Editors: Donna E. Hansel, MD, PhD, division head of pathology and laboratory medicine, MD Anderson Cancer Center, Houston; James Solomon, MD, PhD, assistant professor, Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York; Erica Reinig, MD, assistant professor and medical director of molecular diagnostics, University of Wisconsin-Madison; Marcela Riveros Angel, MD, molecular genetic pathology fellow, Department of Pathology, Oregon Health and Science University, Portland; Maedeh Mohebnasab, MD, assistant professor of pathology, University of Pittsburgh; Alicia Dillard, MD, associate clinical laboratory director, Omniseq/Labcorp, Buffalo, NY; and Richard Wong, MD, PhD, assistant professor of pathology, University of California San Diego.
Role of tripartite fusion and LBD truncation in RARA– and RARG-related atypical acute promyelocytic leukemia
December 2024—Acute promyelocytic leukemia is generally characterized by presence of the PML::RARA fusion. However, a subset of cases with morphological, cytochemical, and immunophenotypic features of acute promyelocytic leukemia (APL) lack this canonical fusion gene and instead present with alternate fusions. These include RARA fusions with partners other than PML and fusions involving other retinoic acid receptor (RAR) genes, such as RARG. Leukemias with these variant fusions often resist all-trans retinoic acid (ATRA) therapy. Specifically, the ATRA sensitivity of RARA fusion genes varies based on its 5′ fusion partner. Interestingly, in some studies, the artificially induced variant RAR bipartite fusion genes responded well to ATRA. These tested bipartite fusion genes involved a single rearrangement between a RAR gene and a single 5′ partner gene (X::RAR) and included NUP98::RARG, PML::RARG, STAT3::RARA, and STAT5B::RARA. To better understand the mechanism behind this variable resistance profile, the authors conducted a comprehensive molecular characterization of APL-like cases harboring variant translocations, referred to as atypical APL or aAPL. They analyzed 27 variant RARA fusion gene-positive aAPL (RARA-aAPL) and 21 RARG fusion gene-positive aAPL (RARG-aAPL) with accessible whole transcriptome sequencing data (WTS), whole genome sequencing data (WGS), or archived samples. WTS data from 42 PML::RARA-positive APL cases and 50 healthy donors were used for comparison. The authors demonstrated that the RAR genes can undergo 5′ and 3′ splicing, resulting in an unexpected novel form of tripartite (X::RAR::X or X::RAR::Y) rather than bipartite (X::RAR) fusions. Unlike bipartite fusions, these tripartite RAR fusion genes exhibited two genetic changes in the RAR gene, showing 5′ and 3′ abnormalities. Notably, the authors identified bipartite and tripartite RARA fusions in aAPL with a 5′ partner gene-associated pattern. In contrast, all analyzed RARG-aAPL cases exhibited tripartite RARG fusions. All observed RARG tripartite and RARA tripartite fusions demonstrated truncation of the ligand-binding domain, whereas bipartite RARA fusions showed it to be intact. The authors theorize that ligand-binding domain truncation prevents RAR proteins from binding with coactivator proteins in response to ATRA. This finding could explain the ATRA resistance observed in all cases of RARG fusion and certain cases of variant RARA fusion. Some of the variant fusions observed by the authors involved 5′ untranslated regions and transposable elements. This study furthers understanding of leukemia fusion genes, highlighting the possibility that leukemogenic fusion genes can be more complicated than the conventional bipartite paradigm. In addition, it sheds light on potential mechanisms of ATRA resistance in aAPL. However, further investigation into the spectrum of variant fusion genes, impact of ligand-binding domain truncation, and mechanisms of resistance in aAPL is warranted. Yet identification and characterization of these more complex fusion events may prove challenging with conventional methodologies, necessitating the use of more specialized techniques and optimized bioinformatic pipelines.
Zhou X, Chen X, Chen J, et al. Critical role of tripartite fusion and LBD truncation in certain RARA– and all RARG-related atypical APL. Blood. 2024;144(14):1471–1485.
Correspondence: Dr. Hongxing Liu at starliu@pku.edu.cn, or Dr. Suning Chen at chensuning@suda.edu.cn, or Dr. Peihua Lu at peihua_lu@126.com
Evaluation of T. cruzi assay to detect parasite DNA in dried blood spots from infants
Chagas disease, caused by the protozoan Trypanosoma cruzi, is a serious public health concern in many areas of the world. Early detection and timely treatment of this pathogen are critical to ensure a higher cure rate and prevent progression to the chronic stage of the disease. Unfortunately, microscopy-based methods for detecting the acute phase of Chagas disease often have low sensitivity and are subject to operator expertise. Real-time polymerase chain reaction (PCR) has proven more reliable, though given its cost and technical and staffing requirements, this testing is often limited to central laboratories. Few countries have incorporated real-time PCR into patient care guidelines. Children born to T. cruzi-infected mothers can undergo serologic testing after nine months, though this delay in testing risks loss to follow-up. The authors recently validated a loop-mediated isothermal amplification (LAMP) test for T. cruzi DNA using an ultrarapid DNA extraction method with dried blood spots on FTA cards. The goal was to develop a methodology for early detection of T. cruzi that would be affordable, sensitive, specific, fast, and user friendly for use in low-complexity laboratories. The procedure was implemented in 10 public health care centers offering maternity care that were located in Paraguay, Bolivia, and Argentina. A total of 14 laboratory technicians across the 10 centers were trained in how to perform the procedure. Operator performance was harmonized across sites using a standardized testing panel that included negative, low-positive, and high-positive controls. Strong agreement (κ=0.924) was observed for the negative and high-positive controls, but only four laboratories demonstrated good agreement (κ>0.8) with the low-positive controls. The authors then evaluated the assay in clinical situations by collecting blood samples from 222 infants born to mothers who were seropositive for T. cruzi infection. Samples were collected at birth or four to 12 weeks, or both. The samples included blood collected via heparinized capillaries for microscopy and dried blood spots in FTA for LAMP, as well as blood anticoagulated in EDTA for real-time PCR. T. cruzi was identified microscopically in four cases. LAMP identified eight positive cases and aligned perfectly with real-time PCR (κ=1), demonstrating higher sensitivity than microscopic observation for early detection of infection in infants. While options for participating in quality assurance programs for certain molecular diagnostic tests can be limited in some settings, the authors highlighted how important such programs are in providing insight into laboratory performance, allowing comparison between labs and bringing to light potential problems related to the assay in question.
Longhi SA, Muñoz-Calderón A, García-Casares LJ, et al; Chagas-Group. Inter-laboratory harmonization study and prospective evaluation of the PURE—Trypanosoma cruzi—loop-mediated isothermal amplification assay for detecting parasite DNA in newborn’s dried blood spots. J Mol Diagn. 2024. doi.org/10.1016/j.jmoldx.2024.08.007
Correspondence: Dr. Silvia Longhi at longhi.ingebi@gmail.com or Dr. Alejandro Gabriel Schijman at aschijman@ingebi-conicet.gov.ar