Search

Supplementary Table 1 from Interaction with Vascular Endothelium Enhances Survival in Primary Chronic Lymphocytic Leukemia Cells via NF-κB Activation and De novo Gene Transcription

Supplementary Figure 1 from Interaction with Vascular Endothelium Enhances Survival in Primary Chronic Lymphocytic Leukemia Cells via NF-κB Activation and De novo Gene Transcription

Supplementary Table 2 from Interaction with Vascular Endothelium Enhances Survival in Primary Chronic Lymphocytic Leukemia Cells via NF-κB Activation and De novo Gene Transcription

Supplementary Figure 2 from Interaction with Vascular Endothelium Enhances Survival in Primary Chronic Lymphocytic Leukemia Cells via NF-κB Activation and De novo Gene Transcription

Background:: There is a significant disparity in global cancer care and out-come between countries. We aimed to provide data on characteristics, average cost of treatment and survival estimates in patients with Hodgkin Lymphoma in Sri Lanka.Methods: All patients diagnosed with Hodgkin Lymphoma between 01.05.2013 and 01.10.2020 were included in the analysis.Findings: Classical Hodgkin Lymphoma(cHL) diagnosed in 85%; 68% presented with B symptoms and 61% had advanced stage of disease. Treatment was discontinued by 23% either before or just after starting treatment of whom 72% percent were females. The complete response (CR) rate of patients who continued treatment was 86% while the estimated five-year survival rate is 92%. Seventeen percent of these patients died but only two percent due to Hodgkin Lymphoma or associated treatment in the group which continued treatment compared to 45% in the group who defaulted treatment (p-value 0.0002). Five-year survival rate of patients who defaulted treatment was 50% while patients who continued treatment have an estimated five-year survival rate of 90%. Average cost of first line treatment was between US$ 2280 and US$ 7642. First treatment failure may incur substantially higher health care costs.Interpretation: This is the only well characterized study on long-term survival of patients with Hodgkin Lymphoma in Sri Lanka. We have shown that it is possible to successfully apply western treatment and supportive care protocols to the local population. This published data will help to bench mark and improve the treatment and develop blood cancer care in the local setting. Funding: None.

BackgroundSepsis is the main cause of non-relapse mortality and there are no published data on applicability of supportive care protocols from high-income countries in Sri Lanka. The aim of the study was to investigate management and mortality of neutropenic episodes among Haemato-Oncology patients. MethodsWe analysed retrospectively clinical characteristics, management, morbidity and mortality of neutropenic adult Haemato-Oncology patients from 01.01.2019 to 31.12.2019. ResultsA total of 169 neutropenic episodes were identified; 115 (68%) of such episodes were related to chemotherapy. Acute leukaemia, Lymphoproliferative disorders and Plasma cell disorders accounted for 23%, 69% and 8% of patients respectively. The median age of patients who had sepsis was 56 while that of those who had no sepsis was 53 (p-value 0.49). Median time to neutropenia was 9 days for those in the sepsis group compared to 8 days in the group that had no sepsis (0.64). Median neutrophil count in the group who had sepsis was 0.06 while it was 0.69 in the group that had no sepsis (p-value 0.000). Median time to commencement of antibiotics was 20 minutes. There were no deaths in the group of chemotherapy induced neutropenic sepsis. ConclusionThis is the only documented study relating to outcome and successful applicability of western supportive care protocols to Sri Lankan patients with neutropenia. In this study we have shown that survival data comparable to developed countries can be achieved in the setting of limited resources with service development, following guidelines and staff training.

Approximately 70% of chronic lymphocytic leukaemia (CLL) patients present with early stage disease, therefore defining which patients will progress and require treatment is a major clinical challenge. Here, we present the largest study of prognostic markers ever carried out in Binet stage A patients (n = 1154) with a median follow-up of 8 years. We assessed the prognostic impact of lymphocyte doubling time (LDT), immunoglobulin gene (IGHV) mutation status, CD38 expression, ZAP-70 expression and fluorescence in situ hybridization (FISH) cytogenetics with regards to time to first treatment (TTFT) and overall survival (OS). Univariate analysis revealed LDT as the most prognostic parameter for TTFT, with IGHV mutation status most prognostic for OS. CD38 expression, ZAP-70 expression and FISH were also prognostic variables; combinations of these markers increased prognostic power in concordant cases. Multivariate analysis revealed that only LDT, IGHV mutation status, CD38 and age at diagnosis were independent prognostic variables for TTFT and OS. Therefore, IGHV mutation status and CD38 expression have independent prognostic value in early stage CLL and should be performed as part of the routine diagnostic workup. ZAP-70 expression and FISH were not independent prognostic markers in early stage disease and can be omitted at diagnosis but FISH analysis should be undertaken at disease progression to direct treatment strategy.

Intravenous alemtuzumab is an effective and well-tolerated treatment for T-cell prolymphocytic leukemia (T-PLL). Alemtuzumab given intravenously as first-line treatment in 32 patients resulted in an overall response rate of 91% with 81% complete responses. Studies in B-cell chronic lymphocytic leukemia have shown subcutaneous alemtuzumab to be equally as effective as intravenous alemtuzumab. The UKCLL05 pilot study examined the efficacy and toxicity of this more convenient method of administration in 9 previously untreated patients with T-PLL. Only 3 of 9 patients (33%) responded to treatment. Furthermore, 2 of 9 patients (22%) died while on treatment. Recruitment was terminated because of these poor results. After rescue therapy with intravenous alemtuzumab and/or pentostatin, median progression-free survival and overall survival were similar to the intravenous group. Alemtuzumab delivered intravenously, but not subcutaneously, remains the treatment of choice for previously untreated T-PLL. This study is registered at www.eudract.ema.europa.eu as #2004-004636-31.

Objectives: Non-steroidal anti-inflammatory drugs have been shown to induce apoptosis in primary B-cell chronic lymphocytic leukaemia (CLL) cells, but the molecular mechanisms that underpin this observation have not been fully elucidated. Here, we have analysed the effect two novel aspirin analogues, 2-hydroxy benzoate zinc (2HBZ) and 4-hydroxy benzoate zinc (4HBZ), on primary CLL samples. Materials and methods: Cytotoxic effects of 2HBZ and 4HBZ were analysed in primary CLL cells derived from 52 patients, and normal B- and T-lymphocytes. Mechanisms of action of these agents were also elucidated. Results: Both analogues induced apoptosis in a dose-dependent and time-dependent manner. Apoptosis was associated with activation of caspase-3 that could be partially abrogated by the caspase-9 inhibitor (Z-LEHD.fmk). Importantly, both agents demonstrated preferential cytotoxicity in CLL cells when compared to normal B- and T-lymphocytes. In terms of their molecular mechanisms of action, 4HBZ and 2HBZ inhibited COX-2 transcription and protein expression and this was associated with upstream inhibition of transcription factor Rel A. Co-culture of CLL cells with CD40 ligand-expressing mouse fibroblasts significantly increased COX-2 expression and inhibited spontaneous apoptosis. Importantly, the most potent analogue, 4HBZ, overcame prosurvival effects of the co-culture system and significantly repressed COX-2. Finally, elevated COX-2 expression was associated with poor prognostic subsets and increased sensitivity to 4HBZ. Conclusions: Our results demonstrate therapeutic potential of 4HBZ and are consistent with a mechanism involving suppression of Rel A nuclear translocation and inhibition of COX-2 transcription.

We performed single-molecule telomere length and telomere fusion analysis in patients at different stages of chronic lymphocytic leukemia (CLL). Our work identified the shortest telomeres ever recorded in primary human tissue, reinforcing the concept that there is significant cell division in CLL. Furthermore, we provide direct evidence that critical telomere shortening, dysfunction, and fusion contribute to disease progression. The frequency of short telomeres and fusion events increased with advanced disease, but importantly these were also found in a subset of early-stage patient samples, indicating that these events can precede disease progression. Sequence analysis of fusion events isolated from persons with the shortest telomeres revealed limited numbers of repeats at the breakpoint, subtelomeric deletion, and microhomology. Array-comparative genome hybridization analysis of persons displaying evidence of telomere dysfunction revealed large-scale genomic rearrangements that were concentrated in the telomeric regions; this was not observed in samples with longer telomeres. The telomere dynamics observed in CLL B cells were indistinguishable from that observed in cells undergoing crisis in culture after abrogation of the p53 pathway. Taken together, our data support the concept that telomere erosion and subsequent telomere fusion are critical in the progression of CLL and that this paradigm may extend to other malignancies.

Multiple myeloma remains incurable with conventional therapeutics. Thus, new treatments for this condition are clearly required. In this study we evaluated the novel NF-κB inhibitor LC-1 in multiple myeloma cell lines and plasma cells derived from multiple myeloma patients. LC-1 was cytotoxic to multiple myeloma cell lines H929, U266, and JJN3, and induced apoptosis in a dose-dependent manner with an overall LD50 of 3.6 μmol/L (±1.8) after 48 hours in culture. Primary multiple myeloma cells, identified by CD38 and CD138 positivity, had a mean LD50 for LC-1 of 4.9 μmol/L (±1.6); normal bone marrow cells were significantly less sensitive to the cytotoxic effects of LC-1 (P = 0.0002). Treatment of multiple myeloma cell lines with LC-1 resulted in decreased nuclear localization of the NF-κB subunit Rel A and the inhibition of NF-κB target genes. In addition, LC-1 showed synergy with melphalan, bortezomib, and doxorubicin (combination indices of 0.72, 0.61, and 0.78, respectively), and was more effective when cells were cultured on fibronectin. These data show that LC-1 has activity in multiple myeloma cell lines and primary multiple myeloma cells, and its ability to inhibit NF-κB seems important for its cytotoxic effects. Furthermore, LC-1–induced transcriptional suppression of survivin and MCL1 provides a potential explanation for its synergy with conventional agents. Mol Cancer Ther; 9(6); 1574–82. ©2010 AACR.

Chronic lymphocytic leukemia is the most common adult leukemia and is currently incurable with conventional chemotherapeutic agents. Over the last few years, significant discoveries have been made regarding the biology that underpins this disease. These new insights have allowed us to develop more rational prognostic tools and identify promising novel therapeutic targets. In this review, we highlight the importance of both constitutive and inducible DNA binding of the transcription factor NF-κB in chronic lymphocytic leukemia. We describe the current knowledge regarding the activity and function of specific NF-κB subunits in this disease, and discuss the complex mechanisms that regulate NF-κB activation in vivo. In addition, we provide compelling evidence for the utility of the NF-κB subunit, Rel A, as a prognostic marker and as a therapeutic target in this disease, and we also describe how this protein may contribute to the drug resistance commonly encountered with this condition.

Purpose We recently demonstrated the biologic importance of the nuclear factor kappa B (NF-κB) subunit Rel A in chronic lymphocytic leukemia (CLL) and hypothesized that Rel A DNA binding would have prognostic significance in this disease. Patients and Methods Rel A DNA binding was quantified in nuclear extracts derived from 131 unselected CLL patient samples using a quantitative DNA-binding enzyme-linked immunosorbent assay–based method. We then investigated the ability of Rel A to predict for the requirement for treatment and survival and compared our findings with other established prognostic markers. Results Rel A DNA binding was strongly associated with advanced Binet stage (P < .0001) but did not correlate with immunoglobulin VH (IgVH) mutation status (P = .25), CD38 expression (P = .87), or zeta-chain–associated protein kinase 70 (ZAP-70) expression (P = .55). It was predictive of time to first treatment (P = .02) and time to subsequent treatment (P = .0001). In addition, Rel A was the most predictive marker of survival both from date of diagnosis (hazard ratio [HR], 9.1; P = .01) and date of entry into the study (HR, 3.9; P = .05) and retained prognostic significance in multivariate analysis for both time to first treatment and overall survival in the presence of Binet stage, IgVH mutation status, CD38, and ZAP-70. Conclusion Rel A is an independent prognostic marker of survival in CLL and seems to have the unique capacity to predict the duration of response to therapy. Prospective assessment of Rel A as a marker of clinical outcome and as a therapeutic target are now warranted.

Purpose: We have recently shown that the novel nuclear factor-κB (NF-κB) inhibitor LC-1 is effective in primary chronic lymphocytic leukemia (CLL) cells. Here we elucidated the mechanism of action of LC-1, evaluated its relative cytotoxicity in prognostic subsets, and investigated its potential synergistic interaction with fludarabine. Experimental Design: Ninety-six fully characterized CLL cases were assessed for in vitro sensitivity to LC-1 and fludarabine. In selected cases, caspase activation, inhibition of Rel A DNA binding, and the transcription of CFLAR, BIRC5, and BCL2 were measured before and after exposure to LC-1. In addition, the efficacy of LC-1 was assessed in the presence of the survival factors CD154 and interleukin-4, and the potential synergistic interaction between LC-1 and fludarabine was evaluated. Results: Cell death was associated with caspase-3 activation mediated via activation of both caspase-8 and caspase-9. Apoptosis was preceded by a reduction of nuclear Rel A DNA binding and inhibition of CFLAR, BIRC5, and BCL2 transcription. Importantly, LC-1 overcame the cytoprotective effects by interleukin-4 and CD40 ligand and was equipotent in CLL cells derived from good and bad prognostic subsets. LC-1 exhibited strong synergy with fludarabine, and the combination produced a highly significant mean dose reduction index for fludarabine of >1,000. Conclusions: In view of imminent first-in-man study of LC-1 in Cardiff, these data show an important mechanistic rationale for the use of LC-1 in this disease. Furthermore, it validates the concept of targeting nuclear factor-κB in CLL and identifies the therapeutic potential of LC-1 in combination with fludarabine even in patients with fludarabine resistance.

Nuclear factor-kappaB (NF-kappaB) has been implicated in a number of malignancies and has been suggested to be a potential molecular target in the treatment of leukaemia. This study demonstrated the constitutive activation of NF-kappaB in human myeloid blasts and a clear correlation between NF-kappaB expression and in vitro cytoprotection. High NF-kappaB expression was found in many of the poor prognostic acute myeloid leukaemia (AML) subtypes, such as French-American-British classification M0 and M7, and the poor cytogenetic risk group. The in vitro effects of LC-1, a novel dimethylamino-parthenolide analogue, were assessed in 62 primary untreated AML samples. LC-1 was found to be cytotoxic to AML cells in a dose-dependent manner, mediated through the induction of apoptosis. The median drug concentration necessary to kill 50% of the cells was 4.5 micromol/l for AML cells, compared with 12.8 micromol/l for normal marrow cells. LC-1 was shown to reduce the five individual human NF-kappaB Rel proteins in a dose-dependent manner. The subsequent inhibition of many NF-kappaB-regulated cytokines was also demonstrated. Importantly, sensitivity to LC-1 was correlated with the basal NF-kappaB activity. Consequently, LC-1 treatment provides a proof of principle for the use of NF-kappaB inhibitors in the treatment of AML.

Bcl-2 family proteins play a critical role in the regulation of apoptosis in chronic lymphocytic leukemia (CLL). However, their association with established prognostic markers is unknown. In this study, we analyzed the expression of Bcl-2, Bax, and Mcl-1 in 185 CLL patients and evaluated their relationship with other prognostic markers, in vitro sensitivity to fludarabine, and clinical outcome. Mcl-1 expression was significantly correlated with stage of disease (P < .001), lymphocyte doubling time (P = .01), VH gene mutation status (P < .001), CD38 expression (P < .001), and ZAP-70 expression (P = .003). In addition, Mcl-1 and Mcl-1/Bax ratios showed strong correlations with in vitro resistance to fludarabine (P = .005 and P < .001, respectively). Furthermore, elevated Mcl-1 expression and Mcl-1/Bax ratios were predictive of time to first treatment in the whole cohort (P < .001 and P < .001, respectively) and in stage A patients only (P = .002 and P = .001, respectively). Taken together, our data show that Mcl-1 is a key controller of in vitro drug resistance and is an important regulator of disease progression and outcome in CLL. It therefore represents a promising therapeutic target in this incurable condition. The close correlation between Mcl-1 expression and VH gene mutation status, CD38 expression, and ZAP-70 expression offers a biologic explanation for their association with adverse prognosis.

As IgM multiple myeloma is a rare poorly characterised disease entity; there is only limited published data on its clinical, microscopic and immunophenotypic features. We report a 72-year-old man misdiagnosed as Waldenström's macroglobulinemia. Also the diagnosis was further complicated by coexisting chronic lymphocytic leukaemia. Following confirmation of IgM myeloma, in view of the patient's deteriorating clinical condition; he was entered into the UK Medical Research Council Myeloma IX trial where he had partial response to chemotherapy. This case highlights the value of detailed immunophenotypic evaluation when clinical and morphological markers are equivocal.

In the general haematology laboratory the patient's samples are usually analysed on an automated machine which will produce the full blood count result, together with a digital plot of the blood cells depending on the cell's volume and density of staining. An abnormal full blood count is a common finding with many different conditions. However, the result may give little information about the actual cause of the abnormality, and therefore further investigations such as a blood film are necessary.

An abnormal full blood count is a common finding in many different medical conditions; however, the cause may often not be immediately apparent. The history and examination of the patient, together with other tests such as the blood film, may help towards the diagnosis. Occasionally it is necessary to further investigate the patient with a bone marrow examination to determine the origin of the abnormalities. The indications for this are described in Table 1.

In agreement with a recently published manuscript, this present study demonstrated that CD38 + sub-populations had increased proliferative activity as evidenced by higher Ki-67 expression (P < 0.0001). This raised the possibility that the CD38 + fraction is exposed to an increased risk of clonal evolution. However, serial fluorescence in situ hybridisation analysis of highly purified CD38 + and CD38 - sub-populations from individual patients revealed no distinct cytogenetic lesions or evidence of preferential clonal evolution in the CD38 + fractions when compared with their CD38 - counterparts (P = 0.13). Furthermore, telomere length analysis revealed that all of the sub-populations had similarly short telomeres (P = 0.31) and comparably low telomerase (TERT) expression (P = 0.75) and telomerase activity (P = 0.88). Subsequent examination of cell-sorted CD38 + and CD38 - sub-populations from paired peripheral blood and bone marrow samples taken on the same day showed no significant difference in CD38, Ki-67, TERT expression or telomere lengths, indicating that these chronic lymphocytic leukaemia cells were derived from a single pool trafficking between these two compartments. Taken together, our data show that chronic lymphocytic leukaemia cells derived from bimodal patients all have extensive proliferative histories and have undergone a similar number of cell divisions that is mirrored by the episodic expression of CD38.

In this study, we characterized nuclear factor κB (NF-κB) subunit DNA binding in chronic lymphocytic leukemia (CLL) samples and demonstrated heterogeneity in basal and inducible NF-κB. However, all cases showed higher basal NF-κB than normal B cells. Subunit analysis revealed DNA binding of p50, Rel A, and c-Rel in primary CLL cells, and Rel A DNA binding was associated with in vitro survival (P = .01) with high white cell count (P = .01) and shorter lymphocyte doubling time (P = .01). NF-κB induction after in vitro stimulation with anti-IgM was associated with increased in vitro survival (P < .001) and expression of the signaling molecule ZAP-70 (P = .003). Prompted by these data, we evaluated the novel parthenolide analog, LC-1, in 54 CLL patient samples. LC-1 induced apoptosis in all the samples tested with a mean LD50 of 2.8 μM after 24 hours; normal B and T cells were significantly more resistant to its apoptotic effects (P < .001). Apoptosis was preceded by a marked loss of NF-κB DNA binding and sensitivity to LC-1 correlated with basal Rel A DNA binding (P = .03, r2 = 0.15). Furthermore, Rel A DNA binding was inversely correlated with sensitivity to fludarabine (P = .001, r2 = 0.3), implicating Rel A in fludarabine resistance. Taken together, these data indicate that Rel A represents an excellent therapeutic target for this incurable disease.

Junior doctors need to acquire many skills in their training for the efficient and successful day-to-day clinical management of patients. These include effective communication skills, which are essential for a doctor to be able to ask the relevant questions in a sympathetic manner to the patient to acquire the precise facts. Correct examination techniques are also needed to allow doctors to effectively spot clinical signs and formulate their thinking to provide a differential diagnosis, and good presentation skills are needed to enable the students to deliver their findings in a systematic and logical manner.

Chronic lymphocytic leukaemia (CLL) is a neoplastic condition of B cells which commonly affects the lymph nodes, liver, spleen and bone marrow. Leukaemic involvement of the kidney is also relatively common in CLL, but characteristically is not associated with renal impairment. Our report describes a patient who developed acute renal failure as the initial presenting feature of CLL. The renal failure was subsequently found to be due to direct leukaemic infiltration. Treatment with chlorambucil and prednisolone resulted in stabilisation of the renal function for approximately 1 year prior to the need for long-term haemodialysis. Leukaemic infiltration of kidney should always be considered when a patient with CLL presents with renal impairment, regardless of the clinical stage, as the renal failure often responds well to chemotherapy.

Chronic lymphocytic leukaemia (CLL) is the most common form of leukaemia in the Western world. The natural history of CLL is extremely variable with a survival time from initial diagnosis that ranges from 2 to more than 20 years. Understanding the clinical diversity and allowing the subclassification of CLL into various prognostic groups not only assists in predicting future outcome for patients, but also helps to direct treatment decisions. Chlorambucil and fludarabine were the standard therapy for CLL for decades. Randomized studies have reported superior overall response and progression-free survival (PFS) for fludarabine compared with alkylator-based therapy and for the fludarabine–cyclophospamide (FC) combination over fludarabine alone. More recently the addition of rituximab to the FC regimen (R-FC) has shown significant improvement in overall response, PFS and overall survival compared with FC alone. However, there are patients for whom this regimen still provides less satisfactory results. Within the above studies CLL patients who have some of the poorer prognostic markers, such as unmutated IgVH genes and/or high beta-2 microglobulin (B2M), and those who fail to achieve a minimal residual disease (MRD) negative remission are likely to have a shorter PFS compared with those without these features. Various strategies have been explored to improve the outcome for such patients. These include the addition of agents to a frontline R-FC regimen, use of consolidation and consideration of maintenance. The only group that can be clearly identified pretreatment for whom conventional fludarabine-based therapies produce significantly inferior response rates, PFS and overall survival are the patients who harbour a genetic fault; deletion or mutation or a combination of deletion and mutation of tumour protein p53 ( TP53). TP53 inactivation is a less common finding at first treatment but becomes much more common in fludarabine-refractory patients. Alemtuzumab and high-dose corticosteroids have been shown to be effective in this group of CLL patients. Trials combining these two agents have shown improved responses, particularly for those patients with bulky nodal disease for whom alemtuzumab alone may be insufficient. Since the duration of responses remains relatively short, suitable patients should be considered for allogeneic stem cell transplantation according to the European Group for Blood and Marrow Transplantation (EBMT) guidelines. Furthermore, there are a number of other new treatments on the horizon, including humanized antibodies directed against novel targets and small-molecule inhibitors.

Chronic lymphocytic leukemia (CLL) cells rapidly undergo apoptosis in vitro, suggesting that the in vivo microenvironment provides crucial antiapoptotic signals. Overexpression of the antiapoptotic proteins Bcl-2 and Mcl-1 is a hallmark of CLL, and their expression is further enhanced in the lymphoid tissues. However, the high levels of Mcl-1 found in peripheral blood samples, coupled with its short half-life, led us to hypothesize that it must be actively maintained in the peripheral circulation. Coculture of CLL cells with human vascular endothelial cells significantly enhanced tumor cell survival, an effect that was not observed with normal B cells. This was associated with elevated levels of the antiapoptotic proteins Bcl-2, Mcl-1, and Bcl-XL and marked increased expression of CD38 and CD49d, both of which are associated with clinically aggressive disease. Because CD38, CD49d, and some Bcl-2 family genes are transcriptional targets for NF-κB, we assessed NF-κB activation following coculture with endothelial cells. DNA binding of the NF-κB subunit Rel A was significantly increased and strongly correlated with changes in transcription of CD38, CD49d, BCL2, MCL1, and BCLXL, effects that were reversed by a peptide inhibitor of Rel A. These effects were not observed following coculture with nonendothelial cell lines. Therefore, CLL cells receive specific survival signals following interaction with endothelial cells mediated through the activation of NF-κB and the induction of downstream target genes. This type of interaction in the peripheral vasculature may explain the constitutive NF-κB activation and the overexpression of Bcl-2 family proteins commonly seen in this disease. Cancer Res; 70(19); 7523–33. ©2010 AACR.

The world of chronic lymphocytic leukemia (CLL) research is awash with prognosticmarkers. However, very few of the current group play a clearly defined role in the pathology of this disease and even fewer represent a tractable therapeutic target. One such marker that fulfils both of these criteria is the integrin CD49d. This molecule been implicated in the capacity of CLL cells to migrate into lymphoid tissues and there is a CD49d blocking antibody, Natalizumab, currently in clinical trials. Here we carried out the largest multi-centre evaluation of CD49d as a prognosticmarker in 652 primary CLL samples. We confirm that CD49d is predictive for time to first treatment (P < 0.0001) and overall survival (P < 0.0001) and increases the prognostic power of CD38, ZAP-70 and IGHV gene mutation status in concordant cases. Furthermore, CD49d retained independentprognostic significance in multivariate analysis. In contrast to previous studies, we showed no correlation between CD49dexpression and in vitro resistance to fludarabine in liquid cultures (P = 0.28) but CD49dhi cells were significantly more resistant than CD49dlo cells when assays were carried out on fibronectin-coated plates (P = 0.03). Furthermore, we showed for the first time that the expression of CD49d is strongly associated with expression of the chemokine receptor CXCR4 suggesting a co-ordinated role for these molecules in the trafficking of CLL cells to the lymphoid tissues. Taken together, our data support the introduction of CD49d into routine immunophenotyping panels for CLL and indicate that the therapeutic targeting of this molecule may prove useful in this disease

Aminopeptidase enzyme inhibition is thought to deplete the free intracellular amino acids needed by malignant cells for growth and development, resulting in profound anti-proliferative and apoptotic effects. In this study, we investigated the effects of the metalloenzyme-inhibitor CHR-2797 (tosedostat), in primary acute myeloid leukemia (AML) cells. CHR-2797 demonstrated marked in vitro cytotoxicity in AML samples and strong synergy with Cytarabine (Ara-C), but significantly less cytotoxicity to normal marrow progenitors. Furthermore mechanistic investigations revealed that CHR-2797 inhibited the intrinsic nuclear, cytoplasmic and cell surface aminopeptidase function of AML blasts in a dose-dependent manner, demonstrating a promising novel approach for AML therapy.

Studies of the role of individual genes in chronic lymphocytic leukemia (CLL) have been hampered by the inability to consistently transfect primary tumor cells. Here, we describe a highly efficient method of genetically modifying primary CLL cells using a VSVG pseudotyped lentiviral vector. We transduced CD38 negative CLL cells with a lentiviral vector encoding CD38 which caused increased surface CD38 expression in all the samples tested (n=17) with no evidence of plasmacytoid differentiation. The mean percentage of positive cells expressing CD38 was 87%+/-8.5% and the mean cell viability 74%+/-17%. This high level of transduction of all the CLL cell samples tested demonstrates the utility of this technique which should prove applicable for the introduction and analysis of other genes in these non-dividing cells.

Transcription factors are a large class of biological molecules that are important for health and disease. Despite that there are challenges to targeting them therapeutically and most approaches alter their activity indirectly. Research at the chemical biology interface has led to the development of new ways of targeting transcription factors including blocking transcription factor dimerisation, targeting specific DNA sequences and DNA decoys. This review discusses these issues with a view to inspiring the development of new agents that could be useful for the treatment of cancer.

B-lymphocytes are essential for the production of antibodies to fight pathogens and are the cells of origin in 95% of human lymphomas. During their activation, and immortalisation by Epstein-Barr virus (EBV) which contributes to human cancers, B-lymphocytes undergo dramatic changes in cell size and protein content. This study was initiated to compare the proteome of two B-cell lines, from the same individual, that reflect different patterns of activation, one is EBV negative and the other is EBV positive. Using isobaric tags, LC-MALDI TOF-TOF and subcellular fractionation, we quantified 499 proteins from B-cells. From a detergent lysed protein extract, we identified 34 proteins that were differentially expressed in EBV-immortalised B-cells. By analysing a nuclear extract, we identified a further 29 differentially expressed proteins with only four proteins shared between the two extracts, illustrating the benefit of subcellular fractionation. This analysis has identified proteins involved in the cytoskeletal phenotype of activated B-cells and the increased antigen recognition in EBV-immortalised cells. Importantly, we have also identified new regulators of transcription and changes in ribonuclear proteins that may contribute to the increased cell size and immortalisation of lymphoblastoid cells.

In agreement with a recently published manuscript, this present study demonstrated that CD38+ sub-populations had increased proliferative activity as evidenced by higher Ki-67 expression (P0.0001). This raised the possibility that the CD38+ fraction is exposed to an increased risk of clonal evolution. However, serial fluorescence in situ hybridisation analysis of highly purified CD38+ and CD38- sub-populations from individual patients revealed no distinct cytogenetic lesions or evidence of preferential clonal evolution in the CD38+ fractions when compared with their CD38- counter-parts (P = 0.13). Furthermore, telomere length analysis revealed that all of the sub-populations had similarly short telomeres (P = 0.31) and comparably low telomerase (TERT) expression (P = 0.75) and telomerase activity (P = 0.88). Subsequent examination of cell-sorted CD38+ and CD38- sub-populations from paired peripheral blood and bone marrow samples taken on the same day showed no significant difference in CD38, Ki-67, TERT expression or telomere lengths, indicating that these chronic lymphocytic leukaemia cells were derived from a single pool trafficking between these two compartments. Taken together, our data show that chronic lymphocytic leukaemia cells derived from bimodal patients all have extensive proliferative histories and have undergone a similar number of cell divisions that is mirrored by the episodic expression of CD38.

Dear Editor, We report a case of steroid refractory human immune deficiency virus-related immune thrombocytopenic purpura (HIV-TP) successfully treated with single dose of intravenous Anti-Rh (Anti D). Although anti-retroviral treatment is the preferred first option in this situation, we treated him with steroids and, subsequently, with Anti D partly according to the patient’s wishes. He showed a durable response (nearly 2 years now) to a single dose of treatment. We propose that Anti D should be considered before antiretroviral treatment, at least in some patients. A 25-year-old man was referred to the haematology unit for further investigation of symptomatic thrombocytopenia. He had a known history of HIV infection for 5 years but has been asymptomatic till this point. The laboratory examination showed platelet count of 30×10/L with normal white cell count and haemoglobin. All other biochemical and haematological parameters were normal. The CD4/CD3 count on peripheral blood was 450/μL. On further investigations, the bone marrow aspirate showed increased megakaryocytes compatible with a diagnosis of peripheral consumption as the cause for low platelet count. A provisional diagnosis of ITP was made and he was treated with steroids without any response. A dose of Anti D gave an excellent initial response to the therapy, acquiring a platelet count of over 200×10/L. Subsequently, the platelet count fluctuated between 185×10/L and 50×10/L; but despite only having a single dose of therapy, he has been asymptomatic and did not need any further treatment. The variability of platelet count during the course of disease is illustrated on Fig. 1. HIV is one of the many causes of secondary ITP. Thrombocytopenia was associated with AIDS even before the discovery of HIV [1]. While a number of different mechanisms have been reported by which HIV infection could produce thrombocytopenia, the ability of effective anti-retroviral therapy to improve platelet counts demonstrates a close association between HIV replication and thrombocytopenia. Probable mechanisms of thrombocytopenia include anti-platelet glycoprotein antibodies; antiHIV antibodies that cross-react with platelet membrane glycoproteins, platelet clearance due to immune complex disease and decreased platelet production/ineffective erythropoiesis. Thrombocytopenia is more common in patients with advanced HIV with CD4 lymphocyte count of less than 200/μL [2]. Interestingly, our patient had thrombocytopenia with normal CD4 count. HIV-TP has been shown to correlate with the degree of HIV viral load [3] and respond well to anti-retroviral treatment [4]. Hence, highly active anti-retroviral therapy (HAART) is considered as the first port of treatment [5]. Our patient did not agree with this option, so we had to consider either steroids, splenectomy, Ann Hematol (2009) 88:1155–1156 DOI 10.1007/s00277-009-0744-4

Abstract 4813 Standard treatments for acute myeloid leukaemia (AML) result in a median survival of approximately 1 year. There is now a realisation that in order to significantly improve outcomes in this disease more targeted therapies that take account of the specific biology of the tumour cell are required. L-Gossypol is a polyphenolic oil cotton seed extract that has anti-tumour activity against a range of haematological malignancies but has never been evaluated in AML cells. It is known to act as a BH3-mimetic, binding to the BH3 pocket of anti-apoptotic proteins and displacing pro-death partners to induce apoptosis. However, knowledge of the molecular events that underpin its downstream effects is limited. In this study we analysed the in vitro effects of L-Gossypol in 50 primary AML samples in order to determine its efficacy and mode of action. Apoptosis was induced in all the samples tested in a dose- and time-dependent manner as evidenced by increased Annexin V / propidium iodide labelling and the activation of caspase-9 and caspase-3. The median LD50 value (the concentration of drug required to kill 50% of the cells) was 27.5μM ± 18.3μM. There was no association between LD50 and age, sex, presenting white cell count, FLT3 mutation status or karyotype. Mechanistically, L-gossypol decreased the DNA binding activity of the NF-κB subunit, Rel A, in a concentration-dependent manner; this inhibition was evident after only 4 hours and preceded the induction of apoptosis. Furthermore, treatment with L-Gossypol inhibited the transcription of the NF-κB-regulated genes CFLAR, BCL2, BIRC5 and MCL1 in the same timeframe. Finally, studies of Mcl-1 protein expression showed down regulation in a dose-dependent manner but this was only apparent after 8 hours exposure to L-Gossypol. Taken together, our data demonstrate that L-Gossypol works, at least in part, through the inhibition of NF-κB and our data provides a rationale for clinical investigations of this agent in AML patients. Disclosures: No relevant conflicts of interest to declare.

Multiple myeloma (MM) remains incurable with conventional therapeutic agents and has a median survival of only 3–5 years. Therefore, there is clearly a need for novel treatment strategies that can change the natural pathology of this condition. The nuclear factor κB (NF-κB) family of transcription factors is constitutively activated in MM cell lines and the majority of MM patients. Since NF-κB has known oncogenic activity in a number of human malignancies, targeted inhibition of this family of proteins may be useful in the treatment of MM. We and others have recently shown that the parthenolide derivative LC-1 has activity in acute myeloid leukaemia (AML) and chronic lymphocytic leukaemia (CLL) cells. Unusually, it induces apoptosis via the activation of both the intrinsic and extrinsic pathways and apoptosis is preceded by marked inhibition of NF-κB. Importantly, LC-1 is more potent against primary AML blasts and CLL lymphocytes than normal bone marrow progenitors and normal B-cells and T-cells. In this study we set out to evaluate LC-1 in MM cell lines and plasma cells derived from MM patients. LC-1 was cytotoxic to MM cell lines H929, U266 and JJN3 and induced apoptosis in a dosedependent manner resulting in an overall LD50 of 3.6mM (±1.8) after 48 hours in culture. Primary myeloma plasma cells, identified by CD38 and CD138 positivity, had a mean LD50 for LC-1 of 5.4mM (±1.6) after 48 hours of in vitro culture. Normal bone marrow cells were significantly less sensitive to the effects of LC-1 under the same conditions (P = 0.0007). Treatment of MM cell lines with LC-1 resulted in a decrease in the nuclear localization of NF-κB, as evidenced by a dose-dependent decrease in the DNA binding capacity of the NF-κB subunit RelA after 4 hours of treatment. To demonstrate whether synergy exists between LC-1 and existing MM therapies, the H929 cell line was treated for 48 hours with LC-1 and doxorubicin (32:1), melphalan (1:1) or bortezimib (1:500) and the combination indices (CI) calculated using the median effect method. A combination index of less than 1 denotes synergy. LC-1 did not show synergy with doxorubicin (CI >1) but was synergistic with melphalan and bortezimib (CI values of 0.53 and 0.59 respectively). Taken together our data clearly demonstrate that LC-1 has activity in MM cell lines and primary MM cells. Its ability to inhibit the nuclear localization of NF-κB is important to its cytotoxic effects. Furthermore, it may also provide an explanation for the synergy demonstrated with melphalan and bortezimib. These results provide a rationale for exploring the potential of LC-1 in clinical studies.

Chronic lymphocytic leukaemia (CLL) is incurable with conventional chemotherapy and all currently available agents eventually fail because of drug resistance or unacceptable toxicities. We have recently shown that the novel NF-κB inhibitor LC-1 is effective in primary CLL cells and is capable of overcoming drug resistance. In this study, we elucidated the mechanism of action of LC-1, evaluated its relative cytotoxicity in prognostic subsets (n=96) and investigated its potential synergistic interaction with fludarabine (n=26). LC-1 induced cell death was associated with caspase-3 activation that was mediated via the upstream activation of both caspase-8 and caspsase-9. Apoptosis was preceded by a reduction in nuclear Rel A DNA binding and down regulation of the anti-apoptotic NF-κB target genes CFLAR, BIRC5 and BCL2. Importantly, LC-1 was able to overcome the cytoprotective effects by IL-4 and CD40 ligand indicating that it would retain its potency in vivo even in the solid tissue micro-environment. LC-1 was equally effective in CLL cells derived from good and bad prognostic subsets and exhibited strong synergy with fludarabine (mean combination index 0.26). The combination produced a highly significant mean dose reduction index of >1000 that would facilitate a theoretical 3 log reduction in the standard clinical dose of fludarabine. Taken together our data confirm the therapeutic potential of LC-1 both as a single agent and in combination with fludarabine and provide a compelling rationale for early clinical trials of this agent in CLL patients.

We recently demonstrated that the NF-kB subunit Rel A is associated with in vitro survival and clinical disease progression in CLL. We therefore hypothesized that Rel A would have prognostic significance in this disease. Rel A DNA binding was quantified in nuclear extracts derived from 131 unselected CLL patient samples using a quantitative DNA binding ELISA-based method. We tested the ability of Rel A to predict for the requirement for treatment and survival and compared our findings with other established prognostic markers. Rel A DNA binding was strongly associated with advanced Binet stage (P

NF-kB is part of a multi-component signalling pathway that regulates the expression of hundreds of genes involved in the control of cell proliferation, cell survival, stress responses, innate immunity and inflammation. The principal constituents of mammalian NF-kB are p65, p50, p52, c-Rel and Rel-B and in their inactive state they are bound in the cytoplasm to the inhibitor protein IKB. Phosphorylation of IKB by IKB kinase has been shown to result in the translocation of NF-kB into the nucleus where it can bind to kB responsive elements. It has been reported that NF-kB is constitutively activated in chronic lymphocytic leukaemia (CLL) cells. However, no previous studies have prospectively quantified the subunits of NF-kB in primary CLL cells or examined their relative expression between patient samples. Therefore, the aim of our study was to perform quantitative subunit analysis of NF-κB in freshly isolated CLL cells and examine their relationship with some of the well established clinical and molecular prognostic markers. NF-κB was evaluated using EMSA (n= 48), supershift assays and p65 subunit-specific ELISA (n=100). Apoptosis was measured at 24h and 48h using annexin V/propidium iodide labelling. We demonstrated a wide variation in NF-κB expression in CLL samples and p50, p65 and c-rel were consistently shown to be the predominant NF-kB subunits. Importantly, we showed a negative correlation between in vitro apoptosis and nuclear p65 NF-kB expression (r2 = −0.22) suggesting that high levels of p65 confer a cytoprotective effect on primary CLL cells. Furthermore, we found that p65 has a positive correlation (r2 = 0.3) with total white cell count (WBC) and CLL samples with a lymphocyte doubling time below 12 months have significantly higher p65 DNA binding (p= 0.02) implicating this NF-κB subunit in disease progression. Finally we have also demonstrated that the NF-κB inhibitor, LC-1, has potent in vitro apoptosis-inducing effects on primary CLL cells. Together, this work supports the use of specific inhibitors of NF-kB, particularly targeting p65, for treatment of CLL.