BACKGROUND:The substantial technological advancements in next-generation sequencing (NGS), combined with dropping costs, have allowed for a swift diffusion of NGS applications in clinical settings. Although several commercial parties report to have broken the $1000 barrier for sequencing an entire human genome, a valid cost overview for NGS is currently lacking. This study provides a complete, transparent and up-to-date overview of the total costs of different NGS applications.

Graft rejection or graft-versus-host (GVH)disease

Donor T cells recognizing hematopoiesis-restricted minor histocompatibility antigens (mHags) HA-1 and HA-2 on malignant cells play a role in the antileukemia effect of donor lymphocyte infusion (DLI) in patients with relapsed leukemia after allogeneic stem cell transplantation. We quantified the contribution of HA-1 and HA-2 specific T cells to the total number of leukemia-reactive T cells in three HA-2 and/or HA-1 positive patients responding to DLI from their mHag negative donors. Clinical responses occurring 5-7 weeks after DLI were accompanied by an increase in percentages HLA-DR expressing T cells within the CD8 þ T cell population. To clonally analyze the leukemia-reactive immune response, T cells responding to the malignancy by secreting IFNc were isolated from peripheral blood, directly cloned, and expanded. Tetramer analysis and specific lysis of peptide-pulsed target cells showed that 3-35% of cytotoxic T lymphocyte (CTL) clones isolated were specific for HA-1 or HA-2. TCR VB analysis showed oligoclonal origin of the HA-1 and HA-2 specific CTL clones. The HA-1 and HA-2 specific CTL clones inhibited leukemic progenitor cell growth in vitro. The relatively high frequency of HA-1 and HA-2 specific T cells within the total number of tumor-reactive T cells illustrates relative immunodominance of mHags HA-1 and HA-2.

Purpose: Preferentially expressed antigen on melanomas (PRAME) is an interesting antigen for T-cell therapy because it is frequently expressed in melanomas (95%) and other tumor types. Moreover, due to its role in oncogenic transformation, PRAME-negative tumor cells are not expected to easily arise and escape fromT-cell immunity. The purpose of this study is to investigate the usefulness of PRAME as target for anticancerT-cell therapies. Experimental Design: HLA-A*0201-subtyped healthy individuals and advanced melanoma patients were screened for CD8 + T cells directed against previously identified HLA-A*0201-binding PRAME peptides by IFN-g enzyme-linked immunosorbent spot assays and tetramer staining. PRAME-specificT-cell clones were isolated and tested for recognition of melanoma and acute lymphoid leukemia (ALL) cell lines. PRAME mRNA expression was determined by quantitative real-time reverse transcription-PCR. Results: In 30% to 40% of healthy individuals and patients, PRA 100-108 -specific CD8 + T cells were detected both after in vitro stimulation and directly ex vivo after isolation by magnetic microbeads. Although CD45RA À memory PRA 100-108 -specific T cells were found in some individuals, the majority of PRA 100-108 -tetramer + Tcells expressed CD45RA, suggesting a naive phenotype. PRA 100-108 -tetramer + T-cell clones were shown to recognize and lyse HLA-A*0201 + and PRAME + melanoma but not ALL cell lines. Quantitative real-time reverse transcription-PCR showed significantly lower PRAME mRNA levels in ALL than in melanoma cell lines, suggesting that PRAME expression in ALL is below the recognition threshold of our PRA 100-108 -tetramer + Tcells. Conclusion: These data support the usefulness of PRAME and in particular the PRA [100][101][102][103][104][105][106][107][108] epitope as target forT-cell therapy of PRAME-overexpressing cancers.Tumor antigens that are used as targets in clinical studies belong to the melanocyte differentiation antigens, cancer-testis antigens, or antigens overexpressed in tumors (1 -5). Cancertestis antigens are expressed in different tumors but not in normal tissues, except for testis, and are therefore useful targets for T-cell therapy. Most cancer-testis antigens, however, are expressed at low frequencies (30-50%). Preferentially expressed antigen on melanomas (PRAME) has been identified as an antigen recognized by an HLA-A24-restricted CTL isolated from a melanoma patient (6). Semiquantitative reverse transcription-PCR (RT-PCR) analysis showed frequent PRAME expression in melanomas (95%) and other tumor types, including lung and breast tumors and leukemias but not in healthy tissues, except for testis and low expression in endometrium, ovaries, and adrenals. DNA microarray analysis revealed the gene encoding PRAME as one of the genes of an expression profile for poor prognosis in breast carcinoma (7). Recently, the function of PRAME has been elucidated by Epping et al. (8). PRAME binds to retinoic acid receptor a, thereby inhibiting retinoic acid -induced differ...

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