Molecular response to ibrutinib-based therapy. (A) Biospecimen collection for all patients enrolled in our study. Included is the time between the tumor and CSF collection (T/C interval). Red diamonds indicate primary tumor tissue that was collected and sequenced (BM: diagnosed by bone marrow biopsy; C: diagnosed by CSF cytology). X, patient refused CSF collection. P, patient off study for disease progression. Blue circles represent sequenced CSF samples, and white circles represent samples with insufficient volume to perform sequencing. (B) Imaging was performed at baseline and at C3 and C5 in 9 patients. Shown is the spider plot of patients with measurable disease, 1 of whom had disease progression after an initial response to therapy (#11) and 7 patients (#5, #7, #9, #12, #13, #14, #15) had a PR > 90% or CRs on MRI. (C) Heat maps of the variant allelic frequencies of all of the mutations present in CSF collected before treatment initiation (baseline), during ibrutinib-based combination therapy (C3, C5), and at progression (PD) in representative patients with sustained response demonstrating a “clearance” of tumor DNA (for all CSF profiles, see supplemental Figure 6). Variant allelic frequency scale = 0 (white) or 1 (dark blue). (D) Heat map of the variant allelic frequencies (baseline, C3, C5, and at progression [PD]) and early progression, demonstrating a persistent clone (#11). (E) Patient with nonmeasurable leptomeningeal disease on MRI (T1 postcontrast sequences) and CSF (cytology and flow cytometry) at baseline. After 2 cycles of study therapy, the MRI changes resolved. No malignant cells and no ctDNA was detectable in the CSF (C3). After completion of the induction therapy (C5), the brain MRI and CSF (cytology and flow cytometry) continued to show a response, whereas ctDNA was detectable in the CSF. Ultimately, the patient developed progression of disease on MRI, CSF cytology, and CSF flow cytometry after 1 month of maintenance ibrutinib. White arrowheads, leptomeningeal involvement in the cerebellar folia; white arrows, leptomeningeal involvement of both trigeminal nerves; red arrows, recurrent leptomeningeal disease affecting both trigeminal nerves. (F) Heat map of the variant allelic frequencies in a case of early progression with reemergence of genetic alterations (#6). Variant allelic frequency scale = 0 (white) or 1 (dark blue).