ALBERT

Description: Identifying mechanisms of tumor-induced immune suppression will aid the development of effective immunotherapeutic strategies. In the present study we examined the molecular basis for impaired T cell responses in follicular lymphoma (FL) and demonstrate impaired T cell immunological synapse formation. Confocal microscopy was used to visualize F-actin polymerization at the immune synapse between tumor-infiltrating CD4 and CD8 T cells and autologous FL tumor cells with and without superantigen as antigen-presenting cells (APCs). We identified a significant reduction in formation of the T cell immune synapse in both CD4 (p

Description: Micro-vessel density (MVD) is a powerful prognostic factor in cancers but its value in haematological malignancies is more controversial. To examine its prognostic value in follicular lymphoma (FL) we assessed number and distribution of blood vessels by high-throughput tissue microarray and automated image analysis measurements in tissue microarrays (TMA) of paraffin-embedded, diagnostic lymph node biopsies taken from fifty-nine FL patients. These patients were selected from those who died from lymphoma progression less than 5 years after diagnosis (short survivor group) (n=34) and those who survived more than 15 years from diagnosis (long survivor group) (n=25). Immunohistochemistry was used in TMAs to study the number and location of vessels staining positive for the endothelial cell markers CD31 and CD34 and pericyte coverage using PDGFR. Interactive quantification using image analysis software was used to provide details of absolute numbers of vessels from each patient, as well as vessel size and their location. Image stacks of immunofluorescence stained sections were obtained using laser-scanning confocal microscopy to trace the tumour vasculature. Results demonstrated that both total vessel count and mean vessel area were significantly different between the two groups. Samples from the long survivor group were significantly more likely to have fewer (p=0.025), but larger vessels (p=0.002) than those from the short surviving group. The differences in vessel size and number were more prominent in inter-follicular vessels compared with those inside the neoplastic follicles. The smaller and more numerous blood vessels seen in the poorer prognostic sub-group likely reflects active, sprouting angiogenesis as confirmed by confocal microscopy. This study validates the use of TMAs to examine angiogenesis and demonstrates the powerful prognostic value of assessing MVD in FL. These results suggest that sprouting angiogenesis represents a therapeutic target in this disease and ongoing studies are investigating the mechanisms contributing to alteration in angiogenesis in different prognostic subgroups in FL and in transformation of this disease. The prognostic significance of MVD assessment in TMA is currently being evaluated in a validation set of FL samples.

Description: Key Points Trisomy 12 CLL cells exhibit upregulated integrin signaling and enhanced VLA-4-directed adhesion and motility. The increased expression of β2-integrins on trisomy 12 CLL cells is modulated by intercurrent NOTCH1 mutations.

Description: Follicular lymphoma has considerable clinical heterogeneity, and there is a need for easily quantifiable prognostic biomarkers. Microvessel density has been shown to be a useful prognostic factor based on numerical assessment of vessel numbers within histologic sections in some studies, but assessment of tumor neovascularization through angiogenic sprouting may be more relevant. We therefore examined the smallest vessels, single-staining structures measuring less than 30 μm2 in area, seen within histologic sections, and confirmed that they were neovascular angiogenic sprouts using extended focal imaging. Tissue microarrays composing diagnostic biopsies from patients at the extremes of survival of follicular lymphoma were analyzed with respect to numbers of these sprouts. This analysis revealed higher angiogenic activity in the poor prognostic group and demonstrated an association between increased sprouting and elevated numbers of infiltrating CD163+ macrophages within the immediate microenvironment surrounding the neovascular sprout.

Description: Transformation of follicular lymphoma (FL) to a more aggressive clinical and histological phenotype, typically diffuse large B-cell lymphoma (DLBCL), occurs frequently. It is associated with a number of recurrent genomic insults, including the acquisition of TP53 mutations in a subset of patients (pts). The use of novel agents targeting p53 and mdm2 appears attractive given the resistance of transformed DLBCL to conventional therapies. Tailoring these therapies will require precise characterisation of mutation status and functional consequence in transformation. The frequency and temporal relationship of TP53 mutation gain to transformation was analysed in DNA from sequentially collated lymph node biopsies taken pre and post transformation (n=91) obtained from 29 pts. A median of 3 samples (range 2–5) was available from each pt (13 taken at FL presentation). Transformation was documented a median of 3.8 years (range 0.2 to 15.2) from diagnosis, and median follow up from diagnosis for all pts at the time of analysis was 6.7 years (range 2 to 19.1). The entire coding sequence of TP53 was screened by PCR, fluorescent-SSCP and sequencing. Loss of heterozygosity (LOH) was examined at 5 common polymorphic sites with in TP53. Immunocytochemistry for p53, mdm2 and p21 was performed on slides obtained from 77 available paraffin blocks. Ten mutations were detected in 8 pts (28%), of which 5 were missense. The remaining was accounted for by two nonsense mutations, a splice mutation, a branch site mutation and a single base insertion. All mutations were within the genomic region covered by primer sets exon 5–7 inclusive. Mutated TP53 was first documented only at the time of histologic transformation in 4 pts, in the remainder latency between documentation in FL sample and transformation was variable (0.5–6 years). For pts with mutations, time from documentation to death ranged from 1 month-12 years (median 37 months), with 2 pts alive 8.5 and 13.5 years following initial documentation. LOH occurred in 2 pts, both at the time of transformation and was associated with short survival (1 and 17 months). Overall survival from diagnosis or histological transformation was not significantly different between pts with mutated TP53 and wtTP53. Five TP53 mutated pts. recurred post transformation (either with FL or DLBCL); in 4 pts the identical mutation was detected at this time. p53 staining was positive in 82% (9/11) of biopsies with missense mutations, and negative in 71% (45/63) with wtTP53. Mdm2 expression was predominantly centroblastic in FL and was correspondingly higher in DLBCL samples (mean 72%; 95% CI 68–76%) compared to FL (mean 58%; 95% confidence interval: 54–62%) (p

Description: Cancer immune evasion is an emerging hallmark of disease progression. We have demonstrated previously that impaired actin polymerization at the T-cell immunologic synapse is a global immune dysfunction in chronic lymphocytic leukemia (CLL). Direct contact with tumor cells induces defective actin polarization at the synapse in previously healthy T cells, but the molecules mediating this dysfunction were not known. In the present study, we show via functional screening assays that CD200, CD270, CD274, and CD276 are coopted by CLL cells to induce impaired actin synapse formation in both allogeneic and autologous T cells. We also show that inhibitory ligand–induced impairment of T-cell actin dynamics is a common immunosuppressive strategy used by both hematologic (including lymphoma) and solid carcinoma cells. This immunosuppressive signaling targets T-cell Rho-GTPase activation. Of clinical relevance, the immunomodulatory drug lenalidomide prevented the induction of these defects by down-regulating tumor cell–inhibitory molecule expression. These results using human CLL as a model cancer establish a novel evasion mechanism whereby malignant cells exploit multiple inhibitory ligand signaling to down-regulate small GTPases and lytic synapse function in global T-cell populations. These findings should contribute to the design of immunotherapeutic strategies to reverse T-cell tolerance in cancer.

Description: Abstract 141 Patients with follicular lymphoma (FL) have an extremely variable clinical course. Although clinical parameters can be used to define prognostic subgroups, there is a need to identify robust biomarkers that not only aid in prognosis, but also help define the underlying pathophysiology of the disease. Previous gene expression profiling studies demonstrated that among the most important prognostic markers were the molecular features of nonmalignant tumor-infiltrating immune cells present in the tumor at diagnosis (Sandeep et al. NEJM 2004). To investigate the molecular mechanisms whereby T cells are altered in the FL microenvironment we studied highly purified, sorted infiltrating CD4 and CD8 T cells from previously cryopreserved single cell suspensions of lymph node (LN) biopsies at the time of diagnosis in patients with FL (n=12) and compared them to those isolated from reactive tonsils (n=7) as well as from peripheral blood (PB) (n=10) of age matched healthy individuals. These tumor infiltrating T cells (TILs) have impaired proliferative and cytotoxic responses and impaired capacity to form immunologic synapses with antigen presenting cells. Co-culture of FL cells in contact with healthy allogeneic T cells induces similar T cell functional defects, demonstrating that it is the FL cells that drive the altered T cell function. To investigate the molecular mechanisms for this, we performed global gene expression profiling using Affymetrix U133Plus2 chips of highly purified (〉95% purity) CD4 and CD8 cells. For both CD4 and CD8 cells, unsupervised analyses distinguished healthy and FL T cell subsets. Using a fold change cut off 〉 2 and false discovery rate of 5%, 280 genes were differentially expressed for CD4 and 717 genes for CD8, with109 genes overlapped for both subsets. The gene array data was validated for RNA using Real-Time Quantitative-PCR and for protein by flow cytometry and immunohistochemistry. Pathway analysis indicated disruption in multiple pathways including cytokine signaling, T cell differentiation, cell proliferation and, actin-based motility/cytoskeleton formation. In both CD4 and CD8, among the most downregulated genes in FL TILs were ACTN1 and IL17A, and the most upregulated genes were PMCH and ETV1. Using Tissue Microarray (TMA) we demonstrate that the intensity of expression in TILs in FL was significantly higher for PMCH (p

Description: Abstract 1654 Background: Mantle cell lymphoma (MCL) is an incurable, aggressive subtype of non-Hodgkin lymphoma in which there is a need for novel targeted therapies. Activation of the PI3K-Akt pathway and its role in the pathogenesis of MCL has been highlighted in a number of studies. Constitutive activation of the PI3K pathway inactivates GSK-3β, a downstream target of Akt, that can phosphorylate cyclin D1 resulting in its nuclear export. There is also evidence that cyclin D1 mRNA stability and translation is enhanced by this pathway. The class Ia PI3K p110 catalytic subunit isoforms α, β and δ are primarily implicated in oncogenesis. While the PI3K p110δ isoform is known to be enriched in lymphocytes, a gain of PIK3CA (the gene encoding PI3K p110α) copy number has been shown to be a frequent alteration in MCL. The expression and relative importance of the individual Class Ia PI3K isoforms has not been documented in this disease. With the development of isoform selective inhibitors, this is an important issue that needs to be addressed. Aims: We studied the expression of class Ia PI3K isoforms in primary MCL with relation to morphological variants and disease status. We also compared the efficacy of PI3K inhibition in MCL cell lines and primary samples using two novel inhibitors, GDC-0941(predominantly p110α/δ-selective) and CAL-101 (δ-selective), both of which are in early phase clinical trials. Methods: Tissue microarrays were constructed from triplicate 1mm cores from 144 MCL biopsies and 16 tonsil controls. The levels of p110α, p110β and p110δ isoforms were then determined by immunohistochemistry using isoform-specific antibodies. The in vitro effect of PI3K inhibitors on cell viability and apoptosis was studied in 4 MCL cell lines, (Jeko-1, Granta519, REC-1 and JVM-2), and 15 primary MCL samples. Expression of the class Ia PI3K isoforms and changes in downstream targets of PI3K were determined by western blotting. Results: P110δ was expressed at a consistently higher level in MCL samples and normal tonsil controls compared to the α and β isoforms, while p110β expression was weak and significantly lower than p110α expression. On comparing expression of isoforms at diagnosis and relapse, p110α expression was significantly increased beyond 1st relapse compared to diagnostic biopsies (p=0.04) and tonsil controls (p=0.02), an observation that was even more apparent in 6 paired samples [p=0.008, median IHC score 19.6 (5.0−53.2) at diagnosis vs. 91.5 (38.6 − 129) beyond 1st relapse]. No significant change was found in the expression of p110β or p110δ between diagnostic and relapse samples. There was no significant difference in expression levels of the 3 isoforms between blastoid and non-blastoid morphological variants. Expression of both the p110α and δ isoforms was detected by western blotting in 4 MCL cell lines, but only Jeko-1 cells were sensitive to inhibition with GDC-0941. CAL-101 produced little or no apoptosis in all 4 cell lines. In primary MCL samples, GDC-0941 was consistently more potent than CAL-101, with decrease in cell viability of 32 vs. 20% at 1μM (p=0.15), 51 vs. 25% at 5μM (p=0.02) and 67 vs. 35% at 10μM (p

Description: Follicular lymphoma (FL) is a clinically heterogeneous disease requiring the need for easily quantifiable prognostic biomarkers. Micro-vessel density has been shown to have prognostic significance in some, but not all studies. Previous analyses has been based on simple numerical assessment of vessels within histological sections, providing a relatively thin (3–5μm) ‘snap-shot’ of what is a three-dimensional, branching network. This inherent limitation, coupled with methodological variation in its assessment, has lead to conflicting results and uncertainty of its prognostic value in many malignancies including follicular lymphoma. To determine if assessment of true tumor neovascularisation through angiogenic sprouting may be of more clinical relevance, we performed immunostaining with two routinely used endothelial cell markers (CD31 and CD34) in an established FL tissue microarray (TMA). After initial analysis, we focused attention on the vessels at the smallest end of the spectrum seen within routine thickness sections. These represent small, single staining structures no greater than 30 μm2 in area. We subsequently used extended focal imaging within thicker sections to trace these vascular structures and confirmed them to be blind-ending angiogenic sprouts. Diagnostic biopsies taken from patients at the extremes of survival of FL were analysed with respect to numbers of these sprouts, and revealed higher angiogenic activity in patients who died from lymphoma progression less than 5 years after diagnosis compared with those surviving greater than 15 years (p=0.025). This effect was only seen with CD31 and not CD34. Image overlay analysis of serial sections demonstrated that lymphatic vessels highlighted with LYVE-1, a specific lymphatic endothelial marker were positive with CD31 and negative for CD34. However, no differences between number or extent of sprouting of lymphatic vessels were seen in the two prognostic groups; therefore revealing true vascular angiogenic sprouting seen with CD31 analysis, and demonstrating that these vascular angiogenic sprouts express CD31, but less frequently express CD34. We further characterised these angiogenic sprouts using double-labelling immunofluorescence to assess pericyte coverage. Results indicated there was largely no pericyte coverage of these vascular structures, suggesting that these vessels may be targeted using anti-angiogenic therapy and not protected by pericytes. The increased angiogenic activity seen in the poorer prognostic subgroup was seen only in the inter-follicular regions and not in the neoplastic follicles. It is therefore unlikely that the increased vascularisation is a direct result of tumour cell- driven angiogenesis as a closer spatial relationship between tumour cells and vessels would be expected. Previous studies have highlighted that increased lymphoma-associated macrophages are associated with adverse outcome; their role in promoting angiogenesis has been well studied. We therefore used automated image analysis to assess numbers of CD163+, an M2 type macrophage marker identifying a subset of lymphoma-associated macrophages. Although there was no difference in absolute number of macrophages seen between the two groups, there was a positive correlation between number of these cells and extent of angiogenic sprouting. Last, we assessed the impact of angiogenic sprouting and time to transformation and identified a trend towards increased angiogenic activity in those patients who transformed within three years of diagnosis. In summary, we have used an improved gauge of angiogenic activity by quantifying angiogenic sprouts in TMA and in routine histological sections, and highlighted the impact of angiogenesis on survival and time to transformation in patients with FL. Further investigation into the mechanisms driving increased angiogenesis and its subsequent impact on survival is currently being undertaken in a validation series using a TMA of 450 patients with FL at our institution.

Description: Abstract 769 Previous global gene expression profiling (GEP) studies have demonstrated non-malignant tumor-infiltrating immune cells present in the tumor at diagnosis were among the molecular features of the most important prognostic markers (Sandeep et al. NEJM 2004). We investigated the molecular mechanisms whereby tumor infiltrating T cells (TILs) are altered in the FL microenvironment and examined GEP of highly purified, sorted infiltrating CD4 and CD8 T cells from previously cryopreserved single cell suspensions of lymph node (LN) biopsies at the time of diagnosis in treatment naive patients with FL (n=12) and compared them to those isolated from reactive tonsils (n=7) as well as peripheral blood (PB) (n=10) of age matched healthy donors. In both CD4 and CD8, among the most downregulated genes in FL TILs were ACTN1 and IL17A, and the most upregulated genes were PMCH, ETV1, TNFRSF9 and NAMPT. PMCH is not expressed in PB T cells, but its expression is highly induced when healthy PB T cells are cultured, either in cell contact or in transwell culture, with FL cells. Using well characterized tissue microarrays of diagnostic FL samples (n=172) we now show that the T-cell expressed genes of PMCH, ETV1 and NAMPT have prognostic significance for survival and time to transformation in FL. Patients with higher percentage (p=0.039, median survival 10.59yr vs 19.20yr) and number (p=0.016, median survival 8.67yr vs 19.01yr) of PMCH expressing cells in intrafollicular and higher percentage (p=0.021, median survival 8.18yr vs 16.5yr) in interfollicular area had significantly longer disease specific survival (DSS) compared with patient with low number of PMCH expressing cells. Patients with higher percentage (p=0.014, median survival 19.01yr vs 11.75) and number (p=0.032, median survival 19.01yr vs 12.41yr) of ETV1 expressing cells in intrafollicular area showed significantly shorter DSS comparing to those with lower percentage and number of ETV1 expressing cells. Furthermore, the higher total MI of NAMPT expression showed significantly longer DSS (p=0.003, median survival 7.62yr vs 18.28yr) as well. Pathway analysis indicated disruption in multiple cellular pathways including actin-based motility/cytoskeleton formation which is in keeping with our previous studies where we have shown altered T cell expression of genes regulating actin in CLL (Gorgun et al, JCI 2005) and AML (De Lieu et al, Blood 2009). Using Time-Lapse Imaging we demonstrated both CD4 and CD8 TILs from patients with FL (n=7) have significantly impaired motility compared to those of healthy TILs from reactive tonsil (n=4) (p