Prognostic Implications of Cellular Senescence in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease for which biologically grounded predictors of outcome remain a clinical need. In addition to tumor cell death, antineoplastic drugs can mediate a long-lasting growth arrest of vital, metabolically active tumor cells termed therapy-induced senescence, but structured investigations into its prognostic and predictive power are lacking. Besides its occurrence in response to chemotherapeutic treatment, cellular senescence can be evoked by replicative stress or activation of oncogenes and serves as an initial barrier to cancer development. Yet, long-term effects of senescent cancer cells on tumor growth are unclear as they are known to mediate inflammation via a senescence-associated secretory phenotype (SASP) and are subjected to epigenetic remodeling, thereby acquiring cancer stemness characteristics. In an ex vivo analysis of AML blast samples from patients at diagnosis, I aimed to characterize basal as well as treatment-evoked senescence and determine its role as a prognostic and predictive biomarker. I established assays to detect and therapeutically induce senescence in a primary AML culture setting. Senescence was assessed by senescence-associated β-galactosidase (SA-β-gal) activity and other senescence markers. Gene expression analyses validated my experimental characterization of AML samples as “senescent”, as evidenced by upregulation of senescence-associated gene expression signatures. For prognostic analysis, clinical outcomes and molecular genetics of AML sample donors were retrieved. I found the intra-individual changes of senescence levels in response to the standard anti-leukemic agent daunorubicin to be positively correlated with better disease-free- and overall patient survival. In line with this, a more favorable molecular risk group, normal karyotype, and NPM1 as well as DNMT3AR882 mutations were associated with higher therapy-induced senescence levels. Other therapeutic AML agents, namely hydroxyurea, decitabine and gemtuzumab ozogamicin were also shown to induce senescence. Finally, in a consecutive ex vivo treatment with daunorubicin (to induce senescence), followed by the “senolytic” (i.e., selectively cytotoxic to senescent cells) BCL2 inhibitors venetoclax and navitoclax, both growth and viability of AML blasts were additionally reduced compared to single-agent treatments only in senescence-capable samples. To the best of my knowledge, this is the first study providing direct evidence that cellular senescence, induced ex vivo in patient-derived AML blasts by chemotherapeutic drugs, could serve as a predictive biomarker of long-term response to standard therapy. I believe that therapy-induced senescence might explain, at least in part, the underlying biology of current paraclinical risk indicators, and, as an outlook, might serve as a guidance for future personalized treatment of AML.Die akute myeloische Leukämie (AML) ist eine heterogene Erkrankung, für die die Entwicklung neuer pathophysiologisch fundierter prädiktiver Biomarker von großer klinischer Notwendigkeit ist. Zusätzlich zu apoptotischem Zelltod von Tumorzellen können antineoplastische Medikamente zu einem dauerhaften Zellzyklusarrest viabler, metabolisch aktiver Tumorzellen führen, welches Phänomen als Therapie-induzierte Seneszenz in verschiedenen Tumorentitäten charakterisiert wurde. Die prognostische und prädiktive Relevanz Therapie-induzierter Seneszenz für den Verlauf von Tumorerkrankungen ist derzeit unklar. Außer einer Induktion durch Chemotherapeutika kann Seneszenz u.a. durch replikativen Stress oder Onkogen-Expression hervorgerufen werden und dient dadurch als initiale zelluläre Barriere gegen maligne Entartung. Die langfristige Bedeutung von im Organismus persistierenden seneszenten Tumorzellen bleibt jedoch unklar, da diese durch ihren Seneszenz-assoziierten sekretorischen Phänotyp (SASP) auch proinflammatorisch wirken und durch epigenetische Veränderungen Krebsstammzelleigenschaften aufweisen können. In ex vivo-Untersuchungen an aus Patient:innenproben zum Zeitpunkt der Diagnosestellung gewonnenen AML-Blasten konnte ich zunächst „basale“ und Therapie-bedingte Seneszenz in der AML charakterisieren um daraufhin Seneszenz als prädiktiven Biomarker zu analysieren. Nach Etablierung von Primärkulturbedingungen für die zytostatische Behandlung (und somit mögliche Seneszenzinduktion) aufgereinigter AML-Blasten konnte ich mit zytochemischen und Fluoreszenz-basierten Assays die Zunahme der Seneszenz-assoziierten-β-Galaktosidase (SA-β-gal)-Aktivität und anderer Seneszenzmarker nachweisen. Durch RNA-Sequenzierung konnte meine experimentelle Klassifikation individueller AML-Proben als „Seneszenz-fähig“ anhand Seneszenz-assoziierter Genexpressionssignaturen bestätigt und weiter charakterisiert werden. Zur Analyse der prädiktiven Bedeutung Therapie-bedingter Seneszenz wurden die einzelnen AML Proben weiter molekulargenetisch untersucht und experimentelle Ergebnisse mit dem jeweiligen klinischen Verlauf individueller Patient:innen korreliert. Ich konnte zeigen, dass die intraindividuelle Induzierbarkeit von Seneszenz durch ex vivo-Behandlung mit dem AML-Standardchemotherapeutikum Daunorubicin positiv mit einem verbesserten erkrankungsfreien Überleben und Gesamtüberleben korrelierte. Zudem waren eine günstigere molekulare Risikogruppe, ein normaler Karyotyp sowie NPM1- und DNMT3AR882-Mutationen mit höheren Leveln Therapie-induzierter Seneszenz assoziiert. Durch die Behandlung mit anderen AML-Therapeutika wie Hydroxyurea, Decitabin oder Gemtuzumab-Ozogamicin konnte ebenfalls Seneszenz ausgelöst werden. Schließlich konnten ich durch eine konsekutive ex vivo-Behandlung mit zunächst Daunorubicin (zur Seneszenzinduktion) und darauffolgend mit den „senolytisch“ wirkenden (d.h. selektiv zytotoxisch gegenüber seneszenten Zellen) BCL2-Inhibitoren Venetoclax und Navitoclax sowohl Zellzahl als auch Viabilität seneszenzfähiger AML-Proben im Vergleich zu einer Therapie mit den Einzelsubstanzen oder zu AML-Proben, welche nicht seneszenzfähig waren, zusätzlich reduzieren. Nach meinem Kenntnisstand konnte im Rahmen dieses Promotionsprojektes erstmals nachgewiesen werden, dass durch ex vivo-Chemotherapie in aus Patient:innen gewonnenen AML-Blasten induzierte zelluläre Seneszenz als prädiktiver Biomarker für das langzeitige Therapieansprechen auf die Standard-Induktionstherapie dienen kann. Möglicherweise erklärt Therapie-induzierte Seneszenz Teilaspekte der etablierten paraklinischen Risikofaktoren zugrundliegenden Tumorbiologie und kann perspektivisch als Marker für personalisierte Behandlungskonzepte in der AML verwendet werden

Nirmatrelvir/ritonavir in COVID-19 patients with haematological malignancies:a report from the EPICOVIDEHA registry

Background: Nirmatrelvir/ritonavir treatment decreases the hospitalisation rate in immunocompetent patients with COVID-19, but data on efficacy in patients with haematological malignancy are scarce. Here, we describe the outcome of nirmatrelvir/ritonavir treatment in a large cohort of the latter patients. Methods: This is a retrospective cohort study from the multicentre EPICOVIDEHA registry (NCT04733729) on patients with haematological malignancy, who were diagnosed with COVID-19 between January and September 2022. Patients receiving nirmatrelvir/ritonavir were compared to those who did not. A logistic regression was run to determine factors associated with nirmatrelvir/ritonavir administration in our sample. Mortality between treatment groups was assessed with Kaplan–Meier survival plots after matching all the patients with a propensity score. Additionally, a Cox regression was modelled to detect factors associated with mortality in patients receiving nirmatrelvir/ritonavir. Findings: A total of 1859 patients were analysed, 117 (6%) were treated with nirmatrelvir/ritonavir, 1742 (94%) were treated otherwise. Of 117 patients receiving nirmatrelvir/ritonavir, 80% had received ≥1 anti-SARS-CoV-2 vaccine dose before COVID-19 onset, 13% of which received a 2nd vaccine booster. 5% were admitted to ICU. Nirmatrelvir/ritonavir treatment was associated with the presence of extrapulmonary symptoms at COVID-19 onset, for example anosmia, fever, rhinitis, or sinusitis (aOR 2.509, 95%CI 1.448–4.347) and 2nd vaccine booster (aOR 3.624, 95%CI 1.619–8.109). Chronic pulmonary disease (aOR 0.261, 95%CI 0.093–0.732) and obesity (aOR 0.105, 95%CI 0.014–0.776) were not associated with nirmatrelvir/ritonavir use. After propensity score matching, day-30 mortality rate in patients treated with nirmatrelvir/ritonavir was 2%, significantly lower than in patients with SARS-CoV-2 directed treatment other than nirmatrelvir/ritonavir (11%, p = 0.036). No factor was observed explaining the mortality difference in patients after nirmatrelvir/ritonavir administration. Interpretation: Haematological malignancy patients were more likely to receive nirmatrelvir/ritonavir when reporting extrapulmonary symptoms or 2nd vaccine booster at COVID-19 onset, as opposed to chronic pulmonary disease and obesity. The mortality rate in patients treated with nirmatrelvir/ritonavir was lower than in patients with targeted drugs other than nirmatrelvir/ritonavir. Funding: EPICOVIDEHA has received funds from Optics COMMIT (COVID-19 Unmet Medical Needs and Associated Research Extension) COVID-19 RFP program by GILEAD Science, United States (Project 2020-8223).</p

MOLNUPIRAVIR COMPARED TO NIRMATRELVIR/RITONAVIR FOR COVID-19 IN HIGH-RISK PATIENTS WITH HAEMATOLOGICAL MALIGNANCY IN EUROPE. A MATCHED-PAIRED ANALYSIS FROM THE EPICOVIDEHA REGISTRY

Introduction: Molnupiravir and nirmatrelvir/ritonavir are antivirals used to prevent progression to severe SARS-CoV-2 infections, which reduce both hospitalization and mortality rates. Nirmatrelvir/ritonavir was authorised in Europe in December 2021, while molnupiravir is not yet licensed in Europe as of February 2022. Molnupiravir may be an alternative to nirmatrelvir/ritonavir, because it displays less frequent drug-drug interactions and contraindications. A caveat connected to molnupiravir derives from the mode of action inducing viral mutations. In clinical trials on patients without haematological malignancy, mortality rate reduction of molnupiravir appeared less pronounced than that of nirmatrelvir/ritonavir. Little is known about the comparative efficacy of the two drugs in patients with haematological malignancy at high-risk of severe COVID-19. Thus, we here assess the effectiveness of molnupiravir compared to nirmatrelvir/ritonavir in our cohort of patients with haematological malignancies. Methods: Clinical data of patients treated either with molnupiravir or nirmatrelvir/ritonavir monotherapy for COVID-19 were retrieved from the EPICOVIDEHA registry. Patients treated with molnupiravir were matched by sex, age (±10 years), and baseline haematological malignancy severity to controls treated with nirmatrelvir/ritonavir. Results: A total of 116 patients receiving molnupiravir for the clinical management of COVID-19 were matched to an equal number of controls receiving nirmatrelvir/ritonavir. In each of the groups, 68 (59%) patients were male; with a median age of 64 years (IQR 53-74) for molnupiravir recipients and 64 years (IQR 54-73) for nirmatrelvir/ritonavir recipients; 57% (n=66) of the patients had controlled baseline haematological malignancy, 13% (n=15) stable, and 30% (n=35) had active disease at COVID-19 onset in each of the groups. During COVID-19 infection, one third of patients from each group were admitted to hospital. Although a similar proportion of vaccinated patients was observed in both groups (molnupiravir n=77, 66% vs nirmatrelvir/ritonavir n=87, 75%), those treated with nirmatrelvir/ritonavir had more often received four doses (n=27, 23%) as compared to patients treated with molnupiravir (n=5, 4%, p&lt;0.001). No differences were detected in COVID-19 severity (p=0.39) or hospitalization (p=1.0). No statistically significant differences were identified in overall mortality rate (p=0.78) or in survival probability (d30 p=0.19, d60 p=0.67, d90 p=0.68, last day of follow up p=0.68). In all patients, deaths were either attributed to COVID-19 or the infection contributed to death as per treating physician's judgement. Conclusions: In high-risk patients with haematological malignancies and COVID-19, molnupiravir showed rates of hospitalization and mortality comparable to those of nirmatrelvir/ritonavir in this matched-pair analysis. Molnupiravir appears to be a plausible alternative to nirmatrelvir/ritonavir for COVID-19 treatment in patients with haematological malignancy

Nirmatrelvir/ritonavir in COVID-19 patients with haematological malignancies: a report from the EPICOVIDEHA registry

Background: Nirmatrelvir/ritonavir treatment decreases the hospitalisation rate in immunocompetent patients with COVID-19, but data on efficacy in patients with haematological malignancy are scarce. Here, we describe the outcome of nirmatrelvir/ritonavir treatment in a large cohort of the latter patients. Methods: This is a retrospective cohort study from the multicentre EPICOVIDEHA registry (NCT04733729) on patients with haematological malignancy, who were diagnosed with COVID-19 between January and September 2022. Patients receiving nirmatrelvir/ritonavir were compared to those who did not. A logistic regression was run to determine factors associated with nirmatrelvir/ritonavir administration in our sample. Mortality between treatment groups was assessed with Kaplan-Meier survival plots after matching all the patients with a propensity score. Additionally, a Cox regression was modelled to detect factors associated with mortality in patients receiving nirmatrelvir/ritonavir. Findings: A total of 1859 patients were analysed, 117 (6%) were treated with nirmatrelvir/ritonavir, 1742 (94%) were treated otherwise. Of 117 patients receiving nirmatrelvir/ritonavir, 80% had received ≥1 anti-SARS-CoV-2 vaccine dose before COVID-19 onset, 13% of which received a 2nd vaccine booster. 5% were admitted to ICU. Nirmatrelvir/ritonavir treatment was associated with the presence of extrapulmonary symptoms at COVID-19 onset, for example anosmia, fever, rhinitis, or sinusitis (aOR 2.509, 95%CI 1.448-4.347) and 2nd vaccine booster (aOR 3.624, 95%CI 1.619-8.109). Chronic pulmonary disease (aOR 0.261, 95%CI 0.093-0.732) and obesity (aOR 0.105, 95%CI 0.014-0.776) were not associated with nirmatrelvir/ritonavir use. After propensity score matching, day-30 mortality rate in patients treated with nirmatrelvir/ritonavir was 2%, significantly lower than in patients with SARS-CoV-2 directed treatment other than nirmatrelvir/ritonavir (11%, p&nbsp;=&nbsp;0.036). No factor was observed explaining the mortality difference in patients after nirmatrelvir/ritonavir administration. Interpretation: Haematological malignancy patients were more likely to receive nirmatrelvir/ritonavir when reporting extrapulmonary symptoms or 2nd vaccine booster at COVID-19 onset, as opposed to chronic pulmonary disease and obesity. The mortality rate in patients treated with nirmatrelvir/ritonavir was lower than in patients with targeted drugs other than nirmatrelvir/ritonavir. Funding: EPICOVIDEHA has received funds from Optics COMMIT (COVID-19 Unmet Medical Needs and Associated Research Extension) COVID-19 RFP program by GILEAD Science, United States (Project 2020-8223)

Age, Successive Waves, Immunization, and Mortality in Elderly COVID-19 Haematological Patients: EPICOVIDEHA Findings

Introduction: elderly patients with haematologic malignancies face the highest risk of severe COVID-19 outcomes. The infection impact in different age groups remains unstudied in detail. Methods: We analysed elderly patients (age groups: 65-70, 71-75, 76-80 and &gt;80 years old) with hematologic malignancies included in the EPICOVIDEHA registry between January 2020 and July 2022. Univariable and multivariable Cox regression models were conducted to identify factors influencing death in COVID-19 patients with haematological malignancy. results: the study included data from 3,603 elderly patients (aged 65 or older) with haematological malignancy, with a majority being male (58.1%) and a significant proportion having comorbidities. The patients were divided into four age groups, and the analysis assessed COVID-19 outcomes, vaccination status, and other variables in relation to age and pandemic waves.tThe 90-day survival rate for patients with COVID-19 was 71.2%, with significant differences between groups. The pandemic waves had varying impacts, with the first wave affecting patients over 80 years old, the second being more severe in 65-70, and the third being the least severe in all age groups. factors contributing to 90-day mortality included age, comorbidities, lymphopenia, active malignancy, acute leukaemia, less than three vaccine doses, severe COVID-19, and using only corticosteroids as treatment. Conclusions: These data underscore the heterogeneity of elderly haematological patients, highlight the different impact of COVID waves and the pivotal importance of vaccination, and may help in planning future healthcare efforts

Lymphocytopenia and Anti-CD38 Directed Treatment Impact the Serological SARS-CoV-2 Response after Prime Boost Vaccination in Patients with Multiple Myeloma

Even though several SARS-CoV-2 vaccines have shown high effectiveness in the prevention of COVID-19 in healthy subjects, vaccination response in patients with plasma-cell-related disorders (PCD) remains widely unknown. Here, we report on an analysis describing the serological response after prime-boost SARS-CoV-2 vaccination in PCD patients, as compared to a healthy control group, and on possible influencing factors of serological responses. Blood samples were analyzed for the presence of quantitative anti-SARS-CoV-2 spike RBD Ig. A total of 82 patients were included; 67 received mRNA-, eight vector-based and four heterologous vaccinations. SARS-CoV-2 antibody titers (SP-AbT) were assessed in a mean of 23 days (SD ± 11 days) after the first and in a mean 21 days (SD ± 9) after prime-boost vaccination. A positive SP-AbT was detected in 31.9% of PCD patients after the first vaccination, and in 88.9% (44/49) after prime-boost vaccination, which was significantly less likely than that in the control group (100%, 78/78) (p = 0.008). Furthermore, we have been able to validate our previously suggested threshold of 30 CD19+ B lymphocytes/µL as being predictive for SP-AbT development. Despite anti-CD38 directed therapy, quadruplet treatment, higher age and missing deep remission, which correlated negatively with SP-AbT appearance, SP-AbT formation is possible in a majority of myeloma patients after prime-boost vaccination

Peripheral and Portal Venous KRAS ctDNA Detection as Independent Prognostic Markers of Early Tumor Recurrence in Pancreatic Ductal Adenocarcinoma

BACKGROUND: KRAS circulating tumor DNA (ctDNA) has shown biomarker potential for pancreatic ductal adenocarcinoma (PDAC) but has not been applied in clinical routine yet. We aim to improve clinical applicability of ctDNA detection in PDAC and to study the impact of blood-draw site and time point on the detectability and prognostic role of KRAS mutations. METHODS: 221 blood samples from 108 PDAC patients (65 curative, 43 palliative) were analyzed. Baseline peripheral and tumor-draining portal venous (PV), postoperative, and follow-up blood were analyzed and correlated with prognosis. RESULTS: Significantly higher KRAS mutant detection rates and copy numbers were observed in palliative compared to curative patients baseline blood (58.1% vs 24.6%; P = 0.002; and P < 0.001). Significantly higher KRAS mutant copies were found in PV blood compared to baseline (P < 0.05) samples. KRAS detection in pre- and postoperative and PV blood were significantly associated with shorter recurrence-free survival (all P < 0.015) and identified as independent prognostic markers. KRAS ctDNA status was also an independent unfavorable prognostic factor for shorter overall survival in both palliative and curative cohorts (hazard ratio [HR] 4.9, P = 0.011; HR 6.9, P = 0.008). CONCLUSIONS: KRAS ctDNA detection is an independent adverse prognostic marker in curative and palliative PDAC patients-at all sites of blood draw and a strong follow-up marker. The most substantial prognostic impact was seen for PV blood, which could be an effective novel tool for identifying prognostic borderline patients-guiding future decision-making on neoadjuvant treatment despite anatomical resectability. In addition, higher PV mutant copy numbers contribute to an improved technical feasibility