Molecular responses in decitabine- and decitabine/venetoclax-treated patients with acute myeloid leukemia and myelodysplastic syndromes.

Not available.

Two Interventions on Pathologic Nodal Staging in a Population-Based Lung Cancer Resection Cohort.

BACKGROUND: Despite its prognostic importance, poor pathologic nodal staging of lung cancer prevails. We evaluated the impact of 2 interventions to improve pathologic nodal staging. METHODS: We implemented a lymph node specimen collection kit to improve intraoperative lymph node collection (surgical intervention) and a novel gross dissection method for intrapulmonary node retrieval (pathology intervention) in nonrandomized stepped-wedge fashion, involving 12 hospitals and 7 pathology groups. We used standard statistical methods to compare surgical quality and survival of patients who had neither intervention (group 1), pathology intervention only (group 2), surgical intervention only (group 3), and both interventions (group 4). RESULTS: Of 4019 patients from 2009 to 2021, 50%, 5%, 21%, and 24%, respectively, were in groups 1 to 4. Rates of nonexamination of lymph nodes were 11%, 9%, 0%, and 0% and rates of nonexamination of mediastinal lymph nodes were 29%, 35%, 2%, and 2%, respectively, in groups 1 to 4 (P < .0001). Rates of attainment of American College of Surgeons Operative Standard 5.8 were 22%, 29%, 72%, and 85%; and rates of International Association for the Study of Lung Cancer complete resection were 14%, 21%, 53%, and 61% (P < .0001). Compared with group 1, adjusted hazard ratios for death were as follows: group 2, 0.93 (95% CI, 0.76-1.15); group 3, 0.91 (0.78-1.03); and group 4, 0.75 (0.64-0.87). Compared with group 2, group 4 adjusted hazard ratio was 0.72 (0.57-0.91); compared with group 3, it was 0.83 (0.69-0.99). These relationships remained after exclusion of wedge resections. CONCLUSIONS: Combining a lymph node collection kit with a novel gross dissection method significantly improved pathologic nodal evaluation and survival.

Clinical whole-genome sequencing and FISH identify two different fusion partners for NUP98 in a patient with acute myeloid leukemia: A case report.

BACKGROUND: Only rare cases of acute myeloid leukemia (AML) have been shown to harbor a t(8;11)(p11.2;p15.4). This translocation is believed to involve the fusion of NSD3 or FGFR1 with NUP98; however, apart from targeted mRNA quantitative PCR analysis, no molecular approaches have been utilized to define the chimeric fusions present in these rare cases. CASE PRESENTATION: Here we present the case of a 51-year-old female with AML with myelodysplastic-related morphologic changes, 13q deletion and t(8;11), where initial fluorescence in situ hybridization (FISH) assays were consistent with the presence of NUP98 and FGFR1 rearrangements, and suggestive of NUP98/FGFR1 fusion. Using a streamlined clinical whole-genome sequencing approach, we resolved the breakpoints of this translocation to intron 4 of NSD3 and intron 12 of NUP98, indicating NUP98/NSD3 rearrangement as the likely underlying aberration. Furthermore, our approach identified small variants in WT1 and STAG2, as well as an interstitial deletion on the short arm of chromosome 12, which were cryptic in G-banded chromosomes. CONCLUSIONS: NUP98 fusions in acute leukemia are predictive of poor prognosis. The associated fusion partner and the presence of co-occurring mutations, such as WT1, further refine this prognosis with potential clinical implications. Using a clinical whole-genome sequencing analysis, we resolved t(8;11) breakpoints to NSD3 and NUP98, ruling out the involvement of FGFR1 suggested by FISH while also identifying multiple chromosomal and sequence level aberrations.

Missense mutations in Myc Box I influence MYC cellular localization, mRNA partitioning and turnover to promote leukemogenesis.

Somatic missense mutations in the phosphodegron domain of the MYC gene ( M YC Box I) are detected in the dominant clones of a subset of acute myeloid leukemia (AML) patients, but the mechanisms by which they contribute to AML are unknown. To unveil unique proprieties of MBI MYC mutant proteins, we systematically compared the cellular and molecular consequences of expressing similar oncogenic levels of wild type and MBI mutant MYC. We found that MBI MYC mutants can accelerate leukemia by driving unique transcriptional signatures in highly selected, myeloid progenitor subpopulations. Although these mutations increase MYC stability, they overall dampen MYC chromatin localization and lead to a cytoplasmic accumulation of the mutant proteins. This phenotype is coupled with increased translation of RNA binding proteins and nuclear export machinery, which results in altered RNA partitioning and accelerated decay of select transcripts encoding proapoptotic and proinflammatory genes. Heterozygous knockin mice harboring the germline MBI mutation Myc p.T73N exhibit cytoplasmic MYC localization, myeloid progenitors' expansion with similar transcriptional signatures to the overexpression model, and eventually develop hematological malignancies. This study uncovers that MBI MYC mutations alter MYC localization and disrupt mRNA subcellular distribution and turnover of select transcripts to accelerate tumor initiation and growth.

Compliance and regulatory considerations for the implementation of the multi-attribute-method by mass spectrometry in a quality control laboratory.

Multi-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under the good manufacturing practice (GMP) regime, due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. This article is the second part of a two-tiered publication aiming at providing guidance for implementation of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) in a QC laboratory. The first part [1] focuses on technical considerations, while this second part provides considerations related to GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG).

Genomic landscape of TP53-mutated myeloid malignancies.

TP53-mutated myeloid malignancies are associated with complex cytogenetics and extensive structural variants, which complicates detailed genomic analysis by conventional clinical techniques. We performed whole-genome sequencing (WGS) of 42 acute myeloid leukemia (AML)/myelodysplastic syndromes (MDS) cases with paired normal tissue to better characterize the genomic landscape of TP53-mutated AML/MDS. WGS accurately determines TP53 allele status, a key prognostic factor, resulting in the reclassification of 12% of cases from monoallelic to multihit. Although aneuploidy and chromothripsis are shared with most TP53-mutated cancers, the specific chromosome abnormalities are distinct to each cancer type, suggesting a dependence on the tissue of origin. ETV6 expression is reduced in nearly all cases of TP53-mutated AML/MDS, either through gene deletion or presumed epigenetic silencing. Within the AML cohort, mutations of NF1 are highly enriched, with deletions of 1 copy of NF1 present in 45% of cases and biallelic mutations in 17%. Telomere content is increased in TP53-mutated AMLs compared with other AML subtypes, and abnormal telomeric sequences were detected in the interstitial regions of chromosomes. These data highlight the unique features of TP53-mutated myeloid malignancies, including the high frequency of chromothripsis and structural variation, the frequent involvement of unique genes (including NF1 and ETV6) as cooperating events, and evidence for altered telomere maintenance.

Complement contributes to antibody-mediated protection against repeated SHIV challenge.

The first clinical efficacy trials of a broadly neutralizing antibody (bNAb) resulted in less benefit than expected and suggested that improvements are needed to prevent HIV infection. While considerable effort has focused on optimizing neutralization breadth and potency, it remains unclear whether augmenting the effector functions elicited by broadly neutralizing antibodies (bNAbs) may also improve their clinical potential. Among these effector functions, complement-mediated activities, which can culminate in the lysis of virions or infected cells, have been the least well studied. Here, functionally modified variants of the second-generation bNAb 10-1074 with ablated and enhanced complement activation profiles were used to examine the role of complement-associated effector functions. When administered prophylactically against simian-HIV challenge in rhesus macaques, more bNAb was required to prevent plasma viremia when complement activity was eliminated. Conversely, less bNAb was required to protect animals from plasma viremia when complement activity was enhanced. These results suggest that complement-mediated effector functions contribute to in vivo antiviral activity, and that their engineering may contribute to the further improvements in the efficacy of antibody-mediated prevention strategies.

Technical considerations for the implementation of the multi-attribute-method by mass spectrometry in a quality control laboratory.

Multi-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under good manufacturing practice (GMP) due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. Here, current literature related to the development and application of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) is compiled with the aim of providing guidance for the implementation of MAM in a QC laboratory. This article, focusing on technical considerations, is the first part of a two-tiered publication, whereby the second part will focus on GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG).

Persistent Molecular Disease in Adult Patients With AML Evaluated With Whole-Exome and Targeted Error-Corrected DNA Sequencing.

PURPOSE: Persistent molecular disease (PMD) after induction chemotherapy predicts relapse in AML. In this study, we used whole-exome sequencing (WES) and targeted error-corrected sequencing to assess the frequency and mutational patterns of PMD in 30 patients with AML. MATERIALS AND METHODS: The study cohort included 30 patients with adult AML younger than 65 years who were uniformly treated with standard induction chemotherapy. Tumor/normal WES was performed for all patients at presentation. PMD analysis was evaluated in bone marrow samples obtained during clinicopathologic remission using repeat WES and analysis of patient-specific mutations and error-corrected sequencing of 40 recurrently mutated AML genes (MyeloSeq). RESULTS: WES for patient-specific mutations detected PMD in 63% of patients (19/30) using a minimum variant allele fraction (VAF) of 2.5%. In comparison, MyeloSeq identified persistent mutations above 0.1% VAF in 77% of patients (23/30). PMD was usually present at relatively high levels (>2.5% VAFs), such that WES and MyeloSeq agreed for 73% of patients despite differences in detection limits. Mutations in DNMT3A, ASXL1, and TET2 (ie, DTA mutations) were persistent in 16 of 17 patients, but WES also detected non-DTA mutations in 14 of these patients, which for some patients distinguished residual AML cells from clonal hematopoiesis. Surprisingly, MyeloSeq detected additional variants not identified at presentation in 73% of patients that were consistent with new clonal cell populations after chemotherapy. CONCLUSION: PMD and clonal hematopoiesis are both common in patients with AML in first remission. These findings demonstrate the importance of baseline testing for accurate interpretation of mutation-based tumor monitoring assays for patients with AML and highlight the need for clinical trials to determine whether these complex mutation patterns correlate with clinical outcomes in AML.

KDM6B protects T-ALL cells from NOTCH1-induced oncogenic stress.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic neoplasm resulting from the malignant transformation of T-cell progenitors. While activating NOTCH1 mutations are the dominant genetic drivers of T-ALL, epigenetic dysfunction plays a central role in the pathology of T-ALL and can provide alternative mechanisms to oncogenesis in lieu of or in combination with genetic mutations. The histone demethylase enzyme KDM6A (UTX) is also recurrently mutated in T-ALL patients and functions as a tumor suppressor. However, its gene paralog, KDM6B (JMJD3), is never mutated and can be significantly overexpressed, suggesting it may be necessary for sustaining the disease. Here, we used mouse and human T-ALL models to show that KDM6B is required for T-ALL development and maintenance. Using NOTCH1 gain-of-function retroviral models, mouse cells genetically deficient for Kdm6b were unable to propagate T-ALL. Inactivating KDM6B in human T-ALL patient cells by CRISPR/Cas9 showed KDM6B-targeted cells were significantly outcompeted over time. The dependence of T-ALL cells on KDM6B was proportional to the oncogenic strength of NOTCH1 mutation, with KDM6B required to prevent stress-induced apoptosis from strong NOTCH1 signaling. These studies identify a crucial role for KDM6B in sustaining NOTCH1-driven T-ALL and implicate KDM6B as a novel therapeutic target in these patients.

Cisternal, Falciform, and Optic Canal Decompression Influencing Optic Nerve Biomechanics: A Microsurgical Anatomic Study.

BACKGROUND: Questions remain regarding optic nerve (ON) physiology, mechanical compliance, and microvasculature, particularly surgical outcomes and atypical visual field defects associated with sellar/parasellar pathology (eg, tumors and aneurysms). OBJECTIVE: To study the microsurgical/histological anatomy of each ON segment and corresponding microvasculature, calculate area of optic-carotid space at each decompression stage, and measure ON tension before/after compression. METHODS: Five cadaveric heads (10 sides) underwent sequential dissection: (1) intradural (arachnoidal) ON dissection; (2) falciform ligament opening; (3) anterior clinoidectomy, optic canal decompression, and ON sheath release. At each step, we pulled the nerve superiorly/laterally with a force meter and measured maximal mobility/mechanical tension in each position. RESULTS: Cisternal ON microvasculature was more superficial and less dense vs the orbital segment. ON tension was significantly lower with higher mobility when manipulated superiorly vs lateromedially. Optic-carotid space significantly increased in size at each decompression stage and with ON mobilization both superiorly and laterally, but the increase was statistically significant in favor of upward mobilization. At decompression step, upward pull provided more space with less tension vs side pull. For upward pull, each step of decompression provided added space as did side pull. CONCLUSION: Opening the optic canal, falciform ligament, and arachnoid membrane decompresses the ON for safer manipulation and provided a wider optic-carotid surgical corridor to access sellar/parasellar pathology. When tailoring decompression, the ON should be manipulated superiorly rather than lateromedially, which may guide surgical technique, help prevent intraoperative visual deterioration, facilitate postoperative visual improvement, and help understand preoperative visual field deficits based on mechanical factors.

Genomic landscape of TP53 -mutated myeloid malignancies.

TP53 -mutated myeloid malignancies are most frequently associated with complex cytogenetics. The presence of complex and extensive structural variants complicates detailed genomic analysis by conventional clinical techniques. We performed whole genome sequencing of 42 AML/MDS cases with paired normal tissue to characterize the genomic landscape of TP53 -mutated myeloid malignancies. The vast majority of cases had multi-hit involvement at the TP53 genetic locus (94%), as well as aneuploidy and chromothripsis. Chromosomal patterns of aneuploidy differed significantly from TP53 -mutated cancers arising in other tissues. Recurrent structural variants affected regions that include ETV6 on chr12p, RUNX1 on chr21, and NF1 on chr17q. Most notably for ETV6 , transcript expression was low in cases of TP53 -mutated myeloid malignancies both with and without structural rearrangements involving chromosome 12p. Telomeric content is increased in TP53 -mutated AML/MDS compared other AML subtypes, and telomeric content was detected adjacent to interstitial regions of chromosomes. The genomic landscape of TP53 -mutated myeloid malignancies reveals recurrent structural variants affecting key hematopoietic transcription factors and telomeric repeats that are generally not detected by panel sequencing or conventional cytogenetic analyses. Key Points: WGS comprehensively determines TP53 mutation status, resulting in the reclassification of 12% of cases from mono-allelic to multi-hit Chromothripsis is more frequent than previously appreciated, with a preference for specific chromosomes ETV6 is deleted in 45% of cases, with evidence for epigenetic suppression in non-deleted cases NF1 is mutated in 48% of cases, with multi-hit mutations in 17% of these cases TP53 -mutated AML/MDS is associated with altered telomere content compared with other AMLs.

The disproportionately large contribution of the Maori and Pacific Islander community to the healthcare burden of gout in Western Sydney.

BACKGROUND: Gout is a common chronic inflammatory disorder due to monosodium urate deposition, which results in severe inflammatory arthritis. It is particularly common in those of Maori or Pacific Islander heritage. There is a significant number of this at-risk ethnic group in western Sydney. AIMS: To determine the healthcare burden of gout in Western Sydney. METHODS: We characterised patients managed in the emergency departments (EDs) of the four Western Sydney Local Health District (WSLHD) hospitals and those admitted for gout as the primary or secondary diagnosis from 1 January 2017 to 31 December 2018. RESULTS: There were 472 patients managed in ED on 552 occasions at a direct cost to the LHD of A$367 835. Those of Maori or Pacific Islander ethnicity comprised 25.2% (n = 119/472), while half (n = 39/80) of those managed in ED for gout on two or more occasions were of Maori or Pacific Islander ethnicity. Overall, 310 patients were admitted with gout as the principal diagnosis on 413 occasions at a cost of A$1.73 million. Seventy-five (24.2%) of the 310 patients were of Maori or Pacific Islander heritage. A total of 584 WSLHD inpatients had gout as a secondary diagnosis. This was associated with 714 admissions. CONCLUSIONS: The disproportionately large healthcare burden of gout in Western Sydney from the relatively small Maori and Pacific Islander population needs attention. Urgent culturally appropriate interventions to address gout are required to address this inequality.

Evaluation of Breast Implant-Associated Anaplastic Large Cell Lymphoma With Whole Exome and Genome Sequencing.

BACKGROUND: Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a rare malignancy originating from the periprosthetic capsule of a textured, most often macrotextured, breast implant. Identified in women whose indications for breast implants can be either aesthetic or reconstructive, the genomic underpinnings of this disease are only beginning to be elucidated. OBJECTIVES: The aim of this study was to evaluate the exomes, and in some cases the entire genome, of patients with BIA-ALCL. Specific attention was paid to copy number alterations, chromosomal translocations, and other genomic abnormalities overrepresented in patients with BIA-ALCL. METHODS: Whole-exome sequencing was performed on 6 patients, and whole-genome sequencing on 3 patients, with the Illumina NovaSeq 6000 sequencer. Data were analyzed with the Illumina DRAGEN Bio-IT Platform and the ChromoSeq pipeline. The Pathseq Genome Analysis Toolkit pipeline was used to detect the presence of microbial genomes in the sequenced samples. RESULTS: Two cases with STAT3 mutations and 2 cases with NRAS mutations were noted. A critically deleted 7-Mb region was identified at the 11q22.3 region of chromosome 11, and multiple nonrecurrent chromosomal rearrangements were identified by whole-genome sequencing. Recurrent gene-level rearrangements, however, were not identified. None of the samples showed evidence of potential microbial pathogens. CONCLUSIONS: Although no recurrent mutations were identified, this study identified mutations in genes not previously reported with BIA-ALCL or other forms of ALCL. Furthermore, not previously reported with BIA-ALCL, 11q22.3 deletions were consistent across whole-genome sequencing cases and present in some exomes.

Electrocorticography Analysis in Patients With Dual Neurostimulators Supports Desynchronization as a Mechanism of Action for Acute Vagal Nerve Stimulator Stimulation.

PURPOSE: Both vagal nerve stimulation (VNS) and responsive neurostimulation (RNS System) are treatment options for medically refractory focal epilepsy. The mechanism of action of both devices remains poorly understood. Limited prior evidence suggests that acute VNS stimulation may reduce epileptiform activity and cause EEG desynchronization on electrocorticography (ECoG). Our study aims to isolate effects of VNS on ECoG as recorded by RNS System in patients who have both devices, by comparing ECoG samples with and without acute VNS stimulation. METHODS: Ten 60-second ECoGs each from 22 individuals at 3 epilepsy centers were obtained-5 ECoGs with VNS "off" and 5 ECoGs with VNS "on." Electrocorticograps containing seizures or loss of telemetry connection artifact were excluded from analysis (total of 169 ECoGs were included). Electrocorticographs were analyzed for differences in spectral content by generating average spectrograms for "on" and "off" states and using a linear mixed-effects model to isolate effects of VNS stimulation. RESULTS: Acute VNS stimulation reduced average power in the theta band by 4.9%, beta band by 3.8%, and alpha band by 2.5%. The reduction in theta power reached statistical significance with a P value of <0.05. CONCLUSIONS: Our results provide evidence that acute VNS stimulation results in desynchronization of specific frequency bands (salient decrease in theta and beta bands, smaller decrease in alpha band) in ECoGs recorded by the RNS device in patients with dual (VNS and RNS) neurostimulators. This finding offers support for desynchronization as a theorized mechanism of action of VNS. Further research may lead to future improved neurostimulator efficacy by informing optimal stimulation programming parameters.

Clonal dynamics of haematopoiesis across the human lifespan.

Age-related change in human haematopoiesis causes reduced regenerative capacity1, cytopenias2, immune dysfunction3 and increased risk of blood cancer4-6, but the reason for such abrupt functional decline after 70 years of age remains unclear. Here we sequenced 3,579 genomes from single cell-derived colonies of haematopoietic cells across 10 human subjects from 0 to 81 years of age. Haematopoietic stem cells or multipotent progenitors (HSC/MPPs) accumulated a mean of 17 mutations per year after birth and lost 30 base pairs per year of telomere length. Haematopoiesis in adults less than 65 years of age was massively polyclonal, with high clonal diversity and a stable population of 20,000-200,000 HSC/MPPs contributing evenly to blood production. By contrast, haematopoiesis in individuals aged over 75 showed profoundly decreased clonal diversity. In each of the older subjects, 30-60% of haematopoiesis was accounted for by 12-18 independent clones, each contributing 1-34% of blood production. Most clones had begun their expansion before the subject was 40 years old, but only 22% had known driver mutations. Genome-wide selection analysis estimated that between 1 in 34 and 1 in 12 non-synonymous mutations were drivers, accruing at constant rates throughout life, affecting more genes than identified in blood cancers. Loss of the Y chromosome conferred selective benefits in males. Simulations of haematopoiesis, with constant stem cell population size and constant acquisition of driver mutations conferring moderate fitness benefits, entirely explained the abrupt change in clonal structure in the elderly. Rapidly decreasing clonal diversity is a universal feature of haematopoiesis in aged humans, underpinned by pervasive positive selection acting on many more genes than currently identified.

Convergent Clonal Evolution of Signaling Gene Mutations Is a Hallmark of Myelodysplastic Syndrome Progression.

Progression from myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (AML) is associated with the acquisition and expansion of subclones. Our understanding of subclone evolution during progression, including the frequency and preferred order of gene mutation acquisition, remains incomplete. Sequencing of 43 paired MDS and secondary AML samples identified at least one signaling gene mutation in 44% of MDS and 60% of secondary AML samples, often below the level of standard sequencing detection. In addition, 19% of MDS and 47% of secondary AML patients harbored more than one signaling gene mutation, almost always in separate, coexisting subclones. Signaling gene mutations demonstrated diverse patterns of clonal evolution during disease progression, including acquisition, expansion, persistence, and loss of mutations, with multiple patterns often coexisting in the same patient. Multivariate analysis revealed that MDS patients who had a signaling gene mutation had a higher risk of AML progression, potentially providing a biomarker for progression. SIGNIFICANCE: Subclone expansion is a hallmark of progression from MDS to secondary AML. Subclonal signaling gene mutations are common at MDS (often at low levels), show complex and convergent patterns of clonal evolution, and are associated with future progression to secondary AML. See related article by Guess et al., p. 316 (33). See related commentary by Romine and van Galen, p. 270. This article is highlighted in the In This Issue feature, p. 265.

Clinical whole-genome sequencing in cancer diagnosis.

Characterizing the genomic landscape of cancers is a routine part of clinical care that began with the discovery of the Philadelphia chromosome and has since coevolved with genomic technologies. Genomic analysis of tumors at the nucleotide level using DNA sequencing has revolutionized the understanding of cancer biology and identified new molecular drivers of disease that have led to therapeutic advances and improved patient outcomes. However, the application of next-generation sequencing in the clinical laboratory has generally been limited until very recently to targeted analysis of selected genes. Recent technological innovations and reductions in sequencing costs are now able to deliver the long-promised goal of tumor whole-genome sequencing as a practical clinical assay.