bluuebunny/crossref_metadata_2025 huggingface dataset:
Implementation of Clinical Pathways for CAR-T Cell Therapy in a Brazilian Private Healthcare Organization
Authors: Carvalho De Camargo Maria Fernanda FERNANDA , Santos Francisca VANOIDE DE BRITO , Seber Adriana , Rabello Chiattone Ricardo , Bigni Ricardo , Maiolino Angelo , Barroso Fernando , Coelho Erika , Silva Erica FRANCISCO , Chimentao Denise , Lobo Cecilia OLIVEIRA , Santos Elisangela , Lucas Ticiane , Lima Patricia BARROS , Abraao Ligia
Published: December 2023

Introduction: the treatment with CAR-T cells in Brazil is challenging because it requires a highly qualified multidisciplinary team trained in new clinical pathways to take care of these patients.

Objective: to describe the implementation of the clinical pathways for CAR-T cell therapy, ensuring safety and quality.

Method: this is an experience report study, performed in four private general hospitals of the Americas from São Paulo, Rio de Janeiro and Ceará states, during the year of 2022. The process of implementing the CAR-T Cell clinical pathway included: creation of a medical committee with the main medical references and specialist nurses from each center, for the elaboration of the main clinical protocols and operational procedures, in addition to the discussion of eligibility cases for therapy with CAR-T Cells. Identification of the multidisciplinary reference team in all phases of treatment, training the team involved in the clinical care of the patient, and finally, establishment of patient clinical pathway before, during and after the CAR-T cell therapy. The implementation process lasted seven months between the qualification of centers by the Therapy provider, internal qualification each center and the first cell infusion with CAR-T Cell performed.

Results: In all hospitals, professionals from the multidisciplinary team (pharmacy, nursing, physicians - hematologists, pediatric hematology-oncologists involved in hematopoietic stem cell transplantation, neurologists, pediatric and adult intensive care physicians) were trained. The Multidisciplinary Care Protocol was elaborated including indications, early and late complications. The Standard Operating Procedures addressed the pre and post infusion care, daily screening, criteria to transfer the patients to the intensive care unit, recognition and management of cytokine release syndrome and neurological toxicities. The main challenges of this process were to create the routines for evaluation and systematic monitoring and to identify and involve the multidisciplinary professionals directly involved in patient care.

Conclusions: the implementation of the program brought improvements to the clinical pathway, positively impacting professional satisfaction, the quality and safety of the patients. The CAR-T cell therapy was performed in a safe and integrated environment, uneventful and successful.
Ex-Vivo Testing Platform to Predict Clinical Response and Resistance to Proteosome Inhibitors, IMiDs and Daratumumab in Multiple Myeloma
Authors: Marin Constanza , Nicola Maribel , Andino Diego , Bernaschini Maria Laura , Conrrero Agustina , Ferreira Gimena , Piaggio Marisa , Remaggi Guillermina , Jarchum Maria Sol , Jarchum Gustavo , Meneces Bustillo Rodrigo , Rossi Blanca de los Milagros , García Juan José , Zaki Tarek Ali , Llorens Candelaria , Gatti Gerardo , Arroyo Daniela , Soria Gastón
Published: December 2023

Treatment of Multiple Myeloma (MM) relies on Standard of Care (SoC) schemes that include immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs) in combination with dexamethasone (Dexa). More recently, monoclonal antibodies such as Daratumumab (Dara) have become complementary therapeutic strategies to treat newly diagnosed and relapsed MM patients. Despite the multiple therapeutic schemes available, MM remains an incurable disease in which patients transit intermittent cycles of relapse and remission that ultimately lead to multi-drug resistance. It has therefore become clear that novel tools for personalized therapy selection are necessary to improve outcomes and reduce trial and error, particularly when selecting later lines of treatment.

At OncoPrecision, we have developed a personalized ex-vivo platform that mimics the tumor microenvironment and promotes the survival of Patient-Derived Cells (PDCs), with the goal to predict the performance of the complete array of FDA-approved drugs, as well as experimental treatments, within 7 days. This technology, which we have named Patient Micro Avatars (PMAs), consists of the co-culture of PDCs with engineered neoplastic (System Control) and stromal (Tox Control) heterologous cells in combination with a multi-tagging approach coupled to high-throughput flow cytometry (Figure: Upper Panel). Our PMAs not only enable us to rank the activity of all approved treatments, but also unveil potential unspecific toxicities of novel treatments on non-tumoral cells, thus unlocking unique insights for early drug development programs.

In this work, we introduce our PMA technology for MM by presenting differential response between patients in a Clinical Study performed in collaboration with two healthcare centers which, through approved IRBs, have provided MM patient samples from bone marrow aspiration. We present ex-vivo profiling results with a comprehensive MM drug matrix which includes SoC treatments used in several lines of therapy. We illustrate the potential of PMAs to identify differential activity from individual agents and synergistic drugs combinations in 13 patients (Figure: Lower Panel). By anticipating vulnerability/resistance to SoC treatments, our platform has the potential to become a first-in-class phenotypic biomarker to serve as a decision-support tool for Physicians to select optimal therapeutic regimes, as well as to elucidate valuable pre-clinical patient-derived insights for the development of innovative therapies for MM.
Rational Combinatorial Targeting By Adapter CAR T Cells (AdCAR-T) Prevents Antigen Escape in Acute Myeloid Leukemia (AML)
Authors: Atar Daniel , Ruoff Lara , Mast Anna-Sophia , Krost Simon , Moustafa-Oglou Moustafa , Scheuermann Sophia , Kristmann Beate , Feige Maximilian , Canak Aysegül , Wolsing Katrin , Schilbach Karin , Zekri Latifa , Ebinger Martin , Nixdorf Daniel , Subklewe Marion , Schulte Johannes , Lengerke Claudia , Werchau Niels , Mittelstaet Joerg , Lang Peter , Handgretinger Rupert , Schlegel Patrick , Seitz Christian Martin
Published: December 2023

Targeting AML by chimeric antigen receptor T cells (CAR-T) has been challenging due to promiscuous expression of AML-associated antigens on healthy hematopoietic stem and progenitor cells as well as a high degree of inter- and intratumoral heterogeneity. Consequently, we did not yet see the dramatic clinical successes as in B-phenotypic malignancies treated with CD19 or BCMA directed CAR-T.

In the present study, we analyzed inter- and intratumoral heterogeneity of AML-associated antigens in 30 primary pediatric AML samples at single cell resolution. We identified CD33, CD38, CD371, IL1RAP and CD123 to be most frequently expressed. Notably, high variability was not only observed across the different patient samples, but also among leukemic cells of the same patient, which indicates that multiplexed targeting approaches will be required to target all leukemic cells. To address this need, we utilized our modular Adapter CAR (AdCAR) platform enabling precise qualitative and quantitative control over CAR-T function. We show highly efficient and target specific activity for newly generated adapter molecules (AMs) against CD33, CD38, CD123, CD135 and CD371, both in vitro and in vivo. Importantly, we prove that inherent intratumoral heterogeneity in antigen expression translates into antigen escape and therapy failure to mono-targeted CAR-T therapy. For the first time, we demonstrate in a PDX model that rational combinatorial targeting by AdCAR-T can cure heterogenic disease.

In conclusion, we elucidate the clinical relevance of heterogeneity in antigen expression in pediatric AML and present a novel concept for precision immunotherapy by combinatorial targeting, utilizing the AdCAR platform.
A Complex Relationship between Clostridium Difficile Infection and Graft Versus Hoste Disease: Fundeni Clinical Institute Experience
Authors: Lipan Lavinia Eugenia , Enache Alexandra Oana , Colita Anca , Craciun Oana , Stefan Laura Diana , Tanase Alina Daniela
Published: December 2023

Introduction: Clostridium difficile infection (CDI) is a common complication in patients who have undergone hematopoietic stem cell transplantation (HSCT) due to prolonged hospitalization, prolonged neutropenia, use of broad-spectrum antibiotics, mucosal damage, changes in gut microbiota and compromised immunity. The incidence of CDI in patients undergoing allogeneic HSCT varies between 12 and 34% in the literature and has gradually increased in recent years in Europe North America due to the NAP1 strain. In the patient who has experienced allogeneic HSCT, diagnostic testing for CDI should include both nucleic acid amplification test (NAAT) and enzyme immunoassays (EIA) for toxins A and B, because detection of toxins A and B tests have a sensitivity of 75-80%. 5 In HSCT patients, CDI appears to be strongly associated with graft versus host disease (GVHD), gut GVHD and/or severe GVHD, but this has not been universally demonstrated.

The aim: For this reason, we wanted to see whether patients who developed a CDI between day 0 and day 100 were at higher risk for gut GVHD or for severe GVHD.

Results: Between January 2017 and August 2022, we performed 290 of allogeneic hematopoietic stem cell transplant procedures, in the Fundeni Clinical Institute. 50/290 developed CDI within the first 100 days after HSCT and before the diagnosis of intestinal GVHD, and 240/290 did not develop CDI. 16/50 (32%) of those with CDI developed acute GVHD, respectively 402/240 (43%) of those without CDI. From these 13/16, respectively 50/102 hade gut involvement. Of the 118 patients with acute GVHD, 13/16 in the group with CDI, respectively 30/102 in the group without CDI were assigned to severe acute GVDH (grade III and IV). Mortality due to gut GVHD in the group with CDI was 46%, respectively 14% in the group without CDI.

In conclusion: CDI is not a risk factor for GVHD, but gut damage is usually associated. CDI infection is associated with severe forms of GVHD, and the implication of the digestive tract in these patients increases mortality. Also, often in the patient with digestive GVHD, CDI can coexist, making them difficult to treat.
Adiponectin Promote TPO Response for Treating ITP By Relating Rab6A-myosin9 Trafficking Vehicle Carrying c-Mpl to Cell Surface
Authors: Zhao Xin , Ma Huixian , Li Guosheng , Sun Tao , Ji Chunyan
Published: December 2023

Impaired megakaryocyte maturation is one of the pathogenic mechanisms of primary immune thrombocytopenia (ITP). Exploring the regulatory mechanisms of megakaryocyte maturation is important for the clinical treatment of ITP. The thrombopoietin (TPO) receptor c-Mpl on the membrane surface of megakaryocytes plays a critical role in the cellular reception of TPO signals and megakaryocyte maturation. But how the distribution of c-Mpl on the membrane surface of megakaryocytes influences the pathogenesis of ITP and the specific regulatory mechanisms are still unclear.

In this project, we found that the distribution of c-Mpl on the membrane surface of megakaryocytes of ITP patients was significantly reduced, confirming that the abnormal distribution of c-Mpl is associated with the pathogenesis of ITP. Further, we applied immunoprecipitation and protein profiling to find the intracellular c-Mpl transport-related protein molecules. Motor protein myosin 9 and G protein-associated protein Rab6A were screened out. Next, we down-regulated the expression of myosin 9 and Rab6A, proved that aberrant expression of both leads to reduced transportation of the c-Mpl to membranes and impaired megakaryocyte maturation, identifying the regulatory complex for c-Mpl transport to membranes was composed of myosin 9 and Rab6A.

Moreover, we confirmed that the formation of the above complex is dependent on GTP activation of Rab6A by transient overexpression of wild-type Rab6A (wt), dominant-negative (T27N) and GTPase-deficient (Q72L) Rab6A, clarified the working mechanism of how the Rab6A-myosin9 trafficking vehicle regulate c-Mpl transport to membranes. At the same time, we demonstrated that the abnormally low expression of Adiponectin in the plasma of ITP patients affects the transportation of the c-Mpl to membranes by inhibiting the interaction between Rab6A and myosin 9, thereby reducing the responsiveness of megakaryocytes to TPO and inhibiting the activation of its corresponding intracellular signaling pathways.

Finally, using a mouse model of ITP disease, we demonstrated that Adiponectin could mediate the transportation of the c-Mpl to membranes by regulating the Rab6A-myosin9 complex and can improve the efficacy of TPO in the treatment of ITP. In conclusion, the present study identified a novel trafficking complex mediating the transportation of the c-Mpl to membranes, clarified the abnormal mechanism of this complex in ITP, and confirms that Adiponectin can regulate this complex to improve the efficacy of TPO, which provides a new idea for the clinical treatment of ITP.
Post-Marketing Observational Study of the Safety and Efficacy of Fibrinogen Concentrate in Congenital Fibrinogen Deficiency in Patients of All Ages
Authors: Peyvandi Flora , Pezeshki Gita , Solomon Cristina
Published: December 2023

Background and Significance: Congenital fibrinogen deficiency (CFD) is a rare coagulation disorder characterized by a lack or low levels of functional fibrinogen leading to inadequate clot formation. Patients with CFD may experience severe or frequent bleeding episodes (BEs), either spontaneously or following trauma or surgery. Human fibrinogen concentrate (HFC; Fibryga® [Octapharma]) can restore hemostasis, and its efficacy and safety has been demonstrated in interventional clinical studies of adult and pediatric patients. The primary objective of this planned observational study is to collect real-world evidence to further assess the safety profile of this HFC, in patients of all ages with congenital fibrinogen deficiency, by characterizing the risks of thrombosis and anaphylaxis following its administration for treatment of bleeding (Figure 1). The observational data collected in this study will provide a more comprehensive safety profile of this HFC in patients of all ages following its marketing authorization.

Study Design and Methods: FORMA-07 (NCT03793426) is a prospective, non-interventional, multicenter, observational, post-marketing study. Patients of all ages with a documented diagnosis of CFD (i.e., congenital afibrinogenemia or hypofibrinogenemia) requiring on demand HFC for BE treatment are eligible for inclusion (Table 1). Exclusion criteria include other coagulation disorders, history of anti-fibrinogen inhibitors, and participation in concurrent clinical studies. This study will examine a highly purified, plasma derived, double virus inactivated/eliminated, lyophilized HFC, which will be used according to individual country approval and labelling, and individually dosed as per the locally approved package insert. Independent ethical committee approval will be sought prior to study start and freely given, written informed consent obtained from all participants, or legal guardians in the case of pediatric patients. This study will be conducted in accordance with the ethical principles laid down in the Declaration of Helsinki. The primary endpoint is the incidence of thromboembolic adverse drug reactions (ADRs) . Serious ADRs, ADRs of special interest (including thromboembolic events and anaphylaxis), and thromboembolic monitoring using the Wells Score will be documented. Hemostatic efficacy will be assessed as a secondary endpoint by the study investigator using a robust 4-point objective scale from 2 to 24 hours following treatment.

FORMA-07 is ongoing and actively enrolling patients of all ages with CFD. The study aims to enroll ~25 patients over 7 years, collecting data from 105 BEs (≥10 major BEs in 10 patients). Results of HFC safety and efficacy will be continually monitored throughout the study. Completion is expected Q4 2027.
The Plasma Proteomic Landscape of Thrombotic Events across Multi-Center Cohorts
Authors: Kreft Iris charlotte , Smit Eva , Camilleri Eleonora , Burggraaf Louise , Van Rein Nienke , Van Vlijmen Bart , Hulshof Anne-Marije , Van Bussel Bas , van Rosmalen Frank , van den Berg Tom , Henskens Yvonne , Ten Cate Hugo , Coutinho Jonathan , Kruip Marieke , Eikenboom Jeroen , Hoogendijk Arie , Cannegieter Suzanne , Van Den Biggelaar Maartje
Published: December 2023

Background: Diagnosis and risk prediction of thrombosis is challenging due to nonspecific symptoms, diverse risk factors, symptom overlap with other conditions and the variability in thrombosis presentations. Little is known on the impact of thrombosis on the plasma proteome across the spectrum of its clinical representations.

Objective: The aim of this study was to explore the plasma proteomic landscape across multi-center thrombosis cohorts. The focus was on identifying protein signatures representative of thrombosis in general or among patients at risk of thrombosis, those with a history of thrombosis, those at acute phase and those with COVID-19 associated thrombosis.

Method: Using state-of-the-art mass spectrometry-based proteomics, we analyzed 420 plasma proteome samples both cross-sectional and longitudinal from 211 patients, including those at risk of thrombosis, in the acute thrombotic phase (cerebral venous sinus thrombosis patients) and with COVID-19-associated thrombosis (Figure 1). Linear models and unsupervised co-expression clustering were used to explore common and distinct protein signatures among the cohorts.

Results: Across the analyzed samples 475 proteins were quantified, with 86% showing a CV below 30%, and we found a strong correlation between protein levels quantified by MS and ELISA. Plasma proteomic alterations exhibited variation across cohorts, with the most pronounced changes observed in COVID-19 patients. While co-expression analysis unveiled distinct protein clusters associated with SARS-CoV-2 signaling pathways (CRP, SAA1) and Vitamin K-dependent (VKA) coagulation proteins (F9, F2, F10, PROS, PROC, PROZ, C4BPA), we did not identify a specific protein cluster related to thrombosis (Figure 2). Notably, VKA-therapy was associated with decreased abundance of VKA-dependent coagulation proteins, with F10 and PROZ showing significant alterations between patients who experienced a thrombosis compared to healthy controls. We observed an inverse correlation between the International Normalized Ratio and abundance of VKA-dependent coagulation proteins, with an increased correlation of F2 and F9 in patients who experienced a thrombotic event. Additionally, our analysis also highlighted potential variations in multi-center cohorts during sample handling, emphasizing the importance of standardized protocols in ensuring the reliability and comparability of results.

Conclusion: Using a comprehensive MS-based proteomics approach, we identified clear plasma proteomic signatures associated with VKA-treatment and COVID-19 , but no distinct thrombosis signature across multi-center thrombosis cohorts.
ABO-Incompatible Platelet Transfusion Is Associated with Ischemic Lesions on Brain MRI in Patients with Intracerebral Hemorrhage
Authors: Carvalho Poyraz Fernanda , Ridha Mohamed , Simonetto Marialaura , Kumar Aditya , Hess Evan , Magid-Bernstein Jessica , Connolly E. Sander , Ghoshal Shivani , Agarwal Sachin , Park Soojin , Claassen Jan , Hod Eldad A. , Roh David
Published: December 2023

Background: We have identified that major ABO-incompatible platelet transfusions are associated with poor intracerebral hemorrhage (ICH) outcomes. The underlying drivers for the relationship of these incompatible platelet units on outcome are unknown, yet do not appear to be related to impaired hemostasis. Conversely, ICH patients are known to encounter remote, “silent” ischemic lesions, thought to be from microthrombosis, during their hospitalization. Though these ischemic lesions are well known to associate with poor ICH outcomes, risk factors for its formation are unknown. We explored whether major ABO-incompatible platelet transfusions are associated with acute ischemic lesions on brain MRI during an ICH hospitalization.

Methods: Consecutive spontaneous ICH patients enrolled into a single-center, prospective cohort study between 2009 and 2018 were assessed. Patients who received a single platelet transfusion within 24 hours of ICH admission and had a brain MRI during the hospitalization were included in the analysis. Major ABO incompatible platelet transfusion (vs minor mismatch/identical) was defined as the exposure. Presence of remote ischemic lesions, defined as diffusion restriction >10 mm away from the hematoma detected on brain MRI, was the outcome. Regression models were used to assess relationships between major ABO-incompatible platelet unit exposure and acute ischemic brain lesions, adjusting for time from ICH onset to MRI or ICH severity.

Results: We identified 40 ICH patients receiving an acute platelet transfusion and having a brain MRI performed during the ICH hospitalization. The mean age was 67.1 (SD 14.1), 37.5% were female, and 20% received a major ABO incompatible platelet unit. Patients receiving a major incompatible platelet unit were more likely to encounter ischemic lesions (62.5% vs 21.9%). We also identified an association of major ABO-incompatible platelets units with acute ischemic brain lesions in our regression models after adjusting for time from ICH symptom onset to MRI (OR 5.45, 95%CI 1.01-29.30, p=0.048) and ICH severity (OR 9.15, 95%CI 1.37-62.67, p=0.024). Additional sensitivity analysis adjusting for patient blood type did not alter this relationship (OR 17.1, 95%CI 1.34-217.9, p = 0.029).

Conclusions: Among ICH patients receiving acute platelet transfusions and MRI neuroimaging, major ABO incompatible units were associated with remote ischemic lesions on brain MRI. Further work is needed to address the generalizability of our data and to clarify relationships of platelet unit characteristics, cerebral microthrombosis, and clinical outcomes after ICH.
High Expression Level of MMP7 and Mir-489-3p/MMP7 Axis Associates with Venetoclax Resistance in Acute Myelogenous Leukemia
Authors: Wang Li , Wang Jishi
Published: December 2023

The BCL2 inhibitor venetoclax used to treat acute myelogenous leukemia (AML). Although this has been a promising therapeutic option for patients with AML, many of these patients develop resistance and relapsed disease. Matrix metalloproteinases (MMPs) were demonstrated to have important implications in the progression and invasiveness of many malignant disorders. In contrast, the biological significance of these molecules in AML is not clear. We determined the levels of MMP-7 in the bone marrow of 76 patients with AML. 70 bone marrow donors served as normal controls. The level of MMP-7 was significantly higher in the AML than in the normal controls. MMP-7 levels were significantly lower in the AML patients who achieved a complete remission (CR) than in those who did not. Through in vitro studies, we observed that MMP7 levels were elevated in AML cell lines (U937, THP-1) and in Venetoclax-resistant AML cell lines (U937R, THP-1R) and that downregulation of MMP7 could enhance Venetoclax efficacy in U937R and THP-1R cells. Our in vitro and in vivo investigations show that miR-489-3p was downregulated in Venetoclax-resistant AML cell lines and promoting level of miR-489-3p expression can directly suppressing MMP-7 expression. Pharmacological inhibition of miR-489-3p markedly reversed the effects of Venetoclax-resistant upon AML proliferation and metastasis. Thus, our study suggests that MMP-7 is an essential factor in AML, and regulator the miR-489-3p/MMP7 axis may be a novel treatment strategy for overcoming Venetoclax resistance in AML.
Deep Immunophenotyping Using High Complexity Flow Cytometry in Acute Myeloid Leukemia
Authors: Pearl Jessica , Bruni Elena , Hsu Haoting , Walling Brandon , Schiavini Paolo , Ritchie Michael , Heidemann Karin abarca , Mancini Maria
Published: December 2023

Acute Myeloid Leukemia (AML) is a highly heterogenous disease with a diverse range of abnormal myeloid subpopulations resulting in challenges in clinical diagnosis and treatment of disease. While sequencing may provide precise information about mutations, the turnaround times make it impractical for routine tracking of disease progression. In contrast, flow cytometry utilizes the expression patterns of cellular markers to determine AML subpopulations with high accuracy. This, coupled with the limited turnaround time, makes flow cytometry an excellent tool for the identification and tracking of disease progression. AML lineage characterization and disease progression are found to be a predictor of both residual disease and the likelihood of relapse further emphasizing the need for effective and accurate characterization of AML populations.

Champions Oncology has developed a high-complexity 18-color flow cytometry panel to identify AML subpopulations at high resolution, named PhenoSeek AML ®. Clinical samples may be shipped to a central location for processing which requires sample populations to be retained during transit. Utilizing Champions' bank of primary AML samples, we have evaluated the stability of key AML subpopulations and found stability to be retained for up to 72 hours after collection. In addition to sample stability, assessing the lower limit of detection is critical for minimum residual disease and relapse screening. Flow cytometry provides an excellent tool for these tests due to its single cell nature, which allows for the interrogation of individual populations with as little as hundreds of cells. Indeed, the dilution of primary AML subpopulations was detected with as little as 10 3-10 4 cells.

Finally, utilizing Champions' cryopreserved never-passaged AML models characterized by both flow cytometry and sequencing, we investigated how the cryopreservation processes impacted the expression of markers associated with specific AML subpopulations. We observed similar expression and detection of AML populations following cryopreservation indicating that these models can be utilized as an excellent tool for further studies.

Collectively, our data indicates that the panel developed at Champions Oncology provides robust detection and deconvolution of AML subpopulations and can be an excellent tool for the tracking of AML disease progression.
Outcomes of Newly Diagnosed Hodgkin Lymphoma Patients in 4 Academic Centers in Paraguay. the Impact to Access to PET-CT Scanning in Survival
Authors: Von Glasenapp Seisha Alana , Enciso Arrua Maria Elvira , Quiroz Alfredo , Paats Aline Nicole , Morel Laura , Jiménez Leticia Rocio , Andino Lidiane , Salinas Victor Ladislao , Valcarcel Bryan , Malpica Castillo Luis Enrique
Published: December 2023

Background: Hodgkin's lymphoma (HL) is a highly curable lymphoproliferative neoplasm. Assessment of HL at baseline (pre-therapy), intermediate (iPET) and end-of-therapy (EOT) by positron emission tomography/computed tomography (PET/CT) is considered a “gold standard” procedure and has become the main tool for clinical decision making in the management of HL. However, inequity in access to diagnostic medical technology in Paraguay has not allowed clinicians to manage these patients as recommended by international guidelines. We describe here the clinical characteristics, access to PET/CT study and outcomes of patients with HL treated in Paraguay.

Methods: We conducted a retrospective cohort study of patients aged ≥17 years with newly diagnosed HL in 4 academic centers in Paraguay between 2015 and 2022, with follow-up until July 2023. Medical records were manually reviewed and data were abstracted in a standardized form. A historical analysis was performed to evaluate the utilization of PET/CT in all stages of HL treatment (baseline, intermediate and EOT). Survival probabilities were estimated using the Kaplan-Meier method.

Results: A total of 145 patients were identified; 101 had sufficient data for analysis (Table 1). Patients were young, with a median age of 29 years (16-79) and a slight male vs. female predominance (56.4% vs. 43.6%). Nodular sclerosis (76.6%) and mixed cellularity (20.2%) were the most frequent HL subtypes. More than half of the patients (58%) had ECOG 0, 50% advanced stage and 55.9% B symptoms. The majority of patients (97%) received an ABVD regimen as induction therapy. Sixty-nine percent of patients did not undergo PET/CT at baseline, most of them (97%) due to lack of insurance coverage, 16.5% underwent iPET and 58.4% underwent PET/CT at EOT. With a median follow-up of 44 months, overall survival (OS) at 4 years was 77% and progression-free survival (PFS) was 46%. In a subgroup analysis to investigate the short 4-year PFS observed in our cohort, the PFS of patients who underwent PET/CT at EOT versus those who did not was 84 versus 36, respectively (Figure 1).

Conclusions: To our knowledge, this is the first study to characterize the clinical characteristics and outcomes of patients with HL in Paraguay. It is striking to see that lack of access to PET/CT had a significant impact on PFS in our patient population. HL is a highly curable disease affecting young people, therefore, minimizing late side effects in this patient population (e.g., secondary comorbidities and malignancies) remains a high priority. The authors' conclusion is to raise awareness of the importance of improving access to diagnostic tools such as PET/CT to provide optimal care for our patients, especially those who do not have access to PET/CT.
Assessment of Ultra-Deep Mass Spectrometry Based Proteomics Compared to Flow Cytometry and RNA-Based Methods for the Discovery and Validation of Therapeutic Targets in Immune Cells
Authors: Au Yu-Wah , Rigter Gemma , Yu Wingyan , Satijn David , Mehnert Martin , Lachaud Amaury , Vowinckel Jakob , Neijssen Joost , Bon Helene
Published: December 2023

Immuno-oncology (IO) has substantially improved the survival of cancer patients over the past several years encouraging the discovery of novel IO targets which are typically proteins expressed on the surface of immune cells. Sensitive quantification of proteins in complex biological samples is routinely achieved by immunoassays that use antibodies specific to target proteins. Such approaches can be a limitation in IO drug discovery since the development of de novo antibodies is associated with long lead times, high costs, and high failure rates, often resulting in the substitution of the protein readout by a surrogate transcript readout using RNA sequencing methods.

Protein quantification using mass spectrometry (MS) is agnostic to any capture agent and removes the barriers of availability or specificity of antibody-based assays. Furthermore, MS proteomics workflows can support large scale discovery studies with wide proteome coverage, and also represent an attractive antibody-free alternative in targeted quantitative studies for protein measurement.

The main purpose of this work was primarily to assess the sensitivity of DIA-based MS-proteomics platform for the deep proteome profiling of human primary immune cells by measuring the maximum protein coverage obtained across cell inputs ranging from 2 million down to single cells ( performance assessment). The secondary purpose of this work was to assess the accuracy of the quantification of targeted MS proteomics workflow in comparison with the gold standard QIFI® flow cytometry approach, and other methods using mRNA readouts in immortalized human hematological cell lines ( accuracy assessment).

We found that our MS-based proteomics workflows achieve high sensitivity in detection of immune cell markers down to few immune cells as input. Additionally, we report a strong correlation of the quantitative data derived from our MS-based proteomics workflows compared to the flow cytometry gold standard (r 2= 0.7354). Notably, we observed that this correlation was significantly higher than using transcriptomics approaches, emphasizing the benefit of substituting immunoassays for MS-based proteomics platforms in IO target discovery and validation.
Transcription Factor Redistributors Pharmacologically Actuate Non-Canonical Gene Networks to Drive AML Differentiation
Authors: Taylor Samuel , Stauber Jacob , Bohorquez Oliver , Schwenger Emily , Sundaravel Sriram , Farahat Abdelbasset , Bartholdy Boris , Boykin David , Poon Gregory , Steidl Ulrich
Published: December 2023

Aberrant transcriptional networks are hallmarks of aging and cancer, yet our ability to target these aberrations is poor. PU.1 is one such transcription factor (TF) who's transcriptional networks are corrupted in disease, including in >50% of Acute Myeloid Leukemia (AML) cases. In this study we investigate an unappreciated pharmacological approach to target the aberrant PU.1 network, by employing novel small molecules which competitively inhibit PU.1:DNA interactions.

We performed an extensive multiomics-driven, molecular characterization of AML cells following exposure to PU.1-DNA binding inhibitors (e.g. DB2115). Unexpectedly, we found that such compounds not only led to a reduction of many canonical PU.1 transcripts, but also (seemingly paradoxically) caused increases in alternative PU.1-driven transcripts. We discovered that this two-sided response was a result of robust cistromic repositioning of PU.1 chromatin binding rather than global inhibition. From kinetic CUT&Tag and ATAC sequencing studies, we identified that PU.1 redistribution involved rapid PU.1 loss (~1hr) followed by delayed PU.1 gain (~4-12hrs), causing subsequent opening of the chromatin, thus demonstrating endogenous PU.1's powerful pioneering ability. Through development of CLICK-on-CUT&Tag, we identified exclusive drug binding at displaced PU.1 binding sites, and identified unique, sequence-specific ETS motifs dictating PU.1 gain or loss. By employing these motifs in eGFP reporter assays and CRISPRd systems, we identified direct PU.1-mediated transcriptional control of known and novel functionally relevant genes, including MYC, POMP and STRAP. Finally, we establish that primary AML samples are sensitive to pharmacological PU.1 redistribution upon exposure to DB2115, and the rewiring of the PU.1 network in these cells drives differentiation down the myeloid lineage (Figure 1).

Overall, we uncover a novel biological phenomenon we describe as: Pharmacological transcription factor (TF) redistribution. “TF Redistributors”, such as DB2115 and next generation derivatives, are an unprecedented investigative and pharmacological tool which allow the study of complex and fast transcription factor dynamics without disrupting the structure or levels of the TF itself. Furthermore, we discover that pharmacological binding-site restriction and consecutive repositioning of the PU.1 network, cascades into an extensive rewiring of transcriptional circuits which ultimately drives myeloid differentiation of AML cells, potentially providing a novel therapeutic intervention for PU.1-corrupted AML.
Efficacy and Safety of the Second CAR-T Therapy in Patients with Refractory/Relapsed Acute B-Cell Lymphoblastic Leukemia
Authors: Liu Sining , Cui Qingya , Li Zheng , Cui Wei , Li Mengyun , Qiu Huiying , Xue Shengli , Chen Suning , Jin Zhengming , Miao Miao , Han Yue , Wang Ying , Zhu Xiaming , Yu Lei , Wu Depei , Tang Xiaowen
Published: December 2023

Background: Chimeric antigen receptor T (CAR-T) cells therapy has shown significant efficacy in relapsed or refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), but a substantial proportion of patients still relapse after a period of remission. The efficacy of a second CAR-T cell infusion is uncertain. The aim of this study was to explore the efficacy and safety of the second CAR-T therapy in R/R B-ALL patients.

Methods: Between August 2018 and October 2022, 35 patients with R/R B-ALL successfully received the second CAR-T treatment. Among them, 18 patients received single CD19 or CD22 CAR-T therapy, 14 patients received tandem CD19/CD22 CAR-T therapy, and 3 patients received sequential CD19 and CD22 CAR-T therapy.

Results: The complete remission (CR) rates were 55.6% (10/18) in patients who received CD19 or CD22 CAR-T therapy, 85.7% (12/14) in patients who received tandem CD19/CD22 CAR-T therapy, and 33.3% (1/3) in patients who received sequential CD19 and CD22 CAR-T therapy (tandem CD19/CD22 vs. others, P=0.045). Tandem CD19/CD22 CAR-T therapy remained one of the significant favorable factors in multivariate logistic regression analysis of CR rate in all patients (hazard ratio, 0.128; 95% CI, 0.017-0.974). A total of 5 patients with severe cytokine release syndrome (Grade ≥ 3) were observed, including 2 patients in the single CD19 CAR-T group, 2 patients in the tandem CD19/CD22 CAR-T group, and 1 patient in the sequential CD19 and CD22 CAR-T group. Of the 23 patients who achieved CR, 4 patients bridged to allogeneic hematopoietic stem cell transplantation (allo-HSCT) and 3 patients received decitabine (DAC) consolidation.With the median follow-up of 25.5 months (range, 1.1 to 33.5), the 2-year overall survival, leukemia-free survival (LFS) and cumulative incidence of relapse rates were 23.7%, 30.5% and 69.5%, respectively. Multivariate Cox regression analyses showed that a better LFS related to the absence of high-risk cytogenetics and genetic characteristics, DAC combination with fludarabine and cyclophosphamide lymphodepletion regimen, and bridging allo-HSCT or DAC consolidation.

Conclusions: Our study showed that the second infusion of tandem CD19/CD22 CAR-T therapy obtains a better response than other infusion strategies. Bridging allo-HSCT or DAC consolidation had a significant survival benefit in patients with CR after the second CAR-T therapy.
Evaluating the Impact of Prophylactic Intrathecal Chemotherapy in Diffuse Large B Cell Lymphoma (DLBCL) Patients with High Central Nervous System International Prognostic Index (CNS-IPI) Scores: A Comprehensive Review and Meta-Analysis
Authors: Al-Banaa Kadhim , Alshaikhli Alfarooq , Poudel Shyam , Shrotria Shiva , Wang Can , Hanona Paul , Gopishetty Swathi , Idogun Precious , Ezekwudo Daniel , Jaiyesimi Ishmael
Published: December 2023

Introduction

Diffuse large B cell lymphoma (DLBCL) with a high CNS-IPI score poses an increased risk for relapse involving the central nervous system (CNS). Despite various studies seeking to validate the use of CNS prophylaxis, including intrathecal chemotherapy, results have been equivocal. This ambiguity may be partly attributed to the lack of prospective data, patient heterogeneity, limited sample sizes, and outdated methodologies in pre-rituximab studies. Consequently, administering CNS prophylaxis with intrathecal chemotherapy is not a standardized practice, and decisions are often based on individual factors such as anatomical location, disease stage, and specific case characteristics.

Methods

This systematic review and meta-analysis were conducted and reported using the PRISMA guidelines. A thorough search was conducted across multiple online databases, including Google Scholar, Science Direct, PubMed, Cochrane, CINAHL, EMBASE, ProQuest Dissertation and Theses, ISI web of knowledge, PsycINFO, and Embase for articles on this topic. The date range of the articles included was between 2013-2023 and were limited to English language, peer-reviewed articles, cohort studies, retrospective studies & randomized controlled trials. The inclusion and exclusion criteria for studies helped to determine suitable studies

Results

Out of 179 studies retrieved from the database search and 2 from other studies' reference lists, 6 met the inclusion criteria for qualitative and quantitative analysis. Data from 2209 patients in these 6 studies was analyzed using a random-effects model to perform a subgroup analysis of the benefits and risks of prophylactic intrathecal chemotherapy for patients with DLBCL and a high CNS-IPI score. The results demonstrated a significant improvement in overall treatment success, with a difference of 0.60 (95% Confidence Interval, 0.45 to 0.93) on a 0-10 Visual Analogue Scale (VAS). The heterogeneity of the studies was determined to be significant (P=0.02), further substantiating the positive impact of prophylactic intrathecal chemotherapy on the therapy success rate for DLBCL patients with high CNS-IPI scores.

Conclusion

Our review elucidates that intrathecal chemotherapy as a prophylactic measure can reduce the rate of CNS relapse and enhance overall survival for patients with DLBCL possessing a high CNS-IPI score. However, it underscores the necessity to thoroughly assess each patient's unique clinical situation when determining if intrathecal chemotherapy is the appropriate therapeutic approach.
VISTA a Potential New Immuno-Oncology Target in Acute Myeloid Leukemia
Authors: Guillaudeux Thierry , Sridhar Shaarwari , Frazier Emily , Ovechkina Yulia , Iadonato Shawn
Published: December 2023

V-domain Immunoglobulin Suppressor of T cell Activation (VISTA/PD-1H) is a B7 family member highly expressed on circulating and intra-tumoral myeloid cells. VISTA is a negative checkpoint protein that inhibits anti-tumor T cell responses. In multiple solid tumors, VISTA expression is associated with poor overall survival and is also described as a potential mediator of resistance to anti-CLTA-4 and anti-PD1 therapies. VISTA expression was also investigated in hematological tumors and described as highly expressed on blasts including leukemic stem cells from AML patients. Therefore, VISTA is a unique and potentially new target for cancer immunotherapy in this indication.

Kineta has developed a fully human monoclonal antibody targeting VISTA, KVA12123, currently evaluated in a Phase 1/2 clinical trial alone and in combination with pembrolizumab in cancer patients with advanced solid tumors. We also believe that VISTA could be a promising target for AML patients. Therefore, we have tested in vivo, in a Kasumi-3 AML disseminated tumor model, the efficacy of our antibody alone or in combination with standard of care therapy, cytarabine and doxorubicin. Like leukemic cells in AML patients, the Kasumi-3 cell line exhibits a high level of VISTA expression on its cell surface. Female NOD-scid mice were first inoculated with Kasumi cells via tail vein injection, and tumor engraftment was evaluated at different time points in the blood, spleen and bone marrow to monitor engraftment using CD34, CD33, CD45 and HLA-DR cell surface expression markers. Then, when tumor burden was high, standard of care treatment (5 doses of 16mg/kg AraC by IV injections and 3 doses of 0.5mg/kg Dox by IV injections) was initiated in indicated groups for 5 cycles, before starting treatment with KVA12 for the rest of the study. Tumor engraftment was tested after chemotherapy alone to evaluate the efficacy before starting treatment with our antibody. Our results showed that untreated mice had higher number of Kasumi-3 cells in the blood when compared to chemotherapy treated mice. Two backbone versions of our antibody were then tested, a human IgG1 - KVA12123 and a human IgG4 - KVA12402, at 30mg/kg IP injections twice a week for 5 weeks. Our data showed that KVA12123 and KVA12402 as single agents and in combination with chemotherapy reduced Kasumi-3 tumor load in the blood, spleen and bone marrow within the groups and significantly improved survival when compared to IgG1 control groups.

These data all together indicate that anti-VISTA targeted therapy should be evaluated in myeloproliferative disorders like AML alone or in combination with standard of care.
In Vivo Generation of Functional CD19 CAR-T Cells Using a Serum-Stable CD3-Targeted Lentiviral Vector
Authors: Krotova Karina , Nenavath Gopal Naik , Packiriswamy Nandakumar , Vandergaast Rianna , Moy Melissa , Ziegler Christopher , Muñoz Alía Miguel A , Peng Kah-Whye , Russell Stephen
Published: December 2023

The high cost and complexity of CAR-T therapy has spurred efforts to generate CAR-T cells directly within patients via systemic infusion of serum-stable lentiviral vector particles that target T cells in situ for CAR transgene delivery. To ensure effective in vivo delivery, these vectors need to show resistance to neutralization by human serum, demonstrate transduction specificity for T cells, and support subsequent expansion of CAR-T cells.

Lentiviral vectors (LV) pseudotyped with VSV G-glycoprotein (G-WT) transduce target cells via the LDL receptor (LDLR), which is ubiquitously expressed at variable levels and is upregulated on activated T cells. G-WT LVs are widely used to generate therapeutic CAR-T cells ex vivo, following purification and ex vivo activation of the T cells.

To generate LVs capable of targeting circulating T cells in vivo, we utilized rational engineering of the VSV-G glycoprotein to design a G-CD3. This modified G protein avoids LDLR recognition, incorporates a CD3-specific single-chain antibody for targeted recognition of T cells, and minimizes LV particle inactivation upon exposure to human serum. G-CD3 LVs were shown to target and transduce resting T cells in peripheral blood mononuclear cell (PBMC) cultures with very high efficiency. The G-CD3 LVs powerfully activated T cells, evident from their increased CD25 expression, and the infection was not hindered in the presence of fresh, complement-active human serum (25%). Additionally, G-CD3 LVs efficiently transduced T cells in fresh human whole blood without apparent off-target transduction of non-T cell lineages.

In vivo evaluation of G-CD3 LV efficiency was performed in humanized NSG mice by intravenous (IV) or intraperitoneal (IP) delivery of G-CD3 LVs encoding anti-CD19 CAR and GFP transgenes. Both IV and IP delivery of G-CD3 LV led to in vivo CAR T cell generation with peak levels of 17% of total circulating human CD45+ cells on day 21 post vector administration. The in vivo generated CD19-CAR-T cells were fully functional as evidenced by the disappearance of endogenous human CD19+ B cells from peripheral blood. Tissue analysis of spleen, bone marrow, and liver at the time of sacrifice (day 39 post LV injection), showed anti-CD19 CAR-T in all three tissues accompanied by an absence or dramatic reduction in tissue-resident human CD19+ B cells. In contrast, CAR-T cells were not detected at any time point in humanized NSG mice injected with G-WT LV, regardless of whether the virus was administered IV or IP.

In summary, these data highlight the potential applicability of fully retargeted, serum stable LVs for in vivo gene delivery or genome editing, offering promising prospects for future human gene therapy applications.