Norbert Ahrens

Purpose:Non–small cell lung cancer (NSCLC) is a fatal disease with poor prognosis. A membrane-bound form of Hsp70 (mHsp70) which is selectively expressed on high-risk tumors serves as a target for mHsp70-targeting natural killer (NK) cells. Patients with advanced mHsp70-positive NSCLC may therefore benefit from a therapeutic intervention involving mHsp70-targeting NK cells. The randomized phase II clinical trial (EudraCT2008-002130-30) explores tolerability and efficacy of ex vivo–activated NK cells in patients with NSCLC after radiochemotherapy (RCT).Patients and Methods:Patients with unresectable, mHsp70-positive NSCLC (stage IIIa/b) received 4 cycles of autologous NK cells activated ex vivo with TKD/IL2 [interventional arm (INT)] after RCT (60–70 Gy, platinum-based chemotherapy) or RCT alone [control arm (CTRL)]. The primary objective was progression-free survival (PFS), and secondary objectives were the assessment of quality of life (QoL, QLQ-LC13), toxicity, and immunobiological responses.Results:The NK-cell therapy after RCT was well tolerated, and no differences in QoL parameters between the two study arms were detected. Estimated 1-year probabilities for PFS were 67% [95% confidence interval (CI), 19%–90%] for the INT arm and 33% (95% CI, 5%–68%) for the CTRL arm (P = 0.36, 1-sided log-rank test). Clinical responses in the INT group were associated with an increase in the prevalence of activated NK cells in their peripheral blood.Conclusions:Ex vivo TKD/IL2-activated, autologous NK cells are well tolerated and deliver positive clinical responses in patients with advanced NSCLC after RCT.

Purpose:Non–small cell lung cancer (NSCLC) is a fatal disease with poor prognosis. A membrane-bound form of Hsp70 (mHsp70) which is selectively expressed on high-risk tumors serves as a target for mHsp70-targeting natural killer (NK) cells. Patients with advanced mHsp70-positive NSCLC may therefore benefit from a therapeutic intervention involving mHsp70-targeting NK cells. The randomized phase II clinical trial (EudraCT2008-002130-30) explores tolerability and efficacy of ex vivo–activated NK cells in patients with NSCLC after radiochemotherapy (RCT).Patients and Methods:Patients with unresectable, mHsp70-positive NSCLC (stage IIIa/b) received 4 cycles of autologous NK cells activated ex vivo with TKD/IL2 [interventional arm (INT)] after RCT (60–70 Gy, platinum-based chemotherapy) or RCT alone [control arm (CTRL)]. The primary objective was progression-free survival (PFS), and secondary objectives were the assessment of quality of life (QoL, QLQ-LC13), toxicity, and immunobiological responses.Results:The NK-cell therapy after RCT was well tolerated, and no differences in QoL parameters between the two study arms were detected. Estimated 1-year probabilities for PFS were 67% [95% confidence interval (CI), 19%–90%] for the INT arm and 33% (95% CI, 5%–68%) for the CTRL arm (P = 0.36, 1-sided log-rank test). Clinical responses in the INT group were associated with an increase in the prevalence of activated NK cells in their peripheral blood.Conclusions:Ex vivo TKD/IL2-activated, autologous NK cells are well tolerated and deliver positive clinical responses in patients with advanced NSCLC after RCT.

Background: More granulocyte concentrates (GCs) could be produced for more patients from the same donor if apheresis bags were split and stored for longer periods of time. Hence, we tested the hypothesis that splitting and extension of storage of GCs do not impair granulocyte function or viability.Study Design and Methods: Granulocyte apheresis concentrates were produced using modified fluid gelatin as a separation enhancer, split into two portions, and stored for 24 and 48 h. Granulocyte function, represented by cell migration, reactive oxygen species (ROS) production, and neutrophil extracellular trap formation (NETosis), was measured by live‐cell imaging. ROS production, adhesive surface protein expression, and viability were measured by flow cytometry.Results: Splitting had no effect on any of the tested parameters. After 24 h of storage, live‐cell imaging showed no significant difference in migration, time to maximum ROS production, time to half‐maximum NETosis, viability, or CD11b expression, but ROS production induced by phorbol 12‐myristate 13‐acetate (PMA) decreased from an initial median fluorescence intensity of 1775–590 artificial units. After 48 h, PMA‐induced ROS production, viability, and migration declined, as reflected by decreases in median total distance (119 vs. 63.5 μm) and median Euclidean distance (30.75 vs. 14.3 μm).Conclusion: Splitting GC products has no effect on granulocyte viability or function, but extended storage >24 h does compromise granulocyte function. The findings confirm that GCs should be transfused within 24 h of collection. Longer storage cannot be recommended.

Zusammenfassung: Die Extrakorporale Photopherese ist ein etabliertes Therapieverfahren für Patienten mit T-Zell vermittelten Erkrankungen. Dabei besteht das Verfahren aus der Gewinnung autologer mononukleärer Zellen, deren Behandlung mit 8-Methoxypsoralen und UVA-Licht und die Retransfusion der behandelten Zellen.Die Wirkmechanismen der Photopherese sind zwar noch nicht vollständig geklärt, ein zentraler Mechanismus stellt jedoch die Apoptose mononukleärer Zellen dar. Das Ziel der Studie war eine Optimierung der Photopherese im Hinblick auf die Behandlung der Zellen mit 8-Methoxypsoralen/UVA und der daraus induzierten verstärkten Apoptose der Lymphozyten. Dabei sind einige Faktoren bekannt, welche die Effektivität der 8-Methoxypsoralen/UVA-Behandlung der Zellen beeinflussen können, wie z.B. der Hämatokrit oder die UVA-Dosis. Unser Fokus lag auf der Verfügbarkeit der photoaktiven Substanz und dem Einfluss der Zellsuspensionsmatrix auf die Apoptose der Lymphozyten.Die Verfügbarkeit von 8-Methoxypsoralen für die Aufnahme in die Zellen reduzierte sich durch Absorption an Kunststoffe der Bestrahlungssysteme sowie durch Bindung an Proteine bei der Verwendung von autologem Plasma bei der Suspendierung der Zellen. Eine Steigerung der Zugabe von 8-Methoxypsoralen auf 340 ng/mL anstelle von 200 ng/mL führte zu einem Anstieg der T-Zell Apoptose, die sich unter Verwendung von physiologischer Kochsalzlösung als Zellsuspensionsmatrix weiter erhöhte. Eine Anpassung des Verfahrens mit NaCl anstelle von Plasma und die Verwendung einer höheren 8-Methoxypsoralen Konzentration führte zu einer gesteigerten Apoptoseinduktion der T-Zellen. Inwiefern sich eine Erhöhung der Apoptose auf die klinische Wirksamkeit auswirkt, bedarf hingegen noch weiterer klinischen Untersuchungen.

Background and Aims: Platelets are prone to activation from handling; they are therefore transported as gently as possible, most commonly by courier. Speedier methods like pneumatic tube system (PTS) transport could improve patient care but may subject platelets to mechanical stress. To test the impact of mechanical stress caused by transport, we compared a PTS with a conveyor box and courier transport on apheresis platelet function.Methods: Fourteen apheresis platelet concentrate triple donations were analyzed by light transmission aggregometry (LTA), rotational thrombelastometry (ROTEM), and flow cytometry before and after indoor transport over 800 m by PTS, conveyor, and courier, respectively, while recording shocks and vibrations with a high‐frequency acceleration data logger. Shock index scores were calculated as shock intensity (g‐force) times frequency.Results: The shock index was 81 for courier, 6279 for conveyor, and 9075 for PTS. Flow cytometry revealed no significant difference in platelet surface expression of CD62p before (16%) and after transport via courier (15%), conveyor (14%), or PTS (16%). LTA with adenosine phosphate and thrombin receptor‐activating peptide‐6 resulted in comparable platelet aggregation for courier, conveyor, and PTS. ROTEM assays showed no relevant differences in coagulation time, clot formation time, and maximum clot firmness between transport modes.Conclusion: Though the mechanical challenge was smallest with courier transport, there were no significant differences in platelet activation or aggregation between the three transport modes. These data contradict restrictions on the use of PTSs for platelet concentrate transport.

Background Granulocyte transfusions (GT) are used to treat progressive systemic or local infections in prolonged neutropenic patients with antibiotic or antifungal resistance. Granulocytes are most commonly collected from whole blood by apheresis using hydroxyethyl starch (HES) as the red blood cell (RBC) sedimentation agent. This is the first study on the safety and efficacy of transfusing granulocytes collected with modified fluid gelatin (MFG) instead of HES to pediatric patients. Methods Clinical data from 46 pediatric and adolescent patients receiving at least one MFG-based granulocyte transfusion and in total 295 granulocyte concentrates from July 2013 to August 2019 at our local university medical center were evaluated retrospectively. Results Forty-one patients (89%) survived at least 21 days after their last granulocyte transfusion. These survivors had lower CRP values and higher leukocyte counts after GT than non-survivors (mean delta of −5.34 mg/dl vs. –11.99 mg/dl and + 0.62 × 10³/μl vs. +0.18 × 10³/μl of all GT, respectively). The neutrophil corrected count increment (CCI) was 68.72 mm²/ml in survivors versus 28.00 mm²/ml in non-survivors. There were no major or severe adverse events. Conclusion This study suggests that modified fluid gelatin is a safe and effective alternative to hydroxyethyl starch for the collection of granulocytes for transfusion to prolonged neutropenic patients with progressive systemic or local infections refractory to antibiotic or antifungal therapy.

Background Plateletpheresis using a leukocyte reduction system (LRS) traps donor WBCs in the LRS chamber, which may lead to lymphopenia, especially in frequent plateletpheresis donors. It seems plausible that this might cause adverse effects. However, current knowledge about potential confounders and donor health impacts is incomplete. Donors and methods Recent platelet donors and donations collected at University Hospital Regensburg from 2016 to 2019 using the Terumo BCT Trima Accel LRS system were retrospectively analyzed and compared with historical platelet donors and donations collected mainly with Fresenius Kabi Amicus non-LRS system from 2010 to 2013. Additionally, recent donors were prospectively surveyed using a health-related topics questionnaire. Results Analysis of 819 recent donors with 11,254 blood counts and 1464 questionnaires and 1011 historical donors with 12,848 blood counts revealed that increased annual platelet donation frequencies were associated with decreased lymphocyte counts in both groups. Median lymphocyte counts in recent donors with no versus ≥24 previous annual donations declined from 2.0 to 1.2 × 10³/μL (p < 2.2 × 10⁻¹⁶), and those in historical donors with no versus ≥24 previous annual donations decreased from 2.0 to 1.5 × 10³/μL (p = 6 × 10⁻⁴), respectively. The questionnaire results showed that donation frequency and lymphopenia were not associated with upper respiratory tract infection (URTI) incidence or duration, but platelet donors who concomitantly donated granulocytes had significantly shorter URTI durations than those who did not (p = .008). Conclusion This study confirmed that plateletpheresis-associated lymphopenia occurs in LRS and to a lesser degree in non-LRS platelet donors, but revealed no evidence of a negative impact on donor health.

Background: Extracorporeal photopheresis (ECP), an apheresis‐based therapy for various immunological diseases, works mainly by inducing apoptosis in lymphocytes. Several factors influence the efficacy of ECP with the photosensitizer 8‐methoxypsoralen (8‐MOP) and ultraviolet light A (UVA). This study aimed to optimize treatment by varying the 8‐MOP starting concentration and the cell suspension medium.Materials and Methods: All patients (n = 13) included in this study received photopheresis as medically indicated. Cells collected with a Spectra Optia apheresis system were suspended in plasma or physiological saline (NaCl) and incubated with 200 ng/ml versus 340 ng/ml photosensitizer before UVA irradiation (Macogenic G2 or UVA PIT system). Lymphocyte apoptosis and caspase activity were analyzed by flow cytometry and fluorimetry, and residual 8‐methoxypsoralen concentrations by liquid chromatography–mass spectrometry.Results: Raising the 8‐MOP starting concentration significantly increased lymphocyte apoptosis, with values of 22% versus 35% (plasma) and 28%–46% (NaCl) at 24 h post‐ECP and 37% versus 86% (plasma) and 74% versus 97% (NaCl) at 48 h for 200 ng/ml versus 340 ng/ml. Pre‐transfusion residual 8‐MOP levels were 168 ng/ml (plasma) and 162 ng/ml (NaCl) versus 290 ng/ml (plasma) and 266 ng/ml (NaCl) for the lower versus higher dose, respectively.Discussion: Hence, 8‐MOP concentration influences the efficacy of photopheresis as lymphocyte apoptosis rates were significantly higher with the higher starting concentration and with NaCl versus plasma. This indicates that increased 8‐MOP starting doses and saline as additional suspension medium could help in improving ECP's efficacy.