Value of hemophagocytosis in the diagnosis of hemophagocytic lymphohistiocytosis in critically ill patients.

BACKGROUND: Ferritin is an established biomarker in the diagnosis of secondary hemophagocytic lymphohistiocytosis (HLH), which is diagnosed by the HLH-2004 criteria. Among these criteria, detection of hemophagocytosis through invasive procedures may delay early life saving treatment. Our aim was to investigate the value of hemophagocytosis in diagnosing HLH in critically ill patients. METHODS: In this secondary analysis of a retrospective observational study, we included all patients aged ≥18 years and admitted to any adult ICU at Charité-Universitätsmedizin Berlin between January 2006 and August 2018, who had hyperferritinemia (≥500 µg/L) and underwent bone marrow biopsy during their ICU course. RESULTS: Two hundred fifty-two patients were included, of whom 31 (12.3%) showed hemophagocytosis. In multivariable logistic regression analysis, maximum ferritin was independently associated with hemophagocytosis. By removing hemophagocytosis from HLH-2004 criteria and HScore, prediction accuracy for HLH diagnosis was only marginally decreased compared to the original scores. CONCLUSIONS: Our results strengthen the diagnostic value of ferritin and underline the importance of considering HLH diagnosis in patients with high ferritin but only four fulfilled HLH-2004 criteria, when hemophagocytosis was not assessed or not detectable. Proof of hemophagocytosis is not required for a reliable HLH diagnosis.

AML with complex karyotype: extreme genomic complexity revealed by combined long-read sequencing and Hi-C technology.

Acute myeloid leukemia with complex karyotype (CK-AML) is associated with poor prognosis, which is only in part explained by underlying TP53 mutations. Especially in the presence of complex chromosomal rearrangements, such as chromothripsis, the outcome of CK-AML is dismal. However, this degree of complexity of genomic rearrangements contributes to the leukemogenic phenotype and treatment resistance of CK-AML remains largely unknown. Applying an integrative workflow for the detection of structural variants (SVs) based on Oxford Nanopore (ONT) genomic DNA long-read sequencing (gDNA-LRS) and high-throughput chromosome confirmation capture (Hi-C) in a well-defined cohort of CK-AML identified regions with an extreme density of SVs. These rearrangements consisted to a large degree of focal amplifications enriched in the proximity of mammalian-wide interspersed repeat elements, which often result in oncogenic fusion transcripts, such as USP7::MVD, or the deregulation of oncogenic driver genes as confirmed by RNA-seq and ONT direct complementary DNA sequencing. We termed this novel phenomenon chromocataclysm. Thus, our integrative SV detection workflow combing gDNA-LRS and Hi-C enables to unravel complex genomic rearrangements at a very high resolution in regions hard to analyze by conventional sequencing technology, thereby providing an important tool to identify novel important drivers underlying cancer with complex karyotypic changes.

General condition and comorbidity of long-term survivors of adult acute lymphoblastic leukemia.

Cure rates in adult acute lymphoblastic leukemia (ALL) improved using pediatric-based chemotherapy and stem cell transplantation (SCT). However, limited data on the health condition of cured adults are available whereas pediatric data cannot be transferred. The GMALL analyzed the health status in survivors of adult ALL retrospectively. Physicians answered a questionnaire on general condition (Eastern Cooperative Oncology Group [ECOG] status) and comorbidity or syndrome occurrence observed after treatment. Five hundred and thirty-eight patients with a median age of 29 (range, 15-64) years at diagnosis were analyzed, median follow-up was 7 (range, 3-24) years. Thirty-one percent had received SCT. ECOG status was 0-1 in 94%, 34% had not developed significant comorbidities. Most frequent comorbidities involved the neurologic system (27%), endocrine system (20%), skin (18%), graft-versus-host-disease (15%), cardiac system (13%), fatigue (13%). SCT impacted ECOG status and comorbidity occurrence significantly. ECOG 0-1 was observed in 86% of SCT and 98% of non-SCT patients (P<0.0001); comorbidity was observed in 87% and 57% respectively (P<0.0001). Our analysis elucidates the spectrum of comorbidities in cured adult ALL patients, with higher risk for transplanted patients, providing stimulations for the design of adequate aftercare programs. Overall, a large proportion of non-SCT patients achieved unrestricted general condition. The data provide a reference for new patient-centered endpoints in future trials.

Gene Gun Her2/neu DNA Vaccination: Evaluation of Vaccine Efficacy in a Syngeneic Her2/neu Mouse Tumor Model.

Genetic vaccination using naked plasmid DNA is an immunization strategy both against infectious diseases and cancer.In order to improve efficacy of DNA vaccines, particularly in large animals and humans, different strategies have been pursued. These vaccination strategies are based on different application routes, schedules and coexpression of immunomodulatory molecules as adjuvants. Our mouse tumor model offers the possibility to investigate Her2/neu DNA vaccines in different settings, that is, intramuscular or intradermal application with or without coexpression of adjuvants. The immunogenicity of predicted peptides for Her2/neu specific memory T cells were screened and confirmed after intramuscular and intradermal application. Protection from tumor growth in tumor challenge experiments and both T cell and humoral immune responses against Her2/neu peptides are used as surrogate parameters for vaccine efficacy.

Midostaurin plus intensive chemotherapy for younger and older patients with AML and FLT3 internal tandem duplications.

We conducted a single-arm, phase 2 trial (German-Austrian Acute Myeloid Leukemia Study Group [AMLSG] 16-10) to evaluate midostaurin with intensive chemotherapy followed by allogeneic hematopoietic-cell transplantation (HCT) and a 1-year midosta urin maintenance therapy in adult patients with acute myeloid leukemia (AML) and fms-related tyrosine kinase 3 (FLT3) internal tandem duplication (ITD). Patients 18 to 70 years of age with newly diagnosed FLT3-ITD-positive AML were eligible. Primary and key secondary endpoints were event-free survival (EFS) and overall survival (OS). Results were compared with a historical cohort of 415 patients treated on 5 prior AMLSG trials; statistical analysis was performed using a double-robust adjustment with propensity score weighting and covariate adjustment. Results were also compared with patients (18-59 years) treated on the placebo arm of the Cancer and Leukemia Group B (CALGB) 10603/RATIFY trial. The trial accrued 440 patients (18-60 years, n = 312; 61-70 years, n = 128). In multivariate analysis, EFS was significantly in favor of patients treated within the AMLSG 16-10 trial compared with the AMLSG control (hazard ratio [HR], 0.55; P < .001); both in younger (HR, 0.59; P < .001) and older patients (HR, 0.42; P < .001). Multivariate analysis also showed a significant beneficial effect on OS compared with the AMLSG control (HR, 0.57; P < .001) as well as to the CALGB 10603/RATIFY trial (HR, 0.71; P = .005). The treatment effect of midostaurin remained significant in sensitivity analysis including allogeneic HCT as a time-dependent covariate. Addition of midostaurin to chemotherapy was safe in younger and older patients. In comparison with historical controls, the addition of midostaurin to intensive therapy led to a significant improvement in outcome in younger and older patients with AML and FLT3-ITD. This trial is registered at clinicaltrialsregistry.eu as Eudra-CT number 2011-003168-63 and at clinicaltrials.gov as NCT01477606.

Precision medicine in myeloid malignancies.

Myeloid malignancies have always been at the forefront of an improved understanding of the molecular pathogenesis of cancer. In accordance, over the last years, basic research focusing on the aberrations underlying malignant transformation of myeloid cells has provided the basis for precision medicine approaches and subsequently has led to the development of powerful therapeutic strategies. In this review article, we will recapitulate what has happened since in the 1980s the use of all-trans retinoic acid (ATRA), as a first targeted cancer therapy, has changed one of the deadliest leukemia subtypes, acute promyelocytic leukemia (APL), into one that can be cured without classical chemotherapy today. Similarly, imatinib, the first molecularly designed cancer therapy, has revolutionized the management of chronic myeloid leukemia (CML). Thus, targeted treatment approaches have become the paradigm for myeloid malignancy, but many questions still remain unanswered, especially how identical mutations can be associated with different phenotypes. This might be linked to the impact of the cell of origin, gene-gene interactions, or the tumor microenvironment including the immune system. Continuous research in the field of myeloid neoplasia has started to unravel the molecular pathways that are not only crucial for initial treatment response, but also resistance of leukemia cells under therapy. Ongoing studies focusing on leukemia cell vulnerabilities do already point to novel (targetable) "Achilles heels" that can further improve myeloid cancer therapy.

Acute myeloid leukemia: negative prognostic impact of early blast persistence can be in part overcome by a later remission prior to post-induction therapy.

In acute myeloid leukemia, there is an ongoing debate on the prognostic value of the early bone marrow assessment in patients receiving intensive therapy. In this retrospective study, we analyzed the prognostic impact of the early response in 1,008 patients with newly diagnosed acute myeloid leukemia, who were treated at our institution with intensive chemotherapy followed by consolidation chemotherapy and/or allogeneic hematopoietic stem cell transplantation (HSCT). We found that early blast persistence has an independent negative prognostic impact on overall survival, eventfree survival and relapse-free survival. This negative prognostic impact may only be overcome in patients showing at least a partial remission at the early bone marrow assessment and who subsequently achieve blast clearance by additional induction chemotherapy prior to consolidation therapy with allogeneic HSCT. In accordance, we propose that the time slope of remission is an additional leukemia-related dynamic parameter that reflects chemosensitivity and thus may inform post-induction therapy decision-making. In addition to patient-related factors, European LeukemiaNet risk group, measurable residual disease monitoring and donor availability, this may particularly apply to European LeukemiaNet intermediate-risk patients, for whom a decision between consolidation chemotherapy and allogeneic HSCT remains challenging in many cases.

Knowledge transfer to enhance the performance of deep learning models for automated classification of B cell neoplasms.

Multi-parameter flow cytometry (MFC) is a cornerstone in clinical decision making for leukemia and lymphoma. MFC data analysis requires manual gating of cell populations, which is time-consuming, subjective, and often limited to a two-dimensional space. In recent years, deep learning models have been successfully used to analyze data in high-dimensional space and are highly accurate. However, AI models used for disease classification with MFC data are limited to the panel they were trained on. Thus, a key challenge in deploying AI into routine diagnostics is the robustness and adaptability of such models. This study demonstrates how transfer learning can be applied to boost the performance of models with smaller datasets acquired with different MFC panels. We trained models for four additional datasets by transferring the features learned from our base model. Our workflow increased the model's overall performance and, more prominently, improved the learning rate for small training sizes.

B cell depletion and signs of sepsis-acquired immunodeficiency in bone marrow and spleen of COVID-19 deceased.

OBJECTIVES: In coronavirus disease 2019 (COVID-19), the adaptive immune response is of considerable importance, and detailed cellular immune reactions in the hematological system of patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are yet to be clarified. METHODS: This study reports the morphological characterization of both bone marrow and spleen in 11 COVID-19 decedents with respect to findings in the peripheral blood and pulmonary SARS-CoV-2 burden. RESULTS: In the bone marrow, activation and left shift were found in at least 55% of patients, which was mirrored by peripheral anaemia, granulocytic immaturity and multiple thromboembolic events. Signs of sepsis-acquired immunodeficiency were found in the setting of an abscess-forming superinfection of viral COVID-19 pneumonia. Furthermore, a severe B cell loss was observed in the bone marrow and/or spleen in 64% of COVID-19 patients. This was reflected by lymphocytopenia in the peripheral blood. As compared to B cell preservation, B cell loss was associated with a higher pulmonary SARS-CoV-2 burden and only a marginal decrease of of T cell counts. CONCLUSIONS: The results of this study suggest the presence of sepsis-related immunodeficiency in severe COVID-19 pneumonia with superinfection. Furthermore, our findings indicate that lymphocytopenia in COVID-19 is accompanied by B cell depletion in hematopoietic tissue, which might impede the durability of the humoral immune response to SARS-CoV-2.

CXCR5 CAR-T cells simultaneously target B cell non-Hodgkin's lymphoma and tumor-supportive follicular T helper cells.

CAR-T cell therapy targeting CD19 demonstrated strong activity against advanced B cell leukemia, however shows less efficacy against lymphoma with nodal dissemination. To target both B cell Non-Hodgkin's lymphoma (B-NHLs) and follicular T helper (Tfh) cells in the tumor microenvironment (TME), we apply here a chimeric antigen receptor (CAR) that recognizes human CXCR5 with high avidity. CXCR5, physiologically expressed on mature B and Tfh cells, is also highly expressed on nodal B-NHLs. Anti-CXCR5 CAR-T cells eradicate B-NHL cells and lymphoma-supportive Tfh cells more potently than CD19 CAR-T cells in vitro, and they efficiently inhibit lymphoma growth in a murine xenograft model. Administration of anti-murine CXCR5 CAR-T cells in syngeneic mice specifically depletes endogenous and malignant B and Tfh cells without unexpected on-target/off-tumor effects. Collectively, anti-CXCR5 CAR-T cells provide a promising treatment strategy for nodal B-NHLs through the simultaneous elimination of lymphoma B cells and Tfh cells of the tumor-supporting TME.

Clinical outcome of older adults with acute myeloid Leukemia: An analysis of a large tertiary referral Center over two decades.

OBJECTIVE: In older adults with acute myeloid leukemia (AML), the overall outcome is still dismal and long-term data on survival are scarce, particularly outside of clinical trials. Here, we assess characteristics, prognostic factors and long-term survival in patients ≥60 years who were treated for AML at our center over the past 17 years. METHODS: 590 older adults with newly diagnosed AML were characterized according to Eastern Cooperative Oncology Group (ECOG) score, Charlson comorbidity index (CCI), European LeukemiaNet (ELN) risk, type of therapy, serum ferritin (SF) and further baseline characteristics. Survival analysis was performed accordingly. RESULTS: Median age was 68 years and most patients were in good general condition. Median follow-up was 55.8 months. Of all patients, 66% received intensive chemotherapy (IC) +/- allogeneic hematopoietic stem cell transplantation (allo-HSCT). The remaining cohort received palliative chemotherapy (PC, 26%) or best supportive care only (BSC, 8%). Enrollment rate for interventional clinical trials was 26%. 5-year overall survival (OS) and relapse-free survival (RFS) were 18% (median 12.5 months) and 11,5% (median 10.0 months). Long-term survival was independently influenced by ECOG score, ELN risk group, baseline SF, previous myocardial infarction, and choice of therapy, but not consistently by age or CCI. Considering therapeutic subgroups, the contribution of particular parameters in predicting OS was most compelling in IC patients, but less consistent with PC or BSC. CONCLUSION: Our results provide thorough insights into prognostication within therapeutic subgroups and emphasize the need for more detailed prognostic algorithms and routine geriatric assessment in the treatment of older adults with AML.

Quantitative proteomics reveals specific metabolic features of acute myeloid leukemia stem cells.

Acute myeloid leukemia is characterized by the accumulation of clonal myeloid blast cells unable to differentiate into mature leukocytes. Chemotherapy induces remission in the majority of patients, but relapse rates are high and lead to poor clinical outcomes. Because this is primarily caused by chemotherapy-resistant leukemic stem cells (LSCs), it is essential to eradicate LSCs to improve patient survival. LSCs have predominantly been studied at the transcript level, thus information about posttranscriptionally regulated genes and associated networks is lacking. Here, we extend our previous report on LSC proteomes to healthy age-matched hematopoietic stem and progenitor cells (HSPCs) and correlate the proteomes to the corresponding transcriptomes. By comparing LSCs to leukemic blasts and healthy HSPCs, we validate candidate LSC markers and highlight novel and potentially targetable proteins that are absent or only lowly expressed in HSPCs. In addition, our data provide strong evidence that LSCs harbor a characteristic energy metabolism, adhesion molecule composition, as well as RNA-processing properties. Furthermore, correlating proteome and transcript data of the same individual samples highlights the strength of proteome analyses, which are particularly potent in detecting alterations in metabolic pathways. In summary, our study provides a comprehensive proteomic and transcriptomic characterization of functionally validated LSCs, blasts, and healthy HSPCs, representing a valuable resource helping to design LSC-directed therapies.

Allogeneic hematopoietic cell transplantation improves outcome of adults with t(6;9) acute myeloid leukemia: results from an international collaborative study.

Acute myeloid leukemia (AML) with t(6;9)(p22;q34) is a distinct entity accounting for 1-2% of AML cases. A substantial proportion of these patients have a concomitant FLT3-ITD. While outcomes are dismal with intensive chemotherapy, limited evidence suggests allogeneic hematopoietic cell transplantation (allo-HCT) may improve survival if performed early during first complete remission. We report on a cohort of 178 patients with t(6;9)(p22;q34) within an international, multicenter collaboration. Median age was 46 years (range: 16-76), AML was de novo in 88%, FLT3-ITD was present in 62%, and additional cytogenetic abnormalities in 21%. Complete remission was achieved in 81% (n=144), including 14 patients who received high-dose cytarabine after initial induction failure. With a median follow up of 5.43 years, estimated overall survival at five years was 38% (95%CI: 31-47%). Allo-HCT was performed in 117 (66%) patients, including 89 in first complete remission. Allo-HCT in first complete remission was associated with higher 5-year relapse-free and overall survival as compared to consolidation chemotherapy: 45% (95%CI: 35-59%) and 53% (95%CI: 42-66%) versus 7% (95%CI: 3-19%) and 23% (95%CI: 13-38%), respectively. For patients undergoing allo-HCT, there was no difference in overall survival rates at five years according to whether it was performed in first [53% (95%CI: 42-66%)], or second [58% (95%CI: 31-100%); n=10] complete remission or with active disease/relapse [54% (95%CI: 34-84%); n=18] (P=0.67). Neither FLT3-ITD nor additional chromosomal abnormalities impacted survival. In conclusion, outcomes of t(6;9)(p22;q34) AML are poor with chemotherapy, and can be substantially improved with allo-HCT.

Measurable residual disease monitoring in acute myeloid leukemia with t(8;21)(q22;q22.1): results from the AML Study Group.

We performed serial measurable residual disease (MRD) monitoring in bone marrow (BM) and peripheral blood (PB) samples of 155 intensively treated patients with RUNX1-RUNX1T1+ AML, using a qRT-PC-based assay with a sensitivity of up to 10-6. We assessed both reduction of RUNX1-RUNX1T1 transcript levels (TLs) and achievement of MRD negativity (MRD-) for impact on prognosis. Achievement of MR2.5 (>2.5 log reduction) after treatment cycle 1 and achievement of MR3.0 after treatment cycle 2 were significantly associated with a reduced risk of relapse (P = .034 and P = .028, respectively). After completion of therapy, achievement of MRD- in both BM and PB was an independent, favorable prognostic factor in cumulative incidence of relapse (4-year cumulative incidence relapse: BM, 17% vs 36%, P = .021; PB, 23% vs 55%, P = .001) and overall survival (4-year overall survival rate BM, 93% vs 70%, P = .007; PB, 87% vs 47%, P < .0001). Finally, during follow-up, serial qRT-PCR analyses allowed prediction of relapse in 77% of patients exceeding a cutoff value of 150 RUNX1-RUNX1T1 TLs in BM, and in 84% of patients exceeding a value of 50 RUNX1-RUNX1T1 TLs in PB. The KIT mutation was a significant factor predicting a lower CR rate and inferior outcome, but its prognostic impact was outweighed by RUNX1-RUNX1T1 TLs during treatment. Virtually all relapses occurred within 1 year after the end of treatment, with a very short latency from molecular to morphologic relapse, necessitating MRD assessment at short intervals during this time period. Based on our data, we propose a refined practical guideline for MRD assessment in RUNX1-RUNX1T1+ AML.

AML: high serum ferritin at initial diagnosis has a negative impact on long-term survival.

Increased serum ferritin (SF) is common in hematologic malignancies; however, its prognostic role in acute myeloid leukemia (AML) is not clearly established. We examined the impact of baseline SF on long-term survival in 137 intensively treated AML patients. Patients and baseline characteristics were retrieved from an AML database at Charité University Medical Center Berlin, Campus Virchow Clinic. After c-reactive protein (CRP)-based adjustment for inflammation, patients were grouped according to their baseline SF level. Survival analysis was performed accordingly. A significant decline in overall survival and relapse-free survival was observed in patients with high SF as compared to those with low SF. Furthermore, elevated baseline SF remained an independent poor prognostic factor within the multivariate analysis and was associated with a significant higher risk of relapse and non-relapse mortality (NRM). In conclusion, our data show that elevated baseline SF has a negative impact on long-term survival in intensively treated AML patients.