Schematic illustrating the application of ultrasensitive ctDNA assessment for the identification of adverse risk in DLBCL at different disease milestones and as a navigation aid to remaining figures. A lymphoma patient is imagined as experiencing these disease milestones over time, depicted as an arrow progressing from left to right. During this temporal sequence, ctDNA can inform risk at diagnosis, during therapy, in surveillance of disease, and at progression or disease transformation, as illustrated in the corresponding figures associated with each milestone. At diagnosis, profiling of tumor DNA obtained from either tissue biopsies (indicated by a scalpel) or plasma (depicted as blood collection tubes) allows for the identification of patients with high tumor burden, non-GCB subtypes, and “double hit” lymphoma. Assessment of ctDNA during and after lymphoma treatment facilitates the detection of both emerging resistance mutations and minimal residual disease (MRD) before progression, with potential for noninvasive prediction of relapse and histological transformation. Tumor evolution in an anecdotal DLBCL patient is illustrated, showing tumor response and clonal evolution over the course of the disease (detectable subclones at diagnosis are shown in blue/gray; an emergent subclone after therapy is shown in red). The profiling of tumor DNA and ctDNA at each milestone is shown by a double-stranded DNA molecule.

(A) Direct comparison of CAPP-Seq with IgHTS for tumor genotyping and ctDNA detection in DLBCL. (B) Change of ctDNA disease burden in response to treatment and during clinical progression in a patient with stage IIAX DLBCL. Shown is the mean AF of all SNVs detected by CAPP-Seq (left y axis) and the number of lymphoma DNA molecules per milliliter of plasma identified by IgHTS (right y axis) over serial time points (x axis). The black arrows highlight ctDNA detection by CAPP-Seq, at which time ctDNA by IgHTS was below the limit of detection (false negative, open circles). In (A) and (B), CAPP-Seq and IgHTS were performed from the same specimens (tissue biopsy or blood draw). IgHTS was performed as part of routine clinical practice by an independent laboratory. ND, not detected; PR, partial response; PD, progressive disease; R-CHOP, rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone; R-DHAP, rituximab, dexamethasone, high-dose cytarabine, and cisplatin; SCT, stem cell transplantation; DeVIC, dexamethasone, etoposide, ifosfamide, and carboplatin. (C) Noninvasive detection of ibrutinib resistance mutations in BTK (C481S, arrows) in a patient with progressive lymphoma, reflecting two independent subclones emerging during therapy. RBL, rituximab, bendamustine, and lenalidomide. (D) Schematic illustrating the two acquired BTK C481S resistance mutations in the patient from (C). Read pileups were rendered with Integrative Genomics Viewer. A major resistance clone harboring the BTK C481S A > T mutation (red, arrow) and a minor clone carrying the BTK C481S C > G mutation (dark green, arrow) were detected during ibrutinib therapy and after disease progression. Shown here are the progression tumor and plasma samples taken at days 217 and 222 with their respective AFs. Germline bases are represented by light green and blue bars at the top. At the bottom, germline bases and amino acid sequences are depicted.

(A) Relationship between LDH and ctDNA concentration from pre-treatment plasma time points. Correlations were determined separately above and below the upper limit of normal (ULN; 340 U/liter). (B) Correlation between MTV, measured from PET/CT imaging, and ctDNA concentrations from pretreatment plasma. Pretreatment LDH and MTV values in (A) and (B) were obtained as close in time as possible to blood draws used for plasma cell–free DNA sequencing (median, 6 days for LDH and 4 days for MTV). r, Pearson correlation coefficient. (C) Association between ctDNA concentration at diagnosis and Ann Arbor stage. Statistical comparison between early-stage (I + II) and late-stage (III + IV) patients was performed using Mann-Whitney U test. Means and SEMs are indicated. (D) Detection of ctDNA in relapsing patients as a function of time. Top: Cumulative fraction of patients with detectable ctDNA as a function of time before relapse. Bottom: Patient level data demonstrating ctDNA detection before relapse (n = 11). Clinical relapses were confirmed radiographically, and corresponding blood draws were taken within 30 days of diagnostic imaging, except for patients DLBCL088 (43 days) and DLBCL071 (78 days). All other blood draws were obtained between radiographic complete response and relapse (14 to 983 days before clinical relapse). Red circle, ctDNA detected; open circle, ctDNA not detected; black bars, imaging studies demonstrating complete response; red bars, imaging studies demonstrating detection of disease. Asterisks highlight patients with an isolated brain relapse. mo, months. (E) Direct comparison of CAPP-Seq and IgHTS for relapse detection at the time of relapse and before relapse. (F) Kaplan-Meier analysis of PFS in patients with at least one ctDNA-positive plasma sample after the end of curative therapy compared to patients without detectable ctDNA after the end of curative therapy. Significance was assessed using the log-rank test.

(A) Top: CAPP-Seq COO classifier scores are shown for each patient’s DLBCL tumor sample (n = 76), ordered on the basis of decreasing log odds scores. Bottom: COO classification of patients in (A) using the Hans immunohistochemistry (IHC) algorithm (n = 59). Cases classified as GCB or non-GCB are shown in orange and blue, respectively. Empty spaces indicate cases with no IHC classification available. (B) PFS from diagnosis in DLBCL cases, as determined by the CAPP-Seq COO classifier on all analyzed DLBCL tumor samples (n = 50). (C) The results of applying univariate Cox proportional hazards regression to analyze PFS in tumor and plasma samples from DLBCL patients. HR, hazards ratio; CI, confidence interval. (D) PFS from diagnosis in DLBCL cases, as determined by Hans algorithm (n = 38). The log-rank test was used in (B) and (D) to determine statistical significance. n.s., not significant. (E) Concordance between COO assignments of the CAPP-Seq classifier applied to tumor samples and applied to corresponding plasma samples (n = 41). Primary central nervous system lymphoma and transformed lymphoma cases were excluded from the patient cohort for the analyses in (B) to (D).

(A) Comparison of mutation profiles from diagnostic tumor samples (“tumor 1”; FL or DLBCL) and follow-up tumor samples (“tumor 2”; transformation or progression) in patients with three distinct NHL types: tFL, ntFL, and rrDLBCL. The fraction of SNVs specific to tumor 2 (x axis) is compared with the proportion of SNVs shared between both tumors (y axis). Each dot represents a single patient. Shaded ovals highlight patients with different histologies, excluding outliers. (B) Network depiction of the mutational divergence between each tumor 1 and tumor 2 pair analyzed in (A). The central node represents tumor 1, and the distance between tumor 1 and each patient’s tumor 2 (edge) is expressed as the fraction of unique mutations to both tumor 1 and tumor 2. Bar graph: percentage of SNVs unique to both tumor 1 and tumor 2 (nonshared mutations) for the median patient in each histological group. (C) Evolution of different types of NHL as determined by comparing diagnostic tumor samples from (A) (“tumor 1”) with follow-up plasma samples. The percentage of SNVs found uniquely in follow-up plasma compared to tumor 1 is shown for the three histologies. (D) Comparison of ctDNA concentrations in follow-up plasma samples from (C). Statistical comparisons in (C) and (D) were performed using the Mann-Whitney U test. Medians and ranges are indicated. (E) Performance metrics for the prediction of histological transformation from plasma. Sn, sensitivity; Sp, specificity; PPV, positive predictive value; NPV, negative predictive value. (F) Biopsy-free detection of an occult aggressive lymphoma subclone (tFL) in a patient histologically diagnosed with ntFL from a left inguinal lymph node biopsy (left, blue solid circle). The tumor site harboring the aggressive subclone (tFL, green dashed circle) was later identified in a retroperitoneal lymph node biopsy (right, green solid circle). Bottom: Venn diagram analysis of mutations found in tumor/plasma pairs at FL and tFL diagnosis. Mutations in key driver genes, such as CARD11 or PIM1, are indicated.