Antimikrobielle Aktivität des Histon H1.2 in vitro\textit {in vitro} und in infizierten Brandwunden

$\bf {Problem:}$ Die Haut ist ein Schutzschild des menschlichen Körpers gegen pathogene Mikroorganismen. Verbrennungen prädisponieren Schwerbrandverletzte zu Wundinfektionen durch unterschiedliche Mikroorganismen. Im Rahmen dieser Studie wurde untersucht, ob rekombinantes humanes Histon H1.2 als Lokaltherapie von Wundinfektionen einsetzbar ist. $\bf {Methode:}$ Es wurden die antimikrobielle Eigenschaft des Histon H1.2 $\textit {in vitro}$ und in einem infizierten Rattenverbrennungsmodell charakterisiert. Zudem wurden die hämolytische und die zytotoxische Aktivität des Histon H1.2 bestimmt. Wir verglichen das Histon H1.2 mit dem antimikrobiellen Peptid Protegrin-1 und mit klinisch eingesetzten Antibiotika und Antimykotika. $\bf {Ergebnis:}$ Das Histon H1.2 zeigte ausnahmslos eine signifikant stärkere antimikrobielle Aktivität im Vergleich zu den klinisch eingesetzten Antibiotika und Antimykotika bei fehlender hämolytischer Aktivität. In den $\textit {in vivo}$ Versuchen wurde eine signifikante Reduktion der Bakterienzahl gezeigt

Liquid biopsy and other non-invasive diagnostic measures in PCNSL

Primary central nervous system lymphoma is a rare but highly aggressive form of non-Hodgkin lymphoma that remains confined to the CNS neuroaxis. The diagnosis of PCNSL requires a high level of suspicion as clinical presentation varies depending on the involved CNS areas. Neurological symptoms and MRI findings may mimic gliomas, demyelinating lesions, or infectious and granulomatous diseases. Almost all PCNSL patients undergo invasive surgical procedures for definite diagnosis. Stereotactic biopsy is still the gold standard in achieving a diagnostic accuracy of 73–97%. Both the potential procedural morbidity and mortality, as well as the time to definite histopathologic diagnosis resulting in delays of treatment initiation, have to be considered. On the contrary, minimally invasive procedures, such as MRI, CSF cytology, and flow cytometry, still have limited value due to inferior specificity and sensitivity. Hence, novel diagnostic approaches, including mutation analyses (MYD88) in circulating tumor DNA (ctDNA) and the determination of microRNAs (miR-21, miR-19b, and miR-92) as well as cytokine levels (IL10 and IL6) in blood, cerebrospinal fluid (CSF), and vitreous fluid (VRF), move into the focus of investigation to facilitate the diagnosis of PCNSL. In this review, we outline the most promising approaches that are currently under clinical consideration

Successful chimeric antigen receptor (CAR) T-cell treatment in aggressive lymphoma despite coronavirus disease 2019 (CoVID-19) and prolonged severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) replication

In patients with compromised immune function, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and coronavirus disease 2019 (CoVID-19) impose particular challenges. Especially in hematological malignancies, including lymphoma, the demands by this novel virus disease are further enhanced during sophisticated treatments, such as chimeric antigen receptor (CAR) T-cell therapy. Here, we present the first case of a patient with refractory diffuse-large B-cell lymphoma, who underwent CAR T-cell treatment in the context of SARS-CoV-2. Irrespective of prolonged and active SARS-CoV-2 infection, T cells were successfully isolated by apheresis and processed to anti-CD19 CAR T cells (axicabtagene-ciloleucel). In light of the aggressive lymphoma course, lymphodepleting chemotherapy and CAR-T cells were administered in early recovery after oxygen-dependent CoVID-19 pneumonia. Except for moderate cytokine release, this cellular immunotherapy was well tolerated. Notably, there is no deterioration of the SARS-CoV-2 infection; however, complete lymphoma response and full clinical recovery were observed. In conclusion, CAR T-cell treatment in aggressive lymphoma in the setting of SARS-CoV-2 infection is feasible and may offer significant therapeutic activity in refractory disease

Liquid biopsy based HER2 amplification status in gastric cancer patients indicates clinical response

$\underline {Gastric}$ $\underline {carcinomas}$ are among the most common cancers in Germany, with approximately 18,000 new cases per year. About 10 years ago, based on results of the $\underline {Trastuzumab}$ for gastric cancer (ToGA) trial, the addition of the $\underline {monoclonal}$ $\underline {antibody}$ trastuzumab to a platinum-fluoropyrimidine chemotherapy $\underline {backbone}$ became the standard-of-care 1st-line $\underline {therapy}$ for human $\underline {epidermal}$ $\underline {growth}$ $\underline {factor}$ $\underline {receptor}$ 2 ($\it HER2$)-positive gastric cancers. Only patients with primary $\it HER2$ $\underline {gene}$ $\underline {amplification}$ benefit from this therapy. Thus, accurate $\it HER2$ gene amplification detection is predictive and critical for therapy selection. As a gold standard the $\it HER2$ status is currently determined in tumor tissue specimens using immune $\underline {histochemistry}$ and $\underline {fluorescent}$ $\underline {in}$ $\underline {situ}$ $\underline {hybridisation}$. However, $\it HER2$ amplification is detectable in only about 20 % of gastric carcinomas. The recent approval of an antibody-drug conjugate $\underline {Trastuzumab}$ $\underline {deruxtecan}$ (T-DXd) and the establishment of a new subgroup of $\it HER2$-low tumors due to the $\underline {bystander}$ $\underline {effect}$ associated with T-DXd increases the relevance of precise $\it HER2$ diagnostics. Aim of this analysis was to determine the $\it HER2$ amplification status from circulating DNA fragments in blood using a $\it HER2$ $\underline {Copy}$ $\underline {Number}$ $\underline {Variation}$ assay to establish a minimal invasive approach. For the present study, a digital droplet PCR-based method was validated relative to established tissue-based methods. Furthermore and most importantly, the changes of $\it HER2$ status during therapy were investigated in seven patients indicating that the changes of $\it HER2$ status and number of $\it HER2$ copies detected in blood can reflect on therapy efficiency and uncover treatment resistance

Patients with primary central nervous system lymphoma not eligible for clinical trials

Patients with primary central nervous system lymphoma (PCNSL) not fulfilling inclusion criteria for clinical trials represent an underreported population. Thirty-four consecutive PCNSL patients seen at our center between 2005 and 2019 with exclusion criteria for therapeutic trials were analyzed (non-study patients) and compared with patients from the G-PCNSL-SG-1 (German PCNSL Study Group 1) study (study patients), the largest prospective multicenter trial on PCNSL, comprising 551 patients. Median follow up was 68 months (range 1–141) in non-study patients and 51 months (1–105) in study patients. Twenty-seven/34 (79.4%) non-study patients received high dose methotrexate (HDMTX), while seven/34 (20.6%) with a glomerular filtration rate (GFR) < 50 mL/min did not. Median overall survival (OS) was six months (95% confidence interval [CI] 0–21 months) in those 34 non-study patients. The 27 non-study patients treated with HDMTX were compared with 526/551 G-PCNSL-SG-1 study patients who had received HDMTX as well. Median OS was 20 months (95% CI 0–45)/21 months (95% CI 18–25) in 27 non-study/526 study patients ($\it p$ = 0.766). Favorable prognostic factors in non-study patients were young age, application of HDMTX and early response on magnet resonance imaging (MRI). If HDMTX-based chemotherapy can be applied, long-term disease control is possible even in patients not qualifying for clinical trials. Initial response on early MRI might be useful for decision on treatment continuation

Altered T-lymphocyte biology following high-dose melphalan and autologous stem cell transplantation with implications for adoptive T-cell therapy

In relapsed and refractory multiple myeloma (MM), adoptive cell therapies (ACT) including CAR-T-cells are under clinical investigation. However, relapse due to T-cell exhaustion or limited persistence is an obstacle. Before ACT are considered in MM, high-dose (HD) melphalan followed by autologous stem-cell transplantation (autoSCT) has been administered in most clinical situations. Yet, the impact of HD chemotherapy on T-cells in MM with respect to ACT is unclear. In this study, T-lymphocytes’ phenotypes, expansion properties, lentiviral transduction efficacy, and gene expression were examined with special respect to patients following HD melphalan. Significant impairment of T-cells’ expansion and transduction rates could be demonstrated. Expansion was diminished due to inherent disadvantages of the predominant T-cell phenotype but restored over time. The quantitative fraction of CD27−/CD28− T-cells before expansion was predictive of T-cell yield. Following autoSCT, the transduction efficacy was reduced by disturbed lentiviral genome integration. Moreover, an unfavorable T-cell phenotype after expansion was demonstrated. In initial analyses of CD107a degranulation impaired T-cell cytotoxicity was detected in one patient following melphalan and autoSCT. The findings of our study have potential implications regarding the time point of leukapheresis for CAR-T-cell manufacturing. Our results point to a preferred interval of more than 3 months until patients should undergo cell separation for CAR-T therapy in the specific situation post-HD melphalan/autoSCT. Monitoring of CD27−/CD28− T-cells, has the potential to influence clinical decision making before apheresis in MM

Digital-droplet PCR for quantification of CD19-directed CAR T-cells

CD19-directed CAR-T-cells (CD19-CAR) have demonstrated remarkable clinical results in patients suffering from refractory or relapsed lymphoma and acute lymphoblastic leukemia. In order to further optimize follow-up, to explain treatment failure, and to control adverse events biomarkers for monitoring of response are urgently needed. Peak expansion and persistence are correlated with response rates and severity of side effects. However, no standardized method or commercially assay for CD19-CAR measurement is established yet. In this study, two primer-probe assays for digital-droplet PCR (ddPCR) were designed and subsequently explored on 54 samples collected from seven patients after CD19-CAR treatment with axi-cel over time. Detection and quantification of CAR-T-cells were feasible and reliable for all patients included. Peak expansion measured with our assay significantly correlated with the grade of neurologic adverse events but not with cytokine release syndrome. All patients with loss of CAR-signal eventually had disease progression. In summary, our novel assay allows monitoring of CAR-T-cells $\textit {in vivo}$ and may add to safety and efficacy of CAR-T treatment

Evolution of RAS mutational status in liquid biopsies during first-line chemotherapy for metastatic colorectal cancer

Treatment options for patients with metastatic colorectal cancer (mCRC) are limited. This particularly affects the largest group of patients with RAS mutations, who are considered ineligible for therapy with antiEGFR antibodies. In this liquid biopsy-based study, we performed the first in-depth analysis of the RAS mutational status in initially RAS-mutated patients during first-line therapy. RAS status of twelve patients with initially RAS-mutated mCRC was monitored longitudinally in 69 liquid biopsy samples. We focused on patients with stable disease (SD) or partial remission (PR) during first-line therapy (11 patients). Detection of fragmented RAS-mutated circulating cell-free tumor DNA (ctDNA) in plasma was performed by digital-droplet PCR (ddPCR) and BEAMing. Patients' total tumor masses were determined by measuring the tumor volumes using CT scan data. All patients with PR or SD at first follow-up showed a significant decrease of RAS mutational load. In ten patients (91%), the ctDNA-based RAS mutational status converted to wild-type in ddPCR and BEAMing. Remarkably, conversions were observed early after the first cycle of chemotherapy. Plasma concentration of ctDNA was controlled by determination of methylated WIF1-promotor ctDNA burden as a second tumor marker for mCRC. Persistent presence of methylated WIF1-promotor fragments confirmed the ongoing release of ctDNA during treatment. In patients with initially RAS-mutated mCRC, RAS mutations rapidly disappeared during first-line therapy in liquid biopsy, independent of type and intensity of chemotherapy and irrespective of anti-VEGF treatments. Following our results demonstrating conversion of RAS-mutational status, potential effectiveness of anti-EGFR antibodies in selected patients becomes an attractive hypothesis for future studies

Computertomography-based prediction of complete response following neoadjuvant chemoradiotherapy of locally advanced rectal cancer

Therapeutic strategies for patients with locally advanced rectal cancer (LARC) who are achieving a pathological complete response (pCR) after neoadjuvant radio-chemotherapy (neoCRT) are being increasingly investigated. Recent trials challenge the current standard therapy of total mesorectal excision (TME). For some patients, the treatment strategy of "watch-and-wait" seems a preferable procedure. The key factor in determining individual treatment strategies following neoCRT is the precise evaluation of the tumor response. Contrast-enhanced computer tomography (ceCT) has proven its ability to discriminate benign and malign lesions in multiple cancers. In this study, we retrospectively analyzed the ceCT based density of LARC in 30 patients, undergoing neoCRT followed by TME. We compared the tumors´ pre- and post-neoCRT density and correlated the results to the amount of residual vital tumor cells in the resected tissue. Overall, the density decreased after neoCRT, with the highest decrease in patients achieving pCR. Densitometry demonstrated a specificity of 88% and sensitivity of 68% in predicting pCR. Thus, we claim that ceCT based densitometry is a useful tool in identifying patients with LARC who may benefit from a "watch-and-wait" strategy and suggest further prospective studies