Inferior Response Research Articles

Pyroelectric-Effect-Assisted Near-Infrared-Driven Photoelectrochemical Biosensor Based on Exponential DNA Amplifier for MicroRNA Detection.

Although near-infrared responsive photoelectrochemical (PEC) biosensors have less damage to biological components compared to UV-visible light, they still reveal an inferior response due to the rapid recombination of photogenerated electron-hole. In this study, a near-infrared-driven PEC biosensor is fabricated for microRNA (miRNA) detection via integrating photoelectricity and pyroelectricity. Upon the introduction of target miRNA-21, the exponential DNA amplifier is triggered based on enzyme-assisted strand displacement amplification (SDA), releasing multiple Ag2S reporter probes to hybridize with capture probes immobilized on a CdS-2-mercaptobenzimidazole (2MBI)-modified photoelectrode. As a result, under the stimulation of NIR, the photoelectric conversion of Ag2S NPs generates the photocurrents. In addition, due to the strong hole acceptor ability of MBI, the pyroelectric effect of CdS-2MBI nanocomposites is enhanced, which generates highly pyroelectro-induced charge separation efficiency and induces the pyroelectric current benefited from the spontaneous polarization of CdS-2MBI caused by the temperature variation under the function of Ag2S nanoheaters. Impressively, this PEC biosensor has achieved the sensitive and selective determination of miRNA-21 with a detection limit as low as 54 fM. Overall, this NIR-driven PEC biosensor based on pyroelectric and photoelectric effects opens up a new horizon for bioanalysis and early disease diagnosis.

Aniline oligomer-assisted exfoliation of MoS2 nanosheets for high-performance room-temperature NO2 gas sensing

Two-dimensional transition sulfides (TMDs) are ideal materials for room-temperature gas sensing due to their high carrier mobility at room temperature. Nevertheless, the inferior response and incomplete recovery of TMDs at room temperature hinder their development in the field of the most advanced gas sensors. Herein, we propose two-dimensional surface p-p heterojunctions constructed via organic conjugated molecules assisted stripping of molybdenum disulfide (MoS2) for high-performance room-temperature nitrogen dioxide (NO2) gas sensing. The aniline tetramer (TANI) was employed as an exfoliation-assistant reagent, adhering evenly to the surface of the exfoliated MoS2 nanosheets, and forming a good MoS2/TANI heterostructure. The sensor built with this heterostructure has a 28.5% response to 1 ppm NO2 at room temperature, outperforming a standalone MoS2 sensor by more than fourfold. The device still has a significant response even at NO2 concentrations as low as 30 ppb. Furthermore, the MoS2/TANI-3 heterostructure gas sensor also exhibits excellent repeatability, long-term stability, superior linearity, and good selectivity at room temperature. Above all, the proposed design technique, as well as the developed structures, can pave the way for the development of high-performance TMDs-based gas sensors.

The effect of post-covid syndrome on the cytokine cascade in patients with type 2 diabetes mellitus

With the increasing prevalence of type 2 diabetes, it is of interest to study the characteristics of the immunological status in this category of patients. According to modern data, in type 2 diabetes, a pro-inflammatory type of immune response is formed with an imbalance between T-helper 1 (Th1), T-helper 2 (Th2), T-helper 17 (Th17) types and T-regulatory cells (Treg). The COVID-19 pandemic makes a certain contribution to changes in the immune response in patients with type 2 diabetes mellitus, since in patients who have had coronavirus infection, significant changes in the composition of circulating immune cells are observed: the proportion of Th17 increases and the number of Tregs decreases. This study is devoted to identifying the features of the cytokine cascade in patients with type 2 diabetes mellitus, depending on the presence of a confirmed history of SARS-CoV-2 infection. The study included 75 patients with type 2 diabetes, in 36 of them the diagnosis was verified in the post-Covid period. The study was conducted at least 6 months after pneumonia caused by SARS-CoV-2 infection. All study participants signed informed consent. The study was approved by the Independent Local Ethics Committee at the State Autonomous Institution of Regional Clinical Hospital “City Clinical Hospital No. 1” of Chelyabinsk, protocol No. 8 dated 04/11/2022, on the basis of which these studies were conducted. Using the multiplex assessment method, the concentrations of 15 cytokines in the blood serum were assessed in patients: GM-CSF, IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17A, MIP-1β, TNFα. The results obtained during the study showed not only a change in the number of cytokines, but also a change in the balance of pro- and anti-inflammatory cytokines, cytokines that regulate forms of development of acquired immunity, in the group of patients with type 2 diabetes in the post-Covid period. In the post-Covid period, in patients with type 2 diabetes mellitus, the concentrations of cytokines secreted by Th1, Th2, Th17 cells, and Tregs sharply decreased. It has been determined that patients with type 2 diabetes mellitus after SARS-CoV-2 infection develop a functionally inferior virus-specific T-cell response, characterized by reduced or suppressed secretion of the cytokines GM-CSF, IFNγ, IL-1β, IL-2, IL -4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17A, MIP-1β, TNFα. A weak T-cell response may increase the likelihood of developing pathological processes associated with an inadequate response to viral antigens (including repeated infections with the SARSCoV-2 virus, exacerbation of chronic infections).

Reduced duration and dosage of venetoclax is efficient in newly diagnosed patients with acute myeloid leukemia

ABSTRACT Objectives The combination of Venetoclax (VEN) and Azacitidine (AZA) increases survival outcomes and yields excellent responses in patients with acute myeloid leukemia (AML). However, dose reduction (or discontinuation) is commonly encountered due to therapy-related toxicity. Thus, this study aimed to investigate the efficiency and safety of a lower dosage of venetoclax for the treatment of AML. Methods This observational study analyzed the characteristics and outcomes of newly diagnosed AML patients who received 100 mg VEN combined with AZA for 14 days at our institution. Results A total of 36 patients were enrolled, and the median age at diagnosis was 64 years. After a median follow-up of 15 (range 4–29) months, the median overall survival (OS) and progression-free survival (PFS) for the whole cohort were 17 (4–29) months and 12 (1–28) months, respectively. Meanwhile, the overall response rate (ORR) was 69.4%, and the CRc rate was 66.7% in the whole cohort. Subgroup analysis revealed that NPM1 mutations and FAB-M5 subtype were associated with higher response rates, whereas the adverse ELN risk group was predictive of an inferior response. Moreover, ASXL1, NPM1, and IDH1/2 mutations negatively impacted PFS. Discussion Our study optimized the administration of venetoclax plus azacytidine for the treatment of AML patients. Response rates were favorable, with median survival in agreement with the findings of earlier reports, offering valuable insights for optimizing VEN-based regimens. Conclusion In summary, the VEN combination regimen is effective for the treatment of newly diagnosed AML patients in the real world despite VEN dose reductions .

ASXL1 Mutation and Leukocytosis Undermine Spleen Response to Pacritinib in Myelofibrosis

Background: Non-transplant treatment options in myelofibrosis (MF) are palliative in scope and include JAK2 inhibitors (JAKi): ruxolitinib, fedratinib, pacritinib, and momelotinib. All four JAKi are effective in reducing spleen size and alleviating symptoms. In addition, pacritinib and momelotinib exhibit erythropoietic effect, attributed to alleviation of ineffective erythropoiesis through inhibition of activin A receptor type-I. In the current study, we sought to identify molecular or clinical predictors of response to pacritinib in MF patients treated outside of clinical trials. Methods: The current study was conducted under an institutional review board approved minimum risk protocol that allowed retrospective collection and analysis of data from patient records (Mayo Clinic, Rochester, MN; Scottsdale, AZ; and Jacksonville, FL). Anemia response assignment was restricted to patients with baseline hemoglobin level of <10 g/dL and required, for transfusion dependent cases, achieving transfusion independence and, for transfusion-independent cases, an increase in hemoglobin of ≥1.5 g/dL, both sustained for a minimum 2 months; spleen response was clinically vetted but was also supported by radiologic imaging when available and required a ≥50% decrease in palpable splenomegaly for spleen size >10 cm or the spleen becoming not palpable for a spleen size 5-10 cm at base line. Symptom response was adjudicated by central review and required a marked or moderate improvement to qualify as response; because of the retrospective nature of the study, symptom burden assessment by formal criteria was not attempted. Leukocyte cutoff level used for response analysis was determined by receiver operating characteristic curve analysis. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results: We identified 36 NGS-annotated patients with MF who received pacritinib therapy between April,2022 and June,2023 (median age 66 years; 56% males). Twenty-five (69%) patients had failed previous JAKi therapy while 11 (31%) were JAKi-naïve. Eighteen (50%) patients were transfusion-dependent while the median (range) counts were 106 x 10(9)/L (14-578) for platelets, 6.6 x 10(9)/L (1.2-112.8) for leukocytes, and 9 x 10(9)/L (5-12.4) for hemoglobin; leukocyte count was ≥15 x 10(9)/L in 11 (31%) patients and hemoglobin <10 g/dL in 29 (81%). Karyotype was unfavorable in 12 (34%) patients. Driver mutation distribution was JAK2 55%, CALR 27%, MPL 11%, and triple-negative 5%. NGS revealed mutations involving ASXL1 36%, U2AF1 13%, IDH1 13%, SF3B1 13%, K/NRAS 5%, SRSF2 8%, and EZH2 5%. Pacritinib was started at 200 mg BID in 9 (25%) patients and at lower doses in the remaining. Median duration of pacritinib therapy was 4 months (range 1-15). Anemia response was documented in 5 (17%) of the 29 patients with baseline hemoglobin <10 g/dL, spleen response in 12 of 28 evaluable (42%) and symptom response in 18 (50%). Improvement in symptoms was always accompanied by both anemia (p=0.008) and spleen (p=0.01) response; by contrast, there was no significant correlation between anemia and spleen responses (p=0.9) (Table 1). Significant correlations with molecular or clinical variables were mostly apparent for spleen response, which was more likely to occur in the absence of ASXL1 mutation (61% vs 10%; p=0.006), with leukocyte count <15 x 10 (9)/L (55% vs 0%; p=0.005), and in JAKi-naïve vs previously-exposed cases (75% vs 30%; p=0.03; Table 1); significance was sustained by all three risk factors during multivariable analysis; the prognostic impact of ASXL1 (p=0.02 vs 0.01) and leukocytosis (p=0.01 vs 0.07) was apparent in both JAKi-naïve vs JAKi-exposed cases. Anemia response was non-significantly (p=0.27) higher in transfusion-independent (27%) vs dependent (11%) cases, at baseline, and was more likely (p=0.04) to occur in patients starting at lower dose levels. Treatment-emergent adverse events included diarrhea 44%, nausea/vomiting 19%, and grade-3/4 thrombocytopenia 38% or neutropenia (11%). Conclusions: ASXL1 mutation and leukocytosis (>15 x 10 9/L) are independently associated with inferior spleen response to pacritinib, in both JAKi-naïve and JAKi-exposed myelofibrosis. These observations are important for individualized treatment selection.

N/KRAS-Mutant AML LSCs Originate from Committed Myelomonocytic Progenitors and Drive Clinical Resistance to Venetoclax

Driver mutations in acute myeloid leukemia (AML) often exhibit distinct temporal acquisition patterns, but the biological basis for this, if any, remains unknown. RAS mutations occur invariably late in AML, upon progression or relapsed/refractory disease, in contrast to their function as early drivers in solid tumors. We developed synthetic leukemogenesis models in human induced pluripotent stem cell (iPSC)- and primary cord blood (CB)-derived human hematopoietic stem/progenitor cells (HSPCs) by introducing the prototypical NRAS G12D mutation alone or with other mutational combinations, using CRISPR, and engraftment of a transplantable leukemia into NSG mice as the readout. RAS mutations alone were not sufficient for leukemogenesis, but required specific cooperating mutations. Subsequently, by creating temporally controlled engineered models, we found that RAS mutations are obligatory late events that can only drive leukemic transformation when acquired after, but not before, cooperating mutations. We provide the mechanistic explanation for this in a requirement for mutant RAS to specifically transform committed granulocyte-monocyte progenitors (GMPs) harboring previously acquired driver mutations, through sorting and transplantation and multi-omics experiments. In contrast, acquisition of RAS mutations in earlier progenitors (common myeloid progenitors, CMP), with or without cooperating mutations, blocked the emergence of GMPs and abolished engraftment or gave rise to a myeloproliferative neoplasm. These results indicate that advanced RAS-mutant (MT) leukemic clones have a different cell type of origin from the ancestral clone. Next, using single-cell transcriptomics in cells isolated from xenografts of two AML-iPSC lines generated from an AML patient with a subclonal KRAS G12D mutation, one capturing the RAS WT major clone and one the KRAS G12D subclone, we show that KRAS G12D leukemia stem cells (LSCs) give rise to leukemia with a more monocytic immunophenotype, while the ancestral clone within the same patient generates more leukemic cells with primitive and neutrophil markers. These results were corroborated by Genotyping of Transcriptomes (GoT) in another AML patient with subclonal NRAS G12D mutation. Recent studies have linked monocytic AML and, independently, RAS pathway mutations to poor responses to Venetoclax (VEN)-containing regiments. In view of the causal link we found between RAS mutations and monocytic disease, we evaluated the associations between monocytic AML, RAS mutations and response to VEN. We reanalyzed data from a cohort of 118 older/unfit newly diagnosed AML patients treated on a prospective clinical trial with VEN and decitabine (DEC) (NCT03404193). All outcomes were comparable between monocytic and non-monocytic groups, including overall response rate, duration of response (DOR) and overall survival. On the other hand, patients with N/KRAS mutations had significantly shorter DOR. These results support the presence of N/KRAS mutations and not the monocytic stage as predictors of inferior responses to VEN regimens. To definitely test this, we treated defined cell types generated from RAS WT and RAS MT iPSC lines - CD34+ LSC-like and CD11b+/CD68+/CD14+ monocytic cells - with VEN. In agreement with previous studies, monocytes were resistant and RAS WT LSCs were sensitive to VEN. However, strikingly RAS MT LSCs were VEN-resistant. Consistent with these, single-cell transcriptomics in patient-derived AML-iPSC cells in vivo, GoT in primary AML cells and bulk transcriptomics in synthetic iPSC-AML cells showed downregulation of BCL2 and upregulation of MCL1 in the RAS MT LSCs. This study provides the first example of a mechanistic explanation of why the timing of mutational acquisition in AML is so strongly biased. It shows for the first time that interaction between an oncogenic driver and the non-genetic developmental hematopoietic hierarchy imposes a specific LSC cell-of-origin restriction and in turn shapes the hierarchical structure of the resulting AML and critically determines therapeutic outcomes in patients. Importantly, our results have direct implications for clinical practice, as they support that RAS MT LSCs drive clinical resistance to VEN and that treatment with VEN in patients with RAS mutations can accelerate disease progression by selection of RAS MT LSCs (Figure).

Fading SARS-CoV-2 humoral VOC cross-reactivity and sustained cellular immunity in convalescent children and adolescents

Cross-reactive cellular and humoral immunity can substantially contribute to antiviral defense against SARS-CoV-2 variants of concern (VOC). While the adult SARS-CoV-2 cellular and humoral immunity and its cross-recognition potential against VOC is broadly analyzed, similar data regarding the pediatric population are missing. In this study, we perform an analysis of the humoral and cellular SARS-CoV-2 response immune of 32 convalescent COVID-19 children (children), 27 convalescent vaccinated adults(C + V+) and 7 unvaccinated convalescent adults (C + V-). Similarly to adults, a significant reduction of cross-reactive neutralizing capacity against delta and omicron VOC was observed 6 months after SARS-CoV-2 infection. While SAR-CoV-2 neutralizing capacity was comparable among children and C + V- against all VOC, children demonstrated as expected an inferior humoral response when compared to C + V+. Nevertheless, children generated SARS-CoV-2 reactive T cells with broad cross-recognition potential. When compared to V + C+, children presented even comparable frequencies of WT-reactive CD4 + and CD8 + T cells with high avidity and functionality. Taking into consideration the limitations of study - unknown disease onset for 53% of the asymptomatic pediatric subjects, serological detection of SARS-CoV-2 infection-, our results suggest that following SARS-CoV-2 infection children generate a humoral SARS-CoV-2 response with neutralizing potential comparable to unvaccinated COVID-19 convalescent adults as well a sustained SARS-CoV-2 cellular response cross-reactive to VOC.

Cooperative Networks between MYC and XPO1 Associated with Decreased T-Cell Presence and a Depleted Tumor Microenvironment May be Addressed By the Synergistic Combination of AZD4573 and Selinexor

Background: Genomically-targeted Diffuse Large B-cell (DLBCL) treatments for refractory or relapsing patients remain a clinical need alongside the growing success of Chimeric Antigen T-cells (CAR-T), which have ushered in durable responses in 40-70% of patients. However, recent studies have identified factors indicative of an inferior CAR-T response. Recent tumor Ecotype studies from Kotlov and Steen reveal that a Depleted or Cold tumor microenvironment (TME) is associated with poor CAR-T response. Notable features of these environments include a decreased presence of native T-cells alongside oncogenic alterations associated with their exhaustion, namely MYC and XPO1. Few precision treatments exist given the difficulty targeting MYC, but two avenues have shown recent success: inhibition of the downstream oncogene CDK9 via AZD4573 and upstream inhibition of a MYC propagator, the nuclear exportin XPO1 via Selinexor. Herein, we report the first observations of synergy between AZD4573 and Selinexor within DLBCL cell lines and data supporting that these genes may serve as accessible markers for inferior tumor immune microenvironments. Methods: We combined progression vs. response results from four previous CAR-T genomics studies, noting MYC status (Jaeger 2020, Jain 2021, Sworder 2021, and Shouval 2022). We next analyzed a 418-patient de-novo DLBCL patient cohort (Xu-Monette 2020) and 48-patient TCGA cohort after GEDIT immune deconvolution for differential expression and component enrichment between patients expressing high levels of MYC and XPO1 (+1 Standard Deviation) compared to normal counterparts. Significantly expressed genes were evaluated via gene set enrichment analyses. We next assayed AZD4573 and Selinexor vs. 7 DLBCL cell lines, 5 of which harbor MYC alterations, across a series of 9 concentrations (78nM-20µM) across 3-9 replicates. We followed these results by combining treatments in the MYC-altered Ly3, DHL6, and DHL4 cell lines, analyzing results using the BLISS synergy model. Results: Integrative analyses support that MYC alterations are associated with significantly reduced CAR-T outcomes. Of the 93 MYC-altered patients within the total 246-patient cohort, 65.59% of them experienced progression during CAR-T treatment compared to just 50.33% of WT CAR-T patients (p = 0.0242). Differential gene expression analyses between de-novo patients expressing high MYC or XPO1 revealed losses in Cytokine Signaling pathways (FDR = 6.96E-18) and gains in Cell Cycle modulators (FDR = 2.525E-20). Importantly, CD8A expression was significantly lower in these patients (FDR < 0.0001) alongside other key reductions of CDKN1A and TNFAIP3. After immune deconvolution, both High-MYC and High-XPO1 patients displayed significant losses of CD8 T-cell association, respectively (FDR = 0.0005, FDR = 0.0396). Notably, High-MYC patients without a CD8 T-cell presence experienced an inferior survival prognosis compared to High-MYC counterparts with a CD8 presence (p = 0.0272). High MYC or XPO1 patients that experienced a failure event before 24-months (EFS24) had significantly lower activated CD4 T-cell presence compared to normal counterparts as well (FDR = 0.0095, FDR = 0.0069). Next, AZD4573 and Selinexor assays vs. DLBCL cell lines demonstrated dose and MYC-dependent cell inhibition, with ED50 values ranging between 58.1 nM to 8.07 µM. Lastly, the molecules were combined vs. Ly3, DHL6 and DHL4 cell lines, resulting in synergistic reduction of cell viability and BLISS synergy scores of 33.76, and 11.86, and 5.04, respectively. Conclusions: Our results support that the cooperation between MYC and XPO1 is associated with T-cell reductions characteristic of a Depleted or Cold TME, a key issue for CAR-T success. We applied the targeted therapies AZD4573 and Selinexor vs. cell line models to address this pathway, with both displaying anti-tumor effects as single agents. Most importantly, we observed synergistic activity in the MYC-harboring Ly3 and DHL6 cell lines, and in the WT DHL4 cell line when these molecules were combined. These results are supported by integrative analyses that highlight the importance of targeting aberrant MYC signaling via CDK9 and XPO1 inhibition, revealing a potential avenue to address inferior response rates faced by patients harboring MYC-positive, Depleted tumor microenvironments that would otherwise face an inferior CAR-T prognosis.

Crebbp Mutations Are Associated with Slow Minimal Residual Response to Upfront Induction Chemotherapy in Pediatric High-Hyperdiploid B-Cell Precursor Acute Lymphoblastic Leukemia Treated in the AIEOP-BFM ALL 2009 Protocol

Introduction High-hyperdiploidy (HHD) represents the most common cytogenetic abnormality found in B-cell precursor acute lymphoblastic leukemia (B-ALL). Despite the overall good outcome of this entity, up to 15% of patients suffer from disease relapse. Genome-wide ALL studies with next-generation sequencing (NGS) approaches identified a distinct pattern of somatic mutations in HHD ALL, enriched in mutations occurring in genes activating the RAS pathway and in CREBBP gene, especially in relapsed patients. Although several studies have attempted to explore the prognostic significance of these mutations that are potentially susceptible of targeted therapies, their impact in uniformly treated patients with newly diagnosed B-ALL is not well characterized, and there is no consensus on their clinical significance. In this study, we aimed to investigate the frequency of recurrent mutations and to define their prognostic role in the setting of newly diagnosed HHD B-ALL, by using end of induction minimal residual disease (MRD) and event-free survival (EFS). Methods We included 101 consecutive patients, aged between 1-17 years, with newly diagnosed HHD B-ALL and enrolled in the AIEOP-BFM ALL 2009 protocol (NCT01117441). HHD was defined by the presence of a DNA index ≥1.16 and lack of ETV6::RUNX1, BCR::ABL1, KMT2A::AFF1 translocations. Diagnostic leukemia samples of 89 patients were sequenced using a validated clinical NGS panel profiling 39 genes involved in leukemia pathogenesis. Mutations affecting RAS pathway ( NRAS, KRAS, PTPN11, NF1, BRAF) and mutations affecting CREBBP gene were included. In addition, digital multiplex ligation-dependent probe amplification (digital MLPA) analysis for the identification of IKZF1 deletion and IKZF1plus phenotype was evaluable for 67 patients. A Fisher exact test and Wilcoxon rank sum test were used for categorical and continuous variables. MRD was assessed by an IG/TR PCR assay at day +33 of induction (timepoint 1, TP1) and at day +78 (timepoint 2, TP2). EFS was estimated by using Kaplan and Meier method and differences between groups were tested using the log-rank test. Resistance to treatment, relapse, death or second malignant neoplasm were considered as events, whichever occurred first. Results Patients' characteristics according to the presence of studied mutations are shown in Table 1. Mutations affecting RAS pathway and CREBBP occurred in 57.3% and 12.3% of HHD B-ALL patients, respectively. Clonal RAS pathway mutations, defined by variant allele frequency (VAF) ≥25%, were found in 16.8% of cases. Among the 11 patients with mutations of CREBBP, 7 had also a mutation affecting KRAS and the association was significant (p=0.03). Seven patients (10.4%) had deletion of IKZF1, of whom 5 (7.4%) were IKZF1plus. There was no association between any mutations or IKZF1 status and patient presenting characteristics, as including sex, age and WBC counts. MRD was evaluable in 84 patients at the end of induction (TP1). MRD level ≥5x10 -4 was found in 36.4% of patients with CREBBP mutations and in 9.6% of those without, and this association was statistically significant (p=0.02). Additionally, MRD negativity was found in 9.1% and 38.4%, respectively. At TP2, MRD was still detectable in 45.5% of patients with CREBBP mutations compared to 20.6% of those with CREBBP wildtype (p=0.08). Similarly, we observed an association between RAS pathway mutations and MRD at TP1 (p=0.01) with a marked difference in MRD negative patients (21.3% vs 51.4% in patients with and without mutations). No association was observed between MRD level and clonal RAS pathway mutations or IKZF1 status. Despite the association between CREBBP mutations and high MRD at TP1, no significant difference in final high-risk group was found (27.3% vs 11.5%, p=0.17). A total of 8 events were observed, of which 6 were relapses. No impact of any mutations or IKZF1 status in term of 5-years EFS was found in our cohort. Conclusion Our study shows that CREBBP mutations in HHD B-ALL are associated with inferior early response to treatment in terms of MRD level at the end of induction (TP1) but not with worse EFS with our whole current chemotherapy backbone. Therefore, the inclusion of CREBBP mutated patients in very low risk protocols should be carefully evaluated. The validation of these findings is ongoing through the analysis of patients treated by other international study groups adopting different chemotherapy backbones.

Immune Responses after One Versus Two Influenza A/B Vaccinations in Patients with Multiple Myeloma

Background: Multiple Myeloma (MM) is a hematological cancer affecting plasma cells, suppressing the immune system and increasing the risk of severe Influenza A/B infection. International guidelines recommend Influenza A/B vaccination on an annual basis. Data from the pre-lenalidomide era highlighted poor vaccination responses, with sufficient seroconversion in 39% of patients with MM after single vaccination and an increase to 55% after additional boosting. Lenalidomide is now widely established in different (maintenance) therapy protocols and other novel agents (e.g. anti-CD38-agents) are incorporated into the therapy lines. Hence, it is of high relevance to characterize the sufficient immune response to Influenza A/B vaccination in this context, evaluate the necessity for a prime and boosting approach, and identify patients with high risk of insufficient response and subsequent hazard of severe disease. Methods: In this retrospective single-center study, we analyzed the serological response from 71 patients with MM treated at our institution who received the annual Influenza A/B vaccination (Influvac Tetra, Mylan, Troisdorf, Germany) in the 2019/20 season. All patients received at least one vaccination with 52 patients receiving a second vaccination. Hemagglutination-inhibition (HI) assay titers for the five most prevalent virus strains were measured before, 21-28 days after the first and 21-28 days after the second vaccination or at observation (42-56 days after first vaccination in case of single vaccination). Peripheral immune cell counts were measured at the initial vaccination event by flow cytometry. Disease, therapy, and patient characteristics were retrieved from the electronical health care system at our institution. Primary endpoint was the sufficient responder status as indicated by ≥1:40 HI titer or an at least four-fold increase in the median HI titers of all strains. All patients declared written informed consent and the study was approved by the local ethics committee. Results: 71 patients with MM were included in the present study who mostly had IgG MM (46.5%), the minority displayed genetic high-risk MM (25.4%), 64.8% were in their first line of therapy and 69% were in good remission (CR/VGPR). Overall, only 5.6% (n=4) showed sufficient titers before vaccination. 63.3% (n=45) achieved a sufficient serological response after one or two Influenza A/B vaccinations. A strong increase in HI titers against all strains was observed after already the first vaccination. In patients receiving a prime and boost approach, significantly higher titers but no significant increase in responder rates were observed after the boost vaccination. Responders were more frequently without current therapy or under maintenance therapy, showed significantly higher rates of CR/VGPR and significantly less immunoparesis. Interestingly, they trended to lower rates of previous anti-CD38 therapy (24.4% versus 50%, p=0.053). Non-responders displayed significantly lower median CD19 + B-cell counts (20.0/µl versus 79.0/µl p=0.001) and reduced median CD4 + T-cell counts (167.5/µl versus 274.0/µl, p=0.033). Multivariate regression analysis highlighted current CR/VGPR (OR 4.1 [95% CI 1.09-16.57], p=0.04) and no immunoparesis (OR 5.37 [95% CI 1.13-33.21], p=0.046) at timepoint of vaccination as independent predictors for sufficient serological response. Those patients achieving a sufficient responder status only after the boost vaccination among all responders displayed significantly shorter time (5.0 versus 26.5 months, p=0.046) since the last high-dose chemotherapy and autologous stem cell transplantation (autoHSCT). Conclusions: Together, 63.3% of patients with MM achieved a sufficient serological response after tetravalent Influenza A/B vaccination without a significant general benefit for a prime and boost approach. Current therapy, uncontrolled MM, present immunoparesis and low CD19 + B-cell counts were associated with inferior response rates. Those patients exceptionally benefitting from a boost vaccination were characterized by recent autoHSCT. Hence, annual one-time Influenza A/B vaccination is applicable for patients with MM and patients directly after autoHSCT can be considered for a prime and boost approach. Further studies are warranted to investigate this in a prospective and randomized fashion.

Targeting myeloid checkpoint Siglec-10 reactivates antitumor immunity and improves anti-programmed cell death 1 efficacy in gastric cancer

ObjectiveImmunotherapy has not yielded satisfactory therapeutic responses in gastric cancer (GC). However, targeting myeloid checkpoints holds promise for expanding the potential of immunotherapy. This study aims to evaluate the critical...

Infusion Product TNFα, Th2, and STAT3 Activities Are Associated with Clinical Responses to Transgenic T-cell Receptor Cell Therapy.

Transgenic T-cell receptor (TCR) T cell-based adoptive cell therapies for solid tumors are associated with dramatic initial response rates, but there remain many instances of treatment failure and disease relapse. The association of infusion product cytokine profiles with clinical response has not been explored in the context of TCR T-cell therapy products. Single-cell antigen-dependent secretomic and proteomic analysis of preinfusion clinical TCR T-cell therapy products revealed that TNFα cytokine functionality of CD8+ T cells and phospho-STAT3 signaling in these cells were both associated with superior clinical responsiveness to therapy. By contrast, CD4+ T-helper 2 cell cytokine profiles were associated with inferior clinical responses. In parallel, preinfusion levels of IL15, Flt3-L, and CX3CL1 were all found to be associated with clinical response to therapy. These results have implications for the development of therapeutic biomarkers and identify potential targets for enrichment in the design of transgenic TCR T-cell therapies for solid tumors.

Early relapse within 18months is a powerful dynamic predictor for prognosis and could revise static risk distribution in multiple myeloma.

The duration of response to treatment is a major prognostic factor, and early relapse (ER) strongly predicts inferior survival in multiple myeloma (MM). However, the definitions of ER in MM vary from study to study and how to dynamically integrate risk distribution is still unsolved. This study evaluated these ER definitions and further investigated the underlying relationship with static risk distribution in 629 newly diagnosed MM (NDMM) patients from the National Longitudinal Cohort of Hematological Diseases in China (NCT04645199). These data indicated that early relapse within 18months (ER18) after initial treatment was the best time point for identifying early progression and dynamic high-risk in MM. The ER18 population (114 of 587, 19.4%) presented with more aggressive biologic features and the inferior response to treatment compared to a reference cohort (p<.001), with a significantly short median overall survival (OS) of 28.9months. Multivariate analyses confirmed the most significant prognostic value of ER18 on OS in the context of International Staging System stage, elevated lactate dehydrogenase, thrombocytopenia, cytogenetic abnormalities, and treatment (hazard ratio, 4.467; p <.001). The authors also described the specific transitions from static risk profile to dynamic risk distribution and then constructed a mixed-risk-pattern to identify four novel populations with distinct survival (p<.001). Additionally, the authors proposed a second-state model that predicts dynamic risk changes, enabling a complementary role to the Revised International Staging System model in facilitating individualized systematic treatment. Collectively, this study concludes that ER18 is a simple and dynamic prognostic predictor in MM. In addition to static risk assessment, dynamic risk plays an important role in survival prediction.

High BCR::ABL1 Expression Defines CD34+ Cells with Significant Alterations in Signal Transduction, Short-Proliferative Potential and Self-Renewal Ability.

Chronic Myeloid Leukemia (CML) is a clonal disorder of the hematopoietic stem cell caused by expression of the BCR::ABL1 oncoprotein. High BCR::ABL1 levels have been associated to proliferative advantage of leukemic cells, blast crisis progression and tyrosine kinase inhibitors (TKIs) inefficacy. We have previously shown that high BCR::ABL1/GUSIS transcripts measured at diagnosis are associated with inferior responses to standard dose Imatinib (IM). However, the mechanisms underlying the higher rates of disease progression and development of TKIs resistance dependent on elevated BCR::ABL1 levels remain unclear. Leukemic cells were collected from CML patients showing, at diagnosis, high or low BCR::ABL1/GUSIS. BCR::ABL1 expression levels were measured using real-time PCR. Short-term culture and long-term culture-initiating cells assays were employed to investigate the role of BCR::ABL1 gene-expression levels on proliferation, clonogenicity, signal transduction, TKIs responsiveness and self-renewal ability. Cell division was performed by carboxyfluorescein-succinimidyl ester (CFSE) assay. We found that BCR::ABL1 oncogene expression levels correlate in both PMNs and CD34+ cells. Furthermore, high oncogene levels increased both proliferation and anti-apoptotic signaling via ERK and AKT phosphorylation. Moreover, high BCR::ABL1 expression reduced the clonogenicity of leukemic CD34+ cells and increased their sensitivity to high doses IM but not to those of dasatinib. Furthermore, we observed that high BCR::ABL1 levels are associated with a reduced self-renewal of primitive leukemic cells and, also, that these cells showed comparable TKIs responsiveness with cells expressing lower BCR::ABL1 levels. Interestingly, we found a direct correlation between high BCR::ABL1 levels and reduced number of quiescent leukemic cells caused by increasing their cycling. Higher BCR::ABL1 levels improving the proliferation, anti-apoptotic signaling and reducing self-renewal properties cause an increased expansion of leukemic clone.

Immunogenicity and Reactogenicity of Coadministration of COVID-19 and Influenza Vaccines

COVID-19 and seasonal influenza vaccines were previously given separately, although their coadministration is warranted for vaccination adherence. Limited data on their coadministration have been published. To compare the reactogenicity and immunogenicity of COVID-19 and influenza vaccinations administered together with those of COVID-19 vaccination alone. This prospective cohort study included health care workers at a large tertiary medical center in Israel who received the Influvac Tetra (Abbott) influenza vaccine (2022/2023), the Omicron BA.4/BA.5-adapted bivalent (Pfizer/BioNTech) vaccine, or both. Vaccination began in September 2022, and data were collected until January 2023. Vaccines were offered to all employees and were coadministered or given separately. Adverse reaction questionnaires were sent, and serologic samples were also collected. Receiving COVID-19 vaccine, influenza vaccine, or both. The main outcomes for the reactogenicity analysis were symptoms following vaccine receipt, assessed by a digital questionnaire: any local symptoms; fever; weakness or fatigue; any systemic symptoms; and their duration. The immunogenicity analysis' outcome was postvaccination anti-spike IgG titer. This study included 2 cohorts for 2 separate analyses. The reactogenicity analysis included 588 participants (of 649 questionnaire responders): 85 in the COVID-19 vaccine-alone group (median [IQR] age, 71 [58-74] years; 56 [66%] female); 357 in the influenza vaccine-alone group (median [IQR] age, 55 [40-65] years; 282 [79%] female); and 146 in the coadministration group (median [IQR] age, 61 [50-71] years; 81 [55%] female). The immunogenicity analysis included 151 participants: 74 participants in the COVID-19 vaccine group (median [IQR] age, 67 [56-73] years; 45 [61%] female) and 77 participants in the coadministration group (median [IQR] age, 60 [49-73] years; 42 [55%] female). Compared with COVID-19 vaccination alone, the risk of systemic symptoms was similar in the coadministration group (odds ratio, 0.82; 95% CI, 0.43-1.56). Geometric mean titers in the coadministration group were estimated to be 0.84 (95% CI, 0.69-1.04) times lower than in the COVID-19 vaccine-alone group. In this cohort study of health care workers who received a COVID-19 vaccine, an influenza vaccine, or both, coadministration was not associated with substantially inferior immune response or to more frequent adverse events compared with COVID-19 vaccine administration alone, supporting the coadministration of these vaccines.

Impact of residual tumor cells in the stem cell collection on multiple myeloma patients receiving autologous stem cell transplantation

Autologous stem cell transplantation (ASCT) is the standard therapy for patients with transplant-eligible multiple myeloma (TEMM). However, the ideal depth of response required before ASCT and the impact of residual tumor cells in the stem cell collection (SCC) on survival remains unclear. Here we collected data of 89 patients with TEMM undergoing ASCT and analyzed the minimal residual disease of SCC (cMRD) and bone marrow (BM) (mMRD) before transplantation. Before ASCT, 31.5% and 76.4% of patients achieved MRD negativity in BM and SCC, respectively. Tumor cells were less in SCC samples than that in BM samples. Neoplastic cells in SCC could be observed in patients with different responses after induction therapy, and there were no significant differences in the percentage and level of cMRD among these subgroups (P > 0.05). No correlation was found between the cMRD status and the response patients achieved after ASCT (P > 0.05). The median follow-up was 26.8 months. mMRD negativity before ASCT was associated with longer PFS (55.9 vs. 27.1 months; P = 0.009) but not OS (not reached vs. 58.9 months; P = 0.115). Patients with different cMRD statuses before ASCT experienced similar PFS (40.5 vs. 76.4 months for negativity vs. positivity; P = 0.685) and OS (not reached vs. 58.8 months for negativity vs. positivity; P = 0.889). These results suggested that detectable cMRD does not significantly predict the inferior post-ASCT response or shorter survival, and patients are eligible to undergo ASCT upon achieving partial response.

Immunosuppressive MFAP2+ cancer associated fibroblasts conferred unfavorable prognosis and therapeutic resistance in gastric cancer.

To explore the predictive merit of MFAP2+ cancer associated fibroblasts (CAFs) infiltration for clinical outcomes and adjuvant chemotherapy or immunotherapy responsiveness in gastric cancer (GC). In this study, several independent cohorts were included respectively to dissect the relationship of clinical outcomes, therapeutic responses and tumor microenvironment with different MFAP2+ CAFs infiltration. Drug sensitivity analysis was conducted to predict the relationship between MFAP2+ CAFs infiltration and targeted drug response. Kaplan-Meier curves and the log-rank test were used to compare clinical outcomes of patients with different MFAP2+ CAFs infiltration. High MFAP2+ CAFs infiltration yielded inferior prognosis in terms of overall survival, progress free survival and recurrence free survival in GC. Patients with low MFAP2+ CAFs infiltration were more likely to gain benefit from adjuvant therapy. Moreover, low MFAP2+ CAFs infiltration could predict a promising response to immunotherapy in GC patients. MFAP2+ CAFs with immunosuppressive features were highly relevant to immune evasive contexture characterized by the dysfunction of CD8+ T cells. We found that MFAP2+ CAFs communicated with T cells, B cells and Macrophages through releasing macrophage migration inhibitor factor (MIF), which further suggested that MFAP2+ CAFs might promote therapeutic resistance through regulating T cells dysfunction and M2 macrophages polarization. Immunosuppressive MFAP2+ CAFs constructed an immune evasive tumor microenvironment characterized by incapacitated immune effector cells, consequently predicting inferior clinical outcomes and response on adjuvant therapy and immunotherapy in patients with GC. The potential of immunosuppressive MFAP2+ CAFs as a therapeutic target for GC deserved thoroughly exploration.

Ingestion of mycoprotein, pea protein, and their blend support comparable postexercise myofibrillar protein synthesis rates in resistance-trained individuals.

Pea protein is an attractive nonanimal-derived protein source to support dietary protein requirements. However, although high in leucine, a low methionine content has been suggested to limit its anabolic potential. Mycoprotein has a complete amino acid profile which, at least in part, may explain its ability to robustly stimulate myofibrillar protein synthesis (MyoPS) rates. We hypothesized that an inferior postexercise MyoPS response would be seen following ingestion of pea protein compared with mycoprotein, which would be (partially) rescued by blending the two sources. Thirty-three healthy, young [age: 21 ± 1 yr, body mass index (BMI): 24 ± 1 kg·m-2] and resistance-trained participants received primed, continuous infusions of l-[ring-2H5]phenylalanine and completed a bout of whole body resistance exercise before ingesting 25 g of protein from mycoprotein (MYC, n = 11), pea protein (PEA, n = 11), or a blend (39% MYC, 61% PEA) of the two (BLEND, n = 11). Blood and muscle samples were taken pre-, 2 h, and 4 h postexercise/protein ingestion to assess postabsorptive and postprandial postexercise myofibrillar protein fractional synthetic rates (FSRs). Protein ingestion increased plasma essential amino acid and leucine concentrations (time effect; P < 0.0001), but more rapidly in BLEND and PEA compared with MYC (time × condition interaction; P < 0.0001). From similar postabsorptive values (MYC, 0.026 ± 0.008%·h-1; PEA, 0.028 ± 0.007%·h-1; BLEND, 0.026 ± 0.006%·h-1), resistance exercise and protein ingestion increased myofibrillar FSRs (time effect; P < 0.0001) over a 4-h postprandial period (MYC, 0.076 ± 0.004%·h-1; PEA, 0.087 ± 0.01%·h-1; BLEND, 0.085 ± 0.01%·h-1), with no differences between groups (all; P > 0.05). These data show that all three nonanimal-derived protein sources have utility in supporting postexercise muscle reconditioning.NEW & NOTEWORTHY This study provides evidence that pea protein (PEA), mycoprotein (MYC), and their blend (BLEND) can support postexercise myofibrillar protein synthesis rates following a bout of whole body resistance exercise. Furthermore, these data suggest that a methionine deficiency in pea may not limit its capacity to stimulate an acute increase in muscle protein synthesis (MPS).

Local and Systemic Immunity During Five Vaccinations Against SARS-CoV-2 in Zanubrutinib-Treated Patients With Chronic Lymphocytic Leukemia.

Patients with chronic lymphocytic leukemia (CLL) are vulnerable to coronavirus disease 2019 (COVID-19) and are at risk of inferior response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination, especially if treated with the first-generation Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib. We aimed to evaluate the impact of the third-generation BTKi, zanubrutinib, on systemic and mucosal response to SARS-CoV-2 vaccination. Nine patients with CLL with ongoing zanubrutinib therapy were included and donated blood and saliva during SARS-CoV-2 vaccination, before vaccine doses 3 and 5 and 2 - 3 weeks after doses 3, 4, and 5. Ibrutinib-treated control patients (n = 7) and healthy aged-matched controls (n = 7) gave blood 2 - 3 weeks after vaccine dose 5. We quantified reactivity and neutralization capacity of SARS-CoV-2-specific IgG and IgA antibodies (Abs) in both serum and saliva, and reactivity of T cells activated with viral peptides. Both zanubrutinib- and ibrutinib-treated patients had significantly, up to 1,000-fold, lower total spike-specific Ab levels after dose 5 compared to healthy controls (P < 0.01). Spike-IgG levels in serum from zanubrutinib-treated patients correlated well to neutralization capacity (r = 0.68; P < 0.0001) and were thus functional. Mucosal immunity (specific IgA in serum and saliva) was practically absent in zanubrutinib-treated patients even after five vaccine doses, whereas healthy controls had significantly higher levels (tested in serum after vaccine dose 5) (P < 0.05). In contrast, T-cell reactivity against SARS-CoV-2 peptides was equally high in zanubrutinib- and ibrutinib-treated patients as in healthy control donors. In our small cohort of zanubrutinib-treated CLL patients, we conclude that up to five doses of SARS-CoV-2 vaccination induced no detectable IgA mucosal immunity, which likely will impair the primary barrier defence against the infection. Systemic IgG responses were also impaired, whereas T-cell responses were normal. Further and larger studies are needed to evaluate the impact of these findings on disease protection.

Cellular immune response of SARS-CoV-2 vaccination in kidney transplant recipients: a systematic review and meta-analysis.

Evidence has demonstrated inferior humoral immune responses after SARS-CoV-2 vaccination in kidney transplant recipients compared to the general population. However, data on cellular immune responses in this population have not been established. We searched the MEDLINE, Scopus, and Cochrane databases and included studies reporting cellular immune response rates in kidney transplant recipients after receiving SARS-CoV-2 vaccines. Studies that reported factors associated with cellular immune responders or non-responders were also included (PROSPERO: CRD42022375544). From a total of 1,494 articles searched, 53 articles were included in the meta-analysis. In all, 21 studies assessed cellular immune response by interferon-γ enzyme-linked immunosorbent spot (IFN-γ ELISPOT), 22 studies used interferon-γ release assay (IGRA), and 10 studies used flow cytometric analysis. The pooled response rate after two doses (standard regimen) and three doses of vaccination was 47.5% (95%CI 38.4-56.7%) and 69.1% (95%CI 56.3-80.6%) from IFN-γ ELISPOT, 25.8% (95%CI 19.7-32.4%) and 14.7% (95%CI 8.5-22.2%) from IGRA, and 73.7% (95%CI 55.2-88.8%) and 86.5% (95%CI 75.3-94.9%) from flow cytometry, respectively. Recipients with seroconversion were associated with a higher chance of having cellular immune response (OR 2.58; 95%CI 1.89-3.54). Cellular immune response in kidney transplant recipients was lower than in dialysis patients (OR 0.24; 95%CI 0.16-0.34) and the general population (OR 0.10; 95%CI 0.07-0.14). Age and immunosuppressants containing tacrolimus or corticosteroid were associated with inferior cellular immune response. Cellular immune response after SARS-CoV-2 vaccination in kidney transplant recipients was lower than in dialysis patients and the general population. Age, tacrolimus, and corticosteroid were associated with poor response. Cellular immune response should also be prioritized in vaccination studies. https://www.crd.york.ac.uk/prospero/, identifier CRD42022375544.