High Ki67 Index Is Associated with Shorter Progression Free Survival in Follicular Lymphoma Patients Treated with Frontline Immunochemotherapy
Authors: Han Jianmin , McCall Chad M , Isom Scott , Smith Wesley , Jenneman Dakota , Begley Stephanie , Bose Rupali , Seegars Mary Beth , Hsi Eric D. , Ghosh Nilanjan 
Published: December 2023

Introduction
The Follicular Lymphoma International Prognostic Index (FLIPI) is a commonly used prognostic tool in follicular lymphoma (FL). However, there is a need for better risk stratification to predict risk of progression after frontline chemoimmunotherapy. Ki67 proliferation index has prognostic value in many lymphomas and is widely available in clinical laboratories. However, the impact of Ki67 on outcomes in FL have not been conclusive. Studies have been limited by the method of quantifying Ki67 (entire specimen or intrafollicular [IF]), heterogeneous treatments, or small single institution studies. Moreover, the impact of Ki67 on predicting disease progression at 24 months (POD24) is unknown. We sought to study the predictive value of Ki67 on PFS in FL treated with Bendamustine Rituximab (BR) or RCHOP with or without Rituximab maintenance (RM) as first line therapy at 2 tertiary care centers.
Methods
This study was conducted with approval by the Wake Forest University Health Sciences IRB. We retrospectively analyzed 122 patients with FL (grade 1-3A) from 2010 to 2021 at Atrium Health (AH) Levine Cancer Institute (n=72) and AH Wake Forest Baptist Medical Center (n=50) who underwent first line therapy with BR or RCHOP and had tissue/slides available for analysis. Clinical data was collected through electronic medical record review.
Archival slides and paraffin-embedded blocks were retrieved. Ki67 immunohistochemistry (IHC) (SP6, Cell Marque, Rocklin, CA), was performed if it was not available from the original work-up (8 cases). CD21 IHC (1F8, Dako, Denmark) was performed in select cases to confirm the presence of follicles in order to assess IF Ki67. Diagnosis, grade, and Ki67 stains were evaluated by 2 hematopathologists. The IF Ki67 index was determined by visual estimate to the nearest 10%. All cases with scores between 20-40% were re-reviewed to achieve a consensus score. Ki67 controls were exchanged between the two centers to ensure equivalent staining.
Comparisons of patient characteristics were done by Kruskal-Wallis test for continuous variables (age) or Chi-square tests (categorical variables). Survival analysis was performed using Kaplan-Meier estimation and log-rank testing. Cox proportional hazards modeling was used for multivariable progression free survival (PFS) analysis.
Results
The clinical and pathologic characteristics are shown in Table 1. No differences in demographics [age, stage, cytologic grade, FLIPI, IF Ki67 status (&lt;30 or ≥30) and treatment selection] were seen between the two centers. 100 cases were grade 1-2 (82.0%) and 22 were grade 3A (18.0%). Consistent with expected treatment patterns in FL, more patients were treated with BR (70.5%) compared to RCHOP (29.5%) and RCHOP was more frequently used in grade 3A compared to grade 1-2 FL patients. As expected, low FLIPI score (0-1) was associated with favorable PFS (P=0.02, Figure 1A), but not OS. However, treatment regimen (BR vs RCHOP), use of RM, and cytologic grade (grade 1-2 vs 3A) were not associated with PFS or OS. The IF Ki67 index was elevated (≥30%) in 46 cases (38%) and high IF Ki67 was associated with shorter PFS (P=0.002, Figure 1B), but was not associated with OS (P=0.11). When stratified by treatment regimen and IF Ki67, these results were maintained ( Figure 1C). There was no association between FLIPI and IF Ki67 (P=0.10). In a multivariable regression analysis, IF Ki67 ≥30 was associated with a significantly higher risk of progression (HR= 2.3, 95% CI 1.3 to 4, P=0.005) after accounting for the effect of FLIPI. In an exploratory analysis, a FLIPI/Ki67 (FKi) score was devised in which one point was given for either FLIPI ≥ 2 or IF Ki67 ≥ 30% giving a possible score of 0, 1 or 2. The FKi score was associated with PFS (P=0.0005, Figure 1D). Moreover, IF Ki67 ≥ 30 was associated with a higher risk of POD 24 (P=0.037). FLIPI ≥2 was also associated with POD24 (P=0.005).
Conclusion
Intrafollicular Ki67 ≥ 30% predicts for increased risk of progression independent of FLIPI in patients with FL treated with frontline immunochemotherapy. Addition of Ki67 to FLIPI is strongly associated with PFS in FL. Ki67 stains were done over an 11 year period in different laboratories, demonstrating the wide applicability and robustness of the Ki67 IHC technique. Larger studies incorporating IF Ki67 in risk stratification models are warranted in FL.