A 9-month posttransplantation course of an allogeneic stem-cell transplant recipient (human cytomegalovirus [HCMV] serostatus, donor positive/recipient negative), in whom ganciclovir (GCV) resistance developed (UL97 mutations M460V, L595S, and C603W) on day 164 after transplantation and who developed HCMV retinitis and fatal HCMV encephalitis is presented. Virus strains isolated from secondary cultures were analyzed by UL97 restriction assays and sequencing and were compared with primary DNA extracts of the same specimens, which resulted in molecular proof of an initial HCMV strain-specific in vitro selection of the in vivo nondominant UL97 L595S-C603 mutant strain from 3 viral variants present in vivo. In addition, compartmentalization of virus present in blood and cerebrospinal fluid was found. The influence of rapidly increasing plasma virus load (to >10(6) copies/mL) and oral administration of GCV on the emergence of GCV resistance is shown. These findings have strong implications for the diagnosis of HCMV drug resistance.

Background: Tumor cells that shed into stool are attractive targets for molecular screening and early detection of colon or pancreatic malignancies. We developed a diagnostic test to screen for 10 of the most common mutations of codons 12 and 13 of the K-ras gene by hybridization to a new biochip array. Methods: DNA was isolated from 26 stool samples by column-based extraction from 9 cell lines. Peptide nucleic acid (PNA)-mediated PCR clamping was used for mutant-specific amplification. We used a biochip, consisting of a small plastic support with covalently immobilized 13mer oligonucleotides. The read out of the biochip was done by confocal time-resolved laser scanning. Hybridization, scanning, and data evaluation could be performed in <2 h. Results: Approximately 80 ng of DNA was obtained from 200-mg stool samples. No inhibition of the PCR by remaining impurities from stool was observed. Mutation detection was possible in 1000-fold excess of wild-type sequence. Discrimination ratios between the mutations were >19 as demonstrated by hybridization with tumor cell line DNA. Stool samples (n = 26) were analyzed in parallel with PNA-PCR, restriction assay for K-ras codon 12 mutations, sequencing, and hybridization to the biochip. Nine mutations were found by hybridization, all confirmed by sequencing. PNA-PCR alone leads to an overestimation of mutations because suppression of the wild type is not effective enough with high concentrations of wild-type DNA. The restriction assay found only four mutations. Conclusions: The K-ras biochip is well suited for fast mutation detection from stool in colorectal cancer screening.

Children with innate immunodeficiencies may be at high risk for early development of ganciclovir-resistant human cytomegalovirus (HCMV) infection after bone marrow transplantation (BMT). For early and frequent monitoring of the occurrence of ganciclovir resistance-associated mutations in codons of the UL97 gene, a panel of previously described restriction assays was expanded for use on codons 591, 592, and 603. This technique enabled detection of suddenly emerging ganciclovir-resistant HCMV after BMT in a 7-year-old child with a T cell defect. Resistance emerged among the isolation of a ganciclovir-sensitive HCMV strain 32 days after transplantation, the first detection of genotypical resistance at day 44, and the isolation of resistant HCMV (ID50>12 microM) at day 54. Simple and yet comprehensive methods for therapy surveillance may be important in this patient group, in which the restriction assays proved useful.

Immunotherapies represent a promising strategy for glioblastoma multiforme (GBM) treatment. Different immunotherapies include the use of checkpoint inhibitors, adoptive cell therapies such as chimeric antigen receptor (CAR) T cells, and vaccines such as dendritic cell vaccines. Antibodies have also been used as toxin or radioactive particle delivery vehicles to eliminate target cells in the treatment of GBM. Oncolytic viral therapy and other immunogenic cell death-inducing treatments bridge the antitumor strategy with immunization and installation of immune control over the disease. These strategies should be included in the standard treatment protocol for GBM. Some immunotherapies are individualized in terms of the medicinal product, the immune target, and the immune tumor–host contact. Current individualized immunotherapy strategies focus on combinations of approaches. Standardization appears to be impossible in the face of complex controlled trial designs. To define appropriate control groups, stratification according to the Recursive Partitioning Analysis classification, MGMT promotor methylation, epigenetic GBM sub-typing, tumor microenvironment, systemic immune functioning before and after radiochemotherapy, and the need for/type of symptom-relieving drugs is required. Moreover, maintenance of a fixed treatment protocol for a dynamic, deadly cancer disease in a permanently changing tumor–host immune context might be inappropriate. This complexity is illustrated using our own data on individualized multimodal immunotherapies for GBM. Individualized medicines, including multimodal immunotherapies, are a rational and optimal yet also flexible approach to induce long-term tumor control. However, innovative methods are needed to assess the efficacy of complex individualized treatments and implement them more quickly into the general health system.

Three seropositive pediatric recipients of allogeneic stem cell transplantation out of a group of 42 patients receiving T-cell–depleted, unrelated transplants and 37 patients receiving T-cell–depleted, haploidentical transplants were monitored longitudinally for human cytomegalovirus (HCMV) infection and the emergence of antiviral drug resistance. Early in the posttransplant course, all 3 patients developed HCMV mutations conferring drug resistance to ganciclovir. One child additionally developed multidrug resistance to foscarnet and cidofovir, with mutations in the viral phosphotransferase gene (UL97) and the DNA-polymerase gene (UL54) being found. These data show that resistant HCMV infection does not necessarily correlate with a severe clinical outcome. The early detection of genotypic resistance up to 129 days before the emergence of phenotypic resistance and the dissociation of resistance patterns among different body sites emphasize the importance of genotypic analyses of different DNA specimens for an efficient antiviral therapy. T-cell–depleted children having transplantation might be at an increased risk for the development of drug resistance.