paired with the apparently normal peri-tumoral liver tissue, were compared to 6 liver biopsies from healthy individuals. Characteristic gene signatures were identified when normal tissue was compared with HCV-related primary HCC, corresponding HCV-positive non-HCC as well as gastrointestinal liver metastases. Pathway analysis classified the cellular and biological functions of the genes differentially expressed as related to regulation of gene expression and post-translational modification in HCV-related primary HCC; cellular Growth and Proliferation, and Cell-To-Cell Signaling and Interaction in HCV-related non HCC samples; Cellular Growth and Proliferation and Cell Cycle in metastasis. Also characteristic gene signatures were identified of HCV-HCC progression for early HCC diagnosis.
Conclusions
A diagnostic molecular signature complementing conventional pathologic assessment was identified.
doi:10.1371/journal.pone.0056153
PMCID: PMC3575468  PMID: 23441164
10.  A signature of immune function genes associated with recurrence-free survival in breast cancer patients 
The clinical significance of tumor-infiltrating immune cells has been reported in a variety of human carcinomas including breast cancer. However, molecular signature of tumor-infiltrating immune cells and their prognostic value in breast cancer patients remain elusive. We hypothesized that a distinct network of immune function genes at the tumor site can predict a low risk versus high risk of distant relapse in breast cancer patients regardless of the status of ER, PR, or HER-2/neu in their tumors. We conducted retrospective studies in a diverse cohort of breast cancer patients with a 1–5 year tumor relapse versus those with up to 7 years relapse-free survival. The RNAs were extracted from the frozen tumor specimens at the time of diagnosis and subjected to microarray analysis and real-time RT-PCR. Paraffin-embedded tissues were also subjected to immunohistochemistry staining. We determined that a network of immune function genes involved in B cell development, interferon signaling associated with allograft rejection and autoimmune reaction, antigen presentation pathway, and cross talk between adaptive and innate immune responses were exclusively upregulated in patients with relapse-free survival. Among the 299 genes, five genes which included B cell response genes were found to predict with >85% accuracy relapse-free survival. Real-time RT-PCR confirmed the 5-gene prognostic signature that was distinct from an FDA-cleared 70-gene signature of MammaPrint panel and from the Oncotype DX recurrence score assay panel. These data suggest that neoadjuvant immunotherapy in patients with high risk of relapse may reduce tumor recurrence by inducing the immune function genes.
doi:10.1007/s10549-011-1470-x
PMCID: PMC3431022  PMID: 21479927
Breast cancer prognosis; Tumor relapse; Tumor microenvironment; Immune response; Neoadjuvant immunotherapy
11.  Identification and characterization of human leukocyte antigen class I ligands in renal cell carcinoma cells 
Proteomics  2011;11(12):2528-2541.
The presentation of tumor antigen-derived peptides by human leukocyte antigen (HLA) class I surface antigens on tumor cells is a key prerequisite to trigger effective T-cell responses in cancer patients. Multiple complementary strategies like cDNA and serological expression cloning, reverse immunology and different ‘ome’-based methods have been employed to identify potential T-cell targets. This report focuses on a ligandomic profiling approach leading to the identification of 49 naturally processed HLA class I peptide ligands presented on the cell surface of renal cell carcinoma (RCC) cells. The source proteins of the defined HLA ligands are classified according to their biological function and subcellular localization. Previously established cDNA microarray data of paired tissue specimen of RCC and renal epithelium assessed the transcriptional regulation for 28 source proteins. In addition, HLA-A2-restricted, peptide-specific T cells directed against a HLA ligand derived from sulfiredoxin-1 (SRXN1) were generated, which were able to recognize and lyse ligand-presenting target cells in a HLA class I-restricted manner. Furthermore, tumor-infiltrating T cells isolated from a RCC patient were also able to kill SRXN1 expressing tumor cells. Thus, this experimental strategy might be suited to define potential candidate biomarkers and novel targets for T-cell-based immunotherapies of this disease.
doi:10.1002/pmic.201000486
PMCID: PMC3517181  PMID: 21595034
Biomarkers; Cell biology; HLA antigens; Peptide ligands; Renal cell carcinoma; T-cell response
12.  Single-Cell Network Profiling of Peripheral Blood Mononuclear Cells from Healthy Donors Reveals Age- and Race-Associated Differences in Immune Signaling Pathway Activation 
A greater understanding of the function of the human immune system at the single-cell level in healthy individuals is critical for discerning aberrant cellular behavior that occurs in settings such as autoimmunity, immunosenescence, and cancer. To achieve this goal, a systems-level approach capable of capturing the response of the interdependent immune cell types to external stimuli is required. In this study, an extensive characterization of signaling responses in multiple immune cell subpopulations within PBMCs from a cohort of 60 healthy donors was performed using single-cell network profiling (SCNP). SCNP is a multiparametric flow cytometry-based approach that enables the simultaneous measurement of basal and evoked signaling in multiple cell subsets within heterogeneous populations. In addition to establishing the interindividual degree of variation within a broad panel of immune signaling responses, the possible association of any observed variation with demographic variables including age and race was investigated. Using half of the donors as a training set, multiple age- and race-associated variations in signaling responses in discrete cell subsets were identified, and several were subsequently confirmed in the remaining samples (test set). Such associations may provide insight into age-related immune alterations associated with high infection rates and diminished protection following vaccination and into the basis for ethnic differences in autoimmune disease incidence and treatment response. SCNP allowed for the generation of a functional map of healthy immune cell signaling responses that can provide clinically relevant information regarding both the mechanisms underlying immune pathological conditions and the selection and effect of therapeutics.
doi:10.4049/jimmunol.1102514
PMCID: PMC3517183  PMID: 22246624
13.  Applying the uncertainty principle to immunology 
Immunotherapy  2011;3(10):1127-1128.
doi:10.2217/imt.11.125
PMCID: PMC3517186  PMID: 21995565
14.  Molecular insights on the peripheral and intra-tumoral effects of systemic high dose rIL-2 (Aldesleukin) administration for the treatment of metastatic melanoma 
Purpose
We have previously shown that within tumors, recombinant interleukin-2 (rIL-2, Aldesleukin) consistently activates tumor-associated macrophages and up-regulates interferon stimulated genes (ISGs) while inducing minimal migration, activation or proliferation of T-cells. These effects are independent of tumor response to treatment. Here, we prospectively evaluated transcriptional alterations induced by rIL-2 in peripheral blood mononuclear cells (PBMCs) and within melanoma metastases.
Experimental Design
We evaluated gene expression changes by serially comparing pre- to post-treatment samples in 14 patients and also compared transcriptional differences among lesions displaying different responsiveness to therapy, focusing on 2 lesions decreasing in size and 2 remaining stable (responding lesions) compared to non-responding ones.
Results
As previously described, the effects of rIL-2 were dramatic within PBMC, while effects within the tumor microenvironment were lesion-specific and limited. However, distinct signatures specific to response could be observed in responding lesions pre-treatment that were amplified following rIL-2 administration. These signatures match the functional profile observed in other human or experimental models in which immune-mediated tissue-specific destruction (TSD) occurs underlying a common pathways leading to rejection. Moreover, the signatures observed in pre-treatment lesions were qualitatively similar to those associated with TSD underlining a determinism to immune responsiveness that depends upon the genetic background of the host or the intrinsic genetic makeup of individual tumors.
Conclusions
This is the first prospectively collected insight on global transcriptional events occurring during high-dose rIL-2 therapy in melanoma metastases responding to treatment.
doi:10.1158/1078-0432.CCR-11-1650
PMCID: PMC3229653  PMID: 21976537
15.  Association between HRAS rs12628 and rs112587690 polymorphisms with the risk of melanoma in the North American population 
HRAS belongs to the RAS genes superfamily. RAS genes are important players in several human tumors and the single-nucleotide polymorphism rs12628 has been shown to contribute to the risk of bladder, colon, gastrointestinal, oral, and thyroid carcinoma. We hypothesized that this SNP may affect the risk of cutaneous melanoma as well. HRAS gene contains a polymorphic region (rs112587690), a repeated hexanucleotide -GGGCCT-located in intron 1. Three alleles of this region, P1, P2, and P3, have been identified that contain two, three, and four repeats of the hexanucleotide, respectively. We investigated the clinical impact of these polymorphisms in a case–control study. A total of 141 melanoma patients and 118 healthy donors from the North America Caucasian population were screened for rs12628 and rs112587690 polymorphisms. Genotypes were assessed by capillary sequencing or fragment analysis, respectively, and rs12628 CC and rs112587690 P1P1 genotypes significantly associated with increased melanoma risk (OR = 3.83, p = 0.003; OR = 11.3, p = 0.033, respectively), while rs112587690 P1P3 frequency resulted significantly higher in the control group (OR = 0.5, p = 0.017). These results suggest that rs12628 C homozygosis may be considered a potential risk factor for melanoma development in the North American population possibly through the linkage to rs112587690.
doi:10.1007/s12032-012-0255-3
PMCID: PMC3505523  PMID: 22618666
HRAS; Polymorphism; Melanoma; rs12628; rs112587690
16.  Linkage of microRNA and Proteome-Based Profiling Data Sets: A Perspective for the Priorization of Candidate Biomarkers in Renal Cell Carcinoma? 
Journal of proteome research  2011;10(1):191-199.
Despite recent advances in the understanding of the biology of renal cell carcinoma (RCC) and the implementation of novel targeted therapies, the overall 5 years’ survival rate for RCC patients remains disappointing. Late presentation, tumor heterogeneity and in particular the lack of molecular biomarkers for early detection and classification represent major obstacles. Global, untargeted comparative analysis of RCC vs tumor adjacent renal epithelium (NN) samples by high throughput analyses both at the transcriptome and proteome level have identified signatures, which might further clarify the molecular differences of RCC subtypes and might allow the identification of suitable therapeutic targets and diagnostic/prognostic biomarkers, but none thereof has yet been implemented in routine clinical use. The increasing knowledge regarding the functional role of noncoding microRNA (miR) in physiological, developmental, and pathophysiological processes by shaping the protein expression profile might provide an important link to improve the definition of disease-relevant regulatory networks. Taking into account that miR profiling of RCC and NN provides robust signatures discriminating between malignant and normal tissues, the concept of evaluating and scoring miR/protein pairs might represent a strategy for the selection and prioritization of potential biomarkers and their translation into practical use.
doi:10.1021/pr1011137
PMCID: PMC3506708  PMID: 21142213
RCC; miR; proteomics; biomarker
17.  Regression of melanoma metastases after immunotherapy is associated with activation of antigen presentation and interferon-mediated rejection genes 
We present the results of a comparative gene expression analysis of 15 metastases (10 regressing and 5 progressing) obtained from 2 melanoma patients with mixed response following different forms of immunotherapy. Whole genome transcriptional analysis clearly indicate that regression of melanoma metastases is due to an acute immune rejection mediated by the upregulation of genes involved in antigen presentation and interferon mediated response (STAT-1/IRF-1) in all the regressing metastases from both patients. In contrast, progressing metastases showed low transcription levels of genes involved in these pathways. Histological analysis showed T cells and HLA-DR positive infiltrating cells in the regressing but not in the progressing metastases. Quantitative expression analysis of HLA-A, B and C genes on microdisected tumoral regions indicate higher HLA expression in regressing than in progressing metastases. The molecular signature obtained in melanoma rejection appeared to be similar to that observed in other forms of immune-mediated tissue-specific rejection such as allograft, pathogen clearance, graft versus host or autoimmune disease, supporting the immunological constant of rejection. We favor the idea that the major factor determining the success or failure of immunotherapy is the nature of HLA Class I alterations in tumor cells and not the type of immunotherapy used. If the molecular alteration is reversible by the immunotherapy, the HLA expression will be upregulated and the lesion will be recognized and rejected. In contrast, if the defect is structural the MHC Class I expression will remain unchanged and the lesion will progress.
doi:10.1002/ijc.26471
PMCID: PMC3504975  PMID: 21964766
melanoma; metastasis; immunotherapy; rejection; HLA; IRF-1
18.  A novel series of conferences tackling the hurdles confronting the translation of novel cancer immunotherapies 
While there has been significant progress in advancing novel immune therapies to the bedside, much more needs to be done to fully tap into the potential of the immune system. It has become increasingly clear that besides practical and operational challenges, the heterogeneity of cancer and the limited efficacy profile of current immunotherapy platforms are the two main hurdles. Nevertheless, the promising clinical data of several approaches point to a roadmap that carries the promise to significantly advance cancer immunotherapy. A new annual series sponsored by Arrowhead Publishers and Conferences aims at bringing together scientific and business leadership from academia and industry, to identify, share and discuss most current priorities in research and translation of novel immune interventions. This Editorial provides highlights of the first event held earlier this year and outlines the focus of the second meeting to be held in 2013 that will be dedicated to stem cells and immunotherapy.
doi:10.1186/1479-5876-10-218
PMCID: PMC3551774  PMID: 23127127
19.  Development and promotion in translational medicine: perspectives from 2012 sino-american symposium on clinical and translational medicine 
Background
Clinical translational medicine (CTM) is an emerging area comprising multidisciplinary research from basic science to medical applications and entails a close collaboration among hospital, academia and industry.
Findings
This Session focused discussing on new models for project development and promotion in translational medicine. The conference stimulated the scientific and commercial communication of project development between academies and companies, shared the advanced knowledge and expertise of clinical applications, and created the environment for collaborations.
Conclusions
Although strategic collaborations between corporate and academic institutions have resulted in a state of resurgence in the market, new cooperation models still need time to tell whether they will improve the translational medicine process.
doi:10.1186/2001-1326-1-25
PMCID: PMC3561049  PMID: 23369198
20.  Concurrent vs Sequential Adjuvant Chemotherapy and Hormone Therapy in Breast Cancer: A Multicenter Randomized Phase III Trial 
Background
The most appropriate timing of chemotherapy and hormone therapy administration is a critical issue in early breast cancer patients. The purpose of our study was to compare the efficacy of concurrent vs sequential administration of adjuvant chemotherapy and tamoxifen.
Methods
Women with node-positive primary breast cancer were randomly assigned to receive tamoxifen (20 mg/d for 5 years) during (concurrent arm) or after (sequential arm) adjuvant chemotherapy. Chemotherapy consisted of alternating regimens of cyclophosphamide, epidoxorubicin, and 5-fluorouracil and cyclophosphamide, methotrexate, and 5-fluorouracil every 21 days for a total of 12 cycles. The primary endpoint was overall survival (OS), and secondary endpoints were toxic effects and disease-free survival (DFS). No provision for interim analyses was made in the original study protocol. Survival curves were estimated by the Kaplan–Meier method. Multivariable Cox regression models, adjusted for age, menopausal status, tumor stage, and lymph node and hormone receptor status, were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). All statistical tests were two-sided.
Results
From 1985 to 1992, 431 patients were randomly assigned and studied according to the intention-to-treat principle. After a maximum of 15.4 years of follow-up (median 12.3 years), the estimated actuarial 10-year OS was equivalent for the two study arms (concurrent arm: 111 patients, 66%, 95% CI = 59% to 72%; sequential arm: 114 patients, 65%, 95% CI = 59% to 72%, P = .86). No differences in DFS and toxic effects were evident. Four interim analyses were performed, but no alpha error adjustment was necessary because of the largely negative results of this final analysis (sequential vs concurrent arm: HR of death = 1.06, 95% CI = 0.78 to 1.44, P = .76; HR of relapse = 1.16, 95% CI = 0.88 to 1.52, P = .36).
Conclusions
No statistically significant differences in OS, DFS, and toxic effects between concurrent and sequential adjuvant chemo- and hormone therapies were observed. Our study does not support the superiority of one schedule of chemo- and hormone-therapy administration over the other. However, because of the limited statistical power of the study, these results must be considered with caution.
doi:10.1093/jnci/djr351
PMCID: PMC3202939  PMID: 21921285
21.  Translational medicine from observation to hypothesis to interpretation 
Journal of Translational Medicine  2012;10(Suppl 2):A44.
doi:10.1186/1479-5876-10-S2-A44
PMCID: PMC3479845
23.  Cancer classification using the Immunoscore: a worldwide task force 
Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the ‘Immunoscore’ into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).
doi:10.1186/1479-5876-10-205
PMCID: PMC3554496  PMID: 23034130
24.  Multiparametric Analyses of Human PBMCs Loaded Ex Vivo with a Candidate Idiotype Vaccine for HCV-Related Lymphoproliferative Disorders 
PLoS ONE  2012;7(9):e44870.
Hepatitis C virus (HCV) has been identified as one of the major risk factors for type II mixed cryoglobulinemia (MC), during the clinical evolution of chronic hepatitis, which may lead to development of B cell non-Hodgkin's lymphoma (NHL). We have previously shown that the candidate idiotype vaccine, based on the IGKV3-20 light chain protein, is able to induce activation and maturation of circulating antigen presenting cells (APCs) in both HCV-positive and HCV-negative healthy control subjects, with production of Th2-type cytokines. Here, the effect of the recombinant IGKV3-20 protein on human peripheral blood mononuclear cells (PBMCs) from HCV-positive subjects, with known blood levels of cryoglobulins, is shown via gene expression profiling analysis combined to multiparameter flow cytometry and multiplex analyses of cytokines.
doi:10.1371/journal.pone.0044870
PMCID: PMC3445594  PMID: 23028651
25.  Association of IFN-γ Signal Transduction Defects with Impaired HLA Class I Antigen Processing in Melanoma Cell Lines 
Purpose
Abnormalities in the constitutive and IFN-γ–inducible HLA class I surface antigen expression of tumor cells is often associated with an impaired expression of components of the antigen processing machinery (APM). Hence, we analyzed whether there exists a link between the IFN-γ signaling pathway, constitutive HLA class I APM component expression, and IFN-γ resistance.
Experimental Design
The basal and IFN-γ–inducible expression profiles of HLA class I APM and IFN-γ signal transduction cascade components were assessed in melanoma cells by real-time PCR (RT-PCR), Western blot analysis and/or flow cytometry, the integrity of the Janus activated kinase (JAK) 2 locus by comparative genomic hybridization. JAK2 was transiently overexpressed in JAK2− cells. The effect of IFN-γ on the cell growth was assessed by XTT [2,3-bis(2-methoxy-4-nitro-S-sulfophenynl)-H-tetrazolium-5-carboxanilide inner salt] assay.
Results
The analysis of 8 melanoma cell lines linked the IFN-γ unresponsiveness of Colo 857 cells determined by lack of inducibility of HLA class I surface expression on IFN-γ treatment to a deletion of JAK2 on chromosome 9, whereas other IFN-γ signaling pathway components were not affected. In addition, the constitutive HLA class I APM component expression levels were significantly reduced in JAK2− cells. Furthermore, JAK2-deficient cells were also resistant to the antiproliferative effect of IFN-γ. Transfection of wild-type JAK2 into JAK2− Colo 857 not only increased the basal APM expression but also restored their IFN-γ sensitivity.
Conclusions
Impaired JAK2 expression in melanoma cells leads to reduced basal expression of MHC class I APM components and impairs their IFN-γ inducibility, suggesting that malfunctional IFN-γ signaling might cause HLA class I abnormalities.
doi:10.1158/1078-0432.CCR-10-2114
PMCID: PMC3426200  PMID: 21248298

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