Sperm and testicular tissue cryopreservation and assisted reproductive technology outcomes in male cancer patients: a 15-year experience

Objective: To explore the characteristics of cancer patients who cryopreserved sperm/testicular tissue samples in the Cryobank of Charite-Universitatsmedizin Berlin between 2004 and 2019, and the ART utilization rate with associated outcomes. Methods: Retrospective data were available for 506 cancer patients, of which 46 (9.1%) had used their samples for artificial reproductive technologies (ART). Corresponding cycle information was collected from external fertility centers. Results: Our cohort included 53/506 (10.5%) patients aged < 18 years at diagnosis. While adolescents and adults mainly banked sperm, adolescents showed higher rates of testicular tissue cryopreservation before (11.8%, 6/51 vs. 6.4%, 26/406) and after treatment (16.7%, 4/24 vs. 7.8%, 13/167). At study conduction, storage had been ended for 44.8% (269/601) of samples. The majority of samples used for ART were requested within the first 3 years after cryopreservation (71.5%, 28/39, range = 0-12 years). Pregnancy rate was 51.4% (19/37 cycles), resulting in 11 singleton births, 3 twin pairs, and 4 miscarriages. Conclusion: With the new advantage of public health insurance coverage of fertility preservation (FP) in Germany, an increased utilization has already been noticed in our center, emphasizing the necessity of further knowledge for individual counseling. Adolescent cancer patients need to be addressed specifically, as these patients show especially low cryopreservation rates

Enhanced detection of circulating tumor DNA by fragment size analysis.

Existing methods to improve detection of circulating tumor DNA (ctDNA) have focused on genomic alterations but have rarely considered the biological properties of plasma cell-free DNA (cfDNA). We hypothesized that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for noninvasive genomic analysis of cancer. We surveyed ctDNA fragment sizes in 344 plasma samples from 200 patients with cancer using low-pass whole-genome sequencing (0.4×). To establish the size distribution of mutant ctDNA, tumor-guided personalized deep sequencing was performed in 19 patients. We detected enrichment of ctDNA in fragment sizes between 90 and 150 bp and developed methods for in vitro and in silico size selection of these fragments. Selecting fragments between 90 and 150 bp improved detection of tumor DNA, with more than twofold median enrichment in >95% of cases and more than fourfold enrichment in >10% of cases. Analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of plasma samples from patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA, with area under the curve (AUC) >0.99 compared to AUC 0.91 compared to AUC < 0.5 without fragmentation features. Fragment size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection and could complement or provide an alternative to deeper sequencing of cfDNA.We would like to acknowledge the support of The University of Cambridge, Cancer Research UK and the EPSRC (CRUK grant numbers A11906 (NR), A20240 (NR), A22905 (JDB), A15601 (JDB), A25177 (CRUK Cancer Centre Cambridge), A17242 (KMB), A16465 (CRUK-EPSRC Imaging Centre in Cambridge and Manchester)). The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement n. 337905. The research was supported by the National Institute for Health Research Cambridge, National Cancer Research Network, Cambridge Experimental Cancer Medicine Centre and Hutchison Whampoa Limited. This research is also supported by Target Ovarian Cancer and the Medical Research Council through their Joint Clinical Research Training Fellowship for Dr Moore. The CALIBRATE study was supported by funding from AstraZeneca

Cellular localization of Kir4.1, THIK-1 and THIK-2 K+ channels in the mammalian kidney

Einleitung: Beeinträchtigungen der Kaliumhomöostase gehören zu den häufigsten Elektrolytstörungen überhaupt und können neben neurologischen Symptomen und kardialen Arrhythmien auch schwerwiegende Störungen der Nierenfunktion verursachen. Die Niere ist für die Feineinstellung der Kaliumausscheidung verantwortlich und fungiert dadurch als zentrales Organ der Kaliumhomöostase. Eine entscheidende Rolle bei der Regulation der Kaliumausscheidung durch die Niere spielen Kaliumkanäle, welche sich sowohl in der apikalen als auch in der basolateralen Membran von Nierenepithelien befinden. Eine möglichst umfassende Kenntnis der Funktion und Regulation dieser Kaliumkanalproteine ist Voraussetzung für ein verbessertes Verständnis der Mechanismen der Kaliumhomöostase und könnte damit zur Entwicklung neuer Strategien bei der Diagnostik und Therapie von Störungen des Kaliumhaushaltes beitragen. Ziel der hier vorliegenden Arbeit war die Charakterisierung der renalen Lokalisation der neu identifizierten Kaliumkanäle Kir4.1, THIK-1 und THIK-2. Methodik: Es wurden immunhistochemische Markierungen, In situ Hybridisierung, Western Blot- und PCR-Analysen in Ratten- und Mausgewebe durchgeführt. Ergebnisse: Kir4.1 wurde in der basolateralen Membran im medullären und kortikalen dicken aufsteigenden Schenkel der Henle´schen Schleife, im distalen Konvolut, im Verbindungstubulus und in den Hauptzellen des kortikalen Sammelrohrs in der Mausniere nachgewiesen. Die 2-Poren-Kaliumkanäle THIK-1 und THIK-2 zeigten, trotz ähnlicher molekularer Struktur, ein unterschiedliches Verteilungsmuster im Nephron sowie speziesabhängige Unterschiede in der subzellulären Lokalisation. THIK-1 wurde im gesamten Nephron und im Sammelrohr exprimiert. THIK-2 fand sich im distalen Nephron. Bei der Untersuchungen der subzellulären Verteilung für THIK-1 und THIK-2 zeigte sich in der Rattenniere ein intrazelluläres Signal. THIK-2 zeigte in der Mausniere eine bevorzugt basolaterale Lokalisation. Aufgrund der schwachen Immunreaktivität in der Mausniere wurde in situ Hybridisierung zur Lokalisation von THIK-1 ausgewählt und konnte damit keine Aussage über die intrazelluläre Verteilung gemacht werden. Schlussfolgerung: Die Ergebnisse der vorliegenden Arbeit zeigen eine basolaterale Expression von Kir4.1 im distalen Nephron und in den Hauptzellen des Sammelrohres. Diese Lokalisationsdaten wurden in späteren Studien durch andere Arbeitsgruppen weitgehend bestätigt und tragen entscheidend zum Verständnis der Pathophysiologie von humanen Erkrankungen wie dem SeSAME/EAST- Syndrom bei. Die Ergebnisse zur Lokalisation der THIK Kanäle ergaben ein uneinheitliches Bild. Während die basolaterale Lokalisation von THIK-2 im distalen Nephron der Maus mit einer funktionellen Rolle bei der Ausbildung des background K-leak vereinbar ist, erscheint dies angesichts der intrazellulären Lokalisation von THIK-1 und THIK-2 in der Ratte als unwahrscheinlich. Weitere Studien sind daher notwendig, um die Bedeutung der beiden Kaliumkanäle für die Funktion der Niere aufzuklären.Introduction: Alterations of potassium homeostasis are among the most frequent electrolyte disorders and may cause severe neurological symptoms, cardiac arrhythmias and renal dysfunction. The kidney is responsible for regulated potassium excretion and is thus essential for the maintenance of potassium balance. Potassium channels in the apical and in the basolateral membrane of renal epithelia play an essential role during transmembrane solute transport and are indispensable for regulated potassium secretion. Whereas the mechanisms of potassium transport in the apical membrane of renal epithelia have been extensively studied, little is known regarding the basolateral membrane compartment. A comprehensive knowledge of basolateral potassium channel proteins will improve our understanding of the mechanisms of potassium homeostasis and of renal function in general and may also facilitate the development of novel strategies for the diagnosis and treatment of disorders of potassium homeostasis. Aim of the present study was to characterize the cellular localization of Kir4.1, THIK-1 and THIK-2 in the kidney. Material and methods: Immunohistochemistry, in situ hybridization, Western blot, and RT-PCR on microdissected nephron segments were used to study the renal expression of Kir4.1, THIK-1 and THIK-2 in rats and mice. Results: Localization studies for Kir4.1 showed strong expression in the basolateral membrane of the thick ascending limb of the loop of Henle, the distal convoluted and the connecting tubule as well as in the cortical collecting duct of mice and rats. Despite a similar molecular structure the tandem pore domain potassium channels THIK-1 and THIK-2 showed different localization along the nephron and species- dependent differences in the subcellular localization between rat and mouse. Whereas THIK-1 was expressed throughout the nephron and the collecting duct, THIK-2 was exclusively localized to the distal nephron. Both, THIK-1 and THIK-2 showed intracellular localization in rat and murine kidney sections. By contrast, evaluation of THIK-2 signals in mice demonstrated expression in the basolateral membrane. Conclusion: In the present study basolateral expression of Kir4.1 in the distal nephron and in the principal cells of the collecting tube was demonstrated. These localization data were largely confirmed in later studies by other groups and contributed to an understanding of the pathophysiology of human diseases including the SeSAME/EAST syndrome. The basolateral localization of THIK-2 in the distal nephron of the mouse kidney is compatible with a functional role of the protein during basolateral potassium transport whereas the intracellular localization of THIK-1 and THIK-2 in the rat kidney argues against an involvement of the two channel proteins in this process. Further studies are therefore needed to characterize the role of the two potassium channels for renal function

Enhanced detection of circulating tumor DNA by fragment size analysis

Existing methods to improve detection of circulating tumor DNA (ctDNA) have focused on genomic alterations but have rarely considered the biological properties of plasma cell-free DNA (cfDNA). We hypothesized that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for noninvasive genomic analysis of cancer. We surveyed ctDNA fragment sizes in 344 plasma samples from 200 patients with cancer using low-pass whole-genome sequencing (0.4×). To establish the size distribution of mutant ctDNA, tumor-guided personalized deep sequencing was performed in 19 patients. We detected enrichment of ctDNA in fragment sizes between 90 and 150 bp and developed methods for in vitro and in silico size selection of these fragments. Selecting fragments between 90 and 150 bp improved detection of tumor DNA, with more than twofold median enrichment in >95% of cases and more than fourfold enrichment in >10% of cases. Analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of plasma samples from patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA, with area under the curve (AUC) >0.99 compared to AUC 0.91 compared to AUC < 0.5 without fragmentation features. Fragment size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection and could complement or provide an alternative to deeper sequencing of cfDNA