ALBERT

Description: Background: Aurora kinases (A, B and C) play a critical role in regulating mitosis and cell division. Aurora kinase A is required for correct spindle assembly while aurora kinase B is involved in histone H3 phosphorylation, chromosome segregation, and cytokinesis. Emerging data suggest these proteins are implicated in the survival and proliferation of both haematologic and solid malignancies, and therefore represent novel therapeutic targets. Several aurora kinase inhibitors have been described, mainly inhibiting both A and B kinases, although inhibition of aurora kinase B is more closely associated with the effects seen with these compounds. We have therefore investigated the activity of AZD1152, a novel, specific inhibitor of aurora kinase activity, with selectivity for aurora B. Methods: Drug activity was studied in a panel of leukaemic cell lines (HL-60, MV411, THP-1, U937) and in primary AML cells (n=4) at concentrations of 0–1000 nM AZD1152 for up to 168 h. Cell number and percent viability were determined using a Viacount assay on a Guava PCA-96 analyser, cell cycle distribution (including apoptotic and polyploid (〉4n) cells) by PI staining with flow cytometry, and the phosphorylation of an aurora kinase B substrate histone H3 by fluorescence immunocytochemistry using a phospho-specific antibody. Results: AZD1152 displayed time- and concentration-dependent activity in cell lines and primary cells. A clear decrease in phospho histone H3 positive cells was apparent by 48 h, with 4n population increased to 80% by 96 h and persisted out to 168 h, with little apoptosis. In primary cells an increase in the G2/M population was evident in some cultures at 48 h, with only small changes in cells with 〉4n DNA content. In cell lines 10 nM AZD1152 resulted in a 28–65% reduction in cell number and a 6–35% reduction in cell viability after 48 h. At 100 nM these values increased to 65–78% and 6–42%, respectively. Notably little further increase in activity was observed at 1000 nM. By 96 h 100 nM AZD1152 reduced cell number by 〉80% in all cell lines, and cell viability by 〉67% in 3 lines (17% reduction in THP-1 cells). In primary AML cells AZD1152 over 48 h had little effect on cell number, but by 120 h this had decreased by 〉50% at 100 nM AZD1152, with a small effect on cell viability. Activity with 48 h exposure to drug followed by 72 h drug free was similar to that seen with a continuous 120 h exposure. Conclusions: The specific aurora kinase inhibitor AZD1152 demonstrates antiproliferative and apoptotic activity in leukaemic cell lines and primary cells at concentrations associated with changes in the phosphorylation of an aurora kinase B substrate protein.

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: Follicular lymphoma (FL) is a B-cell non-Hodgkin lymphoma that is characterized in approximately 85% of cases by the chromosomal translocation t(14;18) involving BCL2. While FL3b generally lack the t(14;18), this translocation is also absent in 15% of FL grades 1, 2 and 3a. The current study was designed to identify the frequency of t(14;18)-negative FL in a series of 166 cases of FL1, 2 and 3a in which global gene expression profiles had been established previously (Dave et al., NEJM351:2159–69, 2004). Furthermore, we sought to compare genetic alterations and gene expression profiles between FL with and without the t(14;18). Combined polymerase chain reaction (PCR) and tissue microarray-based fluorescence in situ hybridization (FISH) identified 17 t(14;18)-negative FL cases in this series (9%). Virtually all FL cases carrying the t(14;18) showed BCL2 expression by immunohistochemistry (Dako, clone 124), whereas 11 of the FL cases without a t(14;18) were BCL2-negative at the protein level. Clinically, there was no difference between the t(14;18)-negative and -positive FL subgroups regarding age and gender distribution as well as in median survival times. Comparative genomic hybridization (CGH) in the 166 FL cases revealed a characteristic pattern of chromosomal gains and losses, as previously described. However, significant differences were observed between the t(14;18)-negative and -positive FL subgroups. Specifically, the t(14;18)-positive FL subgroup showed gains of chromosomes 18q (18%), 8q (12%) and X (13%), as well as losses of 13q (16%) and 10q (16%), whereas none of these aberrations were observed in the t(14;18)-negative FL cases. To compare gene expression between the two groups, we used gene set enrichment analysis (GSEA), BRB array tools and a two-sided t-test. Cell cycle-associated genes were found to be enriched in the t(14;18)-negative FL subset. These differences were even more pronounced in FL cases that lacked both the t(14;18) and BCL2 expression at the protein level. Importantly, genes expressed in non-malignant bystander cells appeared also differentially enriched and a cytotoxic gene expression signature was found to be more prominent in t(14;18)-negative FL. These findings point to a different composition of the non-neoplastic cells in t(14;18)-positive and -negative FL and could indicate subtle differences in the immunological microenvironment of t(14;18)-negative FL.

Description: In an international retrospective clinical study of 1159 adult, non-cutaneous mature T-cell lymphomas, verified by an expert pathology panel, 62 cases of enteropathy-type T-cell lymphoma (ETTCL) were identified, representing 5.3%. Patients in this study were diagnosed at one of the participating centers in North America, Europe, and Asia between 1990 and 2002. ETTCL represented the 6th most common T-cell lymphoma, affecting predominantly the small bowel. Of interest, about a third of the cases of ETTCL were obtained from Norway, as the only participating center from Scandinavia. Twenty out of 52 cases (38%) were associated with celiac disease. As expected, abdominal pain was the most frequently recorded symptom, followed by general symptoms such as fatigue, weakness and anorexia. Overall as well as failure-free 5-year survival were poor, 20% and 4%, respectively. Univariate statistical analysis revealed that high T-IPI score (Gallamini, et al: Blood 103: 2474–2479, 2004) but not high IPI score, high CRP and LDH levels, and tumor size 〉5cm were adverse prognostic factors at p values 5cm were independent prognostic factors. In conclusion, ETTCL is a clinically aggressive and fatal disease for which, currently, no effective treatment exists. T-IPI score, CRP levels and tumor size can be used as prognostic factors.

Description: The survival of patients with Hodgkin lymphoma (HL) has improved over recent decades due to advances in therapy and supportive care. The outcome for patients with relapsed or refractory disease, however, is still unsatisfactory. High dose therapy with stem cell support can salvage many patients but results are poor in patients with chemo-resistant, PET-positive disease pre-transplant. Novel therapies for HL have focused on monoclonal antibodies to antigens such as CD30 but these treatments have produced response rates of only 10% when given as native antibody. Radio-immunotherapy is effective treatment for a range of lymphoproliferative disorders including those that are resistant to other therapies. This phase I study utilised basiliximab, a chimeric antibody to the α-chain of the IL-2 receptor, CD25, conjugated to iodine-131 (131I) in patients with relapsed or refractory lymphomas who were resistant to or intolerant of conventional therapies and who had demonstrable CD25 expression by immunohistochemistry on tissue sections. To determine dose-limiting toxicity an accelerated titration design was used with 6 different doses employed- 370, 740, 1480, 2220 and 2960MBq/m2. Fourteen patients (9M, 5F) with a median age of 38 years (range 28–70) were treated (HL 11 patients, primary mediastinal B-cell lymphoma 1, peripheral T-cell lymphoma NOS 1, adult T-cell leukaemia/lymphoma 1). They had previously received a median of 4 therapies (range 2–8) including autologous stem cell transplant in 9 patients. Five patients were being considered for an autologous or allogeneic stem cell transplantation but had not demonstrated chemosensitivity with standard therapies. All patients were FDG-positive by PET prior to therapy. One patient had a complete response at 740MBq/m2. Six of 9 patients responded to therapy at a dose of 1200MBq/m2 or higher. There were 3 complete responses and 3 partial responses. Two of these patients who had previously not been considered for high dose therapy because of a lack of response have now proceeded to autologous or allogeneic transplant. At a clinically active dose of 1200MBq/m2 the only side effects seen were delayed myelotoxicity; the median platelet count nadir was 31x109/L(range 9–83) and was observed at a median of 38 days (range 33–53) following treatment. Neutropenia (median nadir 1.31x109/L, range 0.9–7.5) was also noted at a median of 53 days (range 37–65) following therapy. One patient was treated at a dose of 2960MBq/m2 and developed prolonged grade 4 neutropenia and thrombocytopenia requiring stem cell support. The patient died of pneumocystis carinii pneumonia. There were no other grade 3 or 4 non-haematologic toxicities noted. The 131I-labelled anti-CD25 antibody basiliximab is well tolerated at doses of 1200MBq/m2 and demonstrates clinical activity at this dose in patients who are refractory to conventional therapies. Further studies are required to determine the long term outcome of patients treated with this agent and to assess its efficacy in the pre-autograft/allograft setting.

Description: Background: FLIPI has been proposed as an accurate, simple, and validated prognostic index on the basis of routinely performed tests (age 〉 60 yrs, Ann Arbor stage III-IV, serum LDH level increased, Hg 〈 12g/dL, and more than four nodal areas involved (Solal-Céligny et al., Blood2004; 104: 1258–1265). FLIPI has reliably predicted outcome for many follicular lymphoma (FL) patients (pts) treated with chemotherapy and rituximab. Currently, monoclonal antibody (Mab) therapy and a number of radioimmunoconjugates are considered important components of the treatment of FL; therefore, evaluating FLIPI in clinical trials of these modalities is of interest for optimizing therapy. Methods: A subset analysis was performed to assess the impact of FLIPI on the outcome of patients with relapsed/refractory FL enrolled in a larger, multi-center, open label, single-arm study of epratuzumab (humanized anti-CD22 Mab) in combination with rituximab (chimeric anti-CD20 Mab) (Strauss et al., ASCO 2004). Results: A total of 32 pts with FL were treated according to this protocol (4 weekly infusions at full dose of each agent), including 16 pts who had received 〉 2 prior chemotherapy regimens and 11 pts who had previously received rituximab. Twenty pts (62%) achieved an objective response (OR), including 8 pts (25%) with complete responses (CR, CRu) and 12 (37%) with partial responses, with a median response duration of 16.5 months (95% CI: 6.3 - 25.4) and median time-to-progression (TTP) of 11 months (95% CI: 9.9 - 19.2). Conclusion: Our data indicate that high OR rates and durable CR/CRu’s can be achieved with a combination of rituximab and epratuzumab in pts with low- (0–1) and intermediate-risk (2) FL, who failed multiple prior therapies. OR, CR rates and TTP are similar to rituximab front-line therapy for pts with low tumor burden FL (Solal-Céligny et al., Blood104: 169a, 2004). The combination of rituximab and epratuzumab was significantly less efficacious for pts with high-risk (3–5) FLIPI (P=.0.0023 for TTP). This small Phase-II study supports the prognostic value of FLIPI for pts with recurrent FL who are treated with MAbs. Prospective use of FLIPI may facilitate the optimal design of randomized trials using rituximab in combination with epratuzumab in pts with FL. Results stratified by FLIPI risk groups FLIPI score (No. of pts) OR (%) CR/CRu (%) Median Duration in months (95% CI) Median TTP in months (95% CI) TTP P-value* N/A - Not available due to patients with long TTPs that are still censored (i.e. not reached progression of disease). * - Patients with high (3–5) FLIPI scores versus others, based on the log-rank test. 0–1 (11) 9 (82) 4 (36) 15.7 (N/A) 19.2 (10.3 – 21.3) 0.0023 2 (9) 6 (67) 3 (33) 18.3 (17.2 – 25.4) 18.8 (10 – 26.7) 3–5 (12) 5 (42) 1 (8) 6.3 (N/A) 7.7 (7.1 – 10.2)

Description: The median survival of FL is 10 years, but has a heterogeneous clinical course and some patients die rapidly from disease, while others survive for decades. Global gene array of FL diagnostic LNs has distinguished immune cell-associated signatures associated with good and poor prognosis. The goal of this study was to attempt to validate the gene array data at the protein level as well as assess the ability to discriminate prognostic groups based upon immunohistochemistry of diagnostic lymph nodes. TMAs were constructed of 1mm cores from initial diagnostic LNs from two groups of FL patients. The first group selected were 35 patients who survived 15 yrs from diagnosis (median survival 20 yrs; median age 46 yrs; median stage 3; median FLIPI 2). Patients in both groups received a variety of standard treatments. Immunohistochemisrty was performed on the TMAs using a panel of antibodies detecting antigens associated with T-cells, including (CD4, CD8α, CD7, CD25, TIA-1, CD45RO, FOXP3) and macrophages including CD68, CD163. The immune infiltrates were scored for immunophenotype, frequency and peri- and inter-follicular distribution. Of the panels completed to date, incidence of FOXP3, perifollicular CD4 and perifollicular CD7 showed greatest discrimination between the two patient groups. CD4, CD7 and FOXP3 in prognostic patient groups Antigen expression Good Prognosis Pts Poor Prognosis Pts Perifollicular CD4 〉5 cells/hpf 18/23 cases (78%) 〉5 cells/hpf 18/35 cases (51%) 5 cells/hpf 26/34 cases (76%) 5 cells/hpf 21/32 cases (66%)

Description: The activity of the proteasome inhibitor bortezomib has been evaluated in follicular lymphoma (FL) and mantle cell lymphoma (MCL) patient samples using a modification of a primary lymphoma culture system based on the CD40-CD40L interaction (Johnson et al, Blood. 1993 5;82(6):1848). Methods: Single B-cell suspensions were isolated from lymph nodes, ascites or peripheral blood from 12 patients with histologically confirmed FL or MCL. After disaggregation and/or density gradient separation, cells were plated at a density of 5x105 cells/well into 96-well plates containing CHO cells transfected with the CDw32 Fc receptor to express the CD40 ligand, which, when irradiated to prevent further proliferation, provide a stromal layer supporting B-cell proliferation. Malignant or normal B-cells were cultured in IMDM medium supplemented with 10% human serum and 2ng/ml IL-4, and could be maintained for up to 8 days whilst retaining close phenotypic resemblance to the original sample, confirmed by flow cytometric quantitation of cell surface markers. After 24hrs drug was added at varying concentrations in triplicate and cell number and viability were assessed after a further 48hrs using the trypan blue exclusion assay. EC50 values for % viability were derived for each sample using Graphpad Prism. Results: MCL cells were significantly more sensitive to bortezomib than FL cells (median EC50 209nM range (122–989nM) n=5, vs 1311nM (153–2211nM) n=8 respectively, Mann-Whitney U-test P