Marker Of Relapse Research Articles

Circulating B Cells Display Differential Immune Regulatory Molecule Expression in Granulomatosis with Polyangiitis.

Granulomatosis with polyangiitis (GPA) is a B cell-mediated, relapsing, autoimmune disease. There is a need for novel therapeutic approaches and relapse markers to achieve durable remission. B cells express immune regulatory molecules that modulate their activation and maintain tolerance. While recent studies show dysregulation of these molecules in other autoimmune diseases, data on their expression in GPA are limited. This study aimed to map the expression of surface immune regulatory molecules on circulating B cell subsets in GPA and correlate their expression with clinical parameters. Immune regulatory molecule expression on circulating B cell subsets was comprehensively examined in active GPA (n=16), GPA in remission (n=16), and healthy controls (HCs, n=16) cross-sectionally using a 35-color B cell-specific spectral flow cytometry panel. Our supervised and unsupervised in-depth analysis revealed differential expression of inhibitory and stimulatory immune molecules on distinct B cell populations in GPA, with the most notable differences observed in active GPA. These differences include the upregulation of FcγRIIB on non-mature B cells, downregulation of CD21 and upregulation of CD86 on antigen-experienced B cells, and elevated CD22 expression on various populations. Additionally, we found a strong association between FcγRIIB, BTLA, and CD21 expression on specific B cell populations and disease activity in GPA. Together, these findings provide novel insights into the immune regulatory molecule expression profile of B cells in GPA, and could potentially form the foundation for new therapeutic approaches and disease monitoring markers.

Plasma miR-122-5p and miR-142-5p and their role in chemoresistance of colon cancer patients.

Chemoresistance represents a major issue affecting cancer therapy efficacy. Because microRNAs (miRNAs) regulate gene expression on multiple levels, their role in chemoresistance development is reasonably certain. In our previous study, miR-122-5p and miR-142-5p were identified as diagnostic, prognostic, and predictive biomarkers for primary and metastatic rectal cancer. The aim of the present study was to investigate whether these miRNAs can also reflect the disease course of colon cancer (CC) patients. Further, we focused on a deeper understanding of their involvement in 5-fluorouracil (5-FU) chemoresistance development. The expression analysis of both miRNAs was analysed in repeated whole plasma samplings (n=3, approximately every 6 months) of CC patients (n=49) by RT-qPCR. Expression levels of both miRNAs were determined in the 5-FU sensitive and resistant CC cell lines. From RNA-seq profiles of both sensitive and 5-FU resistant DLD-1 cell lines, the expression levels of miR-122-5p and miR-142-5p validated target genes were detected and compared. Significant differences in the expression levels of both miRNAs between T0 and T1 or T2 samplings were observed. Further, an association between the occurrence of relapse and miR-122-5p expression levels was noticed. Patients who did not relapse had higher expression of miR-122-5p at T1 (p=0.01; 3.16-fold change) and T2 (p=0.04; 2.79-fold change) samplings in comparison with T0 sampling. Out of all miR-122-5p validated targets (n=102), 25 genes were significantly differentially expressed between sensitive and 5-FU-resistant cell lines. Our data suggest that miR-122-5p may represent a predictive marker of tumour relapse in CC patients. In vitro data suggests that this aspect may be linked to the potential therapeutic targets of miR-122-5p related to 5-FU-based chemoresistance. However, deeper mechanistic studies are still needed for progress toward personalized medicine.

Reconstitution of T cells at three months after stem cell transplant influences the discovery of predictive markers of relapse

Reconstitution of T cells at three months after stem cell transplant influences the discovery of predictive markers of relapse

Assessment of the Circulating PD-1 and PD-L1 Levels and P53 Expression as a Predictor of Relapse in Pediatric Patients with Wilms Tumor and Hypernephroma.

Background/Objectives: Wilms tumor (WT) is the most common form of pediatric renal tumor, accounting for over 90% of cases followed by hypernephroma. Some pediatric patients with WT (10%) experience relapse or metastasis and have poor survival rates. PD-L1 assists cancer cells in escaping damage from the immune system. P53 mutations are found in relapsed WT tumor samples. We hypothesized that testing circulating PD-1 and PD-L1 and P53 expression levels could offer a simple method to predict patient relapse and explore novel treatments for pediatric WTs and hypernephroma. Methods: Flow cytometric detection of cPD-1, cPD-L1, and P53 expression in relapsed and in-remission WT and hypernephroma before and after one year of chemotherapy was performed. Results: Our data shows increased levels of cPD-L1 in relapsed pediatric patients with WT or hypernephroma before and after chemotherapy. There were also slight and significant increases in cPD-1 levels in relapsed groups before chemotherapy. Additionally, we observed significant decreases in P53 expression after one year of chemotherapy in relapsed pediatric patients. Conclusions: Our study found that circulating PD-L1 can be used as a predictor marker for WT and hypernephroma relapse. In conclusion, these circulating markers can assist in monitoring relapse in WT and hypernephroma patients without the need for several biopsies.

Decreased Serum Tryptophan and Severe Ulcerative Damage of Colon Mucosa Identify Inflammatory Bowel Disease Patients With High Risk of Cytomegalovirus Colitis.

Patients with inflammatory bowel disease (IBD) are predisposed to the reactivation of viral infections such as cytomegalovirus (CMV). Clinical discrimination of disease flares and colonic CMV reactivation is difficult in patients with established diagnosis of IBD, and there are no reliable noninvasive diagnostic tools yet. Furthermore, the influence of novel therapeutics including biologicals and Janus kinase inhibitors on the risk of CMV colitis is unclear. The goal of this study was to identify risk factors and clinical determinants of CMV colitis that could serve as minimally invasive markers both for active CMV colitis and relapse. To this end, a retrospective analysis of 376 patients with suspected or confirmed CMV colitis 2016-2023 was performed. Previous administration of systemic steroids increased the odds of CMV colitis to OR 4.6. Biologicals did not change the incidence of CMV colitis but decreased the OR of a relapse to 0.13. Clinical parameters such as severely bloody diarrhea, intense microscopic ulcerative damage, and decreased serum tryptophan correlated with detection of CMV. Importantly, persistent decrease of tryptophan was observed in patients with CMV relapse. Furthermore, tryptophan degradation through the kynurenine pathway was increased in CMV-positive patients. Taken together, we identify decreased serum tryptophan as a novel potential minimally invasive marker to aid identification of IBD patients with active CMV colitis and at high risk for relapse.

Real-world effectiveness of antidepressants, antipsychotics and their combinations in the maintenance treatment of psychotic depression. Evidence from within-subject analyses of two nationwide cohorts.

Psychotic depression (PD) is a severe mental disorder leading to functional disability and high risk of suicide, but very little is known about the comparative effectiveness of medications used in its maintenance treatment. The objective of this study was to investigate the comparative effectiveness of specific antipsychotics and antidepressants, and their combinations, on the risk of psychiatric hospitalization among persons with PD in routine care. Persons aged 16-65 years with a first-time diagnosis of PD were identified from Finnish (years 2000-2018) and Swedish (years 2006-2021) nationwide registers of inpatient care, specialized outpatient care, sickness absence, and disability pension. The main exposures were specific antipsychotics and antidepressants, and the main outcome measure was psychiatric hospitalization as a marker of severe relapse. The risk of hospitalization associated with periods of use vs. non-use of medications (expressed as adjusted hazard ratio, aHR) was assessed by a within-individual design, using each individual as his/her own control, and analyzed with stratified Cox models. The two national cohorts were first analyzed separately, and then combined using a fixed-effect meta-analysis. The Finnish cohort included 19,330 persons (mean age: 39.8±14.7 years; 57.9% women) and the Swedish cohort 13,684 persons (mean age: 41.3±14.0 years; 53.5% women). Individual antidepressants associated with a decreased risk of relapse vs. non-use of antidepressants were bupropion (aHR=0.73, 95% CI: 0.63-0.85), vortioxetine (aHR=0.78, 95% CI: 0.63-0.96) and venlafaxine (aHR=0.92, 95% CI: 0.86-0.98). Any long-acting injectable antipsychotic (LAI) (aHR=0.60, 95% CI: 0.45-0.80) and clozapine (aHR=0.72, 95% CI: 0.57-0.91) were associated with a decreased risk of relapse vs. non-use of antipsychotics. Among monotherapies, only vortioxetine (aHR=0.67, 95% CI: 0.47-0.95) and bupropion (aHR=0.71, 95% CI: 0.56-0.89) were associated with a significantly decreased risk of relapse vs. non-use of both antidepressants and antipsychotics. In an exploratory analysis of antidepressant-antipsychotic combinations, a decreased relapse risk was found for amitriptyline-olanzapine (aHR=0.45, 95% CI: 0.28-0.71), sertraline-quetiapine (aHR=0.79, 95% CI: 0.67-0.93) and venlafaxine-quetiapine (aHR=0.82, 95% CI: 0.73-0.91) vs. non-use of antidepressants and antipsychotics. Benzodiazepines and related drugs (aHR=1.29, 95% CI: 1.24-1.34) and mirtazapine (aHR=1.17, 95% CI: 1.07-1.29) were associated with an increased risk of relapse. These data indicate that, in the maintenance treatment of PD, bupropion, vortioxetine, venlafaxine, any LAI, clozapine, and only few specific antidepressant-antipsychotic combinations areassociated with a decreased risk of relapse. These findings challenge the current recommendation by treatment guidelines to combine an antipsychotic with an antidepressant (without further specification) as standard treatment in PD.

Abstract 3952: Multiomic single-cell tumor evolution models of minimal residual disease in pediatric B-cell acute lymphoblastic leukemia

Abstract Detection of minimal residual disease (MRD) after initial diagnosis (IDX) and therapy is a strong prognostic marker for relapse in pediatric B-cell acute lymphoblastic leukemia (B-ALL). Previous studies have explored clonal genetic and gene expression changes between initial diagnosis and relapse. However, studying tumor cells present at MRD is challenging due to their low abundance. Examining the genomic profiles of leukemia cells that resist induction chemotherapy and understanding their evolutionary relationship to cells present at diagnosis may lead to a better understanding of the cells that eventually seed relapse. We analyzed somatic point mutations, indels, and copy number variants from 41 pediatric B-ALL patients with high-risk B-ALL. We specifically focused on patients from the following four common high-risk subtypes: KMT2A-rearranged, Ph+, Ph-like, and iAMP21, who had detectable disease at the end of induction. Leukemic burden at end-induction (day 29) ranged from 0.03% to 66.00%. Data were generated in partnership with the National Cancer Institute’s Human Tumor Atlas Network and the Children’s Oncology Group. Our data set of 41 patients includes 39 with paired initial diagnostic and end-induction samples that we used to create tumor evolution models based on single cells across disease stages. We produced single-cell multiome gene expression (scRNA) and chromatin accessibility (scATAC) profiles (10x Genomics) with a median of 7428 leukemic blast cells at IDX (range: 2490-14219) and 1907 at MRD (range: 240-10167) after filtering. We called somatic mutations from scRNA and scATAC using SComatic and Monopogen, and we detected copy number changes using Numbat and EpiAneufinder. We also designed a targeted DNA sequencing panel including 335 variants based on B-ALL literature and database searches to produce scDNA (Mission Bio) for 24 patients (16 IDX-MRD pairs) with a median of 2346 cells profiled at IDX (range: 340-8849) and 1828 at MRD (range: 596-5133). We built tumor phylogenies based on somatic SNVs and CNVs with COMPASS. Using multiple tools and complementary single-cell omics data types to detect somatic changes improved our confidence in the events used for tumor evolution tree construction. Our approach allowed us to define recurrent genomic events and patterns across pediatric B-ALL subtypes and to describe the clonal evolution of cells measured at initial diagnostic and end-induction disease stages. We were also able to link cell-specific mutation events to their transcriptional and epigenomic signatures, including mapping mutation events along the B-cell development trajectory and within differentially expressed gene pathways. Some cases showed dramatic shifts in subclonal structure between IDX and MRD that may be related to therapy-induced clonal selection, such as a loss of NRAS hotspot mutation. Citation Format: Steven M. Foltz, Avi Loren, Changya Chen, Rushabh Mehta, Elizabeth Li, Jason Xu, Fatemeh Alikarami, Kathrin M. Bernt, Kai Tan. Multiomic single-cell tumor evolution models of minimal residual disease in pediatric B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3952.

Abstract 980: Plasma tumor fraction is an independent prognostic marker of response to front line chemotherapy in high-grade serous epithelial ovarian cancer: The MITO-16a/MANGO-OV2a/clinical trial experience

Abstract Background. Although 80% of high grade serous ovarian cancer (HGSOC) patients are initially responsive to platinum (Pt)-based chemotherapy, the majority of them experience relapse with a progressive Pt-resistant disease. One of the most challenging issue that hampers the possibility to effectively treat relapsed disease is the lack of biological information about tumor recurrence. To date, the Pt-free interval (PFI), an empirical measure of the time lagging between the end of front-line chemotherapy and relapse, is the only and widely accepted parameter to predict patient outcome and sensitivity to Pt second-line chemotherapy. We have previously demonstrated that analysis of circulating tumor DNA (ctDNA) by low pass whole genome sequencing (sWGS) is a suitable tool to predict patient outcome and to intercept early signs of relapsed disease (Paracchini et al. PMID:33323403). In the present study, we quantified the percentage of tumor fraction (TF) in plasma of patients enrolled within the frame of a phase III clinical trial (MITO-16A/MaNGO-OV2A-EUDRACT number: NCT01706120) to demonstrate, at baseline, the prognostic value of untargeted ctDNA analysis. Methods. 172 eligible patients enrolled with the frame of the MITO-16A/MaNGO-OV2A clinical trial, for whom plasma sample was collected at chemotherapy before chemotherapy, were selected for the study. For each plasma sample, circulating-free DNA (cfDNA) was purified and whole genome libraries (HyperPlus, Roche) sequenced (mean coverage 0.5X). ichorCN algorithm was used to infer the percentage of TF, as previously published (Paracchini et al. PMID:33323403). Results. In 168 out of 172 plasma samples analyzed, the TF was detectable and it ranged from 2.4% to 47%. Cox-model univariate analysis with patients’ overall survival (OS) and Progression-Free Survival (PFS), indicates association of TF levels with both OS and PFS (p<0.001). The prognostic relevance of TF was also confirmed in multivariate analysis, where, considering CA-125, residual tumor, stage of disease, histology and age as co-variables, TF maintained its statistical significance in PFS (HR = 2.1, CI 95%, p-value < 0.003). Conclusion. Retrospective analysis of baseline plasma samples collected within the frame of the MITO-16A/MaNGO-OV2A clinical trial, demonstrated that the percentage of TF is an independent prognostic marker of relapse. This finding confirms the importance of liquid biopsy analysis based on untargeted sWGS tool as an innovative approach to predict response to front line therapy and patient’s outcome Citation Format: Sergio Marchini, Lara Paracchini, Laura Arenare, Giovanni Scambia, Carmela Pisano, Riccardo Zadro, Laura Mannarino, Annamaria Ferrero, Paolo Chiodini, Daniela Califano, Sandro Pignata, Maurizio D'Incalci. Plasma tumor fraction is an independent prognostic marker of response to front line chemotherapy in high-grade serous epithelial ovarian cancer: The MITO-16a/MANGO-OV2a/clinical trial experience [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 980.

Abstract A062: Untargeted analysis of plasma tumor fraction is an independent prognostic marker of response to front line chemotherapy in high-grade serous epithelial ovarian cancer: the MITO-16A/MANGO-OV2A clinical trial experience

Abstract Background: Although 80% of high grade serous ovarian cancer (HGSOC) patients are initially responsive to platinum (Pt)-based chemotherapy, the majority of them experience relapse with a progressive Pt-resistant disease. One of the most challenging issues that hampers the possibility to effectively treat relapsed disease is the lack of biological information about tumor recurrence. To date, the Pt-free interval (PFI), an empirical measure of the time lagging between the end of front-line chemotherapy and relapse, is the only and widely accepted parameter to predict patient outcome and sensitivity to Pt second-line chemotherapy. We have previously demonstrated that analysis of circulating tumor DNA (ctDNA) by low pass whole genome sequencing (sWGS) is a suitable tool to predict patient outcome and to intercept early signs of relapsed disease (Paracchini et al. PMID:33323403). In the present study, we quantified the percentage of tumor fraction (TF) in plasma of patients enrolled within the frame of a phase III clinical trial (MITO-16A/MaNGO-OV2A-EUDRACT number: NCT01706120) to demonstrate, at baseline, the prognostic value of untargeted ctDNA analysis. Methods: 172 eligible patients enrolled with the frame of the MITO-16A/MaNGO-OV2A clinical trial, for whom plasma sample was collected at baseline before chemotherapy, were selected for the study. For each plasma sample, circulating-free DNA (cfDNA) was purified and whole genome libraries (HyperPlus, Roche) sequenced (mean coverage 0.5X). ichorCN algorithm was used to infer the percentage of TF, as previously published (Paracchini et al. PMID:33323403). Results: In 168 out of 172 plasma samples analyzed, the TF was detectable, and it ranged from 2.4% to 47%. Cox-model univariate analysis with patients’ overall survival (OS) and Progression-Free Survival (PFS), indicate association of TF levels with both OS and PFS (p<0.001). The prognostic relevance of TF was also confirmed in multivariate analysis, where, considering CA-125, residual tumor, stage of disease, histology and age as co-variables, TF maintained its statistical significance in PFS (HR = 2.1, CI 95%, p-value < 0.003). Conclusion: Retrospective analysis of baseline plasma samples collected within the frame of the MITO-16A/MaNGO-OVtrialnical trial, demonstrated that the percentage of TF is an independent prognostic marker of relapse. This finding confirms the importance of liquid biopsy analysis based on untargeted sWGS tool as an innovative approach to predict response to front line therapy and patient’s outcome. Citation Format: Sergio Marchini, Lara Paracchini, Laura Arenare, Giovanni Scambia, Carmela Pisano, Riccardo Zadro, Laura Mannarino, Annamaria Ferrero, Paolo Chiodini, Daniela Califano, Piergiacomo Di Gennaro, Sandro Pignata, Maurizio D'Incalci. Untargeted analysis of plasma tumor fraction is an independent prognostic marker of response to front line chemotherapy in high-grade serous epithelial ovarian cancer: the MITO-16A/MANGO-OV2A clinical trial experience [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A062.

WT1 and TP53 as valuable diagnostic biomarkers for relapse after hematopoietic stem cell transplantation in acute myeloid leukemia.

Relapse following hematopoietic stem cell transplantation (HSCT) occurs relatively frequently and is a significant risk factor for mortality in patients with acute myeloid leukemia (AML). Early diagnosis is, therefore, of utmost importance and can provide valuable guidance for appropriate and timely intervention. Here, the diagnostic value of two molecular markers, Wilms tumor 1 (WT1) and tumor suppressor protein p53 (TP53), were studied. Twenty AML patients undergoing HSCT participated in this investigation. Some had relapsed following HSCT, while others were in remission. Peripheral blood (PB) and bone marrow (BM) samples were collected following relapse and remission. WT1 and TP53 messenger RNA (mRNA) expression was evaluated using reverse transcription-quantitative polymerase chain reaction (RT‒qPCR). The diagnostic value of genes was evaluated by utilizing receiver-operating characteristic (ROC) curve analysis. ROC analysis showed WT1 and TP53 as diagnostic markers for relapse after HSCT in AML patients. The mRNA expression level of WT1 was elevated in individuals who experienced relapse compared to those in a state of remission (p value < 0.01). Conversely, the expression level of TP53 mRNA was lower in individuals who had relapsed compared to those in remission (p value < 0.01). WT1 and TP53 possess the potential to serve as invaluable biomarkers in the identification of molecular relapse after HSCT in patients with AML. Further studies for a definitive conclusion are recommended.

Aligning outcomes: DLBCL prognosis at a 4th Level University Hospital in Bogotá is comparable to high-income nations, identification of additional prognostic markers for overall survival and relapse.

Diffuse large B-cell lymphoma (DLBCL), a prevalent non-Hodgkin lymphoma subtype, displays diverse clinical outcomes with persistently high mortality and relapse rates, despite treatment advancements. Notably, the Hispanic demographic lacks consideration in existing prognostic indices for DLBCL. A retrospective cohort study encompassing 112 DLBCL patients diagnosed between 2010 and 2020 was conducted at our institution. Patient data, including overall survival (OS), treatment response, and relapse, were analysed. With a median age of 65 years and a predominant male population (60.7%), both the International Prognostic Index (IPI) and revised IPI correlated with OS. In multivariate analysis, patients with ki-67 ≥ 60% exhibited higher mortality risk (Hazard Ratio: 2.35, 95% confidence intervals (CI) 1.05-5.27, p = 0.039), even when controlled by IPI category and B2-microglobulin levels. The absence of B symptoms served as a protective factor for relapse (p < 0.01, OR: 0.147, 95% CI 0.058-0.376) when controlling for ki-67, CD5, and IPI. Our cohort demonstrated a 5-year OS rate comparable to high-income countries, highlighting the need for tailored prognostic models for Hispanic DLBCL patients. This study identifies easily accessible parameters aligning with regional resource constraints, providing insights into additional prognostic factors for DLBCL in the Hispanic population.

Akkermansia muciniphila and Parabacteroides distasonis as prognostic markers for relapse in ulcerative colitis patients.

Ulcerative colitis is an inflammatory disorder characterized by chronic inflammation in the gastrointestinal tract, mainly in the colon and rectum. Although the precise etiology of ulcerative colitis remains unclear, recent research has underscored the significant role of the microbiome in its development and progression. The aim of this study was to establish a relationship between the levels of specific gut bacterial species and disease relapse in ulcerative colitis. For this study, we recruited 105 ulcerative colitis patients in remission and collected clinical data, blood, and stool samples. Akkermansia muciniphila and Parabacteroides distasonis levels were quantified in the stool samples of ulcerative colitis patients. Binary logistic regression was applied to collected data to predict disease remission. The median time in remission in this cohort was four years. A predictive model incorporating demographic information, clinical data, and the levels of Akkermansia muciniphila and Parabacteroides distasonis was developed to understand remission patterns. Our findings revealed a negative correlation between the levels of these two microorganisms and the duration of remission. These findings highlight the importance of the gut microbiota in ulcerative colitis for disease prognosis and for personalized treatments based on microbiome interventions.

Identification of Clinical-Biological Features of Newly Diagnosed Early Relapse Multiple Myeloma Patients Eligible for Autologous Stem Cell Transplantation

Despite many therapeutics advances, Multiple Myeloma (MM) remains an incurable hematological malignancy with a cohort of patients who relapse early or does not respond to first-line therapy, including autologous stem cell transplantation (ASCT). The proportion of patients with early relapse is stable over time at about 30% and they have a median Overall Survival (OS) less than 3 years. Consequently, clinical and/or biological features that identify patient at high risk for early relapse at diagnosis represent a critical unmet medical need. With the aim of identify possible factors of early relapse (ER), we conducted a retrospective, observational, single center study on a cohort of 74 MM patients who received an upfront ASCT between 2011 and 2021 after a novel agent-based induction at the Hematological and BMT Unit of the University Hospital of Parma (Italy). The primary end point of the study was the assessment of possible predictive markers for ER, defined as progressive disease that occurred within 18 months from the time of ASCT. Clinical, biochemical and cytogenetics features of the cohort of MM patients were evaluated (Table 1). Definitions of response and progression were used according to the International Myeloma Working Group (IMWG) response criteria. All statistical analyses were done by SPSS Statistics. The characteristics of cohorts were summarized using the median value for continuous variates and the frequency for categorical ones. The Pearson chi-square test and Student t-test were used to identify differences between groups for categorical and continuous variables, respectively. Kaplan-Meier Curve with log-rank test were used to analyze survival data. The bivariate logistic analysis was conducted to recognize independent factors predicting ER, subsequently Cox regression analysis was performed to confirm the validity of multivariate analysis for survival data. The cohort characteristics were summarized in table 1. Among the 74 MM patients included in the study, a total of 19 patients (25.5) experienced ER, with mean time to relapse of 15.6 (18.0) months. Univariate statistical analysis identified as possible predictive markers for early relapse at diagnosis: IgA MM (p &amp;lt; 0.05), elevated serum LDH level (p &amp;lt; 0.05), C-CRAB criteria (p &amp;lt; 0.05), high risk cytogenetic aberrations (p &amp;lt; 0.05), stage R-ISS III (p &amp;lt; 0.001) and stage R2-ISS III or IV (p &amp;lt; 0.001). Multivariate statistical analysis confirmed IgA isotype (p &amp;lt; 0.05) and higher stage according to R2-ISS (p &amp;lt; 0.001) as effectively independent predictive risk factors for ER. Time-to-event analysis displayed a mean progression free survival (PFS) from transplant of 24.7 (16.0) months and 32.5 (19.0) months for those with IgA MM and stage R2-ISS III or IV, respectively. In the group of patients with IgA isotype the mean OS from transplant was 49.5 (30.0) months, while the mean OS from diagnosis was 58.0 (35.0) months without statistically significant differences compared to counterpart. In the group of patients with stage R2-ISS III or IV the mean OS from transplant was 48.2 (41.0) months, while the mean OS from diagnosis was 56.5 (49.0) months. Finally, in ER cohort the mean OS from diagnosis was 47.1 (35.0) in comparison with the 75.0 (71.0) months of non-ER cohort. In conclusion, this retrospective study was able to identify the main features of newly diagnosed early relapsed MM patients eligible to ASCT identifying the IgA isotype and the R2-ISS score system as the main predictive prognostic factors for early relapse in this cohort of patients.

Sensitive Molecular Detection of JAK2 V617F Is a Predictive Marker of Relapse in Patients with Myelofibrosis after Allogeneic Stem Cell Transplantation

Background: Myelofibrosis (MF) is a BCR-ABL1-negative myeloproliferative neoplasm (MPN) associated with JAK2 V617F mutation in approximately 50% of patients. Allogeneic stem cell transplantation (alloSCT) is the only curative treatment option for MF but a significant proportion of patients undergoing alloSCT experience relapse. Although measurable residual disease (MRD) is an established predictor of relapse in other hematological malignancies, the dynamics of JAK2 V617F variant allele frequency (VAF) in MF post-alloSCT and its prognostic impact are yet to be clearly delineated. In this study, we assessed the utility of sensitive molecular detection of JAK2 V617F as a marker of post-alloSCT outcomes in MF particularly with respect to the optimal threshold and time points for predicting early relapse. Methods: A retrospective analysis was conducted of consecutive patients with JAK2 V617F-mutated MF receiving alloSCT at our institution between 2016 and 2022. Data on clinicopathological features, alloSCT characteristics, treatment response assessments, graft-versus-host disease (GVHD) and outcomes were collected. JAK2 V617F was enumerated by droplet digital polymerase chain reaction (ddPCR) using the Bio-Rad QX200 ddPCR platform on archived bone marrow or peripheral blood samples at defined time points pre- and post-alloSCT. JAK2 V617F VAF was defined as the percentage of JAK2 V617F mutant copies amongst total JAK2 (mutant plus wild-type) copies, with a routine assay sensitivity of 0.05%. Results: 31 patients with JAK2 V617F-mutated MF underwent alloSCT during the study period (Table 1). Five patients experienced relapse at a median of 14.2 (range: 3.2-22) months post-alloSCT. 13 patients died of infection (n = 5), overt relapse (n = 4) or GVHD (n = 4). After a median duration of follow-up of 17.5 (range: 0.9-77.9) months, the 5-year overall survival (OS) and relapse-free survival (RFS) were 52.8% and 49.6%, respectively. The 2-year cumulative incidences of relapse (CIR) and non-relapse mortality (NRM) were 26.6% and 35.5%, respectively. On univariate analysis, superior OS and RFS were only significantly associated with chronic GVHD ( P = 0.017 and 0.013, respectively). A total of 174 samples were obtained within 1 month of alloSCT (n = 31) and on days +30 (n = 31), +60 (n = 30), +100 (n = 29), +180 (n = 23) and +360 (n = 21) after alloSCT. All patients had detectable JAK2 at the time of alloSCT with a median VAF of 43.8% (range: 0.2-95%). The incidence of JAK2 V617F detection was 45.2% on day +30, 43.3% on day +60, 44.8% on day +100, 34.8% on day +180 and 23.8% on day +360 post-alloSCT. Of 6 patients who received ruxolitinib for GVHD treatment post-alloSCT, one eventually relapsed while the other 5 had undetectable JAK2 prior to ruxolitinib commencement. JAK2 VAF was higher in patients who eventually relapsed than in non-relapsed patients on days +60, +100, +180 and +360 ( P = 0.008, 0.016, 0.001 and &amp;lt;0.001, respectively) (Figure 1). However, JAK2 positivity at each of these time points was not significantly associated with survival outcomes (OS, RFS, NRM or GVHD-free relapse-free survival [GRFS]). Receiver operating characteristic (ROC) analysis indicated that JAK2 VAF on days +100, +180 and +360 post-alloSCT were significant predictors of eventual relapse ( P = 0.022, 0.019 and 0.01, respectively). The optimal JAK2 VAF thresholds at each of these time points were 0.2% (sensitivity 80%; specificity 83.3%; area under curve [AUC] 0.788) at day +100, 7.1% (sensitivity 75%; specificity 94.7%; AUC 0.842) at day +180, and 11.7% (sensitivity 75%; specificity 94.1%; AUC 0.86) at day +360. Comparisons of JAK2 VAF at each time point to the immediately preceding VAF showed that ≥2-fold increases at days +100 (from day +60) and +180 (from day +100) were significantly associated with relapse ( P = 0.002 and 0.001 respectively). Conclusion: Although persistence of JAK2 is common for more than a year post-alloSCT for JAK2 V617F-mutated MF, serial sensitive molecular monitoring of JAK2 at defined time points appears to be a predictive marker of relapse. In this study, doubling of JAK2 VAF at days +100 and +180 post-alloSCT were associated with relapse. These data support the notion that sensitive molecular detection of JAK2 may have a role akin to MRD in informing the pre-emptive use of immunotherapeutic intervention in this setting, including immunosuppression withdrawal and/or donor lymphocyte infusions.

Relapse-Enriched Gene Expression Signature of Prognostic Relevance in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the abnormal proliferation and accumulation of immature myeloid cells. While standard induction therapy achieves remission in a substantial proportion of AML patients, relapse rates remain high, particularly in children (approximately 40%) and adults (around 70%). Despite advancements in AML treatment strategies, the management of relapsed and refractory disease remains significant clinical challenge, resulting in dismal outcomes with a three-year survival rate below 10%. Therefore, there is an urgent need to identify predictive markers for relapse and refractory AML to improve treatment outcomes. To address this urgent need, we evaluated differential gene expression in relapse vs diagnosis and its impact on outcomes. RNA sequencing data from bone marrow samples were obtained from pediatric AML patients enrolled in four Children's Oncology Group clinical trials: AAML0531 (NCT00372593), AAML1031 (NCT01371981), AAML03P19 (NCT00070174), and CCG2961 (NCT00003790) through the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Gene expression data was batch corrected using Combat. RNA-seq data from 1441 patients available at https://portal.gdc.cancer.gov/projects/TARGET-AML were included in the current analysis of which 31 had RNA-seq data from both diagnosis and relapse and 1420 had RNA-seq data from only from diagnosis. In step 1, we identified genes differentially expressed at relapse using RNA-seq data from 31 matched diagnosis and relapse samples using wilcoxon signed rank test. At p-values &amp;lt;0.005, 201 genes were differentially expressed, with 147 upregulated and 54 downregulated at relapse compared to diagnosis (Figure 1). To further establish clinical relevance of these genes, we evaluated their association with event-free survival (EFS) in the 1420 patients with RNA-seq data at diagnosis (N=1420) using Cox proportional hazard model, identifying 47 genes significantly associated with EFS (p&amp;lt;0.05). Of these, 9 were pseudogenes, 4 had lower expression at relapse and 34 were expressed at higher levels in the relapse samples. We evaluated these 34 genes using a LASSO penalized Cox proportional hazards model, with over 1000 iterations of 10-fold cross-validation. Genes passing more than 95% of the iterations were further selected to build a relapse signature which constituted 23 genes.Notably, this signature was comprised of several important genes including TCF3, a transcription factor with known mutations in some B-cell acute lymphoblastic leukemia (B-ALL) cases; BIRC5 (Survivin), an apoptosis inhibitor whose overexpression has been linked to therapeutic resistance across cancers; PAX5, a key gene for B-cell differentiation that has identified aberrations B-ALL; BCL7A, a member of the BCL gene family associated with lymphoid malignancies, as B-cell lymphomas; and PARP1, known for its role in DNA repair and the recent therapeutic implications of its inhibitors in BRCA-mutated solid tumors. Patients were further stratified into high and low relapse risk groups based on relapse signature scores using recursive partitioning. Patients with high relapse signature score had inferior EFS and OS as compared to those with low signature score (EFS, HR: 2.25, 95% CI 0.25-0.99, p&amp;lt;0.001); OS, (HR 2.35,95% CI 0.40-0.99, p&amp;lt;0.001), Figure 1). Multivariable Cox regression analysis further revealed that the relapse score remained a significant predictor of patient outcomes after adjusting for established risk factors including risk group, race, white blood cell count, and age at diagnosis (EFS: HR 1.64, 95% CI, 1.40-1.92, p&amp;lt;0.001 and OS: HR 1.62, 95% CI 1.33-1.97, p&amp;lt;0.001). To validate the signature, we evaluated it in an independent cohort of 147 pediatric AML patients treated on St Jude AML08 clinical trial with RNA seq data available. Consistent with the discovery cohort, patients with high score had worse EFS (HR: 1.83, p=0.045) and tended to have lower OS (HR: 1.71, p=0.12). These results indicate the relapse-enriched gene expression signature can effectively risk-stratify newly diagnosed pediatric AML patients. Ongoing studies are focused on validating this signature in other independent cohorts, as well as integrating it with cytogenetic features and other previously reported signatures of relevance such as LSC to establish its prognostic utility.

Measurable Residual IDH2 before Allogeneic Transplant for Acute Myeloid Leukemia

Introduction: Detection of measurable residual disease (MRD) in AML patients during remission is associated with adverse clinical outcomes after allogeneic hematopoietic cell transplant (alloHCT), including recent strong evidence from our group regarding residual NPM1 and/or FLT3-ITD variants (PMID: 36881031). Large studies examining the significance of other potential MRD targets, including isocitrate dehydrogenase isoform 2 ( IDH2), are needed. IDH2, which is mutated in approximately 12% of AML patients, is of particular interest due to the possibility of targeted clinical intervention. Methods: Patients over the age of 18 in first complete remission (CR1) with IDH2 mutated AML undergoing alloHCT transplant at a CIBMTR site between 2013-2019 with remission blood samples within 100 days of alloHCT and available clinical data were included in this study. Residual variants in IDH2, NPM1, and FLT3genes were detected by ultrasensitive error-corrected next-generation sequencing (NGS-MRD) on genomic DNA from pre-conditioning blood and validated by digital droplet PCR (ddPCR). The primary outcomes were overall survival (OS) and cumulative incidence of relapse with non-relapse mortality as a competing risk. The day of transplant was considered as time 0, and the median follow-up time was calculated for censored patients. Kaplan-Meier estimation (log-rank tests), Cox proportional hazards models, and Fine-Gray regression models were fitted to examine the clinical outcomes. Results: Among the 257 IDH2-mutated AML patients in this study, 57 (23%) experienced relapse, with 41 events occurring within 12 months after alloHCT. The median follow-up time was 25 months. Among 247 patients with pre-transplant remission flow cytometry results reported, 25% were positive, but no difference in rates of relapse or OS was observed between positive and negative patients. NGS MRD analysis detected residual IDH2 variants in remission samples of 130 patients (51%) with variant allele fraction (VAF) ranging from 0.05-56% (median: 3%), and 100% validation by ddPCR. NGS MRD IDH2 positive patients had increased rates of relapse (p = 0.03; 3yr: +11%, 95% CI: 0.2% to 22%) and decreased OS (p &amp;lt; 0.001; 3yr: -25%, 95% CI: -13% to -38%) compared to those who tested negative (Figure 1A). These OS differences were still observed between NGS MRD IDH2 positive and negative patients, regardless of age stratification (under 60yr p &amp;lt; 0.001, 3yr: -28%, 95% CI: -12% to -43%; over 60yr p = 0.04, 3yr: -19%, 95% CI: -1% to -40%); however, only patients over 60yrs had increased rates of relapse (p = 0.007; 3yr: +21, 95% CI: 6% to 36%). The impact of co-mutation status was examined by subdividing the cohort based on the presence of NPM1 or FLT3-ITD mutations at baseline. Residual IDH2 variants in patients without baseline NPM1 or FLT3-ITD co-mutations (n = 151) were associated with decreased OS and increased relapse ( IDH2+ vs IDH2-; OS p = 0.005, 3yr: -23%, 95% CI: -6% to -40%; Relapse p = 0.01, 3yr: +19, 95% CI: 6% to 32%; Figure 1B). In contrast, residual IDH2 variants were not predictive of relapse in co-mutated patients (n=106, p = 0.5). In these patients, the only prognostic marker for relapse was NPM1 and/or FLT3-ITD MRD, with similar relapse risk between IDH2 only positive and MRD negative groups (p &amp;lt; 0.001; 3yr: 83% vs. 11% vs. 15%; Figure 1B). In multivariable analysis, IDH2 MRD positivity was associated with decreased OS (HR 2.5, 95% CI: 1.5-4.2, p &amp;lt; 0.001) and increased rates of relapse (HR 2.1, 95% CI: 1.4-5.7, p = 0.003) after adjusting for other characteristics. Conclusion: In this study, we demonstrate that adult patients with IDH2-mutated AML who have IDH2 variants detected in CR1 prior to first alloHCT experienced increased relapse rates and decreased overall survival. However, this association is significant only in patients without baseline NPM1 and FLT3-ITD mutations. In co-mutated patients, NPM1 and/or FLT3-ITD MRD serve as superior prognostic markers. Subgroup analyses show the potential influence of factors such as conditioning intensity and age on clinical outcomes. Nevertheless, larger studies with balanced subgroups and more comprehensive baseline mutational information, including IDH2 mutation subtype, are needed to better define the clinical relevance of IDH2 MRD in this context.

Gene Panels for Intraocular Lymphoma Can Overcome Diagnostic Difficulties and Enable Precision Medicine

Background: Definitive diagnosis of intraocular lymphoma (IOL) is often challenging, at least partially due to limited accessibility to the lesion and the rarity of tumor cells, with pathologically false-negative results reportedly occurring in approximately 30% of cases. Delayed diagnosis is often an important clinical issue, and the average time to diagnosis is approximately 1.5 years.Because of delayed diagnosis, IOLs are often found only after central nervous system (CNS) invasion has occurred, possibly leading to worse treatment outcomes. In this study, we tried to overcome diagnostic difficulties by using gene panel diagnostics of IOL. Furthermore, genetic analysis of IOLs may be able to predict the risk of recurrence.in Japan, a second-generation Bruton's tyrosine kinase (BTK) inhibitor, tirabrutinib, has been approved for the treatment of relapsed/refractory CNS lympoma. Herein, we present a case of IOL and CNS lesion with MYD88 mutation treated with tirabrutinib as part of precision medicine. Methods: We have developed a digital polymerase chain reaction-based system for multiplex gene panel testing of cell-free DNA (cfDNA) obtained from intraocular liquid biopsies (patent number 7149000, 2022/9/30). Our system can detect specific gene mutations in minute cfDNA by amplifying specific gene loci in bubbles, even from an extremely small number of cells. Using this multiplex gene panel testing, we analyzed genetic mutations in nine regions of four genes, MYD88, CD79B, BTG2, and PIM1, using cfDNA obtained from intraocular liquid biopsies. The diagnostic performance of the novel system was evaluated in 86 samlpes of patients with IOL. We also retrospectively analyzed the prognostic potential of the genetic mutations for IOL. Lastly, in the case of CNS lymphoma and IOL, we presented the MYD88 mutation treated with tirabrutinib. Results: Among the four disease-specific genes, the MYD88 mutation was present in 55 of 86 cases (64%), and frequencies of the CD79B, BTG2, and PIM1 mutations were 47%, 38%, and 29%, respectively. At least one of these mutations was found in 83 of the 86 IOL cases examined (97%), making this assay highly sensitive (Fig. 1). The MYD88 mutation was not associated with recurrence in patients with recurrent and refractory CNS lymphoma and IOL. Conversely,patients with the CD79B mutation had high rates of relapse for CNS lymphoma: the 4-year rate of recurrence-free survival was 35%, compared to 79% in patients without the CD79B mutation (p=0.012). Patients with the BTG2 mutation also had high rates of relapse (Fig. 2). Furthermore, we present a case of CNS lynphoma and IOL. By testing IOL gene panel, only MYD88 mutation was detected in the vitreous sample of IOL patient. We have experienced this case in which the second-generation BTK inhibitor,tirabrutinib, was effective not only for central but also for intraocular lesions. Conclusion: Our panel enables rapid and accurate diagnosis of IOL with only a small sample of intraocular liquid. We believe that this multiplex gene panel testing may aid in the diagnosis of IOL in difficult-to-diagnose patients, including those with chronic uveitis, by preventing CNS invasion due to diagnostic delay. Moreover, genetic profiling panels of IOL can help to stratify clinical outcomes.CD79B and BTG2 mutation is a potent predictive marker for relapse. In the clinical setting, we experienced a case of IOL with MYD88 mutation in which tirabrutinib was effective. Potentially, tirabrutinib also penetrates directly into the eye in intraocular lesions. Oral drug, tirabrutinib as eye dripping drug, might be effective as a therapeutic option for IOL and should be assessed in future clinical trials of precision medicine for IOL.

Mitochondrial DNA in Urinary Large Extracellular Vesicles as a Marker of Relapse in Children with Nephrotic Syndrome

Mitochondrial DNA in Urinary Large Extracellular Vesicles as a Marker of Relapse in Children with Nephrotic Syndrome

Microfibrillar-associated protein 4 as a potential marker of acute relapse in inflammatory demyelinating diseases of the central nervous system: Pathological and clinical aspects

Background: Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein not previously described in the human central nervous system (CNS). Objectives: We determined MFAP4 CNS expression and measured cerebrospinal fluid (CSF) and serum levels. Methods: Tissue was sampled at autopsy from patients with acute multiple sclerosis (MS) (n = 3), progressive MS (n = 3), neuromyelitis optica spectrum disorder (NMOSD) (n = 2), and controls (n = 9), including 6 healthy controls (HC). MFAP4 levels were measured in 152 patients: 49 MS, 62 NMOSD, 22 myelin oligodendrocyte glycoprotein-associated disease (MOGAD), and 19 isolated optic neuritis (ION). Results: MFAP4 localized to meninges and vascular/perivascular spaces, intense in the optic nerve. At sites of active inflammation, MFAP4 reactivity was reduced in NMOSD and acute MS and less in progressive MS. CSF MFAP4 levels were reduced during relapse and at the onset of diseases (mean U/mL: MS 14.3, MOGAD 9.7, and ION 14.6 relative to HC 17.9. (p = 0.013, p = 0.000, and p = 0.019, respectively). Patients with acute ON (n = 68) had reduced CSF MFAP4 (mean U/mL: 14.5, p = 0.006). CSF MFAP4 levels correlated negatively with relapse severity (rho = −0.41, p = 0.017). Conclusion: MFAP4 immunoreactivity was reduced at sites of active inflammation. CSF levels of MFAP4 were reduced following relapse and may reflect disease activity.

Changes of serum osteoprotegerin level in recurrent urolithiasis

Introduction. In our earlier research on the role of genetic factors in the development of recurrent urolithiasis, a significant relationship was found between the presence of single-nucleotide polymorphism rs3134057 in the osteoprotegerin (OPG) gene and the development of this disease. The above-mentioned single-nucleotide polymorphism is intronic, therefore, it is unable to involve the structure and functions of the OPG but to affect its expression. Thus, the study of serum OPG level may have diagnostic significance in recurrent urolithiasis.Objective. To study the relationship of serum OPG level with the presence of recurrent urolithiasis, as well as the effect of single nucleotide polymorphism rs3134057 on the OPG level.Materials &amp; methods. One hundred and fifteen volunteers were included in the study from January 2021 to January 2022. Of the 115 participants, 45 (main group #1) were diagnosed with recurrent urolithiasis and localisation of the main stone in one of the ureters, 28 participants (main group #2) had previously suffered a single episode of acute renal colic followed by surgical treatment or stone self-discharge and were stone-free at the time of the study. Forty-two control group respondents were free of urolithiasis, including family history of urolithiasis. The serum OPG levels was determined by enzyme immunoassay using the Human OPG ELISA kit ab100617 ("Abcam plc", Cambridge, UK) according to the manufacturer's advice.Results. The analysis revealed that the serum OPG levels were significantly higher in the main group #1 than in the control group (median: 9.02 vs. 3.635, p = 0.012). When analyzing the relationship between the concentration of OPG in serum and the AA + AG genotype versus GG according to rs3134057 of the OPG gene, no statistically significant difference was found (p &gt; 0.05). The same result was obtained when comparing groups with AA vs. AG vs. GG genotypes (p &gt; 0.05).Conclusion. The present study showed that an increased serum OPG level in patients with recurrent urolithiasis may indicate the presence of a relapse of the disease. Further study in large samples will finally confirm the significance of OPG as a marker of relapse, including in genetically determined forms.