Recruiting patients in early-phase clinical trials poses significant challenges, particularly when targeting specific patient populations. This emerged during the phase I/II B-pVAC-SARS-CoV-2 trial investigating CoVac-1, a peptide-based T-cell activator developed to provide T-cell-mediated protection against severe COVID-19 in individuals with inherited or acquired B-cell deficiencies who were unable to mount adequate antibody responses following approved vaccination AGAINST SARS-CoV-2. Despite promising phase I results in healthy volunteers and high patient interest, recruitment of immunocompromised subjects proved substantially more difficult than initially anticipated. Strict inclusion and exclusion criteria, combined with the complexity of patient profiles, required assessing 1,024 candidates at one site to finally recruit 61 subjects and extended the recruitment process to over 8 months. Our experience highlights how rigid eligibility criteria and lengthy protocol adaptation processes may unintentionally limit access to promising investigational treatments for patients with urgent unmet medical needs. More flexible, patient-tailored approaches, whenever safety is not compromised, together with more pragmatic approaches to protocol adaptation, for instance driven by expedited consultations with the DSMB, may help improve recruitment, while preserving patient safety and trial integrity, particularly in academic early-phase clinical research.

Despite high cure rates in classic Hodgkin lymphoma (cHL), relapses are observed. Whether relapsed cHL represents second primary lymphoma or an underlying T-cell lymphoma (TCL) mimicking cHL is underinvestigated. To analyze the nature of cHL recurrences, in-depth clonality testing of immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements was performed in paired cHL diagnoses and recurrences among 60 patients, supported by targeted mutation analysis of lymphoma-associated genes. Clonal Ig rearrangements were detected by next-generation sequencing (NGS) in 69 of 120 (58%) diagnoses and recurrence samples. The clonal relationship could be established in 34 cases, identifying clonally related relapsed cHL in 24 of 34 patients (71%). Clonally unrelated cHL was observed in 10 of 34 patients (29%) as determined by IG-NGS clonality assessment and confirmed by the identification of predominantly mutually exclusive gene mutations in the paired cHL samples. In recurrences of >2 years, ∼60% of patients with cHL for whom the clonal relationship could be established showed a second primary cHL. Clonal TCR gene rearrangements were identified in 14 of 125 samples (11%), and TCL-associated gene mutations were detected in 7 of 14 samples. Retrospective pathology review with integration of the molecular findings were consistent with an underlying TCL in 5 patients aged >50 years. This study shows that cHL recurrences, especially after 2 years, sometimes represent a new primary cHL or TCL mimicking cHL, as uncovered by NGS-based Ig/TCR clonality testing and gene mutation analysis. Given the significant therapeutic consequences, molecular testing of a presumed relapse in cHL is crucial for subsequent appropriate treatment strategies adapted to the specific lymphoma presentation.

2534 Background: While being highly efficient in hematological malignancies, bispecific antibodies (bsAbs), alike CAR T cells, are yet not successful in solid tumors. Moreover, all available T cell-mobilizing strategies cause side effects that endanger patients and, in case of bsAbs, limit applicable doses and thus efficacy. We report on the clinical development of CC-1, a PSMAxCD3 bsAb, in an IgG-based format that induces fully target cell-restricted T cell activity (Zekri et al, EMBO Mol Med, 2020). Targeting PSMA, which is expressed on malignant cells and on the neovasculature, improves accessibility of the tumor site for immune effector cells as critical prerequisite for success in solid tumors. Notably, CC-1 binds a unique PSMA epitope which is expressed on malignant cells both of prostate carcinoma (PC) and 50% of patients with squamous cell carcinoma (SCC) of the lung. Methods: A FIH trial evaluating CC-1 with pre-emptive Tocilizumab included patients with metastatic castration resistant prostate carcinoma (mCRPC) (NCT04104607) and consisted of two parts. Dose escalation (n=10-66) using a novel intra-individual dose escalation design to rapidly reach the target dose of 826µg to determine safety, tolerability and maximum tolerated dose (MTD) (Labrenz et al, Pharm Stat, 2022). The dose expansion cohort exposed patients to CC-1 at MTD and explored efficacy to define RP2D (n=14). Our second phase I trial enrolls patients with SCC of the lung (NCT04496674) to receive CC-1 in combination with checkpoint inhibition. Based on the meanwhile available very favorable safety and preliminary efficacy data, a third phase I trial was initiated where CC-1 is evaluated as first line treatment in patients with hormone sensitive biochemical recurrence (BCR) of PC (NCT05646550), where tumor burden is low and accordingly lower side effects and long-lasting efficacy are expected. Results: Recruitment in the dose escalation part of the trial in mCRPC has been completed and the target dose was reached and MTD defined without DLT upon treatment of the 9 th and 14 th patient, respectively. 24 patients completed treatment, with the most frequently observed toxicity being cytokine release syndrome (CRS, max. 2°) (88%). Besides grade 1 to 2 hypertension (46%) and xerostomia (8%), no further CC-1 related toxicities were observed. A rapid and profound decline of elevated PSA levels was observed in all but one patient, with up to 60% reduction compared to baseline. So far 5 patients received multiple treatment cycles at MTD-level. One patient with SCC of the lung has been treated and CC-1 was escalated up to 153µg without occurrence of CRS. In the phase I trial in BCR of PC, the first three patients were enrolled in February 2023. Conclusions: CC-1 is a promising compound with a favourable toxicity profile and promising clinical activity. Details on study designs and updated data from the 3 clinical trials will be presented at the meeting. Clinical trial information: NCT04104607 , NCT04496674 , NCT05646550 .

Individual data of cytokine release and metabolic activity upon OX40 stimulation of AML cells.

TPS5114 Background: Bispecific antibodies (bsAbs), alike CAR T cells, are not yet successful in solid tumors. Recently we developed a PSMAxCD3 bsAb termed CC-1 in an IgG-based format mediating fully target cell-restricted T cell activation and potent antitumor activity (Zekri et al, EMBO Mol Med, 2020). In prostate cancer (PC), PSMA is not only expressed on the malignant cells themselves, but also on tumor vessels. Targeting the latter improves accessibility of the tumor site for immune effector cells, a critical prerequisite for success. CC-1 is under evaluation in a First in Human trial enrolling patients with castration resistant prostate carcinoma (NCT04104607) and, in combination with checkpoint inhibition, in a phase I trial enrolling patients with squamous cell carcinoma of the lung (NCT04104607). On the basis of the meanwhile available very favorable safety and preliminary efficacy data, CC-1 is now being evaluated as first line treatment in patients with biochemical recurrence (BCR) of PC (NCT05646550), where tumor burden is low and accordingly further reduced side effects and sustained efficacy are expected. Methods: This is an ongoing open label, single center phase I clinical trial evaluating CC-1 without androgen deprivation therapy as first line treatment in men with BCR of PC. Key eligibility criteria include low risk of disease progression (PSA-Doubling time > 1 year after prostatectomy or interval to BCR > 18 months after radiation; ISUP grade < 4; PSA ≥ 0.2 ng/ml and < 5 ng/ml) and no androgen deprivation therapy except in a neoadjuvant/adjuvant setting. The clinical trial consists of (i) a dose escalation part (24-42 patients) using a 3+3 design to determine overall safety, tolerability as well as maximum tolerated dose (MTD) and (ii) a dose expansion part, in which 14 patients are treated at the MTD. CC-1 is applied twice-weekly over 21 days as three-hour infusion in up to six 28-day cycles. In the first cycle, step dosing (day 1: 10µg, day 2: 28µg, day 3 and thereafter: target dose) is performed, and 7 patient cohorts receiving dose levels from 78µg to 600µg are evaluated. Premedication includes acetaminophen, dimetindene and cortisone to minimize the risk of infusion reactions and cytokine release syndrome (CRS). The primary objectives are definition of MTD and recommended phase II dose. Assessment of safety is based on the frequency of adverse events according to CTCAE v5.0. Dose limiting toxicities are defined as ≥ 3° adverse drug reactions (e.g. CRS) with predefined exceptions (e.g. temporary laboratory abnormalities). Efficacy is determined by serial PSA-measurements (PSA-response: ≥50% decrease compared to baseline). Furthermore, percentage of patients with no clinical relapse, no salvage and no subsequent antineoplastic therapy will be assessed. Clinical trial information: NCT05646550 .

Differential diagnosis of clonal versus reactive cytopenia and monocytosis, respectively, frequently presents a diagnostic challenge. With the two recent classifications of myeloid disorders, mutational analysis has gained importance as a diagnostic tool. However, reports on its utility on trephine bone marrow biopsies (BMB) are sparse. The aim of our proof of principle study was to determine the suitability of targeted sequencing for the longitudinal evaluation of cytopenia and monocytosis and demonstration of clonal evolution on sequential BMB. Seventy-seven EDTA-decalcified BMB of 33 patients with peripheral cytopenia and/or monocytosis, including at least one follow-up biopsy/patient, were included. Initial morphological diagnoses were idiopathic cytopenia of undetermined significance (ICUS, 8 cases), MDS (without blast increase, 7 cases), MDS with increased blasts/excess blasts (MDS-IB/EB) (11 cases), and CMML (7 cases). Thirty-one genes relevant for myeloid disorders were examined using two custom AmpliSeq NGS panels. Mutations were found in the initial BMB of 5/8 cases of ICUS, thus changing the diagnosis to clonal cytopenia of unknown significance (CCUS), 5/7 MDS, 10/11 MDS-IB/EB, and 7/7 CMML. Clonal evolution was observed in 14/33 (42%) cases, mostly associated with disease progression. None of the wild-type patients acquired mutations during follow-up. NGS-based mutation profiling is a robust diagnostic tool for BMB and provides valuable additional information, especially for cases with no/minimal dysplasia, and for better risk stratification of MDS. Tracking variant allele frequency and appearance of mutations over time allows for observing clonal evolution or relapse.

Correlative analysis of basal cytokine release, proliferation and apoptosis with OX40 expression on AML cells.

Expression of OX40 in CML and MDS.

Immune cell reconstitution after stem cell transplantation is allocated over several stages. Whereas cells mediating innate immunity recover rapidly, adaptive immune cells, including T and B cells, recover slowly over several months. In this study we investigated kinetics and reconstitution of de novo B cell formation in patients receiving CD3 and CD19 depleted haploidentical stem cell transplantation with additional in vivo T cell depletion with monoclonal anti-CD3 antibody. This model enables a detailed in vivo evaluation of hierarchy and attribution of defined lymphocyte populations without skewing by mTOR- or NFAT-inhibitors. As expected CD3+ T cells and their subsets had delayed reconstitution (<100 cells/μL at day +90). Well defined CD19+ B lymphocytes of naïve and memory phenotype were detected at day +60. Remarkably, we observed a very early reconstitution of antibody-secreting cells (ASC) at day +14. These ASC carried the HLA-haplotype of the donor and secreted the isotypes IgM and IgA more prevalent than IgG. They correlated with a population of CD19− CD27− CD38low/+ CD138− cells. Of note, reconstitution of this ASC occurred without detectable circulating T cells and before increase of BAFF or other B cell stimulating factors. In summary, we describe a rapid reconstitution of peripheral blood ASC after CD3 and CD19 depleted haploidentical stem cell transplantation, far preceding detection of naïve and memory type B cells. Incidence before T cell reconstitution and spontaneous secretion of immunoglobulins allocate these early ASC to innate immunity, eventually maintaining natural antibody levels.

In situ follicular neoplasia (ISFN) is the earliest morphologically identifiable precursor of follicular lymphoma (FL). Although it is genetically less complex than FL and has low risk for progression, ISFN already harbors secondary genetic alterations, in addition to the defining t(14;18)(q32;q21) translocation. FL, in turn, frequently progresses to diffuse large B-cell lymphoma (DLBCL) or high-grade B-cell lymphoma (HGBL). By BCL2 staining of available reactive lymphoid tissue obtained at any time point in patients with aggressive B-cell lymphoma (BCL), we identified ten paired cases of ISFN and DLBCL/HGBL, including six de novo tumors and four tumors transformed from FL as an intermediate step, and investigated their clonal evolution using microdissection and next-generation sequencing. A clonal relationship between ISFN and aggressive BCL was established by immunoglobulin and/or BCL2 rearrangements and/or the demonstration of shared somatic mutations for all ten cases. Targeted sequencing revealed CREBBP, KMT2D, EZH2, TNFRSF14 and BCL2 as the genes most frequently mutated already in ISFN. Based on the distribution of private and shared mutations, two patterns of clonal evolution were evident. In most cases, the aggressive lymphoma, ISFN and, when present, FL revealed divergent evolution from a common progenitor, whereas linear evolution with sequential accumulation of mutations was less frequent. In conclusion, we demonstrate for the first time that t(14;18)+ aggressive BCL can arise from ISFN without clinically evident FL as an intermediate step and that during this progression, branched evolution is common.

Background In situ follicular neoplasia (ISFN) is a t(14;18)(q32;q21)(+) precursor lesion of follicular lymphoma (FL), which in turn can transform into diffuse large B-cell lymphoma (DLBCL). For DLBCL that arise de novo, no precursor lesion is known. Given the high frequency of the t(14;18) translocation in de novo DLBCL as well, we investigated whether they can also arise from ISFN without FL as an intermediate step. Objectives To investigate the clonal evolution of ISFN to DLBCL - transformed from FL and de novo. Materials and methods Identification of ISFN lesions in patients with DLBCL was performed by BCL2 staining of reactive lymphoid tissues. ISFN and DLBCL were subsequently analyzed by fluorescence in situ hybridization, clonality analyses, sequencing of the t(14;18) breakpoint, and targeted next-generation sequencing. Results 10 cases with paired ISFN and DLBCL samples were identified, 6 of which were de novo DLBCL and 4 transformed from FL. 3 DLBCL carried MYC-rearrangements in addition to the t(14;18) and were classified as high-grade B-cell lymphoma (HGBL). The clonal relationship of ISFN and DLBCL/HGBL was confirmed for all cases. CREBBP, KMT2D, EZH2, TNFRSF14, and BCL2 were the genes most frequently mutated, with the distribution of private and shared mutations pointing to 2 different scenarios of clonal evolution. In most cases, DLBCL/HGBL, ISFN, and, if also present, FL had evolved divergently from a common progenitor, whereas linear evolution was less frequent. Conclusion We show for the first time that t(14;18)(+) DLBCL/HGBL can arise directly from ISFN without FL as an intermediate step and that during this progression, divergent evolution is common.

Myeloproliferative neoplasms (MPN) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) both harbor the potential to undergo myelodysplastic progression or acceleration and can transform into blast-phase MPN or MDS/MPN, a form of secondary acute myeloid leukemia (AML). Although the initiating transforming events are yet to be determined, current concepts suggest a stepwise acquisition of (additional) somatic mutations—apart from the initial driver mutations—that trigger disease evolution. In this study we molecularly analyzed paired bone marrow samples of MPN and MDS/MPN patients with known progression and compared them to a control cohort of patients with stable disease course. Cases with progression displayed from the very beginning a higher number of mutations compared to stable ones, of which mutations in five (ASXL1, DNMT3A, NRAS, SRSF2 and TP53) strongly correlated with progression and/or transformation, even if only one of these genes was mutated, and this particularly applied to MPN. TET2 mutations were found to have a higher allelic frequency than the putative driver mutation in three progressing cases (“TET2-first”), whereas two stable cases displayed a TET2-positive subclone (“TET2-second”), supporting the hypothesis that not only the sum of mutations but also their order of appearance matters in the course of disease. Our data emphasize the importance of genetic testing in MPN and MDS/MPN patients in terms of risk stratification and identification of imminent disease progression.

Die in situ follikuläre Neoplasie (ISFN) ist eine t(14;18)(q32;q21)+ Vorläuferläsion des follikulären Lymphoms (FL), das seinerseits in ein diffus großzelliges B‑Zell-Lymphom (DLBCL) transformieren kann. Für DLBCL, die de novo entstehen, ist keine Vorläuferläsion bekannt. Angesichts der hohen Frequenz der t(14;18)-Translokation auch in de novo DLBCL haben wir untersucht, ob diese auch ohne intermediäres FL aus der ISFN hervorgehen können. Erforschung der klonalen Evolution der ISFN zum DLBCL – transformiert aus einem FL und de novo. Die Identifikation von ISFN-Läsionen bei Patienten mit DLBCL erfolgte durch BCL2-Färbung reaktiver lymphatischer Gewebe. ISFN und DLBCL wurden anschließend mittels Fluoreszenz-in-situ-Hybridisierung, Klonalitätsanalysen, Sequenzierung des t(14;18)-Bruchpunktes und gezieltem Next Generation Sequencing analysiert. Es wurden 10 Fälle mit gepaarten ISFN- und DLBCL-Proben identifiziert, davon 6 de novo DLBCL und 4 transformiert aus einem FL. Neben der t(14;18) trugen 3 DLBCL auch MYC-Rearrangements und wurden als high-grade B‑Zell-Lymphom (HGBL) klassifiziert. In allen Fällen konnte die klonale Verwandtschaft von ISFN und DLBCL/HGBL bestätigt werden. CREBBP, KMT2D, EZH2, TNFRSF14 und BCL2 waren die am häufigsten mutierten Gene, wobei die Verteilung privater und gemeinsamer Mutationen auf 2 unterschiedliche Szenarien der klonalen Evolution schließen ließ. Meist hatten sich DLBCL/HGBL, ISFN und, falls ebenfalls vorhanden, FL divergent aus einem gemeinsamen Progenitor entwickelt, während eine lineare Evolution selten war. Wir zeigen erstmals, dass t(14;18)+ DLBCL/HGBL auch ohne intermediäres FL direkt aus einer ISFN entstehen können und dass es hierbei häufig zur divergenten Evolution kommt.

The aim of this study was to test the hypothesis that computed tomography texture analysis (CTTA) is accurate for response assessment of Hodgkin lymphoma (HL).A total of 100 patients with HL were identified. CTTA in baseline and interim staging was performed generating volume of interests in lymphoma tissue from which CTTA features including 1st, 2nd, and higher order textural features were extracted. Baseline and interim 2-deoxy-fluor-glucose positron emission tomography results were used to determine therapy response and compared to CTTA in terms of patient outcome.At interim, 1st-order features yielded a significant drop (e.g., entropy of heterogeneity, P=.01) or a significant rise (deviation, P<.001), whereas 2nd and higher order features decreased (e.g., entropy of co-occurrence matrix, P<.001). Patients achieving complete remission at end of treatment had a significantly lower entropy of heterogeneity at baseline and interim compared to patients achieving partial remission (P<.05).CT textural features change in parallel to metabolic therapy response, and are therefore a feasible diagnostic tool for a more accurate response assessment of HL.

Supplemental Digital Content is available in the text.

Post-transplant lymphoproliferative disorders (PTLD) occur after solid organ transplantation (SOT) or hematopoietic stem cell transplantation (HCT) and are frequently associated with Epstein-Barr virus (EBV). Because of the complex immune setup in PTLD patients, the tumor microenvironment (TME) is of particular interest to understand PTLD pathogenesis and elucidate predictive factors and possible treatment options. We present a comparative study of clinicopathological features of 48 PTLD after HCT (n = 26) or SOT (n = 22), including non-destructive (n = 6), polymorphic (n = 23), and monomorphic (n = 18) PTLD and classic Hodgkin lymphoma (n = 1). EBV was positive in 35 cases (73%). A detailed examination of the TME with image analysis-based quantification in 22 cases revealed an inflammatory TME despite underlying immunosuppression and significant differences in its density and composition depending on type of transplant, PTLD subtypes, and EBV status. Tumor-associated macrophages (TAMs) expressing CD163 (p = 0.0022) and Mannose (p = 0.0016) were enriched in PTLD after HCT. Double stains also showed differences in macrophage polarization, with more frequent M1 polarization after HCT (p = 0.0321). Higher counts for TAMs (CD163 (p = 0.0008) and cMaf (p = 0.0035)) as well as in the T cell compartment (Granzyme B (p = 0.0028), CD8 (p = 0.01), and for PD-L1 (p = 0.0305)) were observed depending on EBV status. In conclusion, despite the presence of immunosuppression, PTLD predominantly contains an inflammatory TME characterized by mostly M1-polarized macrophages and cytotoxic T cells. Status post HCT, EBV positivity, and polymorphic subtype are associated with an actively inflamed TME, indicating a specific response of the immune system. Further studies need to elucidate prognostic significance and potential therapeutic implications of the TME in PTLD.

Classical Hodgkin lymphoma is dominated by the non-neoplastic microenvironment, while the neoplastic Hodgkin-Reed-Sternberg cells compose only a minority of cells in the lymphoma tissue. Both the Hodgkin-Reed-Sternberg cells due to their expression of CD30 and PD-L1 and the microenvironment with abundant T cells and expression of PD1 are specifically targeted by new treatment concepts. We aimed to understand the dynamics of therapeutic targets in patients treated with conventional chemotherapy. We analyzed sequential biopsy specimens obtained at diagnosis and at relapse from the same patient for morphology, immunophenotype, and microenvironmental components. The morphological subtype changed between primary and relapse biopsy in 20% of cases. The immunophenotype was stable with respect to CD30, CD3, and LMP1 but variable with respect to CD15 and CD20 expression. Gene expression revealed 8 upregulated and 20 downregulated genes at relapse (p ≤ 0.05) with a consistent logarithmic fold change direction in at least 75% of all cases. For PD1, we found discrepant results between gene expression analysis (decrease at relapse) and number of PD1-positive cells assessed by immunohistochemistry (unchanged at relapse). PD-L1 in the neoplastic cells appeared unchanged between primary diagnosis and relapse. The expression of the therapeutic targets CD30, PD1, and PD-L1 can reliably be assessed in tumor specimen at first diagnosis and is unchanged under conventional chemotherapy.

Peripheral T cell lymphomas (PTCL) are a rare and heterogeneous group of aggressive non-Hodgkin lymphomas, showing a generally poor prognosis. In this retrospective analysis, we aimed to investigate the impact of autologous stem cell transplantation (autoSCT) in PTCL. A retrospective analysis of 58 consecutive unselected PTCL patients aged 21–71 years undergoing autoSCT as first-line consolidation as well as in the relapse setting was performed. The median follow-up time was 67 months. A 5-year overall survival (OS) of 53% and a 5-year progression-free survival (PFS) after autoSCT of 44% was achieved. The overall relapse rate after autoSCT was 50%. On multivariate analysis, standard baseline characteristics such as age, disease stage and international prognostic index (IPI) score failed to predict outcome in our cohort. First-line treatment with autoSCT was not associated with a benefit in OS when compared to patients receiving autoSCT at relapse. Notably, autoSCT seemed to be a suitable approach even for older transplant-eligible patients (aged ≥ 60 years), with a similar 5-year OS of 49% when compared to younger patients. Our study suggests that autoSCT can achieve long-term survival in PTCL patients even after relapse and should also be considered for eligible older patients.

The traditional concept of unidirectional maturation of hematopoietic cells has been called into question due to the recognition of lineage plasticity, which is increasingly found also in the clonal evolution of hematopoietic and lymphoid malignancies. Here we present an unusual case of a patient with TP53-mutated chronic lymphocytic leukemia (CLL) treated with a PI3Kδ inhibitor evolving to clonally related Langerhans cell histiocytosis (LCH) with acquired BRAF V600E and STK11 mutations and loss of expression of PAX-5 and other examined B cell markers. In indolent B cell lymphoma, transformation to a more aggressive high-grade lymphoma occurs frequently during the course of disease and is thought to be caused by clonal evolution. Our case further supports the concept of significant lineage plasticity in lymphomas and raises the question of a potential role of novel pharmacologic agents in clonal evolution.