Chimeric antigen receptor (CAR) T-cell therapy has dramatically shifted the landscape of treatment especially for Non-Hodgkin-Lymphoma (NHL). This study evaluates the role of fluorodeoxyglucose (FDG)-positron emission tomography/computed tomography (PET/CT) in NHL treated with CAR T-cell therapy concerning response assessment and prognosis.We evaluated 34 patients with NHL who received a CAR T-cell therapy between August 2019 and July 2022. All patients underwent a pre-therapeutic FDG-PET/CT (PET-0) 6 days prior and a post-therapeutic FDG-PET/CT (PET-1) 34 days after CAR T-cell therapy. Deauville score (DS) was used for evaluation of response to therapy and compared to a minimum follow-up of 5 months.19/34 (55.9%) patients achieved DS ≤ 3 on PET-1, the remaining 15 (44.1%) patients had DS > 3 on PET-1. 14/19 patients with DS ≤ 3 on PET-1 had no relapsed or refractory (r/r)-disease and were still alive at last follow-up. The other 5 patients had r/r-disease and 4 of these died. Except for two patients who had no r/r-disease, all other patients (13/15) with DS > 3 on PET-1 had r/r-disease and 12 of these subsequently died. Patients with DS ≤ 3 on PET-1 had significantly better progression free survival (PFS; HR: 5.7; p < 0.01) and overall survival (OS; HR: 5.0; p < 0.01) compared to patients with DS > 3 on PET-1. In addition, we demonstrated that patients with DS ≤ 4 on PET-0 tended to have longer PFS (HR: 3.6; p = 0.05).Early FDG-PET/CT using the established DS after CAR T-cell therapy is a powerful tool to evaluate response to therapy.

Inhibition of the proximal B cell receptor (BCR) signaling pathway by BTK inhibitors is highly effective in the treatment of CLL, but drug resistance or intolerance occurs. Here, we investigated c-Jun N-terminal protein kinase 1 (JNK1) as an alternative drug target in the distal BCR pathway. JNK1 was preferentially overexpressed and activated in poor prognostic CLL with unmutated IGHV. Proximal BCR inhibition (BTK, PI3K, or SYK inhibitors) or SYK knockdown efficiently dephosphorylated JNK1, identifying JNK1 as a critical BCR downstream kinase in CLL. JNK1 inhibition induced apoptosis in primary CLL cells, resulting in the downregulation of BCL2, MCL1, and c-JUN. JNK1 inhibition in patient-derived CLL xenografted mice and Eµ-TCL1-tg mice prevented CLL progression, reduced splenic infiltration, and restored T cell function and normal hematopoiesis. JNK1 inhibitors even remained effective in ibrutinib refractory CLL. In conclusion, our study revealed JNK1 as a promising drug target in CLL downstream of the BCR, overcoming ibrutinib resistance, blocking the protective microenvironment, and improving CLL-specific immunosuppressive mechanisms.

S1. KrasG12D recipient mice develop a myelodysplastic syndrome within the transferred WT hematopoietic system. S2. Gating strategy used for the isolation of bone marrow (BM) niche cells. S3. The frequency of BM niche cells is not affected in KrasG12D recipient mice. S4. Differential pathway analysis in MSPCs and endothelial cells of KrasG12D versus WT mice. S5. Differential pathways analysis in CaR and osteoblast cells of KrasG12D versus WT recipient mice. S6. Expression of phosphorylated H2AX (γ-H2AX) in BM of WT or KrasG12D recipient mice. S7. Analysis of patient-derived BM for KRAS mutations by digital droplet polymerase chain reaction (ddPCR).

Peripheral T-cell lymphomas (PTCL) - especially angioimmunoblastic (AITL) and follicular TCL - have a dismal prognosis due to lack of efficient therapies, and patients` symptoms are often dominated by an inflammatory phenotype including fever, night sweats, weight loss and skin rash. In this study, we investigated the role of inflammatory granulocytes and activated cytokine signaling on PTCL-TFH (T-follicular helper type) disease progression and symptoms. We show, that ITK-SYK driven murine PTCLs and primary human PTCL-TFH xenografts both induce inflammation in mice including murine neutrophil expansion and massive cytokine release. Granulocyte/lymphoma interactions were mediated by positive autoregulatory cytokine loops involving INF-γ (CD4+malignant T-cells) and IL-6 (activated granulocytes), ultimately inducing broad JAK kinase activation (Jak1/2/3, Tyk2) in both cell types. Depletion of inflammatory granulocytes via antibodies (Ly6G), genetic granulocyte depletion (LyzM-Cre/MCL1flox/flox) or the deletion of IL-6 within microenvironmental cells blocked inflammatory symptoms, reduced lymphoma infiltration and enhanced mouse survival. Furthermore, unselective JAK kinase inhibitors (ruxolitinib) inhibited both, TCL progression and granulocyte activation in various PTCL mouse models. Our results support the important role of granulocyte-driven inflammation, cytokine-induced granulocyte/CD4+ TCL interactions and the requirement of an intact JAK/STAT signaling pathway for PTCL-TFH development, and support broad JAK kinase inhibition as an effective treatment strategy in early disease stages.

PET/CT targeting the prostate-specific membrane antigen (PSMA) plays a key role in staging of patients with prostate cancer. Moreover, it is used not only for the assessment of adequate PSMA expression of prostate cancer cells before PSMA-targeting radioligand therapy (RLT) but also for restaging during the course of therapy to evaluate response to treatment. Whereas no established criteria exist for systematic response evaluation so far, recently proposed PSMA PET Progression (PPP) criteria might fill this gap. The aim of this study was to assess the feasibility of PPP criteria in patients undergoing PSMA RLT and their prognostic implications. Methods: In this retrospective analysis, PSMA PET/CT scans of 46 patients before and after completion of PSMA RLT were analyzed separately by 2 readers using modified PPP criteria. After interobserver agreement assessment, consensus results (progressive vs. nonprogressive disease) were compared in a multivariate Cox regression model (endpoint, overall survival [OS]). Results: Interobserver agreement using the modified PPP criteria was substantial (Cohen κ = 0.73), with a concordance in 87% of patients. The median OS of all patients after PSMA RLT (n = 46) was 9.0 mo (95% CI, 7.8-10.2 mo). Progression according to the modified PPP criteria was found in 32 patients and was a significant (P ≤ 0.001) prognostic marker for OS, with a hazard ratio of 15.5 (95% CI, 3.4-70.2). Conclusion: Response assessment in patients undergoing PSMA RLT using modified PPP criteria are reproducible and highly prognostic for OS. The modified PPP criteria should be validated in future prospective trials.

Abstract The bone marrow microenvironment (BMME) is key player in regulation and maintenance of hematopoiesis. Oncogenic RAS mutations, causing constitutive activation of multiple tumor-promoting pathways, are frequently found in human cancer. So far in hematologic malignancies, RAS mutations have only been reported to occur in hematopoietic cells. In this study, we investigated the effect of oncogenic Kras expression in the BMME in a chimeric mouse model. We observed that an activating mutation of Kras in the nonhematopoietic system leads to a phenotype resembling myelodysplastic syndrome (MDS) characterized by peripheral cytopenia, marked dysplasia within the myeloid lineage as well as impaired proliferation and differentiation capacity of hematopoietic stem and progenitor cells. The phenotypic changes could be reverted when the BM was re-isolated and transferred into healthy recipients, indicating that the KrasG12D-activation in the nonhematopoietic BMME was essential for the MDS phenotype. Gene expression analysis of sorted nonhematopoietic BM niche cells from KrasG12D mice revealed upregulation of multiple inflammation-related genes including IL1-superfamily members (Il1α, Il1β, Il1f9) and the NLPR3 inflammasome. Thus, pro-inflammatory IL1-signaling in the BMME may contribute to MDS development. Our findings show that a single genetic change in the nonhematopoietic BMME can cause an MDS phenotype. Oncogenic Kras activation leads to pro-inflammatory signaling in the BMME which impairs HSPCs function. Implications: These findings may help to identify new therapeutic targets for MDS.

Background: Anaplastic thyroid carcinoma (ATC) and metastatic poorly differentiated thyroid carcinomas (PDTCs) are rare aggressive malignancies with poor overall survival (OS) despite extensive multimodal therapy. These tumors are highly proliferative, with frequently increased tumor mutational burden (TMB) compared with differentiated thyroid carcinomas, and elevated programmed death ligand 1 (PD-L1) levels. These tumor properties implicate responsiveness to antiangiogenic and antiproliferative multikinase inhibitors such as lenvatinib, and immune checkpoint inhibitors such as pembrolizumab. Patients and Methods: In a retrospective study, we analyzed six patients with metastatic ATC and two patients with PDTC, who received a combination therapy of lenvatinib and pembrolizumab. Lenvatinib was started at 14-24 mg daily and combined with pembrolizumab at a fixed dose of 200 mg every three weeks. Maximum treatment duration with this combination was 40 months, and 3 of 6 ATC patients are still on therapy. Patient tumors were characterized by whole-exome sequencing and PD-L1 expression levels (tumor proportion score [TPS] 1-90%). Results: Best overall response (BOR) within ATCs was 66% complete remissions (4/6 CR), 16% stable disease (1/6 SD), and 16% progressive disease (1/6 PD). BOR within PDTCs was partial remission (PR 2/2). The median progression-free survival was 17.75 months for all patients, and 16.5 months for ATCs, with treatment durations ranging from 1 to 40 months (1, 4, 11, 15, 19, 25, 27, and 40 months). Grade III/IV toxicities developed in 4 of 8 patients, requiring dose reduction/discontinuation of lenvatinib. The median OS was 18.5 months, with three ATC patients being still alive without relapse (40, 27, and 19 months) despite metastatic disease at the time of treatment initiation (UICC and stage IVC). All patients with long-term (>2 years) or complete responses (CRs) had either increased TMB or a PD-L1 TPS >50%. Conclusions: Our results implicate that the combination of lenvatinib and pembrolizumab might be safe and effective in patients with ATC/PDTC and can result in complete and long-term remissions. The combination treatment is now being systematically examined in a phase II clinical trial (Anaplastic Thyroid Carcinoma Lenvatinib Pembrolizumab [ATLEP]) in ATC/PDTC patients.

Granulomas are immune cell aggregates formed in response to persistent inflammatory stimuli. Granuloma macrophage subsets are diverse and carry varying copy numbers of their genomic information. The molecular programs that control the differentiation of such macrophage populations in response to a chronic stimulus, though critical for disease outcome, have not been defined. Here, we delineate a macrophage differentiation pathway by which a persistent Toll-like receptor (TLR) 2 signal instructs polyploid macrophage fate by inducing replication stress and activating the DNA damage response. Polyploid granuloma-resident macrophages formed via modified cell divisions and mitotic defects and not, as previously thought, by cell-to-cell fusion. TLR2 signaling promoted macrophage polyploidy and suppressed genomic instability by regulating Myc and ATR. We propose that, in the presence of persistent inflammatory stimuli, pathways previously linked to oncogene-initiated carcinogenesis instruct a long-lived granuloma-resident macrophage differentiation program that regulates granulomatous tissue remodeling.

JAK2V617F(+) myeloproliferative neoplasms (MPNs) frequently progress into leukemias, but the factors driving this process are not understood. Here, we find excess Hedgehog (HH) ligand secretion and loss of PTCH2 in myeloproliferative disease, which drives canonical and noncanonical HH-signaling. Interestingly, Ptch2(-/-) mice mimic dual pathway activation and develop a MPN-phenotype with leukocytosis (neutrophils and monocytes), strong progenitor and LKS mobilization, splenomegaly, anemia, and loss of lymphoid lineages. HSCs exhibit increased cell cycling with improved stress hematopoiesis after 5-FU treatment, and this results in HSC exhaustion over time. Cytopenias, LKS loss, and mobilization are all caused by loss of Ptch2 in the niche, whereas hematopoietic loss of Ptch2 drives leukocytosis and promotes LKS maintenance and replating capacity in vitro. Ptch2(-/-) niche cells show hyperactive noncanonical HH signaling, resulting in reduced production of essential HSC regulators (Scf, Cxcl12, and Jag1) and depletion of osteoblasts. Interestingly, Ptch2 loss in either the niche or in hematopoietic cells dramatically accelerated human JAK2V617F-driven pathogenesis, causing transformation of nonlethal chronic MPNs into aggressive lethal leukemias with >30% blasts in the peripheral blood. Our findings suggest HH ligand inhibitors as possible drug candidates that act on hematopoiesis and the niche to prevent transformation of MPNs into leukemias.

Introduction: Novel substances such as the next generation IMiD pomalidomide or the recently approved next generation proteasome inhibitor carfilzomib (Cfz) have considerably expanded our treatment options in MM, both of them influencing multiple myeloma (MM) interaction with bone marrow stroma cells (BMSCs), that provides an interesting target for anti MM therapy. More compounds directed at this disease critical crosstalk are currently under investigation, however the development of novel drugs remains inefficient, displayed by a substantial drop out rate of the 376 preclinical single agents tested since 1961 (Rongvaux, Annu Rev Immunol. 2013; Schüler, Expert Opin Biol Ther. 2013; Kortüm, CLML 2014). Our focus in the projected presented here was to develop a novel bone-derived in vitro 3D co-culture model specifically adapted to mimic the BM niche to more closely study the role of bone and BM bystander cells and to perform more reliable ex vivo compound screening in MM.

Missense mutations are the most common F8 gene defects among the patients with non-severe haemophilia A. This type of mutation is typically associated with low (5%) inhibitor risk. In the present retrospective study we analysed the clinical data of 16 haemophiliacs with the T295A missense mutation treated at Bonn Haemophilia Centre. In total, three patients developed inhibitors: two patients experienced low-titer and one high-titer inhibitors. Both patients with low titer inhibitors underwent successful ITI. The third patient, at the age of 81, developed initially low-titer inhibitors (3 BU/ml) after rFVIII therapy because of knee surgery. He experienced spontaneous multiple large skin haematomas and haemarthrosis. Immunosuppressive therapy was not applicable because of the infectious origin of discitis (Th3-Th4). Immunoadsorption was performed, but the inhibitor titer increased up to 42 BU/ml nine weeks after termination. A successful treatment of discitis with antibiotics finally allowed a weekly therapy (four times) with rituximab (375 mg/m²). This resulted in a decrease of inhibitor titre to 0.7 BU/ml eight weeks after the fourth rituximab application. Patient had endogenous FVIII levels of 3–5%. Twelve months after rituximab therapy (after B cells recovery) he relapsed with low-titer inhibitors and therefore was treated with single rituximab dose (375 mg/m²) again. This resulted in his depletion of B cells, measurable endogenous FVIII levels and non measurable inhibitors. This study demonstrated T295A variant to be associated with significantly increased (3/16 patients, 17%) inhibitor development.