Immune cell profiles provide valuable diagnostic information for hematologic and immunologic diseases. Although it is the most widely applied analytical approach, flow cytometry is limited to liquid blood. Moreover, either analysis must be performed with fresh samples or cell integrity needs to be guaranteed during storage and transport. We developed epigenetic real-time quantitative polymerase chain reaction (qPCR) assays for analysis of human leukocyte subpopulations. After method establishment, whole blood from 25 healthy donors and 97 HIV patients as well as dried spots from 250 healthy newborns and 24 newborns with primary immunodeficiencies were analyzed. Concordance between flow cytometric and epigenetic data for neutrophils and B, natural killer, CD3 T, CD8 T, CD4 T, and FOXP3 regulatory T cells was evaluated, demonstrating substantial equivalence between epigenetic qPCR analysis and flow cytometry. Epigenetic qPCR achieves both relative and absolute quantifications. Applied to dried blood spots, epigenetic immune cell quantification was shown to identify newborns suffering from various primary immunodeficiencies. Using epigenetic qPCR not only provides a precise means for immune cell counting in fresh-frozen blood but also extends applicability to dried blood spots. This method could expand the ability for screening immune defects and facilitates diagnostics of unobservantly collected samples, for example, in underdeveloped areas, where logistics are major barriers to screening.

Purpose Newborn screening (NBS) for severe combined immunodeficiency (SCID) is based on the detection of T-cell receptor excision circles (TRECs). TRECs are a sensitive biomarker for T-cell lymphopenia, but not specific for SCID. This creates a palette of secondary findings associated with low T-cells that require follow-up and treatment or are non-actionable. The high rate of (non-actionable) secondary findings and false-positive referrals raises questions about the harm-benefit-ratio of SCID screening, as referrals are associated with high emotional impact and anxiety for parents. Methods An alternative quantitative TREC PCR with different primers was performed on NBS cards of referred newborns (N = 56) and epigenetic immune cell counting was used as for relative quantification of CD3 + T-cells (N = 59). Retrospective data was used to determine the reduction in referrals with a lower TREC cutoff value or an adjusted screening algorithm. Results When analyzed with a second PCR with different primers, 45% of the referrals (25/56) had TREC levels above cutoff, including four false-positive cases in which two SNPs were identified. With epigenetic qPCR, 41% (24/59) of the referrals were within the range of the relative CD3 + T-cell counts of the healthy controls. Lowering the TREC cutoff value or adjusting the screening algorithm led to lower referral rates but did not prevent all false-positive referrals. Conclusions Second tier tests and adjustments of cutoff values or screening algorithms all have the potential to reduce the number of non-actionable secondary findings in NBS for SCID, although second tier tests are more effective in preventing false-positive referrals.

CLL is a rare hematological cancer and is classified as low-malignancy non-Hodgkin lymphoma. While rare, it is the most frequent form of leukemia in Europe and US accounting for approximately a quarter of all leukemias. As it is a slowly growing tumor, its 5-year survival rate is high compared to faster developing B cell tumors and survival has further improved since introduction of Anti-CD20 Antibody therapies. However, overall the 5-year survival of rare cancers (49%) is significantly lower than for their common counterparts (63%), even though hematological cancers improved outcome over the last years. Due to the currently very late and often incidental diagnosis, some cases turn into aggressively progressing cancers prior to treatment and with concomitant poor outcome. Therefore, diagnosis of CLL and other B cell leukemias remains a challenge. To further improve outcome of CLL and other lymphatic leukemias earlier diagnosis is important. To this end, simple home testing could broaden access to diagnostic tools, but no current method would provide adequate remote initial screening of lymphatic leukemia in populations at risk, such as persons older than 65 years. Epigenetic qPCR has been shown to detect and quantify immune cell populations efficiently from liquid and dried blood samples (DBS). This system is based on specifically unmethylated regulatory elements in gene loci associated with certain cell types. These unique methylation patterns allow specific quantification of cells of interest via qPCR. Different epigenetic qPCR systems were developed for total B cells, naïve and memory B cells as well as IgM producing B cells. While these gene loci are fully unmethylated in the respective target cells, they are fully methylated in all relevant control cell types. The cell type specificity of these markers was shown using bisulfite specific sequencing using purified cells from healthy donors. Here, we investigated blood samples from healthy donors (n = 112) as well as patients at the time of their initial CLL diagnosis. The mean total B cell count of healthy donors at 5.7% was clearly lower than the 60.4% obtained for CLL patients. Similarly drastic differences were observed for naïve B (2.3% normal vs. 20.8% CLL) and IgM (3.3% vs. 49.5%) as well as memory B cells (1.6% vs. 32.6%). All differences were statistically significant. To investigate if these mean differences may also lead to differential (and potentially early) diagnosis, we designed receiver operator characteristics and contingency tables, both indicating near perfect segregation between CLL patients and healthy donors in Ig M, memory and all B cells. Our data indicate applicability of epigenetic immune cell counting as easy, potentially DBS- and home-based approach to test increase of immune cells in people potentially at risk of hematological cancers. Citation Format: Janine Jung, Jeannette Werner, Konstantin Schildknecht, Janika Schulze, Steffi Walter, Christoph Sachsenmaier, Deborah Phippard, Barbara Seliger, Claudia Wickenhauser, Sven Olek. Epigenetic immune cell markers for CLL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3334.

Epigenetic immune cell counting is a DNA (de)methylation-based technique which can be used to quantify lymphocyte subsets on dried blood spots (DBS). The foregoing techniques allow for a retrospective investigation of immune cell profiles in newborns. In this study, we used this technique for determining lymphocyte subcounts as a potential biomarker for necrotizing enterocolitis (NEC). We investigated whether this technique can be implemented in the field of neonatology, by testing whether regulatory T cell (Treg) levels are pre-existently low in preterms with NEC. Newborn screening (NBS) cards from 32 preterms with NEC and 32 age- and weight-matched preterm controls, and 60 healthy term newborns, were analyzed. Relative and absolute cell counts were determined for CD3+, CD4+, CD8+, Th17, and Treg T cells. For both relative and absolute cell counts of CD3+, CD4+, CD8+, and Th17 T cells, significant differences were found between healthy term controls and both preterm groups, but not between preterm groups. For Tregs, no significant differences were found in either relative or absolute counts between any of the newborn groups. This study demonstrates the principle of epigenetic immune cell counting to analyze lymphocyte subsets in preterm neonates.

Early detection and differential diagnosis of Non-Hodgkin lymphoma (NHL) is essential for the initiation of suitable therapy options. Biopsy based diagnosis allows allocation to World Health Organization (WHO) sub-classification groups but prognosis often needs identification of specific genetic aberrations or gene expression patterns. Despite this strategy, prognosis is difficult, since diseases, like e.g. mantel cell lymphoma, can be indolent or aggressive. Here we report the characterization of NHL biopsies using epigenetic immune cell quantification. This approach is based on the identification of specifically demethylated regions in individual immune cell types. These regions are then quantified using methylation-sensitive qPCR after bisulfite conversion. This highly reproducible and objective method allows in depth immunophenotyping of the tumor microenvironment. Next to CD4+ and CD8+ T cells, B cells and NK cells more specialized immune cells like regulatory T cells (Tregs), follicular T helper cells, PD-1 expressing cells and Myeloid derived suppressor cells (MDSC) were quantitatively determined in formalin-fixed tissue samples of 251 patients with different B cell Non-Hodgkin lymphomas. As a result, characteristic immune cell distribution patterns were identified for the different lymphoma entities, including chronic lymphocytic leukemia (B-CLL) (n=41), mantle cell lymphoma (MCL) (n=32), follicular lymphoma (FL) (n=33), diffuse large B cell lymphoma (DLBCL) (n=74), marginal zone lymphoma (MZL) (n=40) and B cell acute lymphoblastic leukemia (B-ALL) (n=26). In addition, substantial heterogeneity within the entities was observed that may correlate with disease prognosis. Among others, this is particularly the case for MDSC in B-CLL patients as well as for NK cells and Tregs in MCL and PD-1+ cells in FL patients. Currently clusters within the different disease groups are analyzed with regard to the clinical outcome to investigate if disease prognosis can be predicted by epigenetic immune cell quantification. In conclusion, epigenetic immune cell quantification is a suitable method to analyze the tumor microenvironment of NHL patients with the potential to support patient’s prognosis. Citation Format: Jeannette Werner, Steffi Walter, Christoph Sachsenmaier, Janika Schulze, Deborah Phippard, Sven Olek, Barbara Seliger, Claudia Wickenhauser. Epigenetic immune cell profiling in B-non-Hodgkin lymphoma biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 993.