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Genome sequences from evolving infectious pathogens allow quantification of case introductions and local transmission dynamics. We sequenced 11,357 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Switzerland in 2020—the sixth largest effort globally. Using a representative subset of these data, we estimated viral introductions to Switzerland and their persistence over the course of 2020. We contrasted these estimates with simple null models representing the absence of certain public health measures. We show that Switzerland’s border closures decoupled case introductions from incidence in neighboring countries. Under a simple model, we estimate an 86 to 98% reduction in introductions during Switzerland’s strictest border closures. Furthermore, the Swiss 2020 partial lockdown roughly halved the time for sampled introductions to die out. Last, we quantified local transmission dynamics once introductions into Switzerland occurred using a phylodynamic model. We found that transmission slowed 35 to 63% upon outbreak detection in summer 2020 but not in fall. This finding may indicate successful contact tracing over summer before overburdening in fall. The study highlights the added value of genome sequencing data for understanding transmission dynamics.

Genome sequences from evolving infectious pathogens allow quantification of case introductions and local transmission dynamics. We sequenced 11,357 SARS-CoV-2 genomes from Switzerland in 2020 - the 6th largest effort globally. Using a representative subset of these data, we estimated viral introductions to Switzerland and their persistence over the course of 2020. We contrast these estimates with simple null models representing the absence of certain public health measures. We show that Switzerland’s border closures de-coupled case introductions from incidence in neighboring countries. Under a simple model, we estimate an 86 - 98% reduction in introductions during Switzerland’s strictest border closures. Furthermore, the Swiss 2020 partial lockdown roughly halved the time for sampled introductions to die out. Finally, we quantified local transmission dynamics once introductions into Switzerland occurred, using a novel phylodynamic model. We find that transmission slowed 35 – 63% upon outbreak detection in summer 2020, but not in fall. This finding may indicate successful contact tracing over summer before overburdening in fall. The study highlights the added value of genome sequencing data for understanding transmission dynamics.One Sentence SummaryPhylogenetic and phylodynamic methods quantify the drop in case introductions and local transmission with implementation of public health measures.

Early treatment of polycythemia vera with ultra-low-dose interferon-α 2a is well tolerated and results in complete hematologic and major molecular remission and a strong reduction of all symptoms, especially pruritus and fatigue.

BackgroundIn December 2020, the United Kingdom (UK) reported a SARS-CoV-2 Variant of Concern (VoC) which is now named B.1.1.7. Based on initial data from the UK and later data from other countries, this variant was estimated to have a transmission fitness advantage of around 40-80% [1, 2, 3].AimThis study aims to estimate the transmission fitness advantage and the effective reproductive number of B.1.1.7 through time based on data from Switzerland.MethodsWe generated whole genome sequences from 11.8% of all confirmed SARS-CoV-2 cases in Switzerland between 14 December 2020 and 11 March 2021. Based on these data, we determine the daily frequency of the B.1.1.7 variant and quantify the variant’s transmission fitness advantage on a national and a regional scale.ResultsWe estimate B.1.1.7 had a transmission fitness advantage of 43-52% compared to the other variants circulating in Switzerland during the study period. Further, we estimate B.1.1.7 had a reproductive number above 1 from 01 January 2021 until the end of the study period, compared to below 1 for the other variants. Specifically, we estimate the reproductive number for B.1.1.7 was 1.24 [1.07-1.41] from 01 January until 17 January 2021 and 1.18 [1.06-1.30] from 18 January until 01 March 2021 based on the whole genome sequencing data. From 10 March to 16 March 2021, once B.1.1.7 was dominant, we estimate the reproductive number was 1.14 [1.00-1.26] based on all confirmed cases. For reference, Switzerland applied more non-pharmaceutical interventions to combat SARS-CoV-2 on 18 January 2021 and lifted some measures again on 01 March 2021.ConclusionThe observed increase in B.1.1.7 frequency in Switzerland during the study period is as expected based on observations in the UK. In absolute numbers, B.1.1.7 increased exponentially with an estimated doubling time of around 2-3.5 weeks. To monitor the ongoing spread of B.1.1.7, our plots are available online.

The rapid spread of the SARS-CoV-2 lineages B.1.1.7 (N501Y.V1) throughout the UK, B.1.351 (N501Y.V2) in South Africa, and P.1 (B.1.1.28.1; N501Y.V3) in Brazil has led to the definition of variants of concern (VoCs) and recommendations by the European Center for Disease Prevention and Control (ECDC) and World Health Organization (WHO) for lineage specific surveillance. In Switzerland, during the last weeks of December 2020, we established a nationwide screening protocol across multiple laboratories, focusing first on epidemiological definitions based on travel history and the S gene dropout in certain diagnostic systems. In January 2021, we validated and implemented an N501Y-specific PCR to rapidly screen for VoCs, which are then confirmed using amplicon sequencing or whole genome sequencing (WGS). A total of 3492 VoCs have been identified since the detection of the first Swiss case in October 2020, with 1370 being B1.1.7, 61 B.1.351, and none P.1. The remaining 2061 cases of VoCs have been described without further lineage specification. In this paper, we describe the nationwide coordination and implementation process across laboratories, public health institutions, and researchers, the first results of our N501Y-specific variant screening, and the phylogenetic analysis of all available WGS data in Switzerland, that together identified the early introduction events and subsequent community spreading of the VoCs.

Pathogen genomes provide insights into their evolution and epidemic spread. We sequenced 1,439 SARS-CoV-2 genomes from Switzerland, representing 3-7% of all confirmed cases per week. Using these data, we demonstrate that no one lineage became dominant, pointing against evolution towards general lower virulence. On an epidemiological level, we report no evidence of cryptic transmission before the first confirmed case. We find many early viral introductions from Germany, France, and Italy and many recent introductions from Germany and France. Over the summer, we quantify the number of non-traceable infections stemming from introductions, quantify the effective reproductive number, and estimate the degree of undersampling. Our framework can be applied to quantify evolution and epidemiology in other locations or for other pathogens based on genomic data.One Sentence SummaryWe quantify SARS-CoV-2 spread in Switzerland based on genome sequences from our nation-wide sequencing effort.

SARS-CoV-2, the virus responsible for the current COVID-19 pandemic, is evolving into different genetic variants by accumulating mutations as it spreads globally. In addition to this diversity of consensus genomes across patients, RNA viruses can also display genetic diversity within individual hosts, and co-existing viral variants may affect disease progression and the success of medical interventions. To systematically examine the intra-patient genetic diversity of SARS-CoV-2, we processed a large cohort of 3939 publicly-available deeply sequenced genomes with specialised bioinformatics software, along with 749 recently sequenced samples from Switzerland. We found that the distribution of diversity across patients and across genomic loci is very unbalanced with a minority of hosts and positions accounting for much of the diversity. For example, the D614G variant in the Spike gene, which is present in the consensus sequences of 67.4% of patients, is also highly diverse within hosts, with 29.7% of the public cohort being affected by this coexistence and exhibiting different variants. We also investigated the impact of several technical and epidemiological parameters on genetic heterogeneity and found that age, which is known to be correlated with poor disease outcomes, is a significant predictor of viral genetic diversity.Author SummarySince it arose in late 2019, the new coronavirus (SARS-CoV-2) behind the COVID-19 pandemic has mutated and evolved during its global spread. Individual patients may host different versions, or variants, of the virus, hallmarked by different mutations. We examine the diversity of genetic variants coexisting within patients across a cohort of 3939 publicly accessible samples and 749 recently sequenced samples from Switzerland. We find that a small number of patients carry most of the diversity, and that patients with more diversity tend to be older. We also find that most of the diversity is concentrated in certain regions and positions of the virus genome. In particular, we find that a variant reported to increase infectivity is among the most diverse positions. Our study provides a large-scale survey of within-patient diversity of the SARS-CoV-2 genome.

Accumulating evidence regarding the causative role of human papillomavirus (HPV) in a wide range of malignant and nonmalignant diseases highlights the importance of HPV testing. This study describes and discusses the efficacy and characteristics of 4 well-established and commercially available tests. Here, 181 cytologic specimens from cervical smears were analyzed using the HPV SIGN PQ (Diatech) and the Linear Array (Roche) method. Discrepant results were further studied with the Real Time High-Risk HPV (Abbott) method and the INNO-LiPA (Fujirebio) method. Of 181 cytologic specimens, 61 (34%) showed discrepant results. High-risk HPV was not detected in 9 cases by HPV SIGN PQ, in 16 cases by Linear Array, in 10 cases by Real Time High-Risk HPV, and in 6 cases by INNO-LiPA, respectively. Lack of DNA detection or problems in interpreting the result were seen in 9 cases with HPV SIGN PQ, 8 cases with Linear Array, 3 cases with Real Time High-Risk HPV, and 3 cases with INNO-LiPA, respectively. This study indicates that the choice of HPV detection method has a substantial influence on the HPV risk classification of tested PAP smears and clinical follow-up decisions.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that can be used for vaccination purposes, to induce a specific T-cell response in vivo against melanoma-associated antigens. We have shown that the sequential use of early-acting hematopoietic growth factors, stem cell factor, IL-3 and IL-6, followed by differentiation with IL-4 and granulocyte-macrophage colony-stimulating factor allows the in vitro generation of large numbers of immature DCs from CD34(+) peripheral blood progenitor cells. Maturation to interdigitating DCs could specifically be induced within 24 hr by addition of TNF-alpha. Here, we report on a phase I clinical vaccination trial in melanoma patients using peptide-pulsed DCs. Fourteen HLA-A1(+) or HLA-A2(+) patients received at least 4 i.v. infusions of 5 x 10(6) to 5 x 10(7) DCs pulsed with a pool of peptides including either MAGE-1, MAGE-3 (HLA-A1) or Melan-A, gp100, tyrosinase (HLA-A2), depending on the HLA haplotype. A total of 83 vaccinations were performed. Clinical side effects were mild and consisted of low-grade fever (WHO grade I-II). Clinical and immunological responses consisted of anti-tumor responses in 2 patients, increased melanoma peptide-specific delayed-type hypersensitivity reactions in 4 patients, significant expansion of Melan-A- and gp100-specific cytotoxic T lymphocytes in the peripheral blood lymphocytes of 1 patient after vaccination and development of vitiligo in another HLA-A2(+) patient. Our data indicate that the vaccination of peptide-pulsed DCs is capable of inducing clinical and systemic tumor-specific immune responses without provoking major side effects.

Background and Objective Activity of human cytochrome P450 enzymes (CYPs) shows high inter-and intra-individual variability, which is determined by genetic and non-genetic factors. Using a combination of CYP-specific probe drugs, phenotyping cocktails allow simultaneous assessment of the activity of different CYP isoforms. The objective of this study was to characterize the phenotyping metrics of the Basel cocktail in healthy male subjects with induced and inhibited CYP activity. Methods In a randomized crossover study, the probe drugs for simultaneous phenotyping of CYP1A2 (caffeine), CYP2B6 (efavirenz), CYP2C9 (losartan), 2C19 (omeprazole), CYP2D6 (metoprolol), and CYP3A4 (midazolam) were administered to 16 subjects without pretreatment (baseline), after pretreatment with a combination of CYP inhibitors (ciprofloxacin, ketoconazole, and paroxetine), and after CYP induction with rifampicin. All subjects were genotyped. Pharmacokinetic profiles of the probe drugs and their main metabolites and metabolic ratios 2, 4, 6, and 8 h after probe drug application were determined in plasma and compared with the corresponding area under the plasma concentration-time curve (AUC) ratios. Results The Basel phenotyping cocktail was well tolerated by all subjects independent of pretreatment. Good correlations of metabolic ratios with AUC ratios of the corresponding probe drugs and their metabolites for all three conditions (baseline, CYP inhibition, and CYP induction) were found at 2 h after probe drug administration for CYP3A4, at 4 h for CYP1A2 and CYP2C19, and at 6 h for CYP2B6 and CYP2D6. While CYP inhibition significantly changed AUC ratios and metabolic ratios at these time points for all six CYP isoforms, CYP induction did not significantly change AUC ratios for CYP2C9. For CYP3A4, total 1′-hydroxymidazolam concentrations after pretreatment of samples with β-glucuronidase were needed to obtain adequate reflection of CYP induction by the metabolic ratio. Conclusions Inhibition of CYP activity can be detected with the Basel phenotyping cocktail for all six tested CYP isoforms at the proposed time points. The AUC ratio of losartan:losartan carboxylic acid in plasma does not seem suitable to detect induction of CYP2C9. The observed metabolic ratios for inhibited and induced CYP activity need to be confirmed for extensive metabolizers, and typical ratios for subjects with genetically altered CYP activity will need to be established in subsequent studies. ClinicalTrials.gov-ID: NCT01386593. Electronic supplementary material The online version of this article (doi:10.1007/s40262-015-0294-y) contains supplementary material, which is available to authorized users.

A new molecular subtyping approach was developed which is based on the amplification and sequencing of a repetitive region of the P1 gene of Mycoplasma pneumoniae . It allows the differentiation of all known subtypes and variants of M. pneumoniae as well as the identification of new subtypes directly in clinical samples to characterize endemic and epidemic M. pneumoniae infections.

We performed a phase I/II clinical trial in metastatic melanoma patients with an ultraviolet (UV)-inactivated nonreplicating recombinant vaccinia virus enabling the expression, from a single construct, of endoplasmic reticulum-targeted HLA-A0201-restricted Melan-A/MART-1(27-35), gp100(280-288), and tyrosinase(1-9) epitopes, together with CD80 and CD86 costimulatory proteins. Corresponding soluble peptides were used to boost responses and granulocyte-macrophage colony-stimulating factor was used as systemic adjuvant. Safety and immunogenicity, as monitored with in vitro-restimulated peripheral blood mononuclear cells by cytotoxic T lymphocyte precursor (CTLp) frequency analysis and tetramer staining, were specifically addressed. Of 20 patients entering the protocol, 2 had to withdraw because of rapidly progressing disease. Immune responses were evaluated in 18 patients (stage III, n = 5; stage IV, n = 13) and increases in specific CTLp frequencies were observed in 15. In 16 patients responsiveness against all 3 antigens could be analyzed: 7 (43%), including all stage III cases, showed evidence of induction of CTLs specific for the three epitopes, and 2 (12%) and 4 (25%), respectively, showed reactivity against two or one tumor-associated antigen. In three stage IV patients no specific CTL reactivity could be induced. Increases in CTLp frequency were detected mostly after viral vaccine injections. However, in a majority of patients final CTLp levels were comparable to initial levels. Tetramer characterization of Melan-A/MART-1(27-35)-specific CTLs during the protocol also suggested preferential expansion after recombinant virus administration. Vector-specific humoral responses, frequently undetectable in stage IV patients, did not appear to prevent tumor-associated antigen-specific CTL induction. Aside from a single occurrence of transient grade 3 leukopenia, no major clinical toxicity was reported. Seventeen of 18 patients completed the 3-month trial (one patient died before the last delayed-type hypersensitivity test). Three displayed regression of individual metastases, seven had stable disease, and progressive disease was observed in seven patients. This is the first report on the administration of a UV-inactivated recombinant vaccinia virus coexpressing five transgenes in cancer patients. The results described here, in terms of safety and immunogenicity, support the use of this reagent in active specific immunotherapy.

Background The number of predictive biomarkers that will be necessary to assess in clinical practice will increase with the availability of drugs that target specific molecular alterations. Therefore, diagnostic laboratories are confronted with new challenges: costs, turn-around-time and the amount of material required for testing will increase with the number of tests performed on a sample. Our consortium of European clinical research laboratories set out to test if semi-conductor sequencing provides a solution for these challenges. Methods We designed a multiplex PCR targeting 87 hotspot regions in 22 genes that are of clinical interest for lung and/or colorectal cancer. The gene-panel was tested by 7 different labs in their own clinical setting using ion-semiconductor sequencing. Results We analyzed 155 samples containing 112 previously identified mutations in the KRAS, EGFR en BRAF genes. Only 1 sample failed analysis due to poor quality of the DNA. All other samples were correctly genotyped for the known mutations, even as low as 2%, but also revealed other mutations. Optimization of the primers used in the multiplex PCR resulted in a uniform coverage distribution over the amplicons that allows for efficient pooling of samples in a sequencing run. Conclusions We show that a semi-conductor based sequencing approach to stratify colon and lung cancer patients is feasible in a clinical setting. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1015-5) contains supplementary material, which is available to authorized users.

TAAs of the MAGE family are mostly studied as targets of specific immune responses. Their potential relevance as tumor markers has also been underlined. We used a MAb, 57B, recognizing MAGE-A4 protein in paraffin-embedded sections, to evaluate its expression in bladder cancers by employing TMA including 2,317 samples from 1,849 patients. In 2,090/2,317 cases (90.2%), immunostaining yielded interpretable results. Since for some patients more than 1 sample was available, only interpretable first biopsies (n = 1,628) were considered. MAGE-A4 protein was expressed at significantly (p < 0.001) higher frequency in squamous (25/55, 45.5%) than in adeno (4/15, 26.7%), sarcomatoid (4/14, 28.6%), small cell (5/20, 25%) or transitional cell (281/1,522, 18.5%) carcinomas. In TCCs, overall MAGE-A4 positivity was significantly correlated with invasive phenotype (p < 0.001) and high tumor grade (p < 0.0001). Clinical data from 908 TCC patients were retrospectively evaluated, revealing that strong 57B staining was highly significantly associated with decreased tumor-specific survival (p < 0.0001). These data suggest that evaluation of MAGE-A4 protein expression is useful in the identification of groups of TCCs characterized by severe prognosis, thus possibly providing indications for early MAGE TAA-targeted immunotherapy.

A specific cellular immune response directed against a panel of three defined tumor-associated antigen (TAA) epitopes was induced in metastatic melanoma patients by a prime-boost strategy taking advantage of an innovative recombinant vaccinia virus as evaluated by quantitative assessment of cytotoxic T lymphocytes (CTLs) with corresponding specificity. The immunization protocol consisted of the administration of psoralen-UV-treated and replication-incompetent recombinant vaccinia virus encoding the three immunodominant HLA-A*0201-restricted epitopes Melan-A(27-35), gp100(280-288), and tyrosinase(1-9) together with two costimulatory molecules, B7.1 and B7.2, in the context of systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. Boosts were subsequently applied with corresponding synthetic nonapeptides and GM-CSF. Specific CTL induction was assessed by tetramer staining and CTL precursor (CTLp) frequency evaluation. Within 12 days of injection of the recombinant vector, cytotoxic T cell responses specific for engineered epitopes were detectable in three of three patients. During the vaccination treatment, antigen-specific CTLp frequencies exceeding 1:10,000 peripheral CD8(+) T cells could be observed. Tetramer staining also revealed significant increases in specific CD8(+) T cell numbers. We conclude that active specific antitumor vaccination can raise a concurrent and specific cellular immune response against a panel of molecularly defined antigens, thereby increasing the chance of an immune hit against neoplastic cells with heterogeneous antigen expression. Data from this study emphasize the potency of a recombinant vaccinia virus vector encoding multiple minigenes and costimulatory molecules in the context of exogenously administered GM-CSF. Clinical effectiveness of this immunologically active protocol should therefore be explored in appropriately selected groups of patients.

Background Many current methods for the detection of gene variants relevant for inherited disorders like malignant hyperthermia (MH) rely on the polymerase chain reaction (PCR). A positive PCR result for the genetic region of interest is required for downstream analysis like sequencing, restriction digestion or probe hybridization. However, it is sometimes overlooked that in order to obtain a true genotype, both paternal and maternal alleles must be represented in the PCR product. We present the case of a preferential PCR amplification of one ryanodine receptor subtype 1 (RyR1) mutant allele that led to apparent homozygosity of a proband.

NY-ESO-1 gene encodes a novel member of the cancer/testis (CT) family of human tumour-associated antigens (TAA). Specific monoclonal antibodies (mAb) have identified the corresponding gene product in lysates of tumour cell lines as a 22 kDa protein but no data are available concerning its intracellular location or distribution within neoplastic tissues. We have generated NY-ESO-1 specific mAbs recognizing the target molecule in cytospin preparations and in sections from clinical tumour specimens. These reagents identify NY-ESO-1 TAA in melanoma cell lines expressing the specific gene as a cytoplasmic protein, sharing the intracellular location of most MAGE TAA. In a series of 12 melanoma specimens, specific staining, limited to neoplastic cells, was detectable in the five cases where NY-ESO-1 gene expression was observed. In two of them over 90% of tumour cells showed evidence of positive staining. Lower percentages of positive neoplastic cells ranging between single cells and 50% were observed in the remaining tumours. These data suggest that active specific immunotherapies targeting NY-ESO-1, alone or in combination with other TAA could be of high clinical relevance in sizeable subgroups of melanoma patients. © 2000 Cancer Research Campaign

Granulocyte-macrophage colony stimulating factor (GM-CSF) gene expression and protein production were investigated in colorectal cancer cell lines and surgical specimens. In 6 of 6 established tumor lines, expression of the GM-CSF gene was observed by reverse transcription polymerase chain reaction (RT-PCR). Furthermore, for 2 of the lines, the cytokine was detectable in > or = 100 pg/ml amounts in culture supernatants by enzyme-linked immunosorbent assay tests. Addition of recombinant GM-CSF at doses ranging between 30 pg and 30 ng/ml did not appear to affect the proliferation of colorectal cancer cell lines as measured by 3H-thymidine incorporation. GM-CSF gene expression was then examined in surgical specimens by a densitometry-assisted, semiquantitative RT-PCR technique. In the 10 samples analyzed, significantly higher expression was detectable in tumors, as compared with autologous healthy mucosa sampled in the vicinity (2 cm) or at distance (10 cm) from the neoplastic focus. Immunohistochemistry studies performed on 13 specimens led to the identification of intracytoplasmic GM-CSF in tumor cells in 9 samples. In 6 of these, positivity of stromal fibroblasts and lymphocytes adjacent to the tumor was also observed. In contrast, intracellular GM-CSF was only detectable in 2 cases in untransformed epithelial cells, close to the neoplasm, but never in healthy mucosa at distance from the tumor. Infiltration by dendritic cells (DC) was also investigated. In 5 of 8 colorectal cancers tested, DC aggregates accounted for more than 10% of stromal cells. Lower numbers were detectable in healthy mucosa. However, DC infiltration could not be correlated with the presence of GM-CSF-positive neoplastic cells in the tumor specimens. Remarkably, cultured DC were unable to exert significant cytotoxic activity against colorectal cancer cells in vitro.

In this study, a computer-assisted reverse immunology approach was utilized in order to identify potentially antigenic peptides derived from the differentiation antigen TRP-2, a melanosomal protein frequently expressed in melanoma. Among the seven peptides complying with HLA-A2.1-binding motifs, two induced specific CD8(+) cytotoxic T lymphocytes. HLA-A2.1(+) melanoma cells expressing TRP-2 were lysed by clones specific for TRP-2(360-368) (TLDSQVMSL) peptide, thus identifying it as a naturally processed epitope. Other T-cell clones directed against TRP-2(476-484) (VMGTLVALV) were unable to lyse HLA-matched TRP-2(+) cell lines. The role of intracellular proteolytic processing in the generation of this epitope was investigated by transfecting mini-genes encoding the TRP-2(476-484) peptide alone or carrying N- or C-terminal extensions. Specific T-cell clones recognized target cells expressing the cytotoxic T-lymphocyte (CTL)-defined epitope or its C-terminally extended precursor, but failed to recognize cells expressing the N-terminally extended TRP-2(476-484) peptide, suggesting the presence of a negative processing signal (NPS). Regarding C-terminus-flanking regions, mutational analysis indicates that the GLY485 residue plays a key role in the processing of the TRP-2(476-484) epitope. Interestingly, proteasome inhibitors preventing the generation of the MART-1/Melan-A(27-35) immunodominant melanoma tumor-associated antigen (TAA) promoted detectable presentation of TRP-2(476-484) epitope in HLA-A2.1(+) and TRP-2(+) tumor lines, as witnessed by cytokine release by specific T-cell clones.

In this study we used TEPITOPE, a new epitope prediction software, to identify sequence segments on the MAGE-3 protein with promiscuous binding to histocompatibility leukocyte antigen (HLA)-DR molecules. Synthetic peptides corresponding to the identified sequences were synthesized and used to propagate CD4+ T cells from the blood of a healthy donor. CD4+ T cells strongly recognized MAGE-3281–295 and, to a lesser extent, MAGE-3141–155 and MAGE-3146–160. Moreover, CD4+ T cells proliferated in the presence of recombinant MAGE-3 after processing and presentation by autologous antigen presenting cells, demonstrating that the MAGE-3 epitopes recognized are naturally processed. CD4+ T cells, mostly of the T helper 1 type, showed specific lytic activity against HLA-DR11/MAGE-3–positive melanoma cells. Cold target inhibition experiments demonstrated indeed that the CD4+ T cells recognized MAGE-3281–295 in association with HLA-DR11 on melanoma cells. This is the first evidence that a tumor-specific shared antigen forms CD4+ T cell epitopes. Furthermore, we validated the use of algorithms for the prediction of promiscuous CD4+ T cell epitopes, thus opening the possibility of wide application to other tumor-associated antigens. These results have direct implications for cancer immunotherapy in the design of peptide-based vaccines with tumor-specific CD4+ T cell epitopes.

Granulocyte-macrophage colony stimulating factor (GM-CSF) gene expression and protein production were investigated in colorectal cancer cell lines and surgical specimens. In 6 of 6 established tumor lines, expression of the GM-CSF gene was observed by reverse transcription polymerase chain reaction (RT-PCR). Furthermore, for 2 of the lines, the cytokine was detectable inor = 100 pg/ml amounts in culture supernatants by enzyme-linked immunosorbent assay tests. Addition of recombinant GM-CSF at doses ranging between 30 pg and 30 ng/ml did not appear to affect the proliferation of colorectal cancer cell lines as measured by 3H-thymidine incorporation. GM-CSF gene expression was then examined in surgical specimens by a densitometry-assisted, semiquantitative RT-PCR technique. In the 10 samples analyzed, significantly higher expression was detectable in tumors, as compared with autologous healthy mucosa sampled in the vicinity (2 cm) or at distance (10 cm) from the neoplastic focus. Immunohistochemistry studies performed on 13 specimens led to the identification of intracytoplasmic GM-CSF in tumor cells in 9 samples. In 6 of these, positivity of stromal fibroblasts and lymphocytes adjacent to the tumor was also observed. In contrast, intracellular GM-CSF was only detectable in 2 cases in untransformed epithelial cells, close to the neoplasm, but never in healthy mucosa at distance from the tumor. Infiltration by dendritic cells (DC) was also investigated. In 5 of 8 colorectal cancers tested, DC aggregates accounted for more than 10% of stromal cells. Lower numbers were detectable in healthy mucosa. However, DC infiltration could not be correlated with the presence of GM-CSF-positive neoplastic cells in the tumor specimens. Remarkably, cultured DC were unable to exert significant cytotoxic activity against colorectal cancer cells in vitro.

BACKGROUND AND OBJECTIVE Phenotyping cocktails use a combination of cytochrome P450 (CYP)-specific probe drugs to simultaneously assess the activity of different CYP isoforms. To improve the clinical applicability of CYP phenotyping, the main objectives of this study were to develop a new cocktail based on probe drugs that are widely used in clinical practice and to test whether alternative sampling methods such as collection of dried blood spots (DBS) or saliva could be used to simplify the sampling process. METHODS In a randomized crossover study, a new combination of commercially available probe drugs (the Basel cocktail) was tested for simultaneous phenotyping of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. Sixteen subjects received low doses of caffeine, efavirenz, losartan, omeprazole, metoprolol and midazolam in different combinations. All subjects were genotyped, and full pharmacokinetic profiles of the probe drugs and their main metabolites were determined in plasma, dried blood spots and saliva samples. RESULTS The Basel cocktail was well tolerated, and bioequivalence tests showed no evidence of mutual interactions between the probe drugs. In plasma, single timepoint metabolic ratios at 2 h (for CYP2C19 and CYP3A4) or at 8 h (for the other isoforms) after dosing showed high correlations with corresponding area under the concentration-time curve (AUC) ratios (AUC0-24h parent/AUC0-24h metabolite) and are proposed as simple phenotyping metrics. Metabolic ratios in dried blood spots (for CYP1A2 and CYP2C19) or in saliva samples (for CYP1A2) were comparable to plasma ratios and offer the option of minimally invasive or non-invasive phenotyping of these isoforms. CONCLUSIONS This new combination of phenotyping probe drugs can be used without mutual interactions. The proposed sampling timepoints have the potential to facilitate clinical application of phenotyping but require further validation in conditions of altered CYP activity. The use of DBS or saliva samples seems feasible for phenotyping of the selected CYP isoforms.

Active specific immunotherapy targeting tumor-associated antigens (TAA) requires reagents of high immunogenicity and safety. To address this issue, we constructed a recombinant vaccinia virus carrying a minigene insert encoding the HLA-A2.I-restricted MART.l/melan-A 27-35 melanoma TAA (rVV-M). To facilitate the entry of the antigenic epitope into the endoplasmic reticulum, a sequence coding for adenovirus E3119K leader peptide was added. This rVV-M was made replication-incompetent by treatment with psoralen and UV light. Infection with rVV-M rendered HLA-A2.1 EBV-trans-formed lymphoblastoid cells sensitive to the cytotoxic effects of HLA-class-l-restricted, MART-1/Melan-A 27-35 -specific cytotoxic T lymphocytes (CTL). The capacity of rVV-M to generate HLA-A2.1-restricted MART-1/Melan A-specific CTL was demonstrated from tumor-infiltrating-lymphocyte (TIL) cultures and from healthy donors' peripheral-blood mononuclear cells (PBMC). MART- 1/Melan-A 27-35 specific CTL were generated from TIL after 2 weekly stimulation courses. Infection with rVV-M elicited a higher CTL response than addition of exogenous peptide, whereas, when a similar protocol was used to stimulate PBMC of healthy donors, significant and specific cytotoxic activity could be observed only upon rVV.M infection but not upon exogenous peptide addition. All CTL generated upon rVV-M stimulation were also able to efficiently kill melanoma cell lines expressing both MART-1/Melan-A and HLA-A2.1. In addition, TNF-α production could be induced in rVV-M-stimulated CTL upon co-culture with COS-7 cells transiently transfected with MART-l/Melan-A and HLA-A2.1 genes. This safe and highly immunogenic reagent could be of use in TAA-targeted clinical immunotherapy.

The authors' objective is to identify MAGE-1 tumor antigen in clinical melanoma specimens and to verify the extent of its expression in tumors where evidence of specific gene transcripts can be obtained.The MAGE-1 gene encodes a tumor-associated antigen that can be recognized by specific cytotoxic T lymphocytes. Transcription of the MAGE-1 gene has previously been demonstrated in various malignancies, but the production of the specific gene product and its distribution in neoplastic tissues have not yet been addressed.Total cellular mRNA was extracted from six melanoma biopsies, reverse-transcribed and tested in 25-45 cycles of reverse polymerase chain reaction (rtPCR) in the presence of primers' pairs specific for the beta-actin-positive control gene and for the MAGE-1-encoding gene. Concurrently, portions of these specimens were lysed and probed for MAGE-1 protein by immunoblotting. Additional material from the same biopsies was analyzed following immunohistological staining with MAGE-1-specific monoclonal antibodies.MAGE-1 gene transcription could be demonstrated following 25 cycles of rtPCR in one out of six biopsies and in three more following 35 cycles of rtPCR. 2/6 samples were negative even after 45 cycles of rtPCR. MAGE-1 protein production could be detected by immunoblotting in the lysates from biopsies showing evidence of specific gene transcription. Cells positive for MAGE-1 protein expression could be identified by immunohistochemistry on snap-frozen sections in three of the four tumors displaying specific transcripts. Distribution of positivity ranged between focal cellular areas and single positive cells in the different tumors.The MAGE-1 tumor antigen can be detected by specific monoclonal antibodies in clinical tumor specimens. The pattern of positivity observed in samples showing evidence of MAGE-1 gene expression suggests a relevant heterogeneity regarding MAGE-1 antigen production within individual tumor specimens.

Generation of cytotoxic-T-lymphocyte (CTL) responses against mutated ras peptides from peripheral-blood mononuclear cells (PBMC) was attempted in a group of HLA-A2.1+ healthy donors. Bulk PBMC cultures were stimulated in vitro with a mixture of peptides encompassing 12 Gly --> Val, 61 Gln --> Lys or 61 Gln --> Leu ras mutations and displaying HLA-A2.1 binding motifs, selected by a computer program. A promiscuous tetanus toxoid peptide was also added. Weekly thereafter, PBMC were re-stimulated with peptide pulsed autologous Epstein-Barr virus (EBV)-transformed B cells. After 8 rounds of re-stimulation, reproducible cytotoxic activity against peptide-pulsed target cells was detectable in one donor. The CTL line recognized 2 nonamers encompassing ras 61 Gln --> Leu mutation. Killing was mediated by CD8+ T cells displaying alphabeta TCR and was inhibited by anti-HLA-A2.1 monoclonal antibodies. No killing of tumor cells expressing the specific mutation could be observed. More than 60 CTL clones were generated. Fine specificity studies revealed effective, though differing cytotoxic activity against both 53-LDILDTAGL-61 and 55-ILDTAGLEE-63, but not against 54-DILDTAGLE-62 mutated peptides, in all but one of the clones. None was able to exert effective cytotoxic activity against tumor cells expressing the specific mutation. T-cell-receptor (TCR) usage was then analyzed phenotypically, by reverse-transcription-polymerase-chain-reaction (RT-PCR) and by sequence analysis. This study revealed the monoclonal nature of the CTL response against mutated nonamers, with TCR expressing Vbeta14 gene product in combination with, Jbeta2.7 and Cbeta2.

Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disorder displaying features reminiscent of premature senescence caused by germline mutations in the LMNA gene encoding lamin A and C, essential components of the nuclear lamina. By studying a family with homozygous LMNA mutation (K542N), we showed that HGPS can also be caused by mutations affecting both isoforms, lamin A and C. Here, we aimed to elucidate the molecular mechanisms underlying the pathogenesis in both, lamin A- (sporadic) and lamin A and C-related (hereditary) HGPS. For this, we performed detailed molecular studies on primary fibroblasts of hetero- and homozygous LMNA K542N mutation carriers, accompanied with clinical examinations related to the molecular findings. By assessing global gene expression we found substantial overlap in altered transcription profiles (13.7%; 90/657) in sporadic and hereditary HGPS, with 83.3% (75/90) concordant and 16.7% (15/90) discordant transcriptional changes. Among the concordant ones we observed down-regulation of TWIST2, whose inactivation in mice and humans leads to loss of subcutaneous fat and dermal appendages, and loss of expression in dermal fibroblasts and periadnexial cells from a LMNA(K542N/K542N) patient further confirming its pivotal role in skin development. Among the discordant transcriptional profiles we identified two key mediators of vascular calcification and bone metabolism, ENPP1 and OPG, which offer a molecular explanation for the major phenotypic differences in vascular and bone disease in sporadic and hereditary HGPS. Finally, this study correlates reduced TWIST2 and OPG expression with increased osteocalcin levels, thereby linking altered bone remodeling to energy homeostasis in hereditary HGPS.

We report on the clinical and cytogenetic findings as well as the array-based characterization of an interstitial familial 13q21 deletion initially recognized by standard karyotyping. Although 13q deletions are known to imply a wide variability of clinical consequences, the deletion carriers of the familial deletion in three generations did not reveal a relevant phenotype. The breakpoints and the deletion size in all three carrier individuals were determined by molecular karyotyping confirming a large 14.5 Mb deletion encompassing the 13q21.1-13q21.33 region identical in all three carriers. Gene paucity and the lack of dosage-sensitive genes in the delineated region might explain the apparently innocuous nature of this chromosomal anomaly. The example of this family presents evidence for describing the chromosomal region 13q21.1-13q21.33 as a large euchromatic variant or benign copy number variation without phenotypic consequences. Our data underline the importance of a phenogenetic approach combining clinical and laboratory evidence in the interpretation of segmental chromosomal anomalies especially in genetic counseling related to prenatal diagnosis.

Approximately 7% of non-small cell lung carcinomas (NSCLCs) harbor oncogenic fusions involving ALK, ROS1, and RET. While tumors harboring ALK fusions are highly sensitive to crizotinib, emerging data have demonstrated that individuals with ROS1 or RET fusions may also benefit from inhibitors targeting these two kinases. A new oncogenic fusion involving NTRK1 that can be targeted by kinase inhibitors has been recently described. The two main methods to identify translocations, fluorescent in situ hybridization (FISH) and Immunohistochemistry (IHC) are labor intensive and sensitivity is rather low. Common sample types for lung cancer analysis are biopsies making the study of more than one fusion gene rather difficult. In this study we aimed to develop and validate a workflow based on AmpliSeq™ technology to comprehensively profile ALK, ROS1, RET and NTRK1 chromosomal translocations in lung tumors. Two hundred lung cancer retrospective samples, including tumor biopsy, were collected from 12 laboratories - the OncoNetwork Consortium. All the samples were previously characterized by orthologous techniques (FISH, IHC, RT-PCR and/or MassArray). More than 30 ALK positive samples were selected. The panel targets over 70 fusion transcripts associated with ALK, RET, ROS1, and NTRK1 genes. The panel also includes 5′ and 3′ gene expression assays for each gene as indicators of a translocation event and assays for 5 internal control genes. The workflow is compatible with formalin fixed paraffin embedded (FFPE) tissue, requiring only 10ng of total RNA and able to multiplex up to 16 libraries on a single Ion 318™ chip. A dedicated data analysis pipeline using the Ion Reporter software 4.2 was evaluated. Serial dilutions of cell lines RNA harboring known fusion events, in normal RNA showed that the methodology had 1% mutant RNA limit of detection. A second dilution experiment using RNA extracted from FFPE lung fusion positive samples diluted in a negative FFPE sample showed a detection limit of 15%. The use of a common control fusion positive RNA samples among all the consortia laboratories showed 100% reproducibility. Concordance study was performed using 200 FFPE samples previously characterized with standard method. A concordance of >98% was obtained between the methodologies. The discordant results are currently under study. A negative FFPE samples was run as single test on an Ion 318™ chip on to evaluate the possibility of false positive results due to high number of reads. No false positive was detected in this experiment. In this study we present a workflow that provides a robust, reproducible and accurate comprehensive genetic screening tool well suited for FFPE lung tumor biopsies, in a fast and cost-efficient manner. This may provide a valuable tool for reducing turn-around-time and expense in lung cancer analysis. Citation Format: Pierre Laurent - Puig, Jose Louis Costa, Orla Sheils, Bastiaan Tops, Andrea Mafficini, Delphine Le Corre, Henriette Kurth, Anna Maria Rachiglio, Helene Blons, Eliana Amato, Christoph Noppen, Renato Franco, Anne Reiman, Roy Bastien, Noah Welker, Jose Carlos Machado, Ian Cree, Harriet Feilotter, Marjolijn Ligtenberg, Aldo Scarpa, Nicola Normanno, Kazuto Nishio, Cecily Vaughn. Comprehensive genetic profiling of chromosomal translocations in lung cancer tumors: development and validation of a next-generation sequencing panel in an international multicenter study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4891. doi:10.1158/1538-7445.AM2015-4891

In an ongoing phase I/II study, metastatic melanoma patients were treated with a replication-incompetent recombinant vaccinia virus (rVV) encoding Melan-A(27-35), gp100(280-288), and tyrosinase(1-9) HLA-A*201-restricted epitopes together with B7.1 and B7.2 co-stimulatory molecules. rVV was administered in the context of systemic GM-CSF treatment. Boosts were subsequently administered 2 weeks apart with corresponding synthetic nonapeptides and GM-CSF. Two cycles of treatment were administered 2 weeks apart from each other. Specific immune responses were evaluated by quantitative assessment of cytotoxic T-lymphocyte precursor frequency and tetramer staining. By the time the two cycles had been completed, four out of five patients showed significant (greater than threefold) increases in gp100(280-288)-specific and four out of five, in Melan-A(27-35)-specific tetramer staining of CD8+ cells. Frequencies of CTL precursors specific for gp100(280-288), tyrosinase(1-9) and Melan-A(27-35) were also significantly increased in all five, and in four and four of the five patients, respectively, in some cases within 12 days after the first injection of the recombinant vector. Thus, the innovative vector under investigation is able to raise a concurrent and specific cellular immune response against a panel of molecularly defined antigens, thereby increasing the chance of an immune hit against neoplastic cells displaying heterogeneous antigen expression.

In an ongoing phase I/II study, metastatic melanoma patients were treated with a replication-incompetent recombinant vaccinia virus (rVV) encoding Melan- A27–35, gpl00280–288, and tyrosinase1–9 HLA-A★201-restricted epitopes together with B7.1 and B7.2 co-stimulatory molecules. rVV was administered in the context of systemic GM-CSF treatment. Boosts were subsequently administered 2 weeks apart with corresponding synthetic nonapeptides and GM-CSF. Two cycles of treatment were administered 2 weeks apart from each other. Specific immune responses were evaluated by quantitative assessment of cytotoxic T- lymphocyte precursor frequency and tetramer staining. By the time the two cycles had been completed, four out of five patients showed significant (greater than threefold) increases in gpl00280–288-specific and four out of five, in Melan- A27–35 -specific tetramer staining of CD8+ cells. Frequencies of CTL precursors specific for gpl00280–288, tyrosinase1–9 and Melan-A27–35 were also significantly increased in all five, and in four and four of the five patients, respectively, in some cases within 12 days after the first injection of the recombinant vector. Thus, the innovative vector under investigation is able to raise a concurrent and specific cellular immune response against a panel of molecularly defined antigens, thereby increasing the chance of an immune hit against neoplastic cells displaying heterogeneous antigen expression.

Chromosomal translocations and corresponding gene fusions play an important role in the initiation of tumorigenesis and these processes have been strongly associated with distinct tumor subtypes. The recent association of ALK, ROS and RET fusion transcripts as lung tumor therapy predictive biomarkers has increased the need of a technology that could detect these biomarkers starting from limited amount of material. Life Technologies and OncoNetwork consortium collaborated for the development of a lung fusion panel based on Ion AmpliSeq™ RNA chemistry. The OncoNetwork consortium is comprised of twelve -translational cancer research institutes with many years of experience in adopting the latest molecular techniques for lung therapy research. Material and Methods: Consortia's requirements for the panel development : 1) detect all variants of ALK, ROS1, or RET fusion transcripts described in COSMIC in a single reaction using 10 ng of total RNA 2) Include 5′ and 3′ ALK, ROS1, RET gene expression assays as an indicator of a translocation at this gene. 3) Include endogenous RNA assay controls to determine if the quality of the results could be affected by RNA quality. 4) Provide similar results on archived FFPE samples tested by FISH. Human lung adenocarcinoma cell lines H2228 (EML4-ALK positive), HCC78 (SLC4A2-ROS1 positive ) ,LC-2/ad (CCDC6-RET positive) and Ambion® FirstChoice® Human Brain Reference (HBR) RNA was used as positive and negative control for the study. Formalin fixed paraffin embedded (FFPE) tissue was isolated using different extraction methods. After amplification using Ion AmpliSeq™ RNA chemistry samples were sequenced on the Ion Torrent PGM™ sequencer using the Ion PGM™ 200 Sequencing Kit. The presence of the fusions was confirmed by custom TaqMan® gene expression assays when possible. Preliminary results of the panel on ALK or ROS1 or RET positive cell lines and FFPE archived cancer research samples gave good concordance with FISH results. Expected negative samples were confirmed negative. We found a KIF5B-RET fusion positive sample in a sample not previously tested for RET fusions. Two of the expected positive samples by FISH were found negative due to limited amount to tumor cells present in the sample. Cell line RNA dilutions were performed to determine the panel's limit of detection. We demonstrate a limit of detection of 1 % tumor RNA in the presence of 99 % normal RNA using the panel with 10 ng of RNA extracted by cell lines. Gene expression controls work well as an indicator of the RNA quality and of the translocation presence. The Ion AmpliSeq™ RNA lung cancer fusion panel workflow is easier and faster to perform in comparison to the FISH method. The results obtained to date are highly encouraging for panel to be used in the clinical research setting. More data needs to be analyzed before a final conclusion is made. Citation Format: Susan M. Magdaleno, Angie Cheng, Rosella Petraroli, Orla Sheils, Bastiaan Tops, Delphine Le Corre, Henriette Kurth, Helene Blons, Eliana Amato, Andrea Mafficini, Anna Maria Rachiglio, Anne Reimann, Christoph Noppen, Chrysanthi Ainali, Jin Katayama, Renato Franco, Harriet Feilotter, Jeoffrey Schageman, Ian Cree, Andrew Felton, Jose Luis Costa, Alain Rico, Aldo Scarpa, Jose Carlos Machado, Kazuto Nishio, Nicola Normanno, Marjolijn Ligtenberg, Cecily P. Vaughn, Ludovic Lacroix, Pierre Laurent-Puig. The OncoNetwork Consortium: A global collaborative research study on the development and verification of an Ion AmpliSeq RNA gene lung fusion panel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3575. doi:10.1158/1538-7445.AM2014-3575

The effect on immunogenicity of different tumor T cell epitope formulations was evaluated in vitro using nonreplicating recombinant vaccinia vector expressing two forms of the melanoma-associated MART-1/Melan-A antigen. The first recombinant virus expressed a minigene encoding a fusion product between an endoplasmic reticulum (ER)-targeting signal and the HLA-A201 binding 27-35 peptide. The second viral construct encoded the complete MART-1/Melan-A protein. The capacity of HLA-A201 cells infected with either viral construct to generate and to stimulate MART-1/Melan-A 27-35 specific cytotoxic T-lymphocytes (CTL), was comparatively characterized. The results obtained here with a tumor antigen confirmed the capacity of vaccinia virus-encoded ER-minigene to generate a very strong antigenic signal. In cytotoxicity assays, recognition of target cells infected with high amounts of both recombinant viruses with activated specific CTL clones, resulted in similar lytic activity. With regard to calcium mobilization, TCR down-regulation, IFN-gamma release, and T cell proliferation assays, the targeted epitope elicited 10- to 1000-fold stronger responses. Remarkably, the immunogenic difference between the two formulations, in their respective capacity to generate CTL from naive HLA-A2 peripheral blood mononuclear cells in vitro as measured by tetramer detection, was lower (2- to 3-fold). Recombinant vectors expressing complete antigens have demonstrated their capacity to generate specific responses and such vaccines might take advantage of a broader potential of presentation. However, as demonstrated here for the HLA-A201-restricted MART-1/Melan-A immunodominant epitope, nonreplicative vaccinia virus expressing ER-targeted minigenes appear to represent a significantly more immunogenic epitope vaccine formulation.

To gain new insights into the functional interaction between DC and neoplastic cells, we have analyzed the effects of melanoma and colorectal cancer lines on the chemotaxis and the phenotype of monocyte-derived DC in vitro . Both types of tumor cells displayed effective chemoattractive capacity towards immature, but not mature DC. Furthermore, conditioned medium of discrete melanoma lines induced upregulation of CD80, CD86, MHC class I, and MHC class II molecules on immature DC. However, de novo expression of E-cadherin and strong upregulation of CD15 could also be detected in the absence of CD83 expression. Melanoma-conditioned DC exhibited an increased adhesion capacity to a melanoma cell line in vitro and did not migrate in response to SLC chemokine. Tumor-infiltrating CD15 + cells displaying DC morphology could also be detected by immunohistochemistry in the original tumor specimens from which discrete melanoma cell lines under investigation were derived. Colorectal cancer cell lines, although able to chemoattract immature DC, were apparently unable to modulate their phenotype. Altogether our results suggest that tumor cells can attract immature DC in vitro and, eventually, modulate their phenotype. As a result, DC mobility could be severely impaired.

Hintergrund: Bei Melanomen konnen Tumor-assoziierte Antigene (TAA) Ziele einer spezifischen zytotoxischen T-Zellantwort sein. Das NY-ESO-1-Gen kodiert ein neues TAA sogenanntes „tumorspezifisches Antigen“. Bislang wurde die Antigenexpression nur auf der Transkriptionsebene nachgewiesen, und es war nicht bekannt, ob das entsprechende Protein in Tumorzellen produziert wird. Wir haben deshalb NY-ESO-1-spezifische monoklonale Antikorper (mAk) hergestellt, die den Nachweis des NY-ESO-1-TAA auf Proteinebene ermoglichen.

C-type lectin-like inhibitory receptors are heterodimers consisting of CD94 and NKG2-A-B molecules expressed on NK cells and on a subset of activated T lymphocytes. Their inhibitory effects on NK cytotoxicity and on the NK-like activity of T cell clones have been demonstrated, but no data are currently available on antigen-specific class I-restricted cytotoxic T lymphocytes (CTL). We have generated a panel of HLA-A2.1-restricted CTL clones directed against a nonapeptide derived from a melanoma-associated antigen, dopachrome tautomerase (TRP-2). All clones were CD8+ and TCR alphabeta+. About half of them expressed a CD94bright phenotype, whereas the remaining were CD94dim. Only the CD94bright CTL expressed the NKG2-A-B gene, consistent with the expression of a C-type, lectin-like, inhibitory CD94/NKG2-A-B heterodimer. Both CD94bright and CD94dim clones appeared to require similar amounts of synthetic epitope sensitizing target cells. Addition of anti-CD94 mAb resulted in a significant increase of specific killing by CD94bright, but not by CD94dim clones in the presence of suboptimal concentrations of peptide, whereas, when optimal amounts were used, the mAb did not induce a significant modulation of the cytotoxicity. Antigen-induced inward [Ca2+]i fluxes were unaffected, but an enhancement of TCR down-modulation could be observed in the presence of anti-CD94 mAb at high concentration of antigenic peptide. The analysis of the TCR-Vbeta repertoire of the CTL clones by RT-PCR and immunofluorescence revealed that all clones regardless of CD94 phenotype shared Vbeta22 expression. Most importantly, sequence analysis showed that they all expressed identical Vbeta22 TCR rearranged with Jbeta2.1 and Cbeta2. Taken together, these data indicate that different expression of functionally active lectin-like inhibitory receptors can be detected in CTL clones sharing identical TCR sequence and peptide specificity.

Genes of the MAGE family encode tumor-specific antigens recognized by cytotoxic T-lymphocytes in a variety of neoplasms. We investigated the protein expression of these antigens as related to the gene expression, in esophageal squamous cell carcinoma by using monoclonal antibodies recognizing MAGE gene products. Esophageal squamous cell carcinomas were found to express both MAGE-1 (4 out of 15 samples) and MAGE-3 (7 out of 15 samples) genes, by RT-PCR. Immunoblotting revealed MAGE-1 and MAGE-3 gene products in 2 and 6 out of 15 samples, respectively. Immunohistochemistry performed on 12 samples showed MAGE-1 protein expression, limited to single tumor cells, in 2 cases. MAGE-3 gene product was detectable in 7 cases: in 5 of them over 50% of neoplastic cells were positive. Considering the high percentages of tumor cells expressing MAGE-3 antigen, the use of epitope-based vaccines could be envisaged in patients displaying appropriate HLA-class I phenotype.

The knowledge of molecular alterations involved in colorectal carcinoma (CRC) and non-small-cell lung carcinoma (NSCLC) has significantly increased in the past few years. Molecular subgroups of tumors carrying mutations in druggable genes have been identified in CRC and NSCLC. In this respect, the availability of approaches that are compatible with small amounts of input DNA from FFPE tissue and that allow a rapid screening of a large number of somatic mutations is definitely needed. Ion AmpliSeq™ technology introduces a groundbreaking workflow enabling the rapid sequencing of hundreds of mutations with low allele frequency using as little as 10ng of DNA per reaction. The OncoNetwork Consortium is a European collaborative effort of 8 Cancer Translational Research Institutions to evaluate a custom panel targeting hotspots mutations in 22 genes implicated in CRC and NSCLC. In particular, 7 labs will test the gene panel in 3 phases using 180 retrospective FFPE samples previously tested with orthogonal technologies, and 1 lab will perform confirmatory testing on novel SNPs. According to the established workflow, 10 ng of genomic DNA are used with a single primer pool that includes 86 pairs of primers spanning 15.7kb of targets. Individual libraries are barcoded during the ligation step, quantified and pooled 5 by 5 for amplification on spheres using the Ion OneTouch™ 200 Template Kit v2. Spheres are loaded on Ion 316 chip for sequencing with the Ion PGM™ Sequencer using the Ion PGM™ 200 Sequencing Kit. Results are analyzed using the Ion Reporter™ Software v1.2. In the first phase of the project, 7 labs received the same 5 blind, control FFPE samples. The average coverage obtained for the genes included in the panel ranged between 1.000x and 6.000x. The percentage of reads on target was 88-93% for all samples except for a sample showing a mean of 77%, with an average per base accuracy >98% for all the samples. Data analysis confirmed that all laboratories identified the expected mutations, when using a mapping quality value (MQV) of 4 as first filter. In the second phase of the project, each lab submitted to the consortium 10 previously tested CRC or NSCLC FFPE samples and received back 10 blind samples for sequencing. These samples were expected to contain 5 to 10 variants including point mutations and indels. The six labs that completed this phase were able to detect all the expected variants. Therefore, a 100% true positive rate was found in controls and blind study. The third phase of the project is ongoing and involves the assessment in each participating lab of 10 NSCLC and 5 CRC samples with a low percentage of tumor cells. In conclusion, the preliminary results suggest that the Onconetwork Ion AmpliSeq Colon and Lung Cancer panel is able to detect with high sensitivity and reproducibility the most frequent mutations of CRC and NSCLC by using a just 10 ng of DNA from FFPE samples. Citation Format: Nicola Normanno, Rosella Petraroli, Alain Rico, Henriette Kurth, Bastiaan Tops, Eliana Amato, Andrea Mafficini, Delphine Le Corre, Anna Maria Rachiglio, Anne Reiman, Orla Sheils, Christoph Noppen, Nora Rieber, Jonathan Mangion, Chrysanthi Ainali, Ludovic Lacroix, Ian Cree, Marjolijn Ligtenberg, Andy Felton, Aldo Scarpa, Pierre Laurent Puig. The OncoNetwork Consortium: A European Collaborative Research study on the development of an Ion AmpliSeq gene panel targeting hot spots in colon and lung cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 36. doi:10.1158/1538-7445.AM2013-36

The MAGE-1 gene encodes a protein encompassing a HLA-A1-restricted target epitope for cytolytic T lymphocytes. Monoclonal antibodies directed against the MAGE-1 protein were tested for usage in immunohistology of routine pathology material. Seven formalin-fixed, paraffin-embedded malignant melanomas were studied by the Avidin-Biotin complex (ABC) method with or without different antigen retrieval methods. Native, frozen tissues from the same tumours were used to validate the results by immunohistochemistry on frozen sections, by PCR for mRNA and by protein demonstration in tissue extracts using western blotting. Of 4 monoclonal antibodies tested, mAB 34B and mAB 77B were highly efficient in detecting MAGE-1 protein in deparaffinised sections with the regular ABC method after microwave pretreatment. In a series of an additional 28 patients 75% expressed MAGE-1, 50% in a substantial proportion. Follow-up studies in 6 patients indicate that the expression pattern remains stable but may change substantially within a short range. Immunohistology is thus a rapid and well-established method that might be used to select and monitor HLA-A1 positive patients with malignant melanoma and other candidate tumours for MAGE-1-directed immuno-therapy.

In this study, a computer-assisted reverse immunology approach was utilized in order to identify potentially antigenic peptides derived from the differentiation antigen TRP-2, a melanosomal protein frequently expressed in melanoma. Among the seven peptides complying with HLAA2.1-binding motifs, two induced specific CD8 1 cytotoxic T lymphocytes. HLA-A2.1 1 melanoma cells expressing TRP-2 were lysed by clones specific for TRP-2360 ‐368 (TLDSQVMSL) peptide, thus identifying it as a naturally processed epitope. Other T-cell clones directed against TRP-2476 ‐ 484 (VMGTLVALV) were unable to lyse HLA-matched TRP-2 1 cell lines. The role of intracellular proteolytic processing in the generation of this epitope was investigated by transfecting minigenes encoding the TRP-2476 ‐ 484 peptide alone or carrying N- or C-terminal extensions. Specific T-cell clones recognized target cells expressing the cytotoxic T-lymphocyte (CTL)-defined epitope or its C-terminally extended precursor, but failed to recognize cells expressing the N-terminally extended TRP-2476 ‐ 484 peptide, suggesting the presence of a negative processing signal (NPS). Regarding C-terminusflanking regions, mutational analysis indicates that the GLY485 residue plays a key role in the processing of the TRP-2476 ‐ 484 epitope. Interestingly, proteasome inhibitors preventing the generation of the MART-1/Melan-A27‐35 immunodominant melanoma tumor-associated antigen (TAA) promoted detectable presentation of TRP-2476 ‐ 484 epitope in HLA-A2.1 1 and TRP-2 1 tumor lines, as witnessed by cytokine release by specific T-cell clones. Int. J. Cancer 87: 241‐246, 2000. © 2000 Wiley-Liss, Inc.