No major differences in perinatal and maternal outcomes between uninterrupted embryo culture in time-lapse system and conventional embryo culture.

STUDY QUESTION: Is embryo culture in a closed time-lapse system associated with any differences in perinatal and maternal outcomes in comparison to conventional culture and spontaneous conception? SUMMARY ANSWER: There were no significant differences between time-lapse and conventional embryo culture in preterm birth (PTB, <37 weeks), low birth weight (LBW, >2500 g) and hypertensive disorders of pregnancy for singleton deliveries, the primary outcomes of this study. WHAT IS KNOWN ALREADY: Evidence from prospective trials evaluating the safety of time-lapse incubation for clinical use show similar embryo development rates, implantation rates, and ongoing pregnancy and live birth rates when compared to conventional incubation. Few studies have investigated if uninterrupted culture can alter risks of adverse perinatal outcomes presently associated with IVF when compared to conventional culture and spontaneous conceptions. STUDY DESIGN, SIZE, DURATION: This study is a Swedish population-based retrospective registry study, including 7379 singleton deliveries after fresh embryo transfer between 2013 and 2018 from selected IVF clinics. Perinatal outcomes of singletons born from time-lapse-cultured embryos were compared to singletons from embryos cultured in conventional incubators and 71 300 singletons from spontaneous conceptions. Main perinatal outcomes included PTB and LBW. Main maternal outcomes included hypertensive disorders of pregnancy (pregnancy hypertension and preeclampsia). PARTICIPANTS/MATERIALS, SETTING, METHODS: From nine IVF clinics, 2683 singletons born after fresh embryo transfer in a time-lapse system were compared to 4696 singletons born after culture in a conventional incubator and 71 300 singletons born after spontaneous conception matched for year of birth, parity, and maternal age. Patient and treatment characteristics from IVF deliveries were cross-linked with the Swedish Medical Birth Register, Register of Birth Defects, National Patient Register and Statistics Sweden. Children born after sperm and oocyte donation cycles and after Preimplantation Genetic testing cycles were excluded. Odds ratio (OR) and adjusted OR were calculated, adjusting for relevant confounders. MAIN RESULTS AND THE ROLE OF CHANCE: In the adjusted analyses, no significant differences were found for risk of PTB (adjusted OR 1.11, 95% CI 0.87-1.41) and LBW (adjusted OR 0.86, 95% CI 0.66-1.14) or hypertensive disorders of pregnancy; preeclampsia and hypertension (adjusted OR 0.99, 95% CI 0.67-1.45 and adjusted OR 0.98, 95% CI 0.62-1.53, respectively) between time-lapse and conventional incubation systems. A significantly increased risk of PTB (adjusted OR 1.31, 95% CI 1.08-1.60) and LBW (adjusted OR 1.36, 95% CI 1.08-1.72) was found for singletons born after time-lapse incubation compared to singletons born after spontaneous conceptions. In addition, a lower risk for pregnancy hypertension (adjusted OR 0.72 95% CI 0.53-0.99) but no significant difference for preeclampsia (adjusted OR 0.87, 95% CI 0.68-1.12) was found compared to spontaneous conceptions. Subgroup analyses showed that some risks were related to the day of embryo transfer, with more adverse outcomes after blastocyst transfer in comparison to cleavage stage transfer. LIMITATIONS, REASONS FOR CAUTION: This study is retrospective in design and different clinical strategies may have been used to select specific patient groups for time-lapse versus conventional incubation. The number of patients is limited and larger datasets are required to obtain more precise estimates and adjust for possible effect of additional embryo culture variables. WIDER IMPLICATIONS OF THE FINDINGS: Embryo culture in time-lapse systems is not associated with major differences in perinatal and maternal outcomes, compared to conventional embryo culture, suggesting that this technology is an acceptable alternative for embryo incubation. STUDY FUNDING/COMPETING INTEREST(S): The study was financed by a research grant from Gedeon Richter. There are no conflicts of interest for all authors to declare. TRIAL REGISTRATION NUMBER: N/A.

Preparing for snow-sport events at the Paralympic Games in Beijing in 2022: recommendations and remaining questions.

During the 2022 Winter Paralympic Games in Beijing, the Para snow-sport events will be held at high altitudes and in possibly cold conditions while also requiring adjustment to several time zones. Furthermore, the ongoing COVID-19 pandemic may lead to suboptimal preparations. Another concern is the high rate of injuries that have been reported in the Para alpine and snowboard events. In addition to these challenges, Para athletes various impairments may affect both sports-specific demands and athlete health. However, the group of Para snow-sport athletes is an understudied population. Accordingly, this perspective paper summarises current knowledge to consider when preparing for the Paralympic Games in Beijing and point out important unanswered questions. We here focus specifically on how sport-specific demands and impairment-related considerations are influenced by altitude acclimatisation, cold conditions, travel fatigue and jetlag, complications due to the COVID-19 pandemic, and injury prevention and sports safety considerations. As Para athletes with spinal cord injury, limb deficiency, cerebral palsy and visual impairment account for the majority of the Para snow-sport athletes, the focus is mainly on these impairment groups. In brief, we highlight the extra caution required to ensure athlete health, performance and sports safety among Para athletes participating in the snow-sport events in the 2022 Beijing Paralympic Games. Although there is an urgent need for more high-quality research focusing on Para winter athletes, we hope these non-consensus recommendations will help prepare for the 2022 Beijing Paralympic Winter Games.

Survival After Adrenalectomy for Metastatic Hepatocellular Carcinoma: A 25-year Institutional Experience.

BACKGROUND: Extrahepatic manifestation of hepatocellular carcinoma (HCC) is rare and primarily affects lung, lymph nodes and bone. Metastases to the adrenal glands are relatively infrequent. This 25-year institutional experience aimed for an analysis of factors influencing survival in patients undergoing surgery for HCC adrenal metastasis. METHODS: A retrospective analysis of the institutional database of the Clinic for General-, Visceral- and Transplantation Surgery of the University Medical Center Mainz, Germany, was performed. Patients who underwent surgery for HCC adrenal metastases from January 1995 to June 2020 were included. Pre-, peri- and postoperative factors with potential influence on survival were assessed. RESULTS: In 16 patients (14 males, two females), one bilateral and 15 unilateral adrenalectomies were performed (13 metachronous, three synchronous). Thirteen operations were carried out via laparotomy, and three adrenalectomies were minimally invasive (two laparoscopic, one retroperitoneoscopic). Median overall survival (after HCC diagnosis) was 35 months, range: 5-198. Median post-resection survival (after adrenalectomy) was 15 months, range: 0-75. Overall survival was longer in patients with the primary HCC treatment being liver transplantation (median 66 months) or liver resection (median 51 months), compared to only palliative intended treatment of the primary with chemotherapy (median 35 months) or local ablation (median 23 months). CONCLUSIONS: Surgery is a feasible treatment option for patients with adrenal metastases originating from HCC. In patients who underwent adrenalectomy for HCC adrenal metastasis, overall survival was superior, if primary HCC treatment was potentially curative (liver transplantation or resection).

[Surgical treatment of hepatic tumors-liver resection and transplantation]. / Chirurgische Therapie bei Lebertumoren ­ Leberresektion und -transplantation.

The most frequent primary hepatic malignancies are hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (intrahepatic cholangiocellular adenocarcinoma [iCCA]). For HCC in cirrhosis, liver transplantation offers the advantage of a complete hepatectomy radically removing all tumorous tissue along with the surrounding cirrhotic parenchyma, which is otherwise associated with a very high risk of recurrence. For HCC in non-cirrhotic livers and iCCA, liver resection is the treatment of choice. Nowadays, even extended resections can be performed with low mortality in experienced centers. Surgical therapy is more and more embedded into multimodal treatment concepts and decision making should be interdisciplinary as for other gastrointestinal tumors.

Evaluation of a newly developed 2D parametric parenchymal blood flow technique with an automated vessel suppression algorithm in patients with chronic thromboembolic pulmonary hypertension undergoing balloon pulmonary angioplasty.

AIM: To evaluate the feasibility of two-dimensional parametric parenchymal blood flow (2D-PPBF) to quantify perfusion changes in the lung parenchyma following balloon pulmonary angioplasty (BPA) for treatment of chronic thromboembolic pulmonary hypertension. MATERIALS AND METHODS: Overall, 35 consecutive interventions in 18 patients with 98 treated pulmonary arteries were included. To quantify changes in pulmonary blood flow using 2D-PPBF, the acquired digital subtraction angiography (DSA) series were post-processed using dedicated software. A reference region of interest (ROI; arterial inflow) in the treated pulmonary artery and a distal target ROI, including the whole lung parenchyma distal to the targeted stenosis, were placed in corresponding areas on DSA pre- and post-BPA. Half-peak density (HPD), wash-in rate (WIR), arrival to peak (AP), area under the curve (AUC), and mean transit time (MTT) were assessed. The ratios of the reference ROI to the target ROI (HPDparenchyma/HPDinflow, WIRparenchyma/WIRinflow; APparenchyma/APinflow, AUCparenchyma/AUCinflow, MTTparenchyma/MTTinflow) were calculated. The relative differences of the 2D-PPBF parameters were correlated to changes in the pulmonary flow grade score. RESULTS: The pulmonary flow grade score improved significantly after BPA (1 versus 3; p<0.0001). Likewise, the mean HPDparenchyma/HPDinflow (-10.2%; p<0.0001), APparenchyma/APinflow (-24.4%; p=0.0007), and MTTparenchyma/MTTinflow (-3.5%; p=0.0449) decreased significantly, whereas WIRparenchyma/WIRinflow (+82.4%) and AUCparenchyma/AUCinflow (+58.6%) showed a significant increase (p<0.0001). Furthermore, a significant correlation between changes of the pulmonary flow grade score and changes of HPDparenchyma/HPDinflow (ρ=-0.21, p=0.04), WIRparenchyma/WIRinflow (ρ=0.43, p<0.0001), APparenchyma/APinflow (ρ=-0.22, p=0.03), AUCparenchyma/AUCinflow (ρ=0.48, p<0.0001), and MTTparenchyma/MTTinflow (ρ=-0.39, p<0.0001) could be observed. CONCLUSION: The 2D-PPBF technique is feasible for the quantification of perfusion changes following BPA and has the potential to improve monitoring of BPA.

[Computer-assisted 3D-navigated laparoscopic resection of a vanished colorectal liver metastasis after chemotherapy]. / Die verschwundene Lebermetastase - Neues Einsatzgebiet für die computerassistierte 3D-navigierte laparoskopische Leberesektion?

BACKGROUND: Modern chemotherapy regimen for colorectal cancer results in complete radiologic remission in a substantial number of cases. However, these "vanished liver metastases" (VLM) often contain vital tumor cells, which mandates the resection of such lesions. In these cases, intraoperative identification of VLM can be challenging, in particular in laparoscopic approaches. We describe the first laparoscopic computer-assisted 3D-navigated resection of a VLM. CASE REPORT: A 60-year-old patient with a synchronous liver metastasis (segment IVb) of sigmoid colon cancer (T4 N1 M1) was referred to our center for elective liver resection after laparoscopic sigmoid resection and systemic chemotherapy (FOLFIRI/Panitumumab). The metastasis was not visible anymore on preoperative CT or sonography. Thus, a 3 D reconstruction of the liver was performed. The size of the initial metastasis (before chemotherapy) was transferred into the current CT. A computer-assisted 3D-navigated laparoscopic resection of the metastasis was performed on these fused images. The metastasis was also not clearly visible upon intraoperative ultrasound. Histology of the resected specimen revealed a 0.5 cm metastasis with predominantly vital tumor cells (regression degree 4 of Rubbia-Brandt) and a sufficient resection margin of at least 7 mm. The postoperative course was uneventful. CONCLUSION: Computer-assisted 3D-navigation enabled a safe oncologic resection of a vanished liver metastasis after chemotherapy. This technique was particularly helpful due to the limited haptic feedback of laparoscopic surgery. Further studies are necessary to verify the clinical benefit of computer assisted 3D-navigated liver surgery.

[Vascular reconstruction in hepatic surgery]. / Gefäßrekonstruktionen in der Leberchirurgie.

BACKGROUND: Due to their size or location liver tumors can infiltrate important vascular structures, which are essential for postoperative liver function. OBJECTIVE: To present the technical possibilities and results of current concepts of vascular resection and reconstruction in liver surgery. MATERIAL AND METHODS: A literature search of the Medline and Cochrane databases was performed regarding currently available studies on vascular resection and reconstruction in liver surgery. RESULTS: Portal vein resections are routinely performed by many institutions and can be performed as an end-to-end anastomosis or graft interposition. This is the basis of the en bloc resection concept, especially for Klatskin tumors. Reconstruction of the inferior vena cava as well as the hepatic arteries is technically feasible and is increasingly being reported in smaller series. In particular, the resection of tumors near the hepatic veins may require total vascular exclusion for complete interruption of liver perfusion, which enables resection in the non-perfused liver and by this reduced blood loss. Furthermore, in situ cooling, ante situm and ex situ resections increase both technical resectability and the ischemic tolerance of the liver to more than 60 min. The majority of vascular reconstructions can be performed without a significant increase in morbidity; however, vascular tumor infiltration is associated with impaired long-term survival. CONCLUSION: Based on the experience of transplantation surgery concepts for vascular reconstruction can be safely applied to liver surgery. These concepts contribute to increasing the resectability of liver tumors. Due to the often impaired prognosis of vascular tumor infiltration, the use of these concepts should be individually assessed by weighing the prognosis against the morbidity.

Androgen levels during adjuvant endocrine therapy in postmenopausal breast cancer patients.

OBJECTIVE: To investigate plasma steroid hormone levels in postmenopausal breast cancer patients with and without adjuvant endocrine therapy and in healthy postmenopausal women. METHODS: Steroid hormone levels in postmenopausal breast cancer patients treated with aromatase inhibitors (n = 32) were compared with breast cancer patients treated with tamoxifen (n = 34), breast cancer patients without adjuvant endocrine therapy (n = 15), and healthy postmenopausal women (n = 56). Pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone (DHEA), androstenedione, total testosterone, dihydrotestosterone, estrone and estradiol were measured using liquid chromatography-tandem mass spectrometry. Sex hormone binding globulin was measured by solid-phase chemiluminescent immunometric assays, and the free androgen index was calculated. RESULTS: Aromatase inhibitor users did not differ in dihydrotestosterone, total testosterone, androstenedione, DHEA, or free androgen index levels from healthy controls or untreated breast cancer patients. The highest total testosterone levels were found in tamoxifen-treated women, who had significantly higher plasma concentrations than both women treated with aromatase inhibitors and breast cancer patients without adjuvant treatment. Concentrations of cortisol and cortisone were significantly greater in aromatase inhibitor users as well as tamoxifen users, in comparison with healthy controls and untreated breast cancer patients. Aromatase inhibitor users had lower estrone and estradiol plasma concentrations than all other groups. CONCLUSION: Adjuvant treatment with aromatase inhibitors or tamoxifen was associated with increased cortisol and cortisone plasma concentrations as well as decreased estradiol concentrations. Androgen levels were elevated in tamoxifen-treated women but not in aromatase inhibitor users.

Getting in early: primary skin cancer prevention at 55 German kindergartens.

BACKGROUND: Skin neoplasms are the most frequent types of neoplasms in white populations, and their incidence is increasing. Epidemiological studies have shown that the major environmental aetiological factor for their development is sunlight exposure. Sun protection programmes are urgently needed to raise awareness of the health hazards of ultraviolet radiation. In 2010 the 'SunPass' project was implemented at 55 kindergartens in Germany. This is the first nationwide environmental education programme for sun safety designed to teach children in kindergartens and their caregivers how to protect themselves from overexposure to the sun. OBJECTIVES: An interventional lecture, site inspections and a certification were part of the programme. Effects of these interventions were studied. METHODS: The gain in knowledge and changed sun-behavioural attributes were quantified by questionnaires administered before and after the 'SunPass' interventions. RESULTS: The total number of children was 5424. Sun-protection behaviour after the intervention improved significantly (P < 0·001). Among parents, 22·2% reported one to five sunburns of their child since birth. There was a significant increase in hat use by children in kindergartens (P = 0·029), as well as some significantly improved shade practices. There was a significantly increased demand for protective clothing for children (P < 0·001). The change in sunscreen use in kindergartens was not significant. CONCLUSIONS: Although some aims of the 'SunPass' project were not fulfilled, such as the precise knowledge of skin types and a change of sunscreen use, the study had some positive outcomes in increasing the awareness of skin cancer and its prevention possibilities. The findings of the present study suggest that relatively brief interventions in kindergartens lead to improved sun protection of children. The whole investigation reaching over 5400 children and their parents underlines the importance of learning appropriate sun-protective behaviour in early childhood in order to decrease the risk for skin cancer.

Model of wound healing for esophagogastric anastomoses in rats.

BACKGROUND: Anastomotic leakage after esophageal surgery is a significant cause of morbidity and mortality. Postoperative leakage of esophagogastric anastomosis has been reported in 2-30% of surgical patient, resulting in an increased need for reoperation and a high risk of subsequent esophageal stricture formation and fistula. So far, experimental investigations on major factors influencing the healing of esophageal anastomoses, e.g. neovascularization and collagen deposition, have been hindered by the lack of a functional rodent model. METHODS: We developed a novel technique of gastric tube formation followed by end-to-end esophagogastric anastomosis in a rat model. Standardized anastomoses were carried out in 18 Brown-Norway rats and normal esophagogastric healing was studied by measuring anastomotic breaking strength 5 days after surgery. RESULTS: Five animals showed an insufficiency of the esophagogastric anastomosis as determined by anastomotic leakage testing. Normal anastomotic healing was found in 10 animals. The anastomotic breaking strength was 1.93 ± 0.45 N. CONCLUSION: The rat model for performing esophagogastric anastomoses after gastric tube formation may serve as a functional and useful model in future research studies on microvascular and molecular processes of anastomotic healing.

Higher than expected estradiol levels in aromatase inhibitor-treated, postmenopausal breast cancer patients.

OBJECTIVE: Vaginal estradiol is considered contraindicated in aromatase inhibitor (AI)-treated patients because of the risk of elevated estrogen levels. This leaves limited treatment options for patients experiencing gynecological symptoms. However, in clinical practice, no precise estimation has been performed of circulating estrogens and aromatase index in postmenopausal breast cancer patients on long-lasting AI or tamoxifen treatment. METHODS: Steroid hormones were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) and extraction radioimmunoassay (RIA). Postmenopausal AI-treated patients (n =33) were compared with tamoxifen-treated patients (n =34) and controls without vaginal treatment (n =56), with vaginal estradiol (n =25), or with estriol (n =11) treatment. RESULTS: By use of LC-MS/MS, median (range) estradiol plasma concentrations were 16.7 (2.4-162.6), 31.0 (13.4-77.1), 27.2 (7.8-115.8) and 33.3 (20.3-340.1) pmol/l in AI-treated breast cancer patients, tamoxifen-treated breast cancer patients, postmenopausal controls and postmenopausal controls on vaginal estradiol, respectively. The AI-treated group and subgroups had significantly lower estradiol and estrone concentrations than all other groups (p <0.05). There was extensive interindividual variation in estradiol concentration within the AI-treated group, measured using both LC-MS/MS (2.3-182.0 pmol/l) and extraction RIA (2.4-162.6 pmol/l). The AI-treated group had lower aromatase index compared to all other groups (p <0.05-0.001). CONCLUSION: Circulating estrogen levels may have been underestimated in previous longitudinal studies of AI-treated breast cancer patients. Additional studies are required to further evaluate the role of circulating estrogens in breast cancer patients suffering from gynecological symptoms.

Allogeneic hematopoietic SCT in patients with AML following treosulfan/fludarabine conditioning.

An alternative reduced-toxicity conditioning regimen for allogeneic transplantation, based on treosulfan and fludarabine, has recently been identified. The safety and efficacy of this new conditioning regimen has been investigated prospectively in patients with AML. A total number of 75 patients with AML in CR were treated with 3 × 14 g/m(2) treosulfan and 5 × 30 mg/m(2) fludarabine, followed by matched sibling or unrelated SCT. Patients were evaluated for engraftment, adverse events, GVHD, and for non-relapse mortality, relapse incidence, overall and disease-free survival (DFS). All patients showed primary engraftment of neutrophils after a median of 20 days. Non-hematological adverse events grade III-IV in severity included mainly infections (59%) and gastrointestinal symptoms (7%). Acute GVHD grade II-IV occurred in 21% and extensive chronic GVHD occurred in 16% of the patients. After a median follow-up of 715 days, the 2-year overall and DFS estimates were 61% and 55%, respectively. The 2-year incidences of relapse and non-relapse mortality reached 34% and 11%, respectively. In summary, our data confirm promising safety and efficacy of the treosulfan-based conditioning therapy in AML patients, ClinicalTrials.gov Identifier: NCT01063660.

Fs-laser-induced Ca2+ concentration change during membrane perforation for cell transfection.

Fs-laser based opto-perforation is a gentle method for gene transfer into sensitive cells such as stem cells or primary cells. The high selectivity and the low damage to the cell lead to a high efficiency of transfection. However, there are side effects which induce stress to the cell due to the exchange of intra- and extracellular media as well as the disintegration of the structure of biomolecules resulting from the laser exposure. Moreover, the mechanisms of the optical transfection are still unclear. In this paper, we present our study on calcium (Ca(2+)) homeostasis during cell surgery, especially during laser induced membrane perforation. We show that the manipulation of cells can induce an increase in the cytosolic Ca(2+) concentration. This increase was not observed if the manipulation of the cells was performed in absence of the extracellular calcium indicating the importance of the Ca(2+) uptake. We found, that the uptake of extracellular Ca(2+) strongly depends on the repetition rate and the irradiation time of the laser pulses. The exposure for several seconds to kHz pulses even induces Ca(2+) induced Ca(2+) release. Dependent on the location of perforation, probably in the vicinity of an intracellular Ca(2+) stock, an instantaneous intracellular Ca(2+) release can be induced. Since Ca(2+) could be involved in negative side effect by cell surgery, we propose an application of the optoperforation technique in nominal Ca(2+)-free external solution.

Parametric color coding of digital subtraction angiography.

BACKGROUND AND PURPOSE: Color has been shown to facilitate both visual search and recognition tasks. It was our purpose to examine the impact of a color-coding algorithm on the interpretation of 2D-DSA acquisitions by experienced and inexperienced observers. MATERIALS AND METHODS: Twenty-six 2D-DSA acquisitions obtained as part of routine clinical care from subjects with a variety of cerebrovascular disease processes were selected from an internal data base so as to include a variety of disease states (aneurysms, AVMs, fistulas, stenosis, occlusions, dissections, and tumors). Three experienced and 3 less experienced observers were each shown the acquisitions on a prerelease version of a commercially available double-monitor workstation (XWP, Siemens Healthcare). Acquisitions were presented first as a subtracted image series and then as a single composite color-coded image of the entire acquisition. Observers were then asked a series of questions designed to assess the value of the color-coded images for the following purposes: 1) to enhance their ability to make a diagnosis, 2) to have confidence in their diagnosis, 3) to plan a treatment, and 4) to judge the effect of a treatment. The results were analyzed by using 1-sample Wilcoxon tests. RESULTS: Color-coded images enhanced the ease of evaluating treatment success in >40% of cases (P < .0001). They also had a statistically significant impact on treatment planning, making planning easier in >20% of the cases (P = .0069). In >20% of the examples, color-coding made diagnosis and treatment planning easier for all readers (P < .0001). Color-coding also increased the confidence of diagnosis compared with the use of DSA alone (P = .056). The impact of this was greater for the naïve readers than for the expert readers. CONCLUSIONS: At no additional cost in x-ray dose or contrast medium, color-coding of DSA enhanced the conspicuity of findings on DSA images. It was particularly useful in situations in which there was a complex flow pattern and in evaluation of pre- and posttreatment acquisitions. Its full potential remains to be defined.

Quantified femtosecond laser based opto-perforation of living GFSHR-17 and MTH53 a cells.

Opto-perforation is an interesting alternative to conventional techniques for gene transfer into living cells. The cell membrane is perforated by femtosecond (fs) laser pulses, in order to induce an uptake of macromolecules e.g. DNA. In this study, we successfully transfected a canine cell line (MTH53a) with GFP vector or a vector coding for a GFP-HMGB1 fusion protein. The transfected cells were observed 48 hours after treatment and they were not showing any signs of apoptosis or necrosis. Based on simultaneously measured membrane potential changes during the perforation, we were able to calculate and experimentally verify that the relative volume exchanged is 0.4 times the total cell volume. Thus, for first time a quantitative predication of the amount of uptaken molecules and therefore a quantification of the transfection is possible. Additionally, this method offers new high efficient possibilities for critical transfection approaches involving special cell types, e.g. primary and stem cells.

[Allogeneic blood stem cell transplantation in high-risk patients after conditioning with treosulfan and fludarabine]. / Allogene Blutstammzelltransplantation von Risikopatienten nach Konditionierung mit Treosulfan und Fludarabin.

BACKGROUND AND OBJECTIVE: Allogeneic transplantation can not be offered to many patients due to potential side-effects of conventional conditioning. Dose-reduced conditioning approaches improve tolerability, however, treatment efficacy may be reduced as well. We have, therefore, developed a dose intense but toxicity reduced conditioning regimen based on treosulfan and fludarabine and report first results. PATIENTS AND METHODS: 65 patients with a median age of 50 years were transplanted from related (n = 21) or unrelated donors (n = 44) after conditioning with treosulfan (3 x 10, 3 x 12 or 3 x 14 g/m(2) i. v.) and fludarabine (5 x 30 mg/m(2) i. v.). 21 patients were in complete remission (CR) and 44 patients had not reached a CR at the time of transplantation. 59 of 65 patients were considered unfit for a conventional conditioning regimen. RESULTS: The actuarial overall survival after 3 years is 59.2 %, the event-free survival 40.1 %. Patients with a related donor or transplantation in CR had a better overall (85.4 resp. 74.2 %) and event-free survival (52.2 % resp. 61.9 %). The cumulative incidence of relapse at 3 years was 26.2 %. Non-relapse mortality at day 100 is 17.4 % (cumulative incidence). In stepwise Cox regression analyses for overall survival, event-free survival and non-relapse mortality the covariables transplantation in CR vs. not in CR and the donor status were shown to be influential. CONCLUSIONS: These results with a conditioning therapy of treosulfan and fludarabine indicate that patients despite higher age, concomitant disease or after intensive pretreatment can be successfully transplanted without increased treatment-related mortality.

Dose-escalated treosulphan in combination with cyclophosphamide as a new preparative regimen for allogeneic haematopoietic stem cell transplantation in patients with an increased risk for regimen-related complications.

Treosulphan has recently demonstrated antileukaemic activity and potent haematopoietic stem cell toxicity. Dose-escalated treosulphan (3 x 12 or 3 x 14 g/m2) combined with cyclophosphamide (Cy) was chosen for a new preparative regimen before allogeneic haematopoietic stem cell transplantation in 18 patients (median age 44, range 19-64 years) with haematological malignancies, considered ineligible for other myeloablative preparative regimens. Pharmacokinetic studies demonstrated rapid treosulphan plasma clearance and a dose-dependent increase of its maximum plasma concentrations and area under the concentration-time curves. Rapid and sustained white blood cell and platelet recovery and full donor chimerism was attained in all evaluable patients. Nonhaematological regimen-related CTC grades 3-4 adverse events were transient and predominantly consisted of cardiac (28%), gastrointestinal (39%), and hepatic (39%) toxicities. The 1-year nonrelapse mortality was 22%. Principal causes of transplant-related lethal events were infections in three of four affected patients. Only one patient died from regimen-related cardiac toxicity. The 1-year relapse estimate is 22%, overall and progression-free survival estimates are 67 and 56%, respectively. In conclusion, this new treosulphan and Cy combination is an effective, comparatively well-tolerated myeloablative preparative regimen even in patients with an increased risk for regimen-related toxic complications.

Antileukaemic activity of treosulfan in xenografted human acute lymphoblastic leukaemias (ALL).

Treosulfan (L-threitol-1,4-bis-methanesulphonate; Ovastat(R)) is a bifunctional alkylating drug indicated for the treatment of advanced ovarian carcinoma. Recent data revealed immunosuppressive characteristics and substantial haematopoietic stem cell toxicity after repeated dosing of mice. Therefore, treosulfan is considered to be an alternative conditioning agent to busulfan (for example) administered prior to allogeneic/autologous stem cell transplantation of patients with haematological malignancies. An antineoplastic activity for treosulfan has been previously shown in preclinical models of melanoma, breast, lung and renal-cell carcinomas. Here, in vivo antileukaemic activity of treosulfan is compared with the activity of equitoxic doses of cyclophosphamide or busulfan for the first time using human acute lymphoblastic leukaemia (ALL)-models of paediatric origin xenotransplanted into non-obese diabetic (NOD)/severe combined immunodeficient (SCID) mice. Treosulfan treatment achieved an optimum treated to control (T/C) value of 159% (survival time) against B-ALL-SCID 7 and a T/C value of 0% (tumour growth) against T-ALL-SCID 4 and proB-ALL-SCID 19, respectively. Complete regression of established subcutaneously (s.c.) growing nodules of ALL-SCID 4 and 19 was obvious and long-term survivors without tumour re-growth were observed. Equitoxic doses of busulfan (ALL-SCID 4, 7, 19) or cyclophosphamide (ALL-SCID 19) were less effective with regard to the numbers of complete regressions and the number of cured animals. Side-effects included myelotoxicity and a small reduction in body weight, but these were tolerable. Treosulfan can be considered a highly active antileukaemic drug whose corresponding clinical value is to be tested in appropriate protocols with leukaemic patients.

CD95/CD95 ligand-independent potentiation of treosulfan cytotoxicity by BSO in malignant glioma cells in vitro and in vivo.

The glutathione synthase inhibitor, buthionine sulfoximine (BSO), specifically enhances the cytotoxic effects of treosulfan in human glioma cells. BSO depletes glutathione and greatly enhances treosulfan cytotoxicity in all the 12 human malignant glioma cell lines examined. None of these cell lines showed enhanced susceptibility to CD95L- or tumor necrosis factor (TNF)-alpha-induced apoptosis when glutathione is depleted. The combination of serial systemic BSO applications (300 mg/kg) and a single systemic dose of treosulfan (2.5 g/kg) reduced the growth of intracranially growing rat C6 gliomas in vivo by 73% whereas treosulfan alone reduced tumor growth by 16% and BSO alone had no effect. BSO lowered glutathione levels to 25-30% in peripheral blood mononuclear cells (PBMC) and to 50% in the glioma tissue. The glutathione levels in the non-tumor-bearing contralateral hemisphere were unaffected by systemic BSO treatment. The main side effects of treosulfan, gastrointestinal and bone marrow toxicity, were not significantly enhanced by BSO.