ALBERT

Description: Introduction: The function of tumor-infiltrating T cells in lymphoma is unknown. Previous studies have shown that an increased percentage of tumor-infiltrating CD4+ T cells was associated with a better overall survival in diffuse large B-cell lymphoma (Ansell S. et al. JCO 2001; Xu et al., British Journal of Haematology 2001). Furthermore, the CD4+ T cells observed in one study was associated with a CD3+/HLA-DR + phenotype, suggesting that they were activated T helper cells. Recently, a molecular predictor model for follicular lymphoma was reported based on microarray analysis of gene expression within the cell populations of biopsy specimens. Three groups of “predictor genes” were identified: one representing B-cell differentiation and two groups representing genes expressed by T cells and macrophages, respectively (ASH abstract, 2003). This raised the question of whether it was simply the presence of tumor-infiltrating T cells that correlated with clinical outcome as opposed to specific activation pathways within those cells. Purpose: The purpose of this study was to examine whether the percentage of tumor-infiltrating T cells is correlated with prognosis of follicular lymphoma. Patients and Methods: We identified 293 follicular lymphoma patients with clinical outcomes available within the Stanford lymphoma database, who had undergone an excisional biopsy, and whose tumor biopsy specimens had been analyzed by flow cytometry. These patients were diagnosed between 1977 and 2003. Among these patients, 167 (57%) had follicular small cleaved lymphoma, 112 (38%) had follicular mixed lymphoma, and 14 (5%) had follicular large cell lymphoma. A total of 66 deaths were documented. The overall survival was estimated from the time of diagnosis to the time of death or last follow-up. Biopsies had been analyzed for cell surface marker expression by single parameter flow cytometry. The percentage of CD3+, CD4+, CD8+, CD20+, and HLA−DR+ cells was determined directly. We also estimated activated T cells, defined by HLA−DR+ T cells, by subtracting the value for total B cells from the value for HLA + cells. Cell population percentages were treated as continuous variables and related to overall survival, each in a univariate analysis. Results: We found no statistically significant correlation between the percentage of any cell population and overall survival. For each group (CD3+, CD4+, CD8+, and estimated HLA−DR+ T cells), we used the percentage of the cell population to divide patients into 3 subgroups of equal number of patients: low-, medium-, and high-percentage. We then compared Kaplan-Meier curves of the 3 subgroups for each cell population. We found no significant differences in survival among the 3 subgroups for each of the cell populations, CD3+, CD4+, CD8+, and the estimated activated T cells, after correcting for multiple hypotheses testing. Conclusion: The percentage of tumor-infiltrating T cells or T cell subsets was not correlated with survival outcome in patients with follicular lymphoma. This finding provides the basis for building molecular predictive models based on gene expression analysis, which represents not only the presence of certain cells but their physiologic state as well.

Description: Background: Low-grade lymphomas are considered incurable, but they are among the most immune-responsive of all human cancers. CpG belongs to a new class of immunomodulators, which activates B cells and plasmacytoid dendritic cells through Toll-like Receptor 9 (TLR 9). We have developed a novel immunotherapy approach that combines low-dose radiation with intra-tumoral injection of CpG for recurrent low-grade lymphomas. We hypothesized that CpG would stimulate tumor-antigen uptake and presentation by dendritic cells in vivo. Tumor cells induced to die by irradiation release tumor antigens and recruit dendritic cells. In this setting, CpG injected into the irradiated tumor stimulates dendritic cell uptake and presentation of tumor antigens. Having demonstrated the validity of this hypothesis in animal models, we developed a clinical trial based on this combination regimen for patients with recurrent low-grade lymphoma. Our primary objective was to test the safety of this combination CpG-radiation regimen. The secondary objective was to assess systemic anti-tumor effect by monitoring tumor regression at sites other than the irradiated/injected site. Patients and Methods: Patients with biopsy confirmed low-grade follicular B-cell lymphoma (FL) of any initial stage or mycosis fungoides (MF) with stage IB-IVA were eligible for the study. B-cell lymphoma patients failed at least one prior treatment. Mycosis fungoides patients failed or were intolerant of at least 2 topical or one systemic treatment. Patients had at least one site of disease accessible for intratumoral injection of CpG and had a Karnofsky Performance Status (KPS) of ≥ 70 with adequate organ and bone marrow functions. Patients received low-dose radiotherapy to a single tumor site on days 1 and 2 (2 Gy each day). PF-3512676 (CpG 7909) injections were administered at a dose of 6mg into the same tumor site within the 24 hours before and the 24 hours after the radiation, and on days 8 and 15. Weekly injections of PF-3512676 were then administered at the same dose subcutaneously in the region of previous injections for 6 additional doses. Results: Of seven patients treated to date (5 with FL and 2 with MF), 7/7 experienced grade 1 and 2 injection site reactions, including erythema, swelling and tenderness; 3/7 had grade 1 fevers after the injection; 4/7 experienced myalgia or arthralgia, 6/7 reported fatigue. All adverse reactions were transient. No hematological adverse reactions or SAEs occurred. One patient experienced transient swelling of the lymph nodes draining the CpG injection site. One of 4 FL and 1 of 2 MF patients who have completed treatment achieved a partial response with tumor regression observed at all un-irradiated sites and the remaining patients had stable disease. Conclusion: Intra-tumoral injection of PF-3512676 (CpG 7909), at a fixed dose of 6mg, combined with low-dose radiation (2Gy x 2) is a safe and well tolerated regimen in patients with recurrent low-grade lymphomas. In addition, an anti-tumor effect has been observed. The study is ongoing.

Description: Background: Follicular lymphoma (FL) has variable clinical outcomes. It has been suspected that tumor-infiltrating immune cells affect the biology and outcome of this disease. Using gene expression profiling and immunoperoxide tissue staining techniques, T cells and macrophages have been related to the survival outcome in some studies, but not others. In the current study, we used flow cytometry to analyze T cells and their subsets in follicular lymphoma biopsy specimens and determined whether these cell populations correlated with clinical features and outcomes. Methods: Two hundred and eighty-nine follicular lymphoma patients (pt) presented from 1997 to 2003 underwent an excisional lymph node biopsy prior to any treatment. The median age of pt at diagnosis was 45.7 yrs, median follow-up was 8.6 yrs for living pts, and median survival was 15.7 yrs. All but 8 patients had stage III/IV disease, 5 had stage I/II, and 3 were unknown. The histological grades were: 162 (56%) grade 1, 112 (39%) grade 2, 13 (4.5%) grade 3 and 2 (0.5%) unknown. Among the 289 patients, 41(17%) had low FLIPI score, 150 (63%) intermediate, 48 (20%) high and 50 unknown. All biopsies were analyzed for CD20, CD3, CD4, CD8 and HLA-DR expression by single-parameter flow cytometry. The 289 pts were divided into a training set of 147 and a validation set of 142, stratified by age and era of diagnosis. We used these two factors to stratify the pts because age at diagnosis is the most important prognostic factor for survival, and, in our data set, the era of diagnosis had an impact on survival and on the time from diagnosis to first treatment. For our analysis, we began with the training set and used the percentages of each immune cell population as a continuous variable in a univariate analysis in relation to clinical features and outcomes. We chose 8 phenotypic variables: CD20, CD3, CD4, CD8, HLA-DR, CD4/CD3 ratio, CD8/CD3 ratio, and activated T cells [defined as (HLA-DR-CD20)/CD3]. Five parameters were used as clinical endpoints: overall survival, FLIPI score at diagnosis, the time from diagnosis to first treatment (defined as the time from the first treatment to second treatment), response to CVP as the first treatment and the duration of the benefit from the first treatment (defined as the time interval between initiation of first treatment and initiation of second treatment). Results: The number of pt evaluable for each of the outcome parameters was as follows: 289 for time to first treatment and for overall survival, 239 for FLIPI scores, 164 for response to CVP and 129 for duration of the benefit from the first treatment., Of the 8 variables tested in the training set, only CD4/CD3 ratio and CD8/CD3 ratio were marginally significant for the survival endpoint, with p 0.034 and 0.088, respectively. None of the variables was significant for any of the other endpoints. A multivariate analysis yielded CD4/CD3 as the only significant predictor for survival. When CD4/CD3 was tested in the validation set, it yielded a p value of 0.48. Conclusion: We find no evidence that the percentage of tumor-infiltrating T cells or their subsets is predictive of clinical outcome in follicular lymphoma. Any gene expression signature involving T cells that does relate to clinical outcome could therefore be a property of the activity of the cells rather than a simple reflection of their numbers.