ALBERT

Description: Abstract 750 The non-malignant immune infiltrate comprises the bulk of pathologic tissue in classical Hodgkin lymphoma (CHL). This microenvironment has the potential to induce both malignant cell suppression and support. Increased macrophage infiltration assessed by CD68 expression has been shown to confer adverse prognostic significance (Steidl et al. N Engl J Med, 2010; 362:875-85) while increased FOXP3 expressing T cells are beneficial in this disease (Tzankov et al. Haematologica, 2008; 93: 193–200). However no histological score routinely leads to modification of treatment. Assessing outcomes by the parameters of overall survival (OS), disease specific survival (DSS) and freedom from first line treatment failure (FFTF) at 5 years in a cohort of patients treated at St Bartholomew's Hospital (Barts), London we developed a prognostic score based upon expression of both FOXP3 and CD68 in the CHL microenvironment which defined poor and good risk groups in both early (Stages I and IIA) and advanced stage disease, including a 'poor risk' early stage group with 25% FFTF and a ‘good risk' advanced stage group with 90% FFTF. Immunohistochemical analysis was performed on tissue microarrays (TMAs) from previously untreated patients' diagnostic lymph node biopsies in whom clinical outcome was available. From all 1056 adult patients with HL diagnosed at Barts between 1972 and 2005, high quality formalin-fixed paraffin-embedded tissue was available for 122 (12%), with characteristics representative of the whole group. Median age of the 122 patients was 30 (range 18–80) years, 35% female, 71% advanced stage with median follow up 16.5 (range 2–40) years. Triplicate cores were made from areas of high cellularity, containing malignant cells and avoiding fibrotic, acellular portions, arrayed onto glass slides and stained immunohistochemically for FOXP3 or CD68. Absolute and proportional numbers of FOXP3+ nuclei and CD68+ cell bodies were counted across all intact cores using an automated image analysis system (Ariol), confirmed by expert histopathologists, and means calculated and corrected to a single high powered field (hpf). Recursive partitioning was used to generate optimal cutoff values discriminating prognostic groups and comparisons performed by the chi-square test. Using cutoffs of 15% to define low, intermediate and high CD68 density, 3 prognostic groups were defined, the favourable group having the lowest CD68+ density. OS for low, intermediate and high groups were 89%, 80% and 65% respectively (p=0.02), with FFTF 82%, 64% and 29% (p=0.001). Prognostic significance was maintained in subgroups presenting with advanced stage (FFTF 73%, 63% and 33%, p=0.03), as well as early stage disease (FFTF 92%, 70% and 20%, p=0.01). Using cutoffs of 50 nuclei/hpf to define low, intermediate and high FOXP3+ cell density, 3 prognostic groups were defined, the favourable group having the highest FOXP3+ density. OS for low, intermediate and high groups were 68%, 80% and 94% respectively (p=0.006), with FFTF 50%, 62%, and 84% (p=0.002). Prognostic significance was maintained for both advanced (FFTF: 48%, 60% and 72%, p=0.04) and early stage disease (FFTF: 57%, 67% and 100%, p=0.04). A combined ‘FOXP3/CD68 score' derived from the patient's prognostic group for both markers, for which suitable cores were available on 98 patients, further improved the predictive value of each individually (See Figure). In this model, favourable, intermediate and unfavourable groups had 5 year FFTF of 93%, 62% and 47% (p=0.0002), DSS 93%, 82% and 63% (p=0.03) and OS 93%, 82% and 59% (p=0.002). The score retained prognostic significance for 5 year FFTF and OS in the subgroup of patients presenting with advanced (FFTF: 90%, 59% and 46%, p=0.008; OS: 90%, 80% and 54%, p=0.004) as well as early stage disease (FFTF: 100%, 71% and 25%, p=0.005; OS: 100%, 82% and 75%, trend only, p=ns). We conclude that FOXP3 and CD68 are important independent factors, which in combination have considerable predictive power and will now be validated prospectively. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 951 Gene expression profiling studies in Diffuse Large B cell Lymphoma (DLBCL) have described signatures enriched in genes representing the immune microenvironment. These signatures, together with the cell of origin classification have prognostic significance. However, a comprehensive functional understanding of the role of inflammation and the immune response in DLBCL is still lacking. The use of immunohistochemistry (IHC) to validate and elaborate upon molecular results is appealing. Our objective was to assess the cellular composition of the non-malignant population in DLBCL quantitatively and thoroughly in a single centre TMA of patients with DLBCL with corresponding clinical and survival data. Based on reported findings and known functional interactions a panel of antibodies were selected that included CD68, FOXP3 and TIA-1. IHC was performed on TMAs from high quality formalin-fixed paraffin-embedded diagnostic tissue biopsies of DLBCL from 1977 to 2009. Triplicate cores were made from areas of representative tumour tissue, arrayed onto glass slides and stained for a panel of antibodies, aiming to characterize immune cell subsets. The TMA included tissue from 218 patients: 128 males, 90 females, with a median age of 55 (18-94) years; 23.2% high-int/high risk IPI; with median follow up of 3,1 years. 33% of patients were treated in the rituximab era. Absolute numbers of positive cells were counted across all intact cores using an automated image analysis system (Ariol), confirmed by histopathologists, and means calculated and corrected to a 1 mm2 area. A training-validation set method was used to generate optimal cutoff values discriminating prognostic groups and comparisons performed using the chi-square test. Among nine antibodies examined, statistically significant prognostic information was provided using staining for FOXP3, TIA-1 and CD68. Using single cut-offs of positive cells/mm2 to define low and high marker density, two prognostic groups were defined for each marker. Patients with a more favourable prognosis had the highest cell density for FOXP3, TIA-1 and CD68. To clarify whether there was an additive prognostic relationship we developed a score that incorporated these three markers. This scoring system allowed us to discriminate two subgroups of patients with divergent survival parameters (fig 1).Marker (cutoff value high vs low)Overall Survival at 3yr (high vs low)Progression Free Survival at 3yr (high vs low)Disease Specific Survival at 3yr (high vs low)FOXP3 (450+ cells/mm2)64% vs 44% (p 0.005)55% vs 36% (p 0.001)69% vs 54% (0.01)CD68 (900+ cells/mm2)58% vs 28% (p 0.023)50% vs 20% (p 0.05)66% vs 36% (p 0.005)TIA-1 (2500+ cells/mm2)76% vs 45% (p 0.002)66% vs 41% (p 0.01)80% vs 56% (p 0.005)Combined Score68% vs 39% (p 0.0002)60% vs 32% (p 0.0003)73% vs 49% (p 0.0008) A Cox regression model was developed, incorporating age, IPI, rituximab treatment and IHC score. The score results were validated as independent prognostic markers for OS, PFS and DSS, together with age, IPI (for OS and DSS) and rituximab (for PFS). We confirm that there is heterogeneity in immune cell infiltration at diagnosis in DLBCL, and we provide correlative prognostic data to suggest a corresponding biological relevance. Attributing single cut-off values for immune cell markers is feasible, attractive as a clinical tool, and appears to provide robust prognostic information. In our single centre cohort we found that higher numbers of macrophages, Tregs and cytotoxic T cells correlate independently with improved patient survival. CD68 has been associated with an adverse prognosis in lymphoid malignancies but phenotypic diversity of macrophages may account for this study's finding and will be explored using macrophage subset specific markers. Our multivariate analysis revealed that the IHC results have an independent impact on survival on this cohort of DLBCL patients. We are currently validating this approach in an independent TMA of R-CHOP treated patients. Combining these three different immune cell parameters allowed us to discriminate prognostic subgroups providing further evidence of the impact of the non-malignant immune cells in the biology of DLBCL and validating their role as potential therapeutic targets for intervention. Disclosures: Gribben: Gilead: Honoraria; Mundipharma: Honoraria; GSK: Honoraria; Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria.