Long‐term follow‐up confirms the favourable prognostic impact of high numbers of tumour infiltrating CD3 T‐cells in follicular lymphoma patients treated by rituximab‐maintenance regimen

Sir, It remains debated as to whether the quantification of tumourinfiltrating T lymphocytes (TILs) could help stratify outcome of follicular lymphoma (FL) patients, as suggested by pioneer gene expression profiling studies.1 Among Tcell subsets of particular interest, PD1+ T follicular helper (TFH) cells, FOXP3+ T regulatory cells (Tregs), and CD8+ cytotoxic Tcells were shown to correlate with outcome.2– 5 However, some of these individual biomarkers have been studied before the introduction of rituximab and/or in FL patients with high clinical heterogeneity including earlyand advancedstage as well as untreated patients. Moreover, discrepant immunohistochemical (IHC) data have been presented for each marker6 (for review), so that the reliability of this approach has been questioned.7 In order to overcome the potential biases due to the heterogeneity of treatments that weakened the conclusion of previous studies, we had previously investigated TIL markers in a large series of FL samples from the PRIMA randomized trial, which represents, to our knowledge, the largest cohort of FL samples from rituximabtreated patients reported thus far.8 It has shown a better PFS in previously untreated patients in need of therapy who received 2 years of Rituximab (R) maintenance after immunochemotherapy induction vs observation.8 Pathological tumour material (FL grade 1, 2 or 3A) was processed as previously described.9 Briefly, immunostainings of CD3, CD4, CD8, PD1, ICOS and FOXP3 were automatically quantified in tissue samples of 417, 287, 418, 406, 379 and 369 patients respectively. The optimal IHC cutoff value had been calculated using the XTile software.10 The corresponding mRNA transcripts were quantified from frozen tissues of 148 patients using bulk RNAseq. We could previously show that high CD3 counts and, to a lesser extent, high PD1 counts were associated with better outcome after 5 years of followup.9 However, when a stringent statistical analysis was applied by dividing the whole cohort into a training and a validation set, none of the TIL markers showed significance in both subsets, suggesting that their prognostic value may be mitigated by the efficacy of Rituximab,9 a hypothesis also suggested by recent studies.11 Importantly, the prognostic impact of TILs with longterm followup has never been extensively studied. We report herein a 10 years followup (yFU) update of TILs prognostic correlations in the PRIMA trial. Survival curves were constructed with the Kaplan– Meier method and compared with the logrank test. The present results after 10 yFU show that the better progressionfree survival (PFS) associated with high CD3 counts at 5 yFU (p = 0.011) was still present and significant (p = 0.029) (Figure S1A). The increased rate of PFS events at 10 yFU was 3.2% (n = 5) and 7.9% (n = 21) in the subgroups of pts with low CD3 counts (n = 155) and with high CD3 counts (n = 262), respectively, suggesting that the progression of the disease may be delayed in pts with high CD3 counts. In contrast, the favourable prognostic value of high PD1 counts at 5 yFU (p = 0.044) was no more observed at 10 yFU (p = NS). The status of other TIL markers (CD4, CD8, ICOS, FOXP3), which were devoid of prognostic value at 5 yFU, remained unchanged at 10 yFU in the whole cohort. When considering only Rmaintenance arm pts (n = 177), the favourable influence of high CD3 counts which had been observed at 5 yFU (p = 0.023) was still present at 10 yFU (p = 0.030), but not in the observation/control arm (p = 0.31) (Figure 1A,B). Moreover, a better PFS associated with the Rmaintenance treatment was only visible in pts with high CD3 counts (n = 236; p = 0.0036) but not in pts with low CD3 counts (n = 140; p = 0.28) (Figure 1C,D). Similarly, the Rmaintenance treatment was associated with a better PFS for pts with high CD8 counts (n = 305; p = 0.0069) but not in pts with low CD8 (n = 75; p = 0.064) (Figure 2A,B). In contrast, pts with high FOXP3 counts (n = 169) had no benefit of Rmaintenance whereas pts with low FOXP3 counts (n = 164) had a better PFS when treated by Rmaintenance (p = 0.016) (Figure 2C,D). These results appear almost similar to our previous 5 yFU data. In fact, a better PFS in patients treated with Rmaintenance compared to the control arm had been also observed at 5 yFU in the subsets of pts with high CD3 (n = 236; p = 0.013), high CD8 (n = 305; p = 0.037) and low FOXP3 counts (n = 164; p = 0.037); whereas the 5 yFU PFS did not differ between Rmaintenance and control arm in pts with low CD3 (n = 140; p = 0.23) or high FOXP3 counts (n = 169; p = 0.112). One difference, however, between 5yFU and 10 yFU was that pts Received: 23 March 2023 | Accepted: 13 May 2023