Molecular pathology selected abstracts

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Editors: Donna E. Hansel, MD, PhD, chair of pathology, Oregon Health and Science University, Portland; Richard D. Press, MD, PhD, professor and director of molecular pathology, OHSU; James Solomon, MD, PhD, assistant professor, Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York; Sounak Gupta, MBBS, PhD, senior associate consultant, Mayo Clinic, Rochester, Minn.; Tauangtham Anekpuritanang, MD, molecular pathology fellow, Department of Pathology, OHSU; Hassan Ghani, MD, molecular genetic pathology fellow, Department of Pathology, OHSU; and Fei Yang, MD, assistant professor, Department of Pathology, OHSU. 

Precision treatment of a hypersensitivity condition informed by single-cell transcriptomics

July 2020—Drug-induced hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms (DiHS/DRESS) is a rare but well-known full-body inflammatory skin condition that leads to life-threatening complications if untreated. The authors conducted a case study that involved a 44-year-old male who developed DiHS/DRESS after taking the combined antibiotic trimethoprim-sulfamethoxazole. Following standard treatment for this condition, the patient was started on high-dose prednisone, which provided no benefit. Subsequent tapering of prednisone advanced the disease to a toxic epidermal necrolysis-like condition. The patient was then given etanercept and high-dose intravenous immunoglobulin, without benefit. Next, the patient received cyclosporine, which resulted in some improvement but led to uncontrollable renal hypertension. Tapering of cyclosporine resulted in severe flares of skin inflammation. Lastly, the patient was given mycophenolate mofetil, without benefit. Because the patient was at high risk of developing life-threatening complications, single-cell RNA sequencing (scRNA-seq) was used to elucidate the immunologic pathway of the patient’s disease with the hope that the pathway could be disrupted with monoclonal antibodies or small-molecule inhibitors. The authors performed scRNA-seq on a skin biopsy from the patient and skin biopsies from five healthy volunteers who served as study controls. Each biopsy was dissociated into a single-cell suspension and sequenced, yielding data on 4,676 cells from the patient and 13,542 cells from the five control subjects. Unsupervised clustering and t-distributed stochastic neighbor (t-SNE) algorithms successfully grouped cells by major skin cell subsets. Among these major cell types, the lymphocyte cluster from the diseased skin showed the greatest degree of differentially expressed genes (DEGs). IL2RG, JAK3, and STAT 1, in particular, were overexpressed in the lymphocytes from the diseased skin, and IHC supported increased activity of the JAK–STAT signaling pathway in these skin-infiltrating lymphocytes. Given the systemic nature of DiHS/DRESS, the authors performed a similar analysis of the patient’s peripheral blood, which revealed T-cells with increased expression of CCR4 and CCR10, which function as skin-homing chemokine receptors. Using polymerase chain reaction, the authors also confirmed reactivation of herpesvirus HHV6b, which is found in the majority of DiHS/DRESS cases. Assessed together, the data pointed to several potential therapeutic targets: cell proliferation pathways, HHV6b, the JAK-STAT pathway, and chemokine receptors. The authors ruled out targeting the cell proliferation pathways due to prior failure of mycophenolate, HHV6b due to renal toxicity of cidofovir and ganciclovir, and chemokine receptors due to risk of mogamulizumab-associated toxic epidermal necrolysis. To target the JAK-STAT pathway, the patient was started on tofacitinib, which is a JAK1 and JAK3 inhibitor. After two weeks of therapy, the patient’s peripheral blood showed a marked reduction in the offending T-cells and the skin inflammation resolved. Therefore, the patient was safely tapered off prednisone and also taken off mycophenolate and cyclosporine, which brought the patient’s renal hypertension under control.

Kim D, Kobayashi T, Voisin B, et al. Targeted therapy guided by single-cell transcriptomic analysis in drug-induced hypersensitivity syndrome: a case report. Nat Med. 2020;26(2):236–243.

Correspondence: Dr. Keisuke Nagao at keisuke.nagao@nih.gov

Repurposing PARP inhibitors to treat T-cell lymphoblastic leukemia

Epigenetic changes, which are predominantly the methylation and demethylation of DNA, play an important role in regulating the cell cycle by enhancing, repressing, or silencing the transcriptional activity of genes. These epigenetic “switches” can be regulated by other genes. Some cancers show single-gene mutations that drive epigenetic changes, which in turn cause overexpression of oncogenes or underexpression of tumor-suppressor genes. In recent years, the FDA has approved therapies that target such epigenetic changes. These include panobinostat and romidepsin for the treatment of multiple myeloma and cutaneous T-cell lymphoma, respectively. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by TET1 overexpression leading to epigenetic dysregulation of the cell cycle. The authors investigated the treatment of T-ALL cells using drugs that are FDA approved to treat other cancers. Published gene-expression data sets of human cancer cell lines show that TET1 is highly expressed in T-ALL patients. The authors reproduced this finding using patient samples and leukemic cell lines analyzed via quantitative real-time PCR (qRT-PCR) and gene-expression microarrays. Knockout of TET1 in mice and knockdown in human T-cells did not perturb normal T-cell proliferation, making TET1 an excellent therapeutic target for T-ALL. The authors found that shRNA-mediated depletion of TET1 in T-ALL cells in vitro reduced cell growth and colony numbers compared with scrambled control in liquid culture and colony-forming unit assays, respectively. They also found that TET1 promotes cell growth via maintenance of a 5-hydroxymethylcytosine (5hmC) mark, which regulates the cell cycle, DNA repair genes, and T-ALL associated oncogenes. The authors further demonstrated that PARP enzymes, which are highly expressed in T-ALL cells, establish H3K4me3 marks at the TET1 promoter and that PARP1 interacts with the TET1 protein. The authors concluded that the FDA-approved PARP inhibitor olaparib downregulated TET1 expression in T-ALL cells, opening a promising avenue for treating T-ALL with PARP inhibitors.