Abstract: Refractory/relapsed diffuse large B-cell lymphoma (DLBCL) is associated with poor outcome. The clinical behavior and genetic landscape of DLBCL is heterogeneous and still not fully understood. TP53 mutations in DLBCL have been identified as markers of poor prognosis and are often associated with therapeutic resistance. Chimeric antigen receptor T-cell therapy is an innovative therapeutic concept and represents a game-changing therapeutic option by supporting the patient’s own immune system to kill the tumor cells. We investigated the impact of TP53 mutations on the overall survival of refractory/relapsed DLBCL patients treated with comparable numbers of therapy lines. The minimum number of therapy lines was 2 (median 4), including either anti-CD19 CAR T-cell therapy or conventional salvage therapy. A total of 170 patients with DLBCL and high-grade B-cell lymphoma with MYC, BCL2, and/or BCL6 rearrangements (DHL/THL), diagnosed and treated in our hospital between 2000 and 2021, were included. Twenty-nine of them received CAR T-cell therapy. TP53 mutations were found in 10/29 (35%) and 31/141 (22%) of patients in the CAR T-cell and conventional groups, respectively. Among the 141 patients not treated with CAR T cells, TP53 mutation was an independent prognostic factor for overall survival (OS) (median 12 months with TP53 vs. not reached without TP53 mutation, p 〈 0.005), but in the CAR T cell treated group, this significance could not be shown (median OS 30 vs. 120 months, p = 0.263). The findings from this monocentric retrospective study indicate that TP53 mutation status does not seem to affect outcomes in DLBCL patients treated with CAR T-cell therapy. Detailed evaluation in large cohorts is warranted.

Abstract: Chimeric antigen receptor T (CART) cell therapy targeting the B cell specific differentiation antigen CD19 has shown clinical efficacy in a subset of relapsed/refractory (r/r) diffuse large B cell lymphoma (DLBCL) patients. Despite this heterogeneous response, blood pre-infusion biomarkers predicting responsiveness to CART cell therapy are currently understudied. Methods Blood cell and serum markers, along with clinical data of DLBCL patients who were scheduled for CART cell therapy were evaluated to search for biomarkers predicting CART cell responsiveness. Findings Compared to healthy controls (n=24), DLBCL patients (n=33) showed significant lymphopenia, due to low CD3 + CD4 + T helper and CD3 - CD56 + NK cell counts, while cytotoxic CD3 + CD8 + T cell counts were similar. Although lymphopenic, DLBCL patients had significantly more activated HLA-DR + (P=0.005) blood T cells and a higher frequency of differentiated CD3 + CD27 - CD28 - (28.7 ± 19.0% versus 6.6 ± 5.8%; P & lt;0.001) T cells. Twenty-six patients were infused with CART cells (median 81 days after leukapheresis) and were analyzed for the overall response (OR) 3 months later. Univariate and multivariate regression analyses showed that low levels of differentiated CD3 + CD27 - CD28 - T cells (23.3 ± 19.3% versus 35.1 ± 18.0%) were independently associated with OR. This association was even more pronounced when patients were stratified for complete remission (CR versus non-CR: 13.7 ± 11.7% versus 37.7 ± 17.4%, P=0.001). A cut-off value of ≤ 18% of CD3 + CD27 - CD28 - T cells predicted CR at 12 months with high accuracy (P & lt;0.001). In vitro , CD3 + CD8 + CD27 - CD28 - compared to CD3 + CD8 + CD27 + CD28 + CART cells displayed similar CD19 + target cell-specific cytotoxicity, but were hypoproliferative and produced less cytotoxic cytokines (IFN-γ and TNF-α). CD3 + CD8 + T cells outperformed CD3 + CD4 + T cells 3- to 6-fold in terms of their ability to kill CD19 + target cells. Interpretation Low frequency of differentiated CD3 + CD27 - CD28 - T cells at leukapheresis represents a novel pre-infusion blood biomarker predicting a favorable response to CART cell treatment in r/r DLBCL patients.

Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Abstract: Background. Patients with aggressive hematologic malignancies failing at least two lines of therapy are without further standard treatment options and have a poor prognosis. Identifying effective therapies with genomic-based precision medicine is hampered by intratumor heterogeneity and incomplete understanding of the contribution of various mutations within specific cancer phenotypes. Next-generation functional drug screening (ngFDS) in patient samples promises to overcome these challenges, however, proof of its clinical utility is limited. Methods. We investigated the feasibility and clinical impact of ngFDS measured at single-cell resolution using high-throughput automated microscopy in blood, bone marrow, pleural effusions, or excised lymph node biopsies (Figure 1A and Valdimer G et al. Nat Chem Biol. 2017). First, the accuracy of ngFDS to predict clinical outcome was evaluated in a retrospective cohort of 20 previously untreated patients with acute myeloid leukemia (AML). Then, 48 patients with aggressive hematologic malignancies failing at least two lines of treatment were prospectively evaluated for ngFDS guided therapy, of which 17 could receive ngFDS-guided treatment. Individual ngFDS-guided treatment regimens were selected by a committee (EXALT-board) of hemato-oncologists, pathologist, and pharmacists based on top-candidate treatments identified by ngFDS, considering drug availability as well as safety profiles of single agents and previously reported combinations (Figure 1B). The majority of these patients (12/17) presented with aggressive lymphoma and had seen in median three (2-7) prior treatment lines (Figure 2A). Overall response rate (ORR) and progression-free survival (PFS) of ngFDS-guided treatment were compared with ORR and PFS for the most recent regimen (MRR) on which patients had previously progressed. Results. ngFDS accurately predicted individual clinical response of AML patients to initial therapy. From prospectively analyzed patients receiving ngFDS guided treatment the ORR-ngFDS was 88% (15/17) compared to ORR-MRR of 24% (4/17; P & lt;0.0004). Twelve (70%) of 17 patients had a PFS ratio of ≥1.3 and the mean PFS increased 3.9-fold, from 5.7 weeks to 22.6 weeks (P & lt;0.007) (Figure 2B). Furthermore, analysis of an independent cohort revealed that ngFDS could positively and negatively predict patients' outcome to drug treatment. Conclusions. Automatedmicroscopy-based ngFDS is feasible and accurately predicts clinical response. It can successfully guide personalized treatment of aggressive refractory hematological malignancies. Figure 1 Figure 1. Disclosures Staber: Takeda: Honoraria; Abbie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Honoraria; Amgen: Honoraria; Gilad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees. Snijder: Allcyte: Equity Ownership, Other: founder and shareholder of Allcyte GmbH that holds a worldwide exclusive license for and commercializes the Pharmacoscopy high content imaging technology.. Vladimer: Allcyte Gmbh: Equity Ownership. Krall: Allcyte Gmbh: Equity Ownership. Hoermann: Ariad: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Gilead: Honoraria, Research Funding. Sperr: Teva: Honoraria; Meda: Research Funding; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Phadia: Research Funding; Novartis: Other: Register. Gisslinger: Janssen Cilag: Honoraria; Takeda: Honoraria; Shire: Honoraria; PharmaEssentia: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; AOP Orphan Pharmaceuticals AG: Consultancy, Honoraria. Valent: Incyte: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Deciphera: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Teva: Honoraria; Celgene: Honoraria, Research Funding; Blueprint: Research Funding. Jaeger: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses. Superti-Furga: Allcyte GmbH: Equity Ownership.

Abstract: Background. Anaplastic lymphoma kinase positive systemic anaplastic large cell lymphoma (ALK+ ALCL) represents an aggressive T-cell malignancy that primarily affects children and younger adults. Upon treatment with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or CHOP-like regimens its prognosis is relatively favourable compared to other aggressive T-cell lymphomas, however, the rare event of disease reoccurrence remains particularly challenging. ALCL is CD30 positive, and mono treatment with brentuximab vedotin (BrV) can achieve long term remissions in only few relapsed patients not receiving consolidating stem cell transplantation (Pro B. et al. Blood 2017). A subset of ALCL patients express platelet derived growth factor receptor (PDGFR) alpha or beta in their tumour cells which is induced by ALK via AP-1 transcription factor activation. We previously demonstrated that PDGFR blockade by the Abl/c-Kit/PDGFR kinase inhibitor imatinib is an effective treatment concept for ALK+ ALCL in experimental mouse models (Laimer D. et al. Nature Med. 2012). Here we aimed to assess the prognostic value of PDGFR expression and to probe the clinical value of its targeting by imatinib in ALK+ ALCL patients. Methods. The impact of PDGFR expression to predict clinical outcome was analyzed in samples of 98 ALK+ ALCL patients included in the studies NHL-BFM 90, 95 and ALCL99 between 1992 and 2006. Six patients (age 18-45) with chemo-refractory ALK+ ALCL were prospectively treated with imatinib plus (4) or minus (2) BrV. 33% (2/6) had previously received salvage treatment with stem cell transplantation (SCT). Three of the patients had been enrolled in a prospective single-arm study combining imatinib and BrV, a trial that was terminated due to poor patient recruitment (AGMT ALCL1 trial, EudraCT No.: 2013-003505-26, supported by Takeda®). PDGFR alpha and beta expression analyses were performed on tumour samples of treated patients. Results. ALK+ ALCL patients with high PDGFR expression on tumor cells (n=11) had a significantly lower event-free survival rate (EFS) at 5 years compared to patients with no or low expression (n=87) (27±13% versus 70±5%, respectively, p 〈 0.001, figure 1a). Four of the six patients with relapsed/ refractory ALK+ ALCL responded to imatinib +/- BrV with a rapid metabolic complete remission (CR). At a median clinical follow-up of 52 months (range 20 to108) all responding patients demonstrated an ongoing CR without SCT even after imatinib was discontinued with an average time on imatinib of 49 weeks (range 32 to 130; figure 1b). Half (2/4) of responding patients received no or only 1 infusion of BrV; both non-responding patients had received BrV. Importantly, despite the few patients that could be prospectively treated, PDGFR expression status had a significant impact on clinical course. All patients (4/4) who expressed either PDGFR alpha or beta in tumour cells and in the lymph node microenvironment responded with an ongoing metabolic CR, whereas the two patients whose lymphoma cells had negative PDGFR expression failed to respond (EFS: 4 versus 210 weeks, p = 0.018; figure 1c). Conclusions. 1) PDGFR expression is a poor risk factor in ALK+ ALCL. 2) Imatinib alone or in combination with BrV overcomes chemo-resistance conferred by PDGFR expression. 3) Imatinib treatment can be discontinued with ongoing CR. 4) These observations suggest that PDGFR expressing ALK+ ALCL patients might benefit from early treatment with imatinib. Disclosures Staber: Takeda-Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; MSD: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Klapper:Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Mayerhoefer:Siemens: Research Funding, Speakers Bureau; BMS: Speakers Bureau. Greil:Gilead: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ratiopharm: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Eisai: Honoraria; Genentech: Honoraria, Research Funding; Janssen-Cilag: Honoraria; GSK: Research Funding; Sandoz: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria; MSD: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi Aventis: Honoraria; Boehringer Ingelheim: Honoraria; Mundipharma: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Jaeger:Novartis, Roche, Sandoz: Consultancy; AbbVie, Celgene, Gilead, Novartis, Roche, Takeda Millennium: Research Funding; Amgen, AbbVie, Celgene, Eisai, Gilead, Janssen, Novartis, Roche, Takeda Millennium, MSD, BMS, Sanofi: Honoraria; Celgene, Roche, Janssen, Gilead, Novartis, MSD, AbbVie, Sanofi: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Imatinib - bcr-abl Inhibitor

Abstract: Inhibition of Janus-kinase 1/2 (JAK1/2) is a mainstay to treat myeloproliferative neoplasms (MPN). Sporadic observations reported the co-incidence of B-cell non-Hodgkin lymphomas during treatment of MPN with JAK1/2 inhibitors. We assessed 626 patients with MPN, including 69 with myelofibrosis receiving JAK1/2 inhibitors for lymphoma development. B-cell lymphomas evolved in 4 (5.8%) of 69 patients receiving JAK1/2 inhibition compared with 2 (0.36%) of 557 with conventional treatment (16-fold increased risk). A similar 15-fold increase was observed in an independent cohort of 929 patients with MPN. Considering primary myelofibrosis only (N = 216), 3 lymphomas were observed in 31 inhibitor-treated patients (9.7%) vs 1 (0.54%) of 185 control patients. Lymphomas were of aggressive B-cell type, extranodal, or leukemic with high MYC expression in the absence of JAK2 V617F or other MPN-associated mutations. Median time from initiation of inhibitor therapy to lymphoma diagnosis was 25 months. Clonal immunoglobulin gene rearrangements were already detected in the bone marrow during myelofibrosis in 16.3% of patients. Lymphomas occurring during JAK1/2 inhibitor treatment were preceded by a preexisting B-cell clone in all 3 patients tested. Sequencing verified clonal identity in 2 patients. The effects of JAK1/2 inhibition were mirrored in Stat1−/− mice: 16 of 24 mice developed a spontaneous myeloid hyperplasia with the concomitant presence of aberrant B cells. Transplantations of bone marrow from diseased mice unmasked the outgrowth of a malignant B-cell clone evolving into aggressive B-cell leukemia-lymphoma. We conclude that JAK/STAT1 pathway inhibition in myelofibrosis is associated with an elevated frequency of aggressive B-cell lymphomas. Detection of a preexisting B-cell clone may identify individuals at risk.

Abstract: Strong responses to venetoclax separate T-PLL from other hematologic malignancies in high- throughput drug screening of clinical samples. Two relapsed and refractory T-PLL patients demonstrated clinical response on venetoclax treatment.

Abstract: Personalized medicine aims to match the right drug with the right patient by using specific features of the individual patient's tumor. However, current strategies of personalized therapy matching provide treatment opportunities for less than 10% of patients with cancer. A promising method may be drug profiling of patient biopsy specimens with single-cell resolution to directly quantify drug effects. We prospectively tested an image-based single-cell functional precision medicine (scFPM) approach to guide treatments in 143 patients with advanced aggressive hematologic cancers. Fifty-six patients (39%) were treated according to scFPM results. At a median follow-up of 23.9 months, 30 patients (54%) demonstrated a clinical benefit of more than 1.3-fold enhanced progression-free survival compared with their previous therapy. Twelve patients (40% of responders) experienced exceptional responses lasting three times longer than expected for their respective disease. We conclude that therapy matching by scFPM is clinically feasible and effective in advanced aggressive hematologic cancers. Significance: This is the first precision medicine trial using a functional assay to instruct n-of-one therapies in oncology. It illustrates that for patients lacking standard therapies, high-content assay-based scFPM can have a significant value in clinical therapy guidance based on functional dependencies of each patient's cancer. See related commentary by Letai, p. 290. This article is highlighted in the In This Issue feature, p. 275

Abstract: Pigmentation, catalase activity and biofilm formation are virulence factors that cause resistance of Staphylococcus aureus to environmental stress factors including disinfectants. In recent years, automatic UV-C room disinfection gained greater importance in enhanced disinfection procedures to improve disinfection success in hospitals. In this study, we evaluated the effect of naturally occurring variations in the expression of virulence factors in clinical S. aureus isolates on tolerance against UV-C radiation. Quantification of staphyloxanthin expression, catalase activity and biofilm formation for nine genetically different clinical S. aureus isolates as well as reference strain S. aureus ATCC 6538 were performed using methanol extraction, a visual approach assay and a biofilm assay, respectively. Log10 reduction values (LRV) were determined after irradiation of artificially contaminated ceramic tiles with 50 and 22 mJ/cm2 UV-C using a commercial UV-C disinfection robot. A wide variety of virulence factor expression was observed, indicating differential regulation of global regulatory networks. However, no direct correlation with the strength of expression with UV-C tolerance was observed for either staphyloxanthin expression, catalase activity or biofilm formation. All isolates were effectively reduced with LRVs of 4.75 to 5.94. UV-C disinfection seems therefore effective against a wide spectrum of S. aureus strains independent of occurring variations in the expression of the investigated virulence factors. Due to only minor differences, the results of frequently used reference strains seem to be representative also for clinical isolates in S. aureus.