Showing papers by "Paul P. Lee published in 2022"

Unique single-nucleotide variations, insertions/deletions and copy number variations in myelodysplastic syndrome during treatment resistance and progression revealed by a single-cell DNA sequencing platform

Abstract: Myelodysplastic syndrome (MDS) is a clonal myeloid neoplasm characterized by ineffective hematopoiesis, cytopenia, dysplasia and clonal instability leading to leukemic transformation. Hypomethylating agents are the mainstay of treatment in higher-risk MDS. However, treatment resistance and disease transformation into acute myeloid leukemia (AML) is observed in the majority patients and portend a dismal outcome. The residual cell clones resistant to therapy or cell clones acquiring new genetic aberrations are two of the key events responsible for drug resistance. Bulk tumor sequencing often fail to detect these rare subclones that confer resistance to therapy. In this study, we employed a single-cell DNA (sc-DNA) sequencing approach to study the clonal heterogeneity and clonal evolution in two MDS patients refractory to HMA. In both patients, different single nucleotide variations (SNVs) or insertions and deletions (INDELs) were detected concordant with bulk tumor sequencing. Rare cell clones with mutations undetectable on bulk tumor sequencing, were detected on single-cell DNA sequencing. In addition to SNVs and short INDELs, this study also revealed the presence clonal copy number loss of DNMT3A, TET2, and GAT2 as standalone events or in associated with the small SNVs or INDELs detected during HMA resistance and disease progression.

Moving Beyond COVID-19-How the Pandemic Is Associated With Transformations in Research and Careers of the Next Generation of Vision Scientists.

Abstract: The beginning of the COVID-19 pandemic had a chilling effect on research. Early-stage investigators reported being disproportionately affected owing to lack of a research track record, impediments to research collaboration, loss of training time, and loss of research time for those with young children (especially female researchers). Those were the findings reported 1 year ago in a Viewpoint,1 which summarized the opinions expressed by a panel of vision investigators convened by the Alliance for Eye and Vision Research (AEVR) to educate Congress about their plight. Now, 1 year later, a new panel of young investigators was convened to address, in the middle of the COVID-19 pandemic, how they perceived the pandemic has changed their research and career pathways. With grants from Research to Prevent Blindness and Novartis, AEVR held its Seventh Annual Emerging Vision Scientists (EVS) Day for Congress, which included activities to educate Congressional members and staff about eye and vision research and the potential impacts of the COVID-19 pandemic on the career pathways of these early-stage investigators who have not yet received independent funding (eg, a National Institutes of Health [NIH] investigator-initiated R01 grant). AEVR leadership, including the authors of this Viewpoint, convened a panel of 27 EVSs in 2 recorded teleconferences held on August 31, 2021, and September 1, 2021. The panel consisted of 17 assistant professors, 1 clinical instructor, 7 postdoctoral fellows, and 2 PhD candidates nominated by their institutions and reviewed by AEVR. The panelists ranged from bench to clinical researchers. None of them participated in previous annual EVS days. AEVR staff did not compile how frequently each issue covered here was mentioned. The opinions expressed here are anecdotal, and the authors do not know how common they were to EVSs as a whole or researchers later in their career. Panelists reported COVID-19 pandemic–driven innovations in the design and execution of clinical research. Data science, public health, and epidemiologic researchers spoke about analyzing big data to refine their research objectives or outcome measures (or as a standalone research project). Data sets included the American Academy of Ophthalmology’s IRIS registry,2 the University of Michigan’s SOURCE data set,3 and nationally representative surveys and medical claims data. Execution of clinical trials and other human studies was dramatically transformed. Research design and administration were altered by greater reliance on teleconferencing sessions with multisite collaborators to hone research questions and minimize patient visits to the clinic. The locus of research often was shifted from the clinic to study participants’ homes, where they perceived to be safer from contracting SARS-CoV-2 infection and thus were more inclined to serve as study participants. One panelist put it succinctly by saying their glaucoma research had been realigned from out of the clinic to reach patients in their own homes. The transformation to more home-based research was evident across all phases of research. First, it affected recruitment, which turned out for some to be more labor intensive. Clinical coordinators had restricted work schedules because of the need for physical distancing. Potential study participants were more difficult to find at home as opposed to through community-based activities. Second, the transformation affected screening and enrollment through greater use of web-based questionnaires, use of electronic health record data to minimize the need for a clinic visit, and the use of electronic consent forms and electronic reimbursement. Third, the transformation was manifest in fewer clinic visits, more web-based questionnaires, more telephone check-ins between the more widely spaced clinic visits, more home-based sample collection where feasible (eg, genetic studies), and more complex logistics in scheduling of ophthalmic technicians whose work schedules were staggered owing to physical distancing. Fourth, the transformation affected study participant follow-up by reliance on more remote visits and web-based questionnaires. Bench research was also the subject of innovations designed to overcome restrictions to laboratory access and supply chain limitations on resources (eg, animals or laboratory supplies). Panelists spoke about revisiting old data sets, described as treasure troves, to refine their research hypotheses or for direct study. One panelist described using previous data sets to connect the dots before feeling pressured to collect new data. Panelists spoke about being more flexible and efficient to minimize bench time and leave data analysis and other support functions for home. Some found the time to learn new skills, such as coding genetic data. An unexpected plus of the COVID-19 pandemic for both bench and bedside research was an explosion in research communication and collaboration through teleconferencing software such as Zoom. One panelist said coordinating multiple clinical sites became the norm because of regularly scheduled Zoom update calls. Several said they had become better speakers, with teleconference calls making them more efficient, organized, and disciplined as opposed to holding more relaxed onsite meetings. Another panelist spoke of teleconferences as fostering research synergy. VIEWPOINT

Epigenetic Silencing of PTEN and Epi-transcriptional Silencing of MDM2 Underlied Resistance to Hypomethylating Agents in Myelodysplastic Syndrome

Abstract: In myelodysplastic syndrome (MDS), resistance to hypomethylating agents (HMA) portends a poor prognosis, underscoring the importance of understanding the molecular mechanisms leading to HMA-resistance. In this study, P39 and Kasumi-1 cells and their azacitidine-resistant and decitabine-resistant sublines were evaluated comparatively with transcriptomic and methylomic analyses. Expression profiling and genome-wide methylation microarray showed downregulation of PTEN associated with DNA hypermethylation in P39 cell lines resistant to azacitidine and decitabine. This pattern of PTEN dysregulation was also confirmed in a cohort of patients failing treatment with HMA. DNA hypomethylation of MDM2 was detected with downregulation of MDM2 in HMA resistant cell lines. Long-read sequencing revealed significant RNA hypomethylation of MDM2 resulting in alternative splicing and production of a truncated MDM2 transcript in azacitidine-resistant P39 cells. The expression of this MDM2 truncated transcript was also significantly increased in HMA-resistant patients compared with HMA-responsive patients. In conclusion, epigenetic and epi-transcriptomic dysregulation of PTEN and MDM2 were associated with resistance to hypomethylating agents.