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, molecular pathologist, Sonic Healthcare USA, Rye Brook, NY; and Richard Wong, MD, PhD, assistant professor of pathology, University of California San Diego.
Epigenetic assays as an alternative to flow cytometry for CD4+ T-cell quantification
September 2026—HIV remains a major global public health concern, with an estimated 40.8 million people living with the virus in 2024. The challenge is particularly striking in low- and middle-income countries, where access to diagnostic testing may be limited. The World Health Organization’s universal test-and-treat strategy recommends that patients diagnosed with HIV immediately begin antiretroviral therapy regardless of their CD4 cell count or clinical stage. This strategy has placed greater emphasis on routine viral load monitoring, leading to a marked decline in CD4 testing and the discontinuation of many point-of-care CD4 platforms. However, CD4 testing remains important for identifying patients with advanced disease, who are at greatest risk of opportunistic infection. And many resource-limited settings, where flow cytometry is not readily available, face a significant gap in access to CD4 monitoring. Epigenetic-based characterization of immune-cell populations has emerged as an alternative to flow cytometry. With this method, cell type-specific DNA methylation markers can be used to quantify lymphocyte subsets. A distinct advantage of epigenetic quantitative PCR (qPCR)-based assays is that they can be used with DNA obtained from dried blood spot cards, whereas conventional flow cytometry requires fresh blood. Because dried blood spots can be collected by finger prick, stored at room temperature, and transported without specialized equipment, this approach could improve access to CD4 testing in remote or underserved regions. In a proof-of-concept study, the authors evaluated a new epigenetic qPCR-based assay for measuring CD4 T-cell counts from dried blood spot specimens and compared the results with those from conventional flow cytometry. Residual EDTA whole blood from 150 patients with HIV who had a wide range of CD4 T-cell counts (5–1,382 cells/mL) was spotted onto dried blood spot cards for downstream analysis. Circular 6-mm discs were punched from the dried blood spots using a standard paper punch and processed for DNA extraction and bisulfite conversion. The bisulfite-converted DNA, together with standards and quality controls, underwent qPCR to determine absolute CD4 T-cell counts. The epigenetic qPCR-based assay demonstrated good agreement with flow cytometry, with a concordance correlation coefficient of 0.91. At the clinically relevant threshold of 200 cells/mL, the assay exhibited a sensitivity of 93.8 percent and specificity of 88.9 percent. The authors concluded that the study demonstrates the technical feasibility of using an epigenetic qPCR-based assay to measure CD4 T-cell counts from dried blood spots. This approach offers a promising alternative to conventional flow cytometry and could expand access to CD4 testing in settings with limited laboratory resources. More broadly, this study illustrates how epigenetic assays can be applied beyond oncology by using cell type-specific DNA methylation patterns to quantify immune cell populations and provide clinically meaningful information.
Munir R, Sungu T, Lawrie D, et al. Epigenetic CD4+ T-cell quantification from dried blood spots using a real-time quantitative PCR-based assay. J Mol Diagn. Published online July 1, 2026. doi.org/10.1016/j.jmoldx.2026.05.010
Correspondence: Dr. Lesley Erica Scott at lesley.scott@wits.ac.za or Dr. Riffat Munir at riffat.munir@witsdih.ac.za
Ability of daraxonrasib to improve survival for patients with previously treated metastatic pancreatic cancer
Pancreatic ductal adenocarcinoma is one of the deadliest cancers, with a median overall survival of less than one year. Many patients present with advanced disease at the time of diagnosis. Although targeted therapies have improved outcomes for a small number of patients with rare molecular alterations, the vast majority of patients still receive chemotherapy that often has toxic side effects and limited benefit. More than 90 percent of patients with pancreatic ductal adenocarcinoma (PDAC) harbor KRAS mutations, and most of these mutations involve codon G12. These mutations keep the K-Ras protein in a permanently active state, leading to activation of downstream signaling pathways and tumor growth. Daraxonrasib is a new oral targeted therapy that selectively inhibits the active form of the RAS proteins encoded by NRAS, KRAS, or HRAS. It was evaluated in the phase three RASolute 302 trial with patients whose metastatic PDAC was previously treated. The study enrolled 500 patients from 59 centers in six countries, and 91.8 percent of the patients had tumors harboring a KRAS mutation. Patients were randomly assigned 1:1 to receive daraxonrasib or the investigator’s choice of standard chemotherapy. Treatment continued until disease progression, unacceptable side effects, or withdrawal of consent. The study results demonstrated a clear clinical benefit from use of daraxonrasib. Those patients with KRAS G12 alterations who received daraxonrasib had a longer median overall survival (13.2 months) and progression-free survival (7.3 months) compared with those who received chemotherapy (6.7-month overall survival, 3.5-month progression-free survival). A similar survival benefit was seen in the overall study population, which included patients with KRAS G12 mutations and a small proportion of patients without G12 mutations. Treatment-related adverse events were common in both groups. The most frequently reported adverse events included rash, diarrhea, stomatitis, nausea, vomiting, fatigue, and anemia. Treatment-related adverse events leading to discontinuation of treatment were higher in patients receiving chemotherapy (11.2 versus 1.2 percent). The RASolute 302 trial represents an important advance in the treatment of pancreatic cancer. The authors concluded that by targeting a molecular alteration present in the vast majority of pancreatic cancers, daraxonrasib significantly improves survival compared with standard chemotherapy. However, because relatively few patients without KRAS G12 mutations were enrolled in the study, it may be necessary to evaluate the effectiveness of the treatment in other, less common, molecular subgroups.
O’Reilly EM, Wainberg ZA, Hendifar AE, et al. Daraxonrasib or chemotherapy in previously treated metastatic pancreatic cancer. N Engl J Med. Published online May 31, 2026. doi.org/10.1056/NEJMoa2605555
Correspondence: Dr. Eileen M. O’Reilly at oreillye@mskcc.org, Dr. Zev A. Wainberg at zwainberg@mednet.ucla.edu, or Dr. Brian M. Wolpin at brian_wolpin@dfci.harvard.edu