Interfollicular CD10 Expression and Follicular PD1 Tumor-Infiltrating Lymphocytes As Biologic Risk Factors in Patients with Previously Untreated Follicular Lymphoma Receiving Rituximab-Based Biologic Therapy: An Alliance Correlative Science Study (CALGB 50901, 50402, 50701, 50803, 50401)

Follicular lymphoma (FL) is an indolent B-cell neoplasm of germinal center origin. �Standard treatment regimens consist of anti-CD20 therapy with or without chemotherapy and while high response rates are common, relapses and progression ultimately occur. �Alliance (formerly CALGB) has been testing rituximab-based biologic therapies in a series of phase 2 trials for previously untreated (CALGB 50901, 50402, 50701, 50803) or recurrent (CALGB 50401) FL. �Clinical risk factors such as the FL international prognostic index (FLIPI) have been identified but there is need for prognostic and predictive biomarkers. �In this exploratory analysis, we studied a series of immunohistochemical (IHC) markers of lymphoma cells (CD10, BCL2, BCL6, MUM1, Ki67) and the microenvironment (CD68, cytotoxic T cells, Tregs, Tfh) using tissue microarrays (TMAs) and correlated them with study endpoints to define markers of interest for future phase 3 trials.

FL diagnosis and grade were histologically confirmed by central review. �Grade 3b cases were excluded. �TMAs were constructed from 1mm diameter formalin-fixed paraffin-embedded tissue cores. �IHC stains were performed using standard laboratory methods. �The following stains were scored as follows by 3 hematopathologists via consensus review: Blimp1 - >5% positive; follicular (F)-BCL2 - >20% positive; F and interfollicular (IF)-CD10, F and IF-BCL6 - positive vs negative; MUM1, FoxP1, F and IF-PD1, granzyme B - % cells positive; FoxP3 - F, perifollicular or diffuse staining pattern; and CD68+ macrophages - # positive cells/high power field (hpf). �Ki67 proliferation index (% positive cells) was evaluated using image analysis software (Definiens, Carlsbad, CA). �Results were correlated with response to therapy and progression-free survival (PFS). �Associations with response were measured using chi-square analysis. �PFS was estimated using the Kaplan-Meier method and differences in PFS were compared using the log-rank test.

Patients in the 4 trials of untreated FL (N=241) were analyzed in aggregate for the purposes of biomarker analysis, and included 124 men and 117 women (median age 56 yrs). �All had stage III, IV, or bulky stage II (single mass ≥7cm in any dimension) disease.� By FLIPI, 58 (25%) had low-risk, 134 (57%) had intermediate-risk and 44 (19%) had high-risk disease. �CALGB 50901 patients were slightly older with a greater proportion of intermediate-risk disease.� CALGB 50803 patients had significantly better response and PFS.

Among patients with available tissue, IF-CD10 positivity was seen in 79/127 (62%) and was associated with shorter PFS compared to IF-CD10-negative cases (2 yr PFS: 59% vs 80%, p=0.0063). �High F-PD1 expression (>33%) was seen in 14/121 (12%) and was associated with longer PFS compared to cases with ≤33% staining (2 yr PFS: 92% vs 65%, p=0.026). �There was marginal association with response for CD68+ macrophages using the median of 31 cells/hpf as a cut-off: 58/65 patients (89%) with >31 cells/hpf showed a response vs 49/64 (77%) with ≤31 cells/hpf, p=0.056. �The remaining IHC parameters did not show a significant correlation with response or PFS. �In patients with recurrent FL (CALGB 50401), there was no association with IF-CD10 or F-PD1 staining suggesting their lack of relevance in the relapse setting, but only few high F-PD1 cases were observed. �Using a biologic risk score of 1 point each for IF-CD10 positivity or F-PD1 ≤33%, untreated patients with a score of 2 had a worse PFS (median 2.9 yrs) vs those with a score of 1 (median 4.8 yrs) or 0 (median not reached), p=0.0045 (Fig 1). �In multivariable Cox proportional hazards model analysis, each biologic risk factor was independent of the FLIPI risk group.� Likewise, the biologic risk score was also independent of FLIPI: p=0.021, HR 0.39 (95% CI: 0.20, 0.76) for score 1 vs 2; and p=0.0005, HR 5.3 (95% CI: 1.76, 16.01) for FLIPI high vs low and HR 1.7 (95% CI: 0.59, 4.92) for FLIPI intermediate vs low, respectively.

IF-CD10 positivity appears to be a poor prognostic biomarker in FL in the untreated but not relapse setting, while high F-PD1 expression appears to be a favorable prognostic feature in untreated FL patients treated with biologic agents. These are promising biomarkers for risk stratification in FL that warrant further validation in future trials.

Support: NCI grants U10CA180821, U10CA180882

Fig 1: PFS in Untreated FL Patients Using a Biologic Risk Score Combining IF-CD10 and F-PD1 Expression