The generation and expansion of functionally competent NK cells
is of great interest for their application in immunotherapy of cancer. Since CD33 constitutes a promising target for immunotherapy of ...myeloid malignancies, NK cells expressing a CD33-specific chimeric antigen receptor (CAR) were generated. Unexpectedly, we noted that CD33-CAR NK cells could not be efficiently expanded
due to a fratricide-like process in which CD33-CAR NK cells killed other CD33-CAR NK cells that had upregulated CD33 in culture. This upregulation was dependent on the stimulation protocol and encompassed up to 50% of NK cells including CD56
NK cells that do generally not express CD33
. RNAseq analysis revealed that upregulation of CD33
NK cells was accompanied by a unique transcriptional signature combining features of canonical CD56
(CD117
, CD16
) and CD56
NK cells (high expression of granzyme B and perforin). CD33
NK cells exhibited significantly higher mobilization of cytotoxic granula and comparable levels of cytotoxicity against different leukemic target cells compared to the CD33
subset. Moreover, CD33
NK cells showed superior production of IFNγ and TNFα, whereas CD33
NK cells exerted increased antibody-dependent cellular cytotoxicity (ADCC). In summary, the study delineates a novel functional divergence between NK cell subsets upon
stimulation that is marked by CD33 expression. By choosing suitable stimulation protocols, it is possible to preferentially generate CD33
NK cells combining efficient target cell killing and cytokine production, or alternatively CD33
NK cells, which produce less cytokines but are more efficient in antibody-dependent applications.
Activation of innate immune sensors by endogenous DNA and RNA can lead to autoimmune and autoinflammatory diseases. Quantification of the unperturbed phosphoprotein content in immune cells provides ...insight into the spontaneous activity of immune signaling pathways triggered by nucleic acid recognition. Here, we present a phosphoflow protocol for measuring phosphoproteins in mouse models of autoimmunity that incorporates strategies to preserve native phosphoprotein levels during sample collection and to reliably detect low signaling activity common in chronic disease states.
For complete details on the use and execution of this protocol, please refer to Jütte et al. (2021).
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•Phosphoflow protocol to quantify basal immune signaling activity•Strategies to preserve native phosphorylation levels and account for background signal•Reliable detection of small changes in phosphoprotein content in chronic diseases
Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Activation of innate immune sensors by endogenous DNA and RNA can lead to autoimmune and autoinflammatory diseases. Quantification of the unperturbed phosphoprotein content in immune cells provides insight into the spontaneous activity of immune signaling pathways triggered by nucleic acid recognition. Here, we present a phosphoflow protocol for measuring phosphoproteins in mouse models of autoimmunity that incorporates strategies to preserve native phosphoprotein levels during sample collection and to reliably detect low signaling activity common in chronic disease states.
The majority of patients with recurrent or metastasized head and neck squamous cell carcinoma (HNSCC) do not benefit from immune checkpoint blockade (ICB) while several patients experience severe and ...persistent immune-mediated side effects. Therefore, predictive biomarkers are urgently needed to allow for a personalized treatment. In this study, we investigated DNA methylation of the immune checkpoint gene CTLA4 with regard to its predictive value.
We analyzed CTLA4 promoter methylation in tumors of HNSCC patients (N = 29) treated with ICB at the University Medical Center Bonn with regard to response to ICB and progression-free survival. We further analyzed a second cohort (N = 138) of patients that did not receive ICB with regard to CTLA4 promoter methylation, CTLA-4 protein expression, and immune cell infiltrates. Finally, we tested inducibility of CTLA-4 protein expression in HNSCC cells using the DNA methyltransferase inhibitor decitabine.
Lower CTLA4 promoter methylation correlated with response to ICB and prolonged progression-free survival. We could show that not only tumor infiltrating immune cells, but also HNSCC cells harbor cytoplasmic and nuclear CTLA-4 expression. CTLA4 promoter methylation inversely correlated with infiltrates of CD3
, CD4
, CD8
, and CD45
immune cells. CTLA4 methylation did not correlate with protein expression in tumors, however, decitabine treatment led to decreased CTLA4 methylation and an induction of CTLA4 mRNA and CTLA-4 protein expression in HNSCC cell lines.
Our results indicate that CTLA4 DNA hypomethylation is a predictive biomarker for response to ICB in HNSCC. Our study warrants further analyses of the predictive value of CTLA4 DNA methylation in clinical trials of anti-PD-1 and/or anti-CTLA-4 immunotherapy in HNSCC.
Abstract
Objectives
Reliable predictive biomarkers for response to immune checkpoint inhibition (ICI) are lacking. Pretreatment serum albumin, a known prognostic and predictive factor in ICI‐treated ...patients, has been proposed as a potential pharmacokinetic surrogate marker for anti‐PD1/PD‐L1 antibodies, as it shares a homeostatic pathway with IgG. However, this hypothesis is currently based on theoretical considerations and limited evidence from retrospective data. Therefore, we comprehensively investigated the prognostic and predictive value of pretreatment albumin and its relationship with anti‐PD‐L1 IgG levels.
Methods
We analysed pretreatment albumin and atezolizumab serum levels and clinical response in four trials (IMvigor210, IMvigor211, IMmotion151 and OAK) of patients with metastatic lung‐, renal‐ or urothelial cancer who received atezolizumab alone or in combination.
Results
A total of 3391 patients were analysed. Correlation between serum albumin and atezolizumab levels was weak (Pearson's coefficient 0.23). We found a strong prognostic value for pretreatment serum albumin across all trials. Both atezolizumab serum levels and serum albumin were independently correlated with overall survival. Importantly, in the three randomised phase III clinical trials, the survival benefit for immunotherapy compared with the active comparator arm was limited to patients with pretreatment serum albumin > 35 g L
−1
.
Conclusion
Our data do not support the hypothesis that albumin serves as a surrogate for atezolizumab pharmacokinetics. However, we show that albumin on its own exerts strong prognostic value for patients treated with immunotherapy. As benefit from immunotherapy was limited to patients with normal/elevated serum albumin levels, baseline albumin could potentially be used as a predictive marker for immune checkpoint inhibition.
Immigration has taken the central stage in world politics, especially in the developed countries like Germany, where the continuous flow of immigrants has been well documented since 1960s. ...Strikingly, emerging data suggest that migrant patients have a poorer response to the treatment and lower survival rates in their new host country, raising concerns about health disparities. Herein, we present our investigation on the treatment response rate and cancer survival in German patients with and without an immigrant background that were treated at our comprehensive cancer center in Germany.
Initially, we considered 8162 cancer patients treated at the Center for Integrated Oncology (CIO), University Hospital Bonn, Germany (April 2002-December 2015) for matched-pair analysis. Subsequently, the German patients with a migration background and those from the native German population were manually identified and catalogued using a highly specific name-based algorithm. The clinical parameters such as demographic characteristics, tumor characteristics, defined staging criteria, and primary therapy were further adjusted. Using these stringent criteria, a total of 422 patients (n = 211, Germans with migration background; n = 211, native German population) were screened to compare for the treatment response and survival rates (i.e., 5-year overall survival, progression-free survival, and time to progression).
Compared to the cohort with migration background, the cohort without migration background was slightly older (54.9 vs. 57.9 years) while having the same sex distribution (54.5% vs. 55.0% female) and longer follow-up time (36.9 vs. 42.6 months). We did not find significant differences in cancer survival (5-year overall survival, P = 0.771) and the response rates (Overall Remission Rate; McNemar's test, P = 0.346) between both collectives.
Contrary to prior reports, we found no significant differences in cancer survival between German patients with immigrant background and native German patients. Nevertheless, the advanced treatment protocols implemented at our comprehensive cancer center may possibly account for the low variance in outcome. To conduct similar studies with a broader perspective, we propose that certain risk factors (country-of-origin-specific infections, dietary habits, epigenetics for chronic diseases etc.) should be considered, specially in the future studies that will recruit new arrivals from the 2015 German refugee crisis.
Prognosis of patients with irresectable cholangiocarcinoma is still poor. The ABC-02 trial established the current first line (1L) standard systemic chemotherapy (CT) with gemcitabine/platinum ...derivate for advanced cholangiocarcinoma. However, the majority of patients needed therapy adaptions. Thus, the aim of this study was to evaluate 1L and second line (2L) therapy regimens and the impact of therapy adaptions in an unselected real-life cohort of patients with advanced cholangiocarcinoma.
This is a single institution retrospective analysis of patients with irresectable cholangiocarcinoma who were treated with gemcitabine/platinum derivate from 2010 to 2018. Overall survival (OS), progression-free survival (PFS) and toxicity were analyzed for all patients, especially with regard to CT de-escalation.
Fifty-eight patients receiving gemcitabine/platinum derivate were included in the analysis. Median OS and PFS were 12.2 and 6.9 months. Interestingly, 41 patients (71%) needed therapy de-escalation. However, despite reduced CT exposition, there was no-significant difference in OS (10.8 months vs. 15.6 months, p = 0.127), and patients suffered from less adverse events during CT. 21 (36%) patients reached 2L CT, most often with FOLFIRI (57%). Survival beyond the end of 1L CT was 7.1 months with 2L CT vs. 2.9 months with BSC.
In our study, the combination of gemcitabine/platinum derivate showed similar OS and PFS as randomized prospective phase II/III trials. Therapy regimen adaptions were needed in the majority of patients. However, individualized modifications of the therapy regimen allowed better tolerance as well as continuation of therapy and did not significantly influence median OS. Furthermore, our study revealed a potential survival benefit with 2L CT for selected patients.
Total body irradiation (TBI) remains an important component in many conditioning regimens before allogeneic hematopoietic stem cell transplantation (allo‐HSCT). Because of its frequent toxicity, ...patient selection is crucial, making it of interest to identify factors improving engraftment. In this retrospective single center analysis, the characteristics of 48 adult such patients were studied. Mean overall survival (OS) was 22.2 months after allo‐HSCT. Interestingly, people with an interval ≥3 days between TBI completion and allo‐HSCT showed improved OS, when compared to a shorter interval (p = 0.10). Peripheral blood kinetics after successful engraftment also differed, with a longer interval resulting in a higher platelet count and lower leukocyte and neutrophil (p < 0.05) count. These data suggest that the exact timing of TBI before allo‐HSCT might directly impact a patient's survival and could help single out those at higher risk of graft failure who might benefit from an altered conditioning regimen.
Patients with longer interval between TBI completion and allo‐HSCT showed improved overall survival, when compared to a shorter interval. Exact timing of TBI before allo‐HSCT might directly impact a patient's survival and could help single out those at higher risk of graft failure who might benefit from an altered conditioning regimen.
Most of the patients with head and neck squamous cell carcinoma (HNSCC) are diagnosed with locally advanced disease. Standards of care for curative-intent treatment of this patient group are either ...surgery and adjuvant radio(chemo)therapy (aRCT) or definitive chemoradiation. Despite these treatments, especially pathologically intermediate and high-risk HNSCC often recur. The ADRISK trial investigates in locally advanced HNSCC and intermediate and high risk after up-front surgery if the addition of pembrolizumab to aRCT with cisplatin improves event-free sur-vival compared to aRCT alone. ADRISK is a prospective, randomized controlled investiga-tor-initiated (IIT)-phase II multicenter trial within the German Interdisciplinary Study Group of German Cancer Society (IAG-KHT). Patients with primary resectable stage III and IV HNSCC of the oral cavity, oropharynx, hypopharynx and larynx with pathologic high (R1, extracapsular nodal extension) or intermediate risk (R0 <5 mm; N≥2) after surgery will be eligible. Two hun-dred forty patients will be randomly assigned (1:1) to either standard aRCT with cisplatin (standard arm) or aRCT with cisplatin + pembrolizumab (200 mg iv, in 3-week cycle, max. 12 months) (interventional arm). Endpoints are event-free and overall survival. Recruitment started in August 2018 and is ongoing.
Abstract
Background
Management of psoriasis patients with arthralgia suffering from suspected psoriatic arthritis (PsA) requires an interdisciplinary approach involving dermatologists and ...rheumatologists. The aim of the study was to analyze the specialized dermatological-rheumatological management of these patients before and after foundation of a PsA center.
Methods
A retrospective cohort study of all dermatological-rheumatological consultations during two periods was conducted. Period one, from April 1st, 2016 to February 28th, 2018 versus period two, from March 1st, 2018 to January 31st, 2020, after foundation of a PsA center. Clinical data on patient characteristics including psoriasis subtypes, clinical symptoms and signs, disease activity scores, classification criteria and comorbidities as well as patient journey were extracted and analyzed.
Results
Four hundred four consultations were studied. Close collaboration in a PsA center lead to a relevantly shortened patient journey concerning rheumatological complaints: period 1: median (IQR): 36.0 (10.0–126.0) months, period 2: median (IQR): 24.0 (6.0–60.0) months. Established scores and classification criteria such as GEPARD or CASPAR did not assist in diagnosis of PsA. Arthralgia (
p
= 0.0407), swollen joints (
p
= 0.0151), morning stiffness (
p
= 0.0451) and dactylitis (
p
= 0.0086) helped to distinguish between osteoarthritis and PsA.
Conclusions
Clinical signs and symptoms, scores and classification criteria usually assessed were less helpful than expected in diagnosis of PsA. Close collaboration in a specialized PsA center yielded the fastest way of diagnosis.
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