Validation of DNA methylation to predict outcome in acute myeloid leukemia by use of xMELP

Epigenetic dysregulation involving alterations in DNA methylation is a hallmark of various types of cancer, including acute myeloid leukemia (AML). Although specific cancer types and clinical aggressiveness of tumors can be determined by DNA methylation status, the assessment of DNA methylation at multiple loci is not routinely performed in the clinical laboratory. We recently described a novel microsphere-based assay for multiplex evaluation of DNA methylation. In the current study, we validated and used an improved assay [termed expedited microsphere HpaII small fragment Enrichment by Ligation-mediated PCR (xMELP)] that can be performed with appropriate clinical turnaround time. Using the xMELP assay in conjunction with a new 17-locus random forest classifier that has been trained using 344 AML samples, we were able to segregate an independent cohort of 70 primary AML patients into methylation-determined subgroups with significantly distinct mortality risk (P = 0.009). We also evaluated precision, QC parameters, and preanalytic variables of the xMELP assay and determined the sensitivity of the random forest classifier score to failure at 1 or more loci. Our results demonstrate that xMELP performance is suitable for implementation in the clinical laboratory and predicts AML outcome in an independent patient cohort

Changes in Paracrine Interleukin-2 Requirement, CCR7 Expression, Frequency, and Cytokine Secretion of Human Immunodeficiency Virus-Specific CD4(+) T Cells Are a Consequence of Antigen Load

Virus-specific CD4(+) T-cell responses are thought to be required for the induction and maintenance of many effective CD8(+) T-cell and B-cell immune responses in experimental animals and humans. Although the presence of human immunodeficiency virus (HIV)-specific CD4(+) T cells has been documented in patients at all stages of HIV infection, many fundamental questions regarding their frequency and function remain. A 10-color, 12-parameter flow cytometric panel was utilized to examine the frequency, memory phenotype (CD27, CCR7, and CD45RA), and cytokine production (interleukin-2 [IL-2], gamma interferon, and tumor necrosis factor alpha) of CD4(+) T cells specific for HIV antigens as well as for adenovirus, Epstein-Barr virus (EBV), influenza H1N1 virus, influenza H3N2 virus, cytomegalovirus, varicella-zoster virus (VZV), and tetanus toxoid in normal controls, long-term nonprogressors (LTNP), and HIV-infected patients with progressive disease on or off therapy. The HIV-specific CD4(+) T-cell responses in LTNP and patients on therapy were similar in frequency, phenotype, and cytokine production to responses directed against adenovirus, EBV, influenza virus, and VZV. HIV-specific CD4(+) T cells from patients off antiretroviral therapy demonstrated a shift towards a CCR7(−) CD45RA(−) phenotype and a reduced percentage of IL-2-producing cells. The alterations in cytokine production during HIV viremia were found to be intrinsic to the HIV-specific CD4(+) T cells and caused a requirement for IL-2 supplied exogenously for proliferation to occur. These observations suggest that many previously described changes in HIV-specific CD4(+) T-cell function and phenotype are a consequence of high levels of antigen in viremic patients. In addition, defects in function and phenotype of HIV-specific CD4(+) T cells are not readily discernible in the context of antiretroviral therapy but rather are similar to responses to other viruses

Exploring Influencing Factors of Anxiety Improvement Following Mindfulness-Based Music Therapy in Young Adults with Cancer

The purpose of this secondary analysis was to explore physiological, psychological, and situational influencing factors that may affect the impact of a mindfulness-music therapy intervention on anxiety severity in young adults receiving cancer treatment. Young adults receiving cancer treatment for ≥ eight weeks were recruited from adult and pediatric oncology outpatient centers at Dana-Farber Cancer Institute. Participants were asked to attend up to four, in-person (offered virtually via Zoom video conference after the onset of the COVID-19 pandemic) 45-min mindfulness-based music therapy sessions over twelve weeks with a board-certified music therapist. Participants completed questionnaires about anxiety, stress, and other cancer treatment-related outcomes before and after participating in the intervention. Changes in anxiety (i.e., PROMIS Anxiety 4a) over time were compared among baseline physiological (e.g., age or sex), psychological (e.g., stress), and situational influencing (i.e., intervention delivery format) factors using Wilcoxon-rank sum tests. Thirty-one of the 37 enrolled participants completed the baseline and post-intervention measures and were eligible for inclusion in the secondary analysis. Results revealed that higher baseline physical functioning (median change = −6.65), anxiety (median change=-5.65), fatigue (median change = −5.6), sleep disturbance (median change = −5.6), female sex (median change = −5.15), or virtual intervention delivery (median change = −4.65) were potential physiological, psychological, or situational influencing factors associated with anxiety improvement following mindfulness-based music therapy. Additional investigation into physiological, psychological, or situational influencing factors associated with anxiety response will help to tailor the design of future mindfulness-music therapy interventions to decrease psychological distress and address the unique psychosocial concerns among young adults receiving cancer treatment. Trial Registration ClinicalTrials.gov Identifier: NCT0370922

Outcome of relapsed or refractory acute B-lymphoblastic leukemia patients and <i>BCR-ABL</i>-positive blast cell crisis of B-lymphoid lineage with extramedullary disease receiving inotuzumab ozogamicin

Acute lymphoblastic leukemia (ALL) can relapse in the extramedullary compartment, with or without medullary involvement. Response to treatment may be individual. We evaluated response to inotuzumab ozogamicin in 31 patients with relapsed/refractory B-ALL with extramedullary disease. Median age was 31 years (range, 19-81). All patients were heavily pretreated, including allogeneic hematopoietic stem cell transplantation (HSCT; n=18). Overall response rate after two cycles of inotuzumab ozogamicin was 84% (complete remission, 55%; partial remission, 29%; resistant disease, 13%; early death, 3%). The median follow-up was 29 months and median overall survival was 12.8 months. One-year and 2-year overall survival rates were 53% (95% CI: 37-76%) and 18% (95% CI: 8-43%), respectively. Age had no impact on overall survival when assessed as a continuous variable or dichotomized at 60 years. Twelve patients proceeded to allogeneic HSCT (complete remission, n=6; partial remission, n=3; resistant disease, n=3). Prior to allogeneic HSCT, eight patients received two or fewer cycles and four patients received three or four cycles of inotuzumab ozogamicin. Sinusoidal obstruction syndrome was reported in three patients, including one after transplantation. Allogeneic HSCT, evaluated as a time-dependent variable, had no impact on overall survival. Inotuzumab ozogamicin seems to be effective as a debulking strategy in relapsed/refractory ALL with extramedullary disease. However, inotuzumab ozogamicin followed by allogeneic HSCT seems not to be effective in maintaining long-term disease control

A Clinical Measure of DNA Methylation Predicts Outcome in De Novo AML

Abstract Background: Despite advances, the majority of patients (pts) with acute myeloid leukemia (AML) will die of their disease. Current genetics-based risk classification schemes inadequately predict outcome. We sought to determine if a novel methylation-based biomarker could enhance risk stratification in AML. Methods: Using a novel methylation assay (xMELP) validated for the clinical laboratory, we performed DNA methylation assessment at 17 previously identified prognostic loci to calculate a summary methylation statistic (M-score, Wertheim et al. Clin Chem 2015) for 166 pts with de novo AML treated at the University of Pennsylvania (UPENN). Targeted next-generation sequencing (NGS) of 33 hematologic malignancy-associated genes was performed for a 136 pt subset. The association of M-score with other prognostic variables and outcome [complete remission (CR) and overall survival (OS)] was evaluated. Median follow-up was 68.1 mos (range 1.4-150.2) among 38 survivors and 10.5 mos (range 0.1-95.2) among those (n=128) deceased. The optimal M-score for identifying groups with differing OS in the total UPENN cohort defined a binary classifier. The classifier was validated in UPENN subgroups and in a separate ECOG ACRIN E1900 trial cohort. Results: The mean and median M-score for the UPENN cohort was 92.3 (95% confidence interval [CI], 87.4-97.2) and 91.4 (range, 30.8-97.3), respectively; the M-score was not significantly associated with age, gender, specimen type, blast percent, NPM1, or FLT3-ITD. Patients with favorable cytogenetics had a lower mean M-score than those with non-favorable cytogenetics; there was no difference between intermediate and unfavorable cytogenetic groups. Univariate analyses demonstrated that a 10-unit increase in M-score was associated with a 10% increase in the hazard of death (HR 1.1; P<.0001) and a 20% increase in the odds of failing to achieve CR (OR 1.2, P<.0001). In multivariable Cox analysis, higher M-score (HR 1.1, P=.011) and older age (P=.001) were significantly associated with increased hazard of death, while NPM1+/FLT3-ITD-status (P=.031) was associated with decreased hazard of death. In a multivariable logistic analysis, higher M-score (HR, 1.1, P=.034) and older age (P=.007) were associated with increased odds of failing to achieve CR, while favorable cytogenetics (P=.030) was associated with achievement of CR. Based on the maximization of the log-rank statistic, the optimal M-score cutpoint was 86, which defined a binary risk classifier (hazard of death for high vs low M-score: unadjusted HR 2.5, P<.0001; adjusted HR 1.9, P=.003). Median OS was 26.6 vs. 10.6 mos for low and high M-score groups (Figure). The CR rates for low and high M-score groups were 84% and 61%, respectively. The performance of the M-score classifier was confirmed in pts ≤ 60 yrs with intermediate cytogenetics (log-rank P=.001, OS in high vs low M-score groups: 36.4 vs 14.0 mos) and among pts who achieved CR with initial therapy (log-rank P<.00001, OS 43.9 vs 17.2 mos). The M-score classifier identified groups with different outcome regardless of allogeneic transplant. The M-score classifier was validated in the E1900 cohort (n=383). The mean and median M-scores were similar to the UPENN cohort and M-score was significantly associated with OS on multivariable analysis (P<0.0001). The M-score classifier identified E1900 subgroups with different OS (log-rank P<.00001; OS 29.5 vs 12.6 mos), a finding confirmed in the intermediate cytogenetics group, in those aged <50 and ≥50 yrs, and among pts assigned to both standard and high dose daunorubicin. Notably, high dose daunorubicin benefited patients with high M-scores (P=.001), but not those with low M-scores (P=.328). We also evaluated the prognostic value of the M-score in the context of an extended molecular profile. Random forest analysis of the UPENN cohort showed that M-score and age are the most robust predictors of OS, while a subset of recurrent mutations (FLT3-ITD, NPM1, IDH2, TET2, TP53, NRAS, CEBPA) also contribute to prognosis, but to a lesser degree. Conclusion: The M-score and associated classifier represent promising tools for clinical decision-making in AML and deserve further investigation. The prognostic value of the M-score may be superior to mutational analysis. Optimal methods for integration of M-score, clinical and genetic information are being defined. Figure 1. Figure 1. Disclosures Loren: Merck: Research Funding. Levine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees

Diminished Production of Monocyte Proinflammatory Cytokines during Human Immunodeficiency Virus Viremia Is Mediated by Type I Interferons

The effect of human immunodeficiency virus (HIV) infection and high-level HIV replication on the function of monocytes was investigated. HIV-positive patients had elevated levels of spontaneous production of some or all of the monocyte proinflammatory cytokines measured (interleukin-1β [IL-1β], IL-6, and tumor necrosis factor alpha [TNF-α]) compared to uninfected controls. In patients on therapy with high frequencies of monocytes producing proinflammatory cytokines, this frequency was diminished in the context of viremia during an interruption of therapy. Diminished production of proinflammatory cytokines during viremia was restored by culture with autologous CD4(+) T cells or monocytes from an on-therapy time point or lipopolysaccharide (LPS). Microarray analysis demonstrated that diminished monocyte production of proinflammatory cytokines was correlated with elevated type I interferon-stimulated gene transcripts. The addition of exogenous alpha 2A interferon diminished the spontaneous production of IL-1β, IL-6, and TNF-α but did not affect responses to LPS, recapitulating the changes observed for HIV-viremic patients. These results suggest that monocyte function is diminished during high-level HIV viremia and that this effect is mediated by chronic stimulation by type I interferons. This effect on monocytes during viremia may play a role in diminished innate or adaptive immune system functions in HIV-infected patients. In addition, the restoration of these functions may also play a role in some immune reconstitution syndromes observed during initiation of therapy