Oral azacitidine prolongs survival of patients with AML in remission independent of measurable residual disease status

Measurable residual disease (MRD) in patients with acute myeloid leukemia (AML) in remission after intensive chemotherapy is predictive of early relapse and poor survival. Post-remission maintenance therapy that prolongs MRD negativity or converts MRD positive (MRD+) patients to MRD negative (MRD-) status may delay or prevent relapse and improve overall survival (OS). In the phase 3 QUAZAR AML-001 trial, oral azacitidine (Oral-AZA; formerly CC-486), a hypomethylating agent, significantly prolonged OS and relapse-free survival (RFS) compared with placebo in patients aged ≥55 years with AML in first remission after intensive chemotherapy who were not candidates for hematopoietic stem cell transplantation. In this trial, MRD (≥0.1% leukemic cells in bone marrow) was assessed by multiparameter flow cytometry in serial samples collected at baseline and on day 1 of every 3 cycles. As expected, baseline MRD status was significantly associated with both OS and RFS. Multivariate analyses showed Oral-AZA significantly improved OS and RFS vs. placebo independent of baseline MRD status. Oral-AZA treatment also extended the duration of MRD negativity by 6 months vs. placebo, and resulted in a higher rate of conversion from MRD+ at baseline to MRD- during treatment: 37% vs. 19%, respectively. In the Oral-AZA arm, 24% of MRD responders achieved MRD negativity >6 months after treatment initiation. While presence or absence of MRD was a strong prognostic indicator of OS and RFS, there were added survival benefits with Oral-AZA maintenance therapy compared with placebo, independent of patients' MRD status at baseline. NCT01757535 Clinicaltrials.gov

Antibody Responses to Phlebotomus papatasi Saliva in American Soldiers With Cutaneous Leishmaniasis Versus Controls

Leishmania major, transmitted in Iraq by the bite of a sand fly Phlebotomus papatasi, causes cutaneous leishmaniasis (CL). The sand fly saliva is immunogenic, with both systemic humoral and cellular human immune responses resulting from natural exposure. 248 Americans who developed L. major infection in Iraq were sex, race/ethnicity, year of Iraq deployment-matched to controls without CL. Using a case-control study design, we compared sand fly saliva-specific human IgG levels and recognized antigens between the two groups. Serologic responses to Ph. papatasi salivary gland homogenate were studied with ELISA and Western blot, using serial samples obtained from before travel, during CL treatment (CL) or at time of return to US (controls), as well as (for CL cases) six to 24 months after return to non-endemic US. The mean change in optical density (MCOD), reflecting the change in sand fly saliva-specific IgG before and after exposure in Iraq, was 0.296 (range -0.138 to 2.057) in cases and 0.151 (range -0.454 to1.085) in controls, p<0.001. Low levels of sand fly saliva specific antibody were noted in CL cases by 7-8 months after return to the US. The most frequently recognized Ph. papatasi salivary antigens were MW30 (PpSP32) and MW64, although other salivary proteins recognized were MW12/14, 15, 18, 28, 32, 36, 42, 44, 46, 52. Logistic regression suggested that MW15, 28 and 42 were associated with the largest effect on the MCOD. MW30 was the most frequently recognized antigen suggesting a role as biomarker for sand fly exposure and CL risk. Anti-Ph. papatasi saliva IgG waned within months of return to the US. We also discuss vector antigenic saliva proteins in the context of CL presentation and identify some salivary antigens that may correlate with less lesion area, ulcer versus papule/plaque, race among those with CL.

Atypical Prolonged Viral Shedding With Intra-Host SARS-CoV-2 Evolution in a Mildly Affected Symptomatic Patient

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is caused by a respiratory virus with a wide range of manifestations, varying from asymptomatic to fatal cases, with a generally short outcome. However, some individuals present long-term viral shedding. We monitored 38 individuals who were mildly affected by the SARS-CoV-2 infection. Out of the total studied population, three (7.9%) showed atypical events regarding the duration of positivity for viral RNA detection. In one of these atypical cases, a previously HIV-positive male patient presented a SARS-CoV-2 RNA shedding and subgenomic RNA (sgRNA) detected from the upper respiratory tract, respectively, for 232 and 224 days after the onset of the symptoms. The SARS-CoV-2 B.1.1.28 lineage, one of the most prevalent in Brazil in 2020, was identified in this patient in three serial samples. Interestingly, the genomic analyses performed throughout the infectious process showed an increase in the genetic diversity of the B.1.1.28 lineage within the host itself, with viral clearance occurring naturally, without any intervention measures to control the infection. Contrasting widely spread current knowledge, our results indicate that potentially infectious SARS-CoV-2 virus might be shed by much longer periods by some infected patients. This data call attention to better adapted non-pharmacological measures and clinical discharge of patients aiming at preventing the spread of SARS-CoV-2 to the population.

Persistence of High Levels of Serum Complement C5a in Severe COVID-19 Cases After Hospital Discharge

Evidence supports a role of complement anaphylatoxin C5a in the pathophysiology of COVID-19. However, information about the evolution and impact of C5a levels after hospital discharge is lacking. We analyzed the association between circulating C5a levels and the clinical evolution of hospitalized patients infected with SARS-CoV-2. Serum C5a levels were determined in 32 hospitalized and 17 non-hospitalized patients from Clinica Universidad de Navarra. One hundred and eighty eight serial samples were collected during the hospitalization stay and up to three months during the follow-up. Median C5a levels were 27.71 ng/ml (25th to 75th percentile: 19.35-34.96) for samples collected during hospitalization, versus 16.76 ng/ml (12.90-25.08) for samples collected during the follow-up (p<0.001). There was a negative correlation between serum C5a levels and the number of days from symptom onset (p<0.001). C5a levels also correlated with a previously validated clinical risk score (p<0.001), and was associated with the severity of the disease (p<0.001). An overall reduction of C5a levels was observed after hospital discharge. However, elevated C5a levels persisted in those patients with high COVID-19 severity (i.e. those with a longest stay in the hospital), even after months from hospital discharge (p=0.020). Moreover, high C5a levels appeared to be associated with the presence of long-term respiratory symptoms (p=0.004). In conclusion, serum C5a levels remain high in severe cases of COVID-19, and are associated with the presence of respiratory symptoms after hospital discharge. These results may suggest a role for C5a in the long-term effects of COVID-19 infection.

Viable Severe Acute Respiratory Syndrome Coronavirus 2 Isolates Exhibit Higher Correlation With Rapid Antigen Assays Than Subgenomic RNA or Genomic RNA

Background: Rapid identification and effective isolation are crucial for curbing the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To meet this requirement, antigen-detection rapid diagnostic tests (Ag-RDTs) are essential.Methods: Between February 2020 and August 2020 we performed a cohort study of patients with confirmed COVID-19. The clinical performance of Ag rapid fluorescence immunoassay (FIA) and Ag Gold was evaluated and compared in parallel with genomic and subgenomic real-time reverse transcription-polymerase chain reaction (rRT-PCR) and cell culture-based assays.Results: In total, 150 samples were tested. Of these, 63 serial samples were obtained from 11 patients with SARS-CoV-2 and 87 from negative controls. Serial respiratory samples were obtained 2 days prior to symptom onset (-2) up to 25 days post-symptom onset. Overall, for rRT-PCR-positive samples (n = 51), the detection sensitivity of Ag rapid FIA and Ag Gold was 74.5% and 53.49%, respectively, with a specificity of 100%; however, for samples with low cycle threshold (Ct) values, Ag rapid FIA and Ag Gold exhibited a sensitivity of 82.61% (Ct ≤ 30, 5.6 log10RNA copies/mL) and 80% (Ct ≤ 25, 6.9 log10RNA copies/mL), respectively. Despite low analytical sensitivity, both Ag-RDTs detected 100% infection in cell culture-positive samples (n = 15) and were highly effective in distinguishing viable samples from those with subgenomic RNA (66.66%). For both Ag-RDTs, all samples that yielded discordant results (rRT-PCR + ve/Ag-RDT -ve) were also negative by culture.Conclusion: The data suggest that Ag-RDTs reliably detect viable SARS-CoV-2; thus, they may serve as an important tool for rapid detection of potentially infectious individuals.

DNMT3A and TET2 mutant Clonal Hematopoiesis May Drive a Proinflammatory State and Predict Enhanced Response to Immune Checkpoint Inhibitors

Background. DNA methylation is a key epigenetic process involved in development, aging, and cancer. Mutations in DNMT3A and TET2 in the hematopoietic stem cell compartment lead to increased self-renewal. In addition to mutations in ASXL1, collectively, these DTA mutations are recognized as an aging phenomenon, known as the most common Clonal hematopoiesis of Indeterminate Potential (CHIP) mutations and alone are not predictive of increased risk for hematopoietic malignancy. Recently, DNMT3A mutations in donor hematopoietic cells were suggested to be associated with enhanced T-cell activity in allografted patients. Additionally, role of DNMT3A mutations in creating a proinflammatory state in cardiovascular disease setting and associated elevation of T-cell markers in the myocardium have been recently explored (Sano S et al. Circ Res. 2018). Since an inflamed tumor microenvironment is associated with improved immune checkpoint inhibitors (CPI) activity, we sought to determine the impact of CHIP (a proinflammatory state) on response to CPI and CPI's effects on clonal dynamics. Additionally, while classical chemotherapy (CTX) can create selective external pressure providing survival advantage to mutant stem cells, the selective pressure of T-cell activating therapies on hematopoietic stem cells is unclear. Methods. To study the relationship between CHIP and CPI, we used paired peripheral-blood samples taken before and after treatment with CPI therapy in patients (pts) with melanoma (MEL; n= 32) and non-small cell lung cancer (NSCLC; n=109). Serial samples (or post CPI samples) were evaluable in 5 MEL pts and 6 NSCLC pts. Error-corrected sequencing of a targeted panel of genes recurrently mutated in clonal hematopoiesis (CH) was performed on peripheral blood genomic DNA. Statistical comparisons between baseline and serial sample VAFs were performed using two-sided fisher's exact test, with a p < 0.05 considered significant. Results. In both the MEL and NSCLC cohort, baseline samples were collected before extensive therapy exposure. 90% (29/32) of the MEL cohort had no CTX or targeted therapy prior to the baseline sample; 28% (9/32) had prior radiotherapy (RT). 10% (11/109) of the NSCLC cohort samples had prior CTX, but only 2 of these were treated for more than 1 month before sample collection. CH was frequent in these minimally pre-treated patient samples; 28.1% (9/32) and 37.6% (41/109) of the baseline MEL and NSCLC samples, respectively. As expected, DTA mutations were the most common events in these cohorts. Samples with CH were from patients of older age, but had normal hematological parameters with exception of increased RDW (p=0.022). Primary tumor responses in this cohort were defined as durable (receipt of ≥12 CPI cycles) or not durable (<12 cycles). DNMT3Amut patients (VAF ≥1%, n=5) had more durable responses, i.e. higher median number of CPI cycles (21 cycles, range:10-40) compared to non-DNMT3Amut pts (7 cycles, range:1-13; p= NS). Additionally, pts with larger DNMT3Amut clones (figure 1- MEL cohort) tended to receive higher numbers of CPI cycles. In the serial sample analysis, we observed that mutations in DNMT3A and TET2 increased in size with longer CPI exposures (Figure 2, MEL cohort); pts 2, 3 and 5 received 13, 15 and 18 CPI cycles respectively, while pt 4 with the most notable clonal expansion in DNMT3A received 40 CPI cycles. All serial samples in MEL cohort showed a statistically significant change in VAF from baseline. In the serial sample analysis of NSCLC pts, we observed that those with ≥ 3 months of CPI exposure demonstrated decreases in clone size for non-DTA gene mutations such as SRCAP, STK11 and TPM1 (Table 1), but increases or stability in DNMT3A and TET2 mutations (Table 1). However, this VAF increase in DNMT3A and TET2 mutations in NSCLC cohort was not statistically significant. Conclusions. In this small cohort of pts with MEL and NSCLC, the presence of DNMT3A/TET2 CH was associated with longer checkpoint inhibitor exposure and increased allelic frequency over time. These findings need further validation in larger cohorts and delineation of the relationship between DTA mutations such as DNMT3A and enhanced immune activity. Acknowledgement: Data and samples for this study were provided by the Data Bank and BioRepository (DBBR), which is funded by the National Cancer Institute (P30 CA016056) and is a Roswell Park Cancer Institute Cancer Center Support Grant shared resource. Figure 1 Figure 1. Disclosures Griffiths: Taiho Oncology: Consultancy, Honoraria; Alexion Pharmaceuticals: Consultancy, Research Funding; Novartis: Honoraria; Boston Biomedical: Consultancy; Astex Pharmaceuticals: Honoraria, Research Funding; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Apellis Pharmaceuticals: Research Funding; Genentech: Research Funding; Takeda Oncology: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Hassane: Tempus Labs, Inc: Current Employment. Guzman: SeqRx: Consultancy; BridgeMedicines: Consultancy; Cellectis: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Wang: Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Genentech: Membership on an entity's Board of Directors or advisory committees; Kite Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Kura Oncology: Consultancy, Honoraria, Other: Advisory board, steering committee, Speakers Bureau; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Other: Advisory Board; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Other: Advisory Board; Mana Therapeutics: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Other: Advisory Board, Speakers Bureau; Stemline Therapeutics: Consultancy, Honoraria, Other: Advisory board, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Advisory board; DAVA Oncology: Consultancy, Speakers Bureau; Rafael Pharmaceuticals: Other: Data safety monitoring committee; Gilead: Consultancy, Honoraria, Other: Advisory board; Daiichi Sankyo: Consultancy, Honoraria, Other: Advisory board; PTC Therapeutics: Consultancy, Honoraria, Other: Advisory board; Genentech: Consultancy; MacroGenics: Consultancy.

Anti-RA33 antibodies are present in a subset of patients with immune checkpoint inhibitor-induced inflammatory arthritis

Background Patients with inflammatory arthritis (IA) associated with immune checkpoint inhibitor (ICI) treatment for cancer are typically seronegative for anti-CCP antibodies and rheumatoid factor, but little is known about the presence of other autoantibodies described in early inflammatory arthritis in this patient population. We investigated the prevalence and characteristics of anti-RA33 antibodies in patients with ICI-induced IA. Methods Anti-RA33 ELISAs were performed on sera from four groups of patients: 79 with ICI-induced IA, 52 with rheumatoid arthritis (RA), 35 treated with ICIs without IA during follow-up, and 50 healthy controls. Anti-RA33 positivity and titer, clinical and demographic data were compared across groups. Results Anti-RA33 antibodies were found in 9/79 (11.4%) patients with ICI-induced IA but in 0/35 patients treated with ICIs who did not develop IA (0%; p=0.04). Of the 9 patients positive for anti-RA33, two had sera available from before ICI treatment; anti-RA33 antibodies were present in both pre-ICI treatment. In RA patients, 7.7% were positive for anti-RA33 antibodies as were 2% of healthy controls. In ICI-induced IA, anti-RA33 antibodies were associated with anti-CCP antibodies (p=0.001). We found no statistically significant differences in other clinical characteristics in those with and without anti-RA33 antibodies, but we observed trends toward anti-RA33 antibodies being more common in women and those receiving prior radiation therapy. Conclusions Anti-RA33 antibodies are present in a subset of patients with ICI-induced IA and may be a biomarker for developing IA. Additional studies evaluating serial samples before and after ICI treatment will further establish the temporal relationship of these antibodies to IA development.

Composition and Dynamics of H1N1 and H7N9 Influenza A Virus Quasispecies in a Co-infected Patient Analyzed by Single Molecule Sequencing Technology

A human co-infected with H1N1 and H7N9 subtypes influenza A virus (IAV) causes a complex infectious disease. The identification of molecular-level variations in composition and dynamics of IAV quasispecies will help to understand the pathogenesis and provide guidance for precision medicine treatment. In this study, using single-molecule real-time sequencing (SMRT) technology, we successfully acquired full-length IAV genomic sequences and quantified their genotypes abundance in serial samples from an 81-year-old male co-infected with H1N1 and H7N9 subtypes IAV. A total of 26 high diversity nucleotide loci was detected, in which the A-G base transversion was the most abundant substitution type (67 and 64%, in H1N1 and H7N9, respectively). Seven significant amino acid variations were detected, such as NA:H275Y and HA: R222K in H1N1 as well as PB2:E627K and NA: K432E in H7N9, which are related to viral drug-resistance or mammalian adaptation. Furtherly, we retrieved 25 H1N1 and 22 H7N9 genomic segment haplotypes from the eight samples based on combining high-diversity nucleotide loci, which provided a more concise overview of viral quasispecies composition and dynamics. Our approach promotes the popularization of viral quasispecies analysis in a complex infectious disease, which will boost the understanding of viral infections, pathogenesis, evolution, and precision medicine.

Allelic Complexity of KMT2A Partial Tandem Duplications in Acute Myeloid Leukemia and Myelodysplastic Syndromes

KMT2A partial tandem duplication (KMT2A-PTD) at 11q23.3 is associated with adverse risk in AML and MDS, is a potential therapeutic target, and is an attractive marker of measurable residual disease. High initial KMT2A-PTD RNA levels have been linked to poor prognosis, but mechanisms regulating KMT2A-PTD expression are not well understood. While it has been reported that KMT2A-PTD affects only a single allele, it has been theorized but not proven that duplications or genomic gains of a monoallelic KMT2A-PTD may occur, thereby potentially driving high expression and disease progression. Copy neutral loss of heterozygosity (CN-LOH) of 11q has also been described and is known to be associated with mutations in CBL but has not been reported to involve KMT2A-PTD. In this study, we identified 94 patients with KMT2A-PTDs using targeted DNA next-generation sequencing (NGS) and found that 16% (15/94) had complex secondary events, including CN-LOH and selective gain involving the KMT2A-PTD allele. High copy numbers indicating complexity were significantly enriched in AML versus MDS and correlated with higher RNA expression. Moreover, in serial samples, complexity was associated with relapse and secondary transformation. Taken together, we provide approaches to integrate quantitative and allelic assessment of KMT2A-PTDs into targeted DNA NGS and demonstrate that secondary genetic events occur in KMT2A-PTD by multiple mechanisms that may be linked to myeloid disease progression by driving increased expression from the affected allele.

Gut Bacterial Dysbiosis and Instability is Associated with the Onset of Complications and Mortality in COVID-19

Objective: There is a growing debate about the involvement of the gut microbiome in COVID-19, although it is not conclusively understood whether the microbiome has an impact on COVID-19, or vice versa, especially as analysis of amplicon data in hospitalized patients requires sophisticated cohort recruitment and integration of clinical parameters. Here, we analyzed fecal and saliva samples from SARS-CoV-2 infected and post COVID-19 patients and controls considering multiple influencing factors during hospitalization. Design: 16S rRNA gene sequencing was performed on fecal and saliva samples from 108 COVID-19 and 22 post COVID-19 patients, 20 pneumonia controls and 26 asymptomatic controls. Patients were recruited over the first and second corona wave in Germany and detailed clinical parameters were considered. Serial samples per individual allowed intra-individual analysis. Results: We found the gut and oral microbiota to be altered depending on number and type of COVID-19-associated complications and disease severity. The occurrence of individual complications was correlated with low-risk (e.g., Faecalibacterium prausznitzii) and high-risk bacteria (e.g., Parabacteroides). We demonstrated that a stable gut bacterial composition was associated with a favorable disease progression. Based on gut microbial profiles, we identified a model to estimate mortality in COVID-19. Conclusion: Gut microbiota are associated with the occurrence of complications in COVID-19 and may thereby influencing disease severity. A stable gut microbial composition may contribute to a favorable disease progression and using bacterial signatures to estimate mortality could contribute to diagnostic approaches. Importantly, we highlight challenges in the analysis of microbial data in the context of hospitalization.