This is a single institution retrospective cohort study of patients diagnosed with WM in or after 2010. The study was approved by the institutional review board and was conducted in compliance with the Declaration of Helsinki. To be eligible, patients must have received treatment for WM and had PB immunophenotyping via flow cytometry performed at diagnosis. CL was defined as detectable clonally restricted B-cells that matched the actual or expected CD5-CD10- B-cell immunophenotype of LPL. Any ambiguous cases that did not fit the diagnosis of the WM variant of LPL (e.g., non-IgM paraprotein and absence of bone marrow involvement) were excluded.

Demographic and clinical patient data were obtained from the electronic medical record. Laboratory values were collected for variables known to be associated with WM prognosis, including hemoglobin, platelet count, β2-microglobulin, monoclonal IgM, albumin, and LDH (7, 8). The International Prognostic Scoring System for WM (IPSSWM) was used to assign a prognostic score for each patient (7). Treatment response was assessed via The International Working Group on Waldenström Macroglobulinemia criteria (9).

Patients who met the study inclusion criteria were classified based on the presence or absence of CL at the time of diagnosis into two groups: those with positive PB flow cytometry (CL+) and those with negative PB flow cytometry (CL-). The primary endpoint of this study was to evaluate progression-free survival (PFS) between the two groups. Secondary endpoints included evaluation of overall survival (OS) and diagnosis to treatment interval (DTI).

PFS was defined as the time from the start of first-line therapy until lymphoma relapse/progression or death from any cause, censoring from the last clinical assessment time. OS was defined as the time from the start of first-line therapy until death from any cause or censoring at the last clinical assessment. DTI was defined as the time from diagnosis to initiation of first-line systemic therapy Skeletal involvement was defined as any of these findings including osteopenia, marrow space widening, endosteal erosions, osteolytic lesions, etc. (10–12) Complex karyotype was defined as the presence of at least three chromosomal aberrations in at least two cells.

Demographic and disease characteristics were summarized using median and range for continuous variables, and frequency and percentage for categorical variables, compared among study groups using the Wilcoxon signed rank test, and Chi-square test, or Fisher’s exact test, respectively. PFS was estimated using the Kaplan-Meier method and compared between two groups using the log-rank test. Cox proportional hazard regression models were used to estimate the hazard ratios for risk of progression or death. The multivariable Cox model was built including all the variables associated with PFS in the univariable model with p<0.10. The proportional hazard assumption was checked using Schoenfeld residuals after fitting Cox models. OS was compared using the log-rank test. Analyses were performed using Stata version 17 (StataCorp, College Station, TX), and all statistical tests were two-sided with a type 1 error of <0.05 indicating statistical significance. All estimates were reported with 95% confidence intervals (95% CI).

Among the 308 patients with WM, 87 had flow performed at diagnosis and after excluding patients who did not meet the eligibility criteria, 69 cases remained that were included in the final analysis (see Consort diagram, Figure S1 ). Among these 42 (61%) had detectable CL at diagnosis. The median age at diagnosis for all included patients was 63 years. There was male predomiance and most of the patients were white and had good performance status (ECOG performance status of 0-1). There were no statistically significant differences in IgM level, presence of B symptoms, skeletal or bone marrow involvement, MYD88 or CXCR4 mutations, or IPSSWM prognostic scores between the CL+ and CL- groups. 64% of the patients received immunochemotherapy as first-line therapy relative to 22% who received rituximab monotherapy. This was not significantly different between the CL- and CL+ groups. The most commonly administered immunochemotherapy was BR (bendamustine and rituximab) in the current study (n=32), which was numerically higher in the CL+ group (n=22, 52%) compared to CL- group (n=10, 37%). The median follow-up was 4.27 years. Table 1 shows the baseline characteristics of the patient population according to the presence or absence of CL.

There were no cases of positive hepatitis C viral antibody in our study. While there were no patients with active hepatitis B viral (HBV) infection based on the serological studies, there were 5 patients (2 in CL+ and 3 in CL-) with positive HBV core antibody but undetectable HBV DNA level (by PCR). All these patients were given antiviral prophylaxis (entecavir or tenofovir) during treatment and for at least 6 months following the last treatment and 12 months following the last rituximab infusion. None of the patients in our study had HBV reactivation.

The overall response rate (ORR) was 74% (n=51) with 42% (n=29) achieving a complete response or very good partial response and 32% (n=22) achieving a partial response. The ORR was numerically higher in CL+ group compared to CL- group (81% versus 61%, respectively), while the CR+VGPR rates were comparable between the two groups (40% versus 42%, respectively). The breakdown of the response rate between the CL- and CL+ groups is shown in Table S1 .

The median PFS was 6.3 years (95% CI=2.2 to not reached [NR]) in the CL- group compared to NR in the CL+ group (95% CI=3.8 to NR), which was not statistically significant (log-rank p=0.35; Figure 1 ). The 3- and 5-year PFS estimates in the CL+ group were 73% (95% CI=55-85%) and 62% (95% CI=43-76%) compared to 63% (95% CI=40-79%) and 56% (95% CI=33-74%) in the CL- group, respectively (log-rank p=0.35, Figure 1 ).

When evaluating the outcomes based on the type of first-line systemic therapy received, there was no significant difference in median PFS (in years) between CL+ and CL- groups among those who received rituximab monotherapy (NR [95% CI: 0.76 to NR] vs NR [95% CI=0.05 to NR]) (p=0.35, Figure S2A ) or rituximab plus combination chemotherapy (R-chemotherapy) (NR [95% CI=3.10 to NR] vs 6.33 [95% CI=2.16-8.01]) (p=0.20, Figure S2B ). In the univariate Cox model, factors associated with inferior PFS included age and IPSSWM prognostic score. After adjusting for these factors in a multivariable Cox model, presence of CL was not associated with significantly inferior PFS (HR=0.50, 95% CI=0.22-1.10, p=0.08, Table 2 ). Only IPSSWM (HR=4.42, 95% CI=1.35 – 14.42, p=0.01, Table 2 ) remained associated with significantly inferior PFS in the multivariable analysis.

Patients in the CL- group had a significantly longer median DTI (4.1 months; 95% CI=0.84-21.6) compared to the CL+ group (1.1 months; 95% CI=0.06-0.18) (p=0.048; Figure S3 ). The percentage of patients who started treatment at 1 year in the CL+ versus CL- groups was 83% vs 63%, respectively. CL was associated with a shorter DTI in the univariate analysis, however, after adjusting for other factors in the multivariable analysis this association was no longer significant ( Table S2 ).

There was no significant difference in median OS between the CL- group (13.0 years; 95% CI=2.4 to NR) and CL+ group (NR; 95% CI: 6.4 to NR), (p =0.15; Figure 2 ). The 3- and 5-year OS estimates in the CL+ group were 94% (95% CI=79-99%) and 86% (95% CI=67-95%) compared to 71% (95% CI=48-85%) and 65% (95% CI=41-81%) in the CL- group, respectively (log-rank p=0.15, Figure 2 ).