Concepts and terms for dose/volume parameters in carbon-ion radiotherapy: Conclusions of the ULICE taskforce.

PURPOSE: The Union of Light Ion Centers in Europe (ULICE) program addressed the need for uniting scientific results for carbon-ion radiation therapy obtained by several institutions worldwide in different fields of excellence, and translating them into a real benefit to the community. Particularly, the concepts for dose/volume parameters developed in photon radiotherapy cannot be extrapolated to high linear energy transfer particles. METHODS AND MATERIALS: The ULICE-WP2 taskforce included radiation oncologists involved in carbon-ion radiation therapy and International Commission on Radiation Units and Measurements, radiation biologists, expert physicists in the fields of carbon-ion radiation therapy, microdosimetry, biological modeling and image-guided radiotherapy. Consensual reports emerged from multiple discussions within both the restricted group and the wider ULICE community. Public deliverables were produced and disseminated to the European Commission. RESULTS: Here we highlight the disparity in practices between treating centers, then address the main topics to finally elaborate specific recommendations. Although it appears relatively simple to add geometrical margins around the clinical target volume to obtain the planning target volume as performed in photon radiotherapy, this procedure is not appropriate for carbon-ion radiation therapy. Due to the variation of the radiation quality in depth, there is no generic relative biological effectiveness value for carbon-ions outside of an isolated point, for a given fractionation and specific experimental conditions. Absorbed dose and "equieffective dose" for specified conditions must always be reported. CONCLUSIONS: This work contributed to the development of standard operating procedures for carbon-ion radiation therapy clinical trials. These procedures are now being applied, particularly in the first phase III international, multicenter trial (PHRC Étoile).

TALEN-mediated editing of endogenous T-cell receptors facilitates efficient reprogramming of T lymphocytes by lentiviral gene transfer.

Adoptive immunotherapy with T lymphocytes expressing transgenic T-cell receptors (TCRs) has shown significant clinical efficacy in various malignant diseases. However, concurrent expression of endogenous and transgenic TCRs in one and the same T cell may impair efficacy and cause safety problems owing to mispairings. The most elegant approach to address these issues is the complete shutoff of the endogenous receptor chains by genome editing. To this end, we designed TCR-α and TCR-ß-specific pairs of transcription activator-like effector nucleases (TALENs). TALENs were delivered into T cells using an optimized messenger RNA-electroporation protocol. Based thereon, we obtained precise and highly efficient knockout (KO) in Jurkat (TCR-α: 59.7 ± 4.0%, TCR-ß: 37.4 ± 7.3%) as well as primary T cells (TCR-α: 58.0 ± 15.0%, TCR-ß: 41.0 ± 17.6%). Moreover, a successive KO strategy for the endogenous TCR chains combined with subsequent transduction of the respective chains of an Influenza virus-specific model TCR led to complete reprogramming of T cells with strongly improved expression and functionality of transgenic TCRs. In conclusion, we have developed novel means for the efficient genome editing in primary T lymphocytes.

Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis.

Vector-encoded fluorescent proteins (FPs) facilitate unambiguous identification or sorting of gene-modified cells by fluorescence-activated cell sorting (FACS). Exploiting this feature, we have recently developed lentiviral gene ontology (LeGO) vectors (www.LentiGO-Vectors.de) for multi-gene analysis in different target cells. In this study, we extend the LeGO principle by introducing 10 different drug-selectable FPs created by fusing one of the five selection marker (protecting against blasticidin, hygromycin, neomycin, puromycin and zeocin) and one of the five FP genes (Cerulean, eGFP, Venus, dTomato and mCherry). All tested fusion proteins allowed both fluorescence-mediated detection and drug-mediated selection of LeGO-transduced cells. Newly generated codon-optimized hygromycin- and neomycin-resistance genes showed improved expression as compared with their ancestors. New LeGO constructs were produced at titers >10(6) per ml (for non-concentrated supernatants). We show efficient combinatorial marking and selection of various cells, including mesenchymal stem cells, simultaneously transduced with different LeGO constructs. Inclusion of the cytomegalovirus early enhancer/chicken beta-actin promoter into LeGO vectors facilitated robust transgene expression in and selection of neural stem cells and their differentiated progeny. We suppose that the new drug-selectable markers combining advantages of FACS and drug selection are well suited for numerous applications and vector systems. Their inclusion into LeGO vectors opens new possibilities for (stem) cell tracking and functional multi-gene analysis.

Radiotherapy alone for invasive vaginal cancer: outcome with intracavitary high dose rate brachytherapy versus conventional low dose rate brachytherapy.

PURPOSE: Our aim was to compare the role of remote afterloaded high-dose-rate brachytherapy (HDRB) with traditional low-dose-rate brachytherapy (LDRB) for patients with invasive primary vaginal carcinoma. METHODS: The study group comprised 190 patients with invasive carcinoma of the vagina. The patients were staged according to the International Federation of Gynecology and Obstetrics (FIGO) staging system. Eighty patients were treated with intracavitary high-dose rate iridium 192 brachytherapy with or without external beam therapy. These patients are compared with a historical group of 110 patients treated with intracavitary low-dose-rate radium 226 or cesium 137 brachytherapy with or without external beam therapy. RESULTS: No significant differences were found for stages, tumor grade or location between the two groups. Crude 5-year survival for all patients was 41% in the former LDRB group, 81% in stage I and 43% in stage II. Overall actuarial 3-year survival and disease-specific survival rates for all patients in the HDRB series were 51% and 66%, respectively. Disease-specific 3-year survival attained 83% in stage I and 66% in stage II. There were no significant differences in local and distant recurrences between the treatment modalities. The comparison of treatments with or without external beam radiation and of complications showed no significant differences between the HDRB and LDRB series. CONCLUSION: With HDRB and its advantages of decreased radiation exposure and patient immobilization and precise positioning, treatment results to be obtained are at least similar to traditional LDRB for primary vaginal cancer.

Portal imaging based definition of the planning target volume during pelvic irradiation for gynecological malignancies.

PURPOSE: Geometrical accuracy in patient positioning can vary substantially during external radiotherapy. This study estimated the set-up accuracy during pelvic irradiation for gynecological malignancies for determination of safety margins (planning target volume, PTV). METHODS AND MATERIALS: Based on electronic portal imaging devices (EPID), 25 patients undergoing 4-field pelvic irradiation for gynecological malignancies were analyzed with regard to set-up accuracy during the treatment course. Regularly performed EPID images were used in order to systematically assess the systematic and random component of set-up displacements. Anatomical matching of verification and simulation images was followed by measuring corresponding distances between the central axis and anatomical features. Data analysis of set-up errors referred to the x-, y-,and z-axes. Additionally, cumulative frequencies were evaluated. RESULTS: A total of 50 simulation films and 313 verification images were analyzed. For the anterior-posterior (AP) beam direction mean deviations along the x- and z-axes were 1.5 mm and -1.9 mm, respectively. Moreover, random errors of 4.8 mm (x-axis) and 3.0 mm (z-axis) were determined. Concerning the latero-lateral treatment fields, the systematic errors along the two axes were calculated to 2.9 mm (y-axis) and -2.0 mm (z-axis) and random errors of 3.8 mm and 3.5 mm were found, respectively. The cumulative frequency of misalignments < or =5 mm showed values of 75% (AP fields) and 72% (latero-lateral fields). With regard to cumulative frequencies < or =10 mm quantification revealed values of 97% for both beam directions. CONCLUSION: During external pelvic irradiation therapy for gynecological malignancies, EPID images on a regular basis revealed acceptable set-up inaccuracies. Safety margins (PTV) of 1 cm appear to be sufficient, accounting for more than 95% of all deviations.

Reproducibility of irregular radiation fields for malignant lymphoma.

PURPOSE: Radiation treatment for malignant lymphoma requires large field irradiation with irregular blocks according to the individual anatomy and tumor configuration. For determination of safety margins (PTV) we quantitatively analysed the accuracy of field and block placement with regard to different anatomical regions. PATIENTS AND METHODS: Forty patients with malignant lymphoma were irradiated using the classical supra-/infradiaphragmatic field arrangements. Treatment was performed with 10-MeV photons and irregularly shaped, large opposing fields. We evaluated the accuracy of field and block placements during the treatment courses by comparing the regularly performed verification-with the simulation films. Deviations were determined with respect to the field edges and the central axis, along the x- and z-axis. RESULTS: With regard to the field edges, mean deviations of 2.0 mm and 3.4 mm were found along the x- and z-axis. The corresponding standard deviations were 3.4 mm and 5.5 mm, respectively. With regard to the shielding blocks, mean displacement along the x- and z-axis was 2.2 mm and 3.8 mm. In addition, overall standard deviations of 5.7 mm (x-axis) and 7.1 mm (z-axis) were determined. During the course of time an improved accuracy of block placement was notable. CONCLUSION: Systematic analysis of port films gives information for a better defining safety margins in external radiotherapy. Evaluation of verification films on a regular basis improves set-up accuracy by reducing displacements.

High-affinity SV40 T-antigen binding sites in the human genome.

We describe two different approaches to isolate human genomic sequences possessing high-affinity binding sites for the simian virus 40 (SV40) large T antigen. First, SV40 T antigen was added to Sau3A-restricted human DNA; the resulting T-antigen-DNA complexes were collected after repeated passages through nitrocellulose filters. The second approach involves the specific immunoprecipitation of chromatin fragments, generated by Sau3A treatment of nuclear chromatin from SV40-transformed human cells. The DNA fragments obtained were cloned in plasmid vectors for further investigation. Using the filter binding approach we isolated four different fragments with high-affinity binding sites. The binding site in one fragment was related to the strong T-antigen binding site I in the SV40 genome. The other three fragments contained multiple recognition pentamers, GA(G)GGC. Only one fragment with a high-affinity binding site was identified among the immunoprecipitable chromatin fragments. This DNA fragment belongs to the L1 family of human repetitive DNA. We present evidence suggesting that a significant fraction of human L1 elements possesses T-antigen binding sites. L1-related sequences appear as extrachromosomal elements in an SV40-transformed human cell line, and the amount of extrachromosomal L1 DNA was found to increase after fusion of transformed cells to permissive monkey cells.