Quantification of DAPK1 Promoter Methylation in Bone Marrow and Peripheral Blood as a Follicular Lymphoma Biomarker  Manuela Giachelia, Valentina Bozzoli, Francesco D'Alò, Maria Chiara Tisi, Giuseppina Massini, Elena Maiolo, Francesco Guidi, Elisa Cupelli, Maurizio Martini, Luigi M. Larocca, Maria Teresa Voso, Giuseppe Leone, Stefan Hohaus  The Journal of Molecular Diagnostics  Volume 16, Issue 4, Pages 467-476 (July 2014) DOI: 10.1016/j.jmoldx.2014.03.003 Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Flowchart of study population according to treatment. R-chemo, rituximab-based immunochemotherapy (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone; rituximab, vincristine, and prednisone; and rituximab, fludarabine, and mitoxantrone); W&W, watchful waiting. The Journal of Molecular Diagnostics 2014 16, 467-476DOI: (10.1016/j.jmoldx.2014.03.003) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 DAPK1 methylation levels in patients and control samples. DAPK1 methylation levels were significantly higher in lymph node, BM, and PB samples from patients with FL compared with those from controls (CTR). DAPK1 methylation levels are shown in a dot plot on a logarithmic scale. Dashed lines represent the cutpoints for aberrant methylation in the respective tissues; solid lines indicate medians. Differences in DAPK1 methylation levels between samples from patients with FL and controls were significant (P < 0.05 for lymph node; P < 0.0001 for BM; P < 0.0001 for PB). The Journal of Molecular Diagnostics 2014 16, 467-476DOI: (10.1016/j.jmoldx.2014.03.003) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Kaplan-Meier curves for HL survival according to DAPK1 methylation levels at diagnosis. Aberrant DAPK1 methylation in BM at diagnosis was associated with inferior PFS. Kaplan-Meier estimates of PFS in 82 patients with FL treated with immunochemotherapy according to DAPK1 methylation. n = 50 patients with aberrant DAPK1 methylation above the cutoff of 0.2%; n = 32 patients without aberrant methylation. P < 0.05 (log-rank test). meth., methylation. The Journal of Molecular Diagnostics 2014 16, 467-476DOI: (10.1016/j.jmoldx.2014.03.003) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Longitudinal study of individual DAPK1 methylation levels in nine separate patients with FL. Samples analyzed at diagnosis, during therapy, and at follow-up evaluations indicated an association of DAPK1 methylation levels with disease activity. DAPK1 methylation levels are shown on a logarithmic scale. A–G: Patients with elevated DAPK1 methylation levels showed normalization of DAPK1 methylation levels when remission was induced, and an increase in the case of relapse (patients A, B, and C) or progression (patients D and E), whereas patients who remained in complete remission maintained DAPK1 methylation levels in the normal range (patients F and G). H and I: Patients with no aberrant DAPK1 methylation signal at diagnosis showed no aberrant DAPK1 methylation during follow-up, either in remission (patient H) or at relapse (patient I). Black diamonds indicate positive BM histology; white diamonds indicate the absence of BM histology positivity. Dashed lines indicate the cutoff for aberrant DAPK1 methylation (0.2%). Shaded areas indicate treatment periods. CR, complete response; PD, progressive disease; PR, partial disease; Rel, relapse. The Journal of Molecular Diagnostics 2014 16, 467-476DOI: (10.1016/j.jmoldx.2014.03.003) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions