This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3′ massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixation and then processed for either standard RNA-Seq or MACE RNA-Seq analysis. To assess the impact of FFPE preparation on MACE, a third part was fixed in formalin and processed for paraffin embedding, and its transcriptional profile was compared with the unfixed specimens analyzed by MACE. To investigate the impact of FFPE storage time on MACE results, 24 FFPE-treated conjunctival samples from 24 patients were analyzed as well. Nineteen thousand six hundred fifty-nine transcribed genes were detected by both MACE and standard RNA-Seq on fresh tissue, while 3251 and 2213 transcripts were identified explicitly by MACE or RNA-Seq, respectively. Standard RNA-Seq tended to yield longer detected transcripts more often than MACE technology despite normalization, indicating that the MACE technology is less susceptible to a length bias. FFPE processing revealed negligible effects on MACE sequencing results. Several quality-control measurements showed that long-term storage in paraffin did not decrease the diversity of MACE libraries. We noted a nonlinear relation between storage time and the number of raw reads with an accelerated decrease within the first 1000 days in paraffin, while the numbers remained relatively stable in older samples. Interestingly, the number of transcribed genes detected was independent on FFPE storage time. RNA of sufficient quality and quantity can be extracted from FFPE samples to obtain comprehensive transcriptome profiling using MACE technology. We thus present MACE as a novel opportunity for utilizing FFPE samples stored in histological archives.

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Summary Recently, in a monocentre study mycophenolate mofetil (MMF) was demonstrated to be efficacious and safe in penetrating high‐risk keratoplasty. Here, preliminary results of a randomized multicentre trial are presented. To date, 86 of 140 scheduled patients undergoing high‐risk penetrating keratoplasty have already been randomized into the two study groups: 48 into the MMF group and 38 into the control group. All 86 patients received fluocortolon 1 mg/kg body weight/day, tapered within 3 weeks, and topical prednisolone acetate 1% tapered within 5 months. MMF was administered at a daily oral dose of 2 × 1000 mg for the first 6 postoperative months. Thereafter, MMF was tapered within 2 weeks. The proportion of grafts with immune reactions and side‐effects were the main outcome measures. Within an average follow up of 9.2 ± 6.6 months two patients developed reversible endothelial immune reactions in the MMF group after cessation of MMF application. In the control group, five reversible and three irreversible immune reactions were observed within an average follow up of 10.1 ± 7.6 months. According to Kaplan and Meier analysis, the ratio of grafts without immune reactions was estimated 89% 1 year postoperatively in the MMF group, in contrast to only 67% in the control group (P = 0.03; log‐rank test). Fifteen patients experienced side‐effects, especially gastroenterotoxicity, tachycardia, arthralgia or systemic infections. All attributable side‐effects were reversible. Systemic MMF may be an effective and safe immune modulating drug in the prophylaxis of immune reactions after penetrating high‐risk keratoplasty.

This study characterizes the transcriptome and the cellular tumor microenvironment (TME) of conjunctival melanoma (CM) and identifies prognostically relevant biomarkers. 12 formalin-fixed and paraffin-embedded CM were analyzed by MACE RNA sequencing, including six cases each with good or poor clinical outcome, the latter being defined by local recurrence and/or systemic metastases. Eight healthy conjunctival specimens served as controls. The TME of CM, as determined by bioinformatic cell type enrichment analysis, was characterized by the enrichment of melanocytes, pericytes and especially various immune cell types, such as plasmacytoid dendritic cells, natural killer T cells, B cells and mast cells. Differentially expressed genes between CM and control were mainly involved in inhibition of apoptosis, proteolysis and response to growth factors. POU3F3, BIRC5 and 7 were among the top expressed genes associated with inhibition of apoptosis. 20 genes, among them CENPK, INHA, USP33, CASP3, SNORA73B, AAR2, SNRNP48 and GPN1, were identified as prognostically relevant factors reaching high classification accuracy (area under the curve: 1.0). The present study provides new insights into the TME and the transcriptional profile of CM and additionally identifies new prognostic biomarkers. These results add new diagnostic tools and may lead to new options of targeted therapy for CM.