Background: Cancer immunotherapy via immune-checkpoint inhibition (ICI) by antibodies against cytotoxic Tlymphocyte-associated protein 4 (CTLA-4) and cell death protein 1 (PD-1) have significantly improved the outcome of metastasized melanoma and of a rapidly increasing number of other cancer types. The anti-tumor effect is often accompanied by immune-related adverse events (irAE). Hematological irAE, specifically neutropenia, are rarely observed. However, neutropenia is associated with high morbidity and mortality due to infection complications. Thus, early detection and treatment is crucial. Methods: We present the clinical course of two patients with severe neutropenia after ICI therapy and demonstrate the difficulty of the diagnosis when a comedication of metamizole, a well-known analgesic drug used to treat cancer pain, is present. Further, we provide a comprehensive descriptive and statistical analysis of published data on diagnostics, treatment and infection complication in patients with at least grade 4 neutropenia by a systematic database search. Results: Finally, 34 patients were analyzed, including the two case reports from our cohort. The median onset of neutropenia was 10.5 weeks after first ICI administration (interquartile range: 6 weeks). In 76% (N = 26), a normalization of the neutrophil count was achieved after a median duration of neutropenia of 13 days. In a subsample of 22 patients with detailed data, the infection rate was 13%, proven by positive blood culture in 3 cases, but 68% (N = 15) presented with fever > 38°C. Treatment regime differed relevantly, but mainly included G-CSF and intravenous corticosteroids. Death was reported in 14 patients (41%), 3 of whom (9%) were associated with hematological irAE but only two directly associated with neutropenia. Conclusion: With an increasing number of cancer patients eligible to ICI therapy, the incidence of severe hematological toxicities may rise substantially over the next years. Clinicians working in the field of cancer immune therapies should be aware of neutropenia as irAE to provide immediate treatment.

Antibody-based immunotherapy represents a promising strategy to specifically target and eliminate chemoresistant leukemic cells in acute myeloid leukemia (AML). We evaluated a single-chain bispecific CD33/CD3 BiTE® antibody (AMG 330) for its suitability as immunotherapy in AML. A prerequisite for successful immunotherapeutic approaches using this molecule is the expression of CD33 on AML blasts including leukemic stem cells (LSCs). Therefore, we quantified CD33 expression on AML blasts and LSCs by flow cytometry (mean fluorescence intensity, MFI) and correlated expression intensity with cytogenetic and molecular disease characteristics in order to identify patient cohorts possibly most suited for CD33-targeted therapies. CD33 expression was detected in >99% of patient samples (n=621, MFI ratio ≥ 1.5) although highly variable. A strong correlation between high CD33 expression levels and NPM1 mutations (p<0.001) was found. In contrast, low CD33 expression levels were significantly associated with complex karyotypes and t(8,21) translocations (p<0.001). Furthermore, LSCs within the CD34+/CD38- compartment displayed CD33 at higher levels than healthy donor stem cells (p=0.047). Importantly, colony formation of CD34+/Lin-cells from healthy donors was not affected after pre-incubation with AMG 330 and T-cells. A major hurdle for measuring cytotoxic effects on AML blasts has long been that primary AML patient samples show progressive cell death within a few days ex-vivo. To simulate the natural setting of target and T-cells in AML patients, we developed a long-term culture system for AML blasts that allowed us to observe these co-cultures for up to 5 weeks. Thus, we were able to show effective elimination of AML blasts within primary samples by AMG 330-activated and expanded residual CD3+/CD45RA-/CCR7+ memory T-lymphocytes. While the functional relevance of CD33 expression levels was shown by faster lysis kinetics of CD33BRIGHT vs. CD33DIM AML cell lines in an in-vitro cytotoxicity assay potent anti-leukemic activity on primary AML blasts was observed irrespective of CD33 expression level. At low effector to target ratios (up to 1:79), the recruited T-cells lysed autologous blasts completely in the majority of samples. Further T-cell analysis showed that naive T-cells (CD45RA+/CCR7+) were not expanded by AMG 330; neither were terminally differentiated T-cells (CD45RA+/CCR7-), probably due to their poor proliferative capacity. We did not observe an increase in percentage of CD3+/CD4+/CD25+/FoxP3+regulatory T-cells in the presence of AMG 330, suggesting that these cells may not have impacted AMG 330-mediated T-cell activity in our experiments. Compared to control cultures, T-cells were shown to up-regulate the activation markers CD25, PD-1, TIM3 and LAG3 upon response to AMG 330, which was partially reversible after complete target cell elimination. However, PD-1 up-regulation did not correlate with an up-regulation of PD-L1 on AML blasts despite substantial INFγ secretion by activated T-cells. This study provides the first correlation of CD33 expression levels to a comprehensive genotype analysis in adult AML patients. While CD33 expression may vary by AML biologic subgroup, AMG 330 exposure led to lysis of AML blasts even in samples with low levels of expression. Targeting CD33 using AMG 330 in primary AML samples led to efficient T-cell activation and expansion as expected from the mechanism of action of BiTE® antibodies. The remarkable ex-vivo activity of AMG 330 supports further development of AMG 330 as an immunotherapy for patients with AML. Disclosures: Kufer: AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Kischel:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Zugmaier:Amgen Research (Munich) GmbH: Employment; Amgen Inc.: Equity Ownership. Baeuerle:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Riethmüller:AMGEN Inc.: Equity Ownership.

No abstract

No abstract

Background: Comparison of a blood or tissue sample with a matched germline control is a key step for accurate detection of somatic mutations. This is particularly important for tracking minimal residual disease (MRD), since alterations erroneously considered to be tumor derived may lead to false detection. Use of blood leukocytes as a germline control is critical not only for appropriate censoring of constitutional allelic variants, but also for addressing those associated with clonal hematopoiesis (CH). However, such matched germline is not always available, or may be suboptimal when contaminated with tumor cells. We hypothesized that accurate tumor genotyping might be feasible without a matched germline, using a dedicated algorithmic framework relying on clonal relationships of alleles of interest (i.e., phylons). Methods: Here, we introduce RePhyNER (Recursive Phylon Nomination, Enumeration, and Recovery), an algorithm for addressing this challenge in serial liquid biopsies. RePhyNER relies on the assumption that when comparing two specimens from the same individual (e.g., before and after therapy), the genomic variants of interests (e.g., tumor somatic mutations) behave differently in their allelic levels when compared to variants needing to be censored (e.g., germline constitutional alleles or CH). Using the change in mean allelic level of all variants in ≥2 samples, RePhyNER relies on a recursive approach to test each candidate somatic variant’s corresponding change gradient against this distribution using count-based statistics. Results: We used simulations to assess the importance of multiple parameters including fraction of contaminating variants or alleles and mean allelic fold-change. These simulations showed substantially superior performance of a recursive approach and revealed that for ≥2-fold changes in the mean VAF in a pair of samples, RePhyNER accurately removed &gt;99% of contaminating alleles while preserving ~100% of true variants. We then applied RePhyNER to 108 plasma cell-free DNA (cfDNA) samples from 47 patients with classic Hodgkin Lymphoma (cHL) profiled by PhasED-Seq and compared MRD-detection performance with or without matched germline. RePhyNER substantially improved specificity by ~30% (68% vs 99%), and Precision by ~50% (46% vs 96%), while only modestly reducing sensitivity (100% vs 91%). We next applied PhasED-Seq to a cohort of cHL patients (n=65) without matched germline information. MRD positivity within the first 2 cycles of therapy was associated with inferior outcome when using RePhyNER (P&lt;0.05 vs ns), correctly reclassifying 26% of detected patients to undetected. Conclusions: RePhyNER enables germline-free and accurate genotyping for MRD detection. Notably, RePhyNER obviates the need for additional germline profiling overcoming key limitations described above and avoids the additional associated costs of sequencing. Citation Format: Mohammad Shahrokh Esfahani, Stefan Alig, Emily Hamilton, Joseph Schroers-Martin, Brian Sworder, Jan Boegeholz, Mari Olsen, Chih Long Liu, David Kurtz, Maximilian Diehn, Ash Alizadeh. RePhyNER: Overcoming limitations of access to matched germline for serial liquid biopsy applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1045.