Search

Despite the practice-changing advances achieved in the prognostic stratification and treatment of chronic lymphocytic leukemia (CLL), a large fraction of the world population resides in countries where access to many of these advances remains unavailable or subject to severe constraints. Although some of these countries display incidence rates of CLL that are lower than those of developed Western countries, a large number of patients are expected to be diagnosed with CLL in these regions every year. In this article, we review issues regarding management of CLL in some less-resourced countries, with a focus on the evidence basis for epidemiological and clinical information on this disease, the availability of diagnostic and therapeutic resources, and participation in clinical trials. Going forward, challenges that still need to be addressed include the development of unified countrywide registries, guidelines for management applicable to each country, wider availability of prognostic tools, access to new drugs, and policies that ensure these drugs are affordable to all patients worldwide.

The treatment landscape of chronic lymphocytic leukemia (CLL) has witnessed immense changes in the past decade. Several newer target therapies and their combinations with anti-CD 20 therapies have got approval for management of CLL in the treatment-naïve and relapsed/refractory setting. Also, the availability of newer diagnostic techniques has helped differentiate the disease into high- and low-risk CLL which acts not just as a prognostic marker but also helps decide the best drug management that can be administered to the patients. Targeted therapy has largely overtaken chemoimmunotherapy in the management of CLL, except for a small subset of the population (young and fit with IGHV mutation). However, with targeted therapy, there is also an issue of previously uncommon treatment-emergent adverse events, the duration of therapy, and financial toxicity. The aim of this review article is to gather results from all landmark CLL trials and discuss the feasibility of incorporating Acalabrutinib in the CLL landscape from an Indian perspective.

Cardiac toxicity following snakebite envenomation has been previously observed, but not studied in detail, especially the involvement in neurotoxic bites. This prospective observational case study evaluates the incidence of cardiac toxicity along with the difference between vasculotoxic and neurotoxic bites and analysing the predictors for development of cardiotoxicity.96 patients who had snake bite envenomation were evaluated for features of cardiotoxicity with clinical features, ECG, echocardiogram and troponin-I levels.Cardiac toxicity was observed in 42.7% of patients, the majority were either ECG changes, noted in 34.3% and rise in troponin-I, noted in 21.9% of patients. Other changes included echocardiographic changes in 4.2%, and Takotsubo cardiomyopathy in 1%. There was no significant difference in the incidence of cardiotoxicity between the neurotoxic (41.7%) and vasculotoxic (42.9%) (p value =1) snake bites, even though the predominant changes seen in neurotoxic snake bites were ECG changes. There were no deaths in the current study. None of the demographic or clinical parameters studied could predict the development of cardiac events.Cardiac toxicity is a well defined complication of poisonous snake bite and incidence is more frequent than previously thought. Both vasculotoxic and neurotoxic snake bites are associated with cardiac toxicity and is not associated with increase in mortality.

East Asians, Asian Indians and Amerindians have a five to ten-fold lower age-adjusted incidence rate (AAIR) of chronic lymphocytic leukaemia (CLL) compared with persons of predominately European descent. The data we review suggest a genetic rather than environmental basis for this discordance. All these populations arose from a common African Black ancestor but different clades have different admixture with archaic hominins including Neanderthals, Denisovans and Homo erectus, which may explain different CLL incidences. There are also some differences in clinical laboratory and molecular co-variates of CLL between these populations. Because the true age-adjusted incidence rate in African Blacks is unknown it is not possible to determine whether modern Europeans acquired susceptibility to CLL or the other populations lost susceptibility and/or developed resistance to developing CLL. We also found other B-cell lymphomas and T- and NK-cell cancers had different incidences in the populations we studied. These data provide clues to determining the cause(s) of CLL.

ARCTIC was a multicenter, randomized-controlled, open, phase IIB non-inferiority trial in previously untreated chronic lymphocytic leukemia (CLL). Conventional frontline therapy in fit patients is fludarabine, cyclophosphamide and rituximab (FCR). The trial hypothesized that including mitoxantrone with low-dose rituximab (FCM-miniR) would be non-inferior to FCR. A total of 200 patients were recruited to assess the primary end point of complete remission (CR) rates according to IWCLL criteria. Secondary end points were progression-free survival (PFS), overall survival (OS), overall response rate, minimal residual disease (MRD) negativity, safety and cost-effectiveness. The trial closed following a pre-planned interim analysis. At final analysis, CR rates were 76 FCR vs 55% FCM-miniR (adjusted odds ratio: 0.37; 95% confidence interval: 0.19-0.73). MRD-negativity rates were 54 FCR vs 44% FCM-miniR. More participants experienced serious adverse reactions with FCM-miniR (49%) compared to FCR (41%). There are no significant differences between the treatment groups for PFS and OS. FCM-miniR is not expected to be cost-effective over a lifetime horizon. In summary, FCM-miniR is less well tolerated than FCR with an inferior response and MRD-negativity rate and increased toxicity, and will not be taken forward into a confirmatory trial. The trial demonstrated that oral FCR yields high response rates compared to historical series with intravenous chemotherapy.

Background Hyponatremia, the most common electrolyte disturbance in clinical practice affecting up to 15%–28% of admitted patients, is defined as a serum sodium concentration ([Na+]) Methods A retrospective case note analysis was carried out among patients admitted under a palliative care unit of a tertiary referral hospital in South India, over a period of 6 months. The sodium level on admission and during the inpatient stay, along with symptoms, drug history and cause of hyponatremia was evaluated. Results Out of 11 patients who presented with hyponatremia, median age was 59;7 patients were male. All the patients had a cancer diagnosis, with 28% had cancer of GI tract,91% had stage IV disease. The median sodium level on admission was 125;82% had hypotonic hyponatraemia. 37% were asymptomatic on presentation,while 18% had generalised weakness.Median number of drugs for each patient was 4 and a number of drugs were implicated to cause hyponatremia.55% of patients were found to have a diagnosis of SIADH,the other causes were extrarenal solute loss(18%), Pseuohyponatremia (18%) and cirrhosis (9%).Only 45% were treated with Tolvaptan,while the rest had sodium replacement, fluid restriction and other supportive care.27% died during the admission and 36% died within the next 6 months of discharge. Conclusions SIADH is not always the cause of hyponatremia in palliative care patients, hence there is a need for accurate diagnosis of patients to enable a targeted management strategy.

Background Capillary leak syndrome (CLS) has been previously observed as a complication of Daboia russelii bite but not clearly defined or studied in length. This observational case-control study evaluates the mortality along with associated clinical and laboratory features. Methods Twenty-five patients who developed CLS were compared with 25 patients without CLS following Daboia russelii (Russell's viper) bite. Results Development of CLS is associated with a significantly high risk of mortality; 11 (44%) patients with CLS died compared with 1 (4%) control (odds ratio 18.8 [95% confidence interval 2.2 to 161.99], p=0.002). Disease-defining manifestations included myalgia (22 [88%]), thirst (20 [80%]), parotid swelling (15 [60%]), conjunctival chemosis (19 [76%]) and hypotension (22 [88%]), which were unobserved in controls. Although several clinical and laboratory parameters were found to be predictive for development of CLS in univariate analysis, none of them had independent predictive value in multivariate analysis. Similarly, development of parotid swelling was the only factor with independent predictive value for mortality in multivariate analysis. Even though the number of vials of snake antivenom used is more in CLS, it seems unlikely to improve the mortality in CLS. Conclusions This study proves that CLS is a well-defined complication of Russell's viper bite with high mortality but with clear predictors for the development of CLS and mortality.

BackgroundThe conventional frontline therapy for fit patients with chronic lymphocytic leukaemia (CLL) is fludarabine, cyclophosphamide and rituximab (FCR). Rituximab (Mabthera®, Roche Products Ltd) targets the CD20 antigen, which is expressed at low levels in CLL. The standard dose of rituximab in CLL (375 mg/m2in cycle 1 and 500 mg/m2in cycles 2–6) was selected based on toxicity data only. Small doses of rituximab (as low as 20 mg) have biological activity in CLL, with an immediate reduction in circulating CLL cells and down-regulation of CD20. Phase II trials had suggested improved efficacy with the addition of mitoxantrone to FCR. The key assumption for the Attenuated dose Rituximab with ChemoTherapy In CLL (ARCTIC) trial was that the addition of mitoxantrone to fludarabine, cyclophosphamide and low-dose rituximab would be more effective than conventional FCR.ObjectivesTo assess whether fludarabine, cyclophosphamide, mitoxantrone and low-dose rituximab (FCM-miniR) (100 mg of rituximab per cycle) was non-inferior to FCR in frontline CLL. Complete response (CR) rate was the primary end point, with the secondary end points being progression-free survival (PFS), overall survival (OS), overall response rate, eradication of minimal residual disease (MRD), safety and cost-effectiveness.DesignARCTIC was a UK multicentre, randomised, controlled, open, Phase IIB non-inferiority trial in previously untreated CLL. A total of 206 patients with previously untreated CLL who required treatment, according to the International Workshop on Chronic Lymphocytic Leukaemia criteria, were to be randomised to FCR or FCM-miniR. There was an independent Data Monitoring and Ethics Committee (DMEC) with a pre-planned interim efficacy assessment on 103 participants.ResultsThe DMEC’s interim analysis led to early trial closure. Although the response rates in both arms were higher than anticipated, FCM-miniR had a lower CR rate than FCR. This was partly attributable to the higher toxicity associated with mitoxantrone. A total of 100 participants completed FCR, 79 completed FCM-miniR and 21 commenced FCM-miniR but switched to FCR following DMEC recommendations. The CR rate for participants receiving FCR was 76%, compared with 55% for FCM-miniR (adjusted odds ratio 0.37; 95% confidence interval 0.19 to 0.73). Key secondary end points also showed that FCR was superior, with more participants achieving MRD negativity (57% for FCR vs. 46% for FCM-miniR). More participants experienced a serious adverse reaction with FCM-miniR compared with FCR (50% vs. 41%). At a median of 37.3 months’ follow-up, the PFS and OS rates are good compared with previous studies, with no significant difference between the treatment arms. The economic analysis indicates that because FCM-miniR is less effective than FCR, FCM-miniR is not expected to be cost-effective over a lifetime horizon, producing a mean cost-saving of –£7723, a quality-adjusted life-year loss of –0.73 and a resulting incremental net monetary loss of –£6780.ConclusionsFCM-miniR is less well tolerated, with poorer response rates, than FCR, partly owing to the additional toxicity associated with mitoxantrone. In view of this, FCM-miniR will not be taken forward into a larger definitive Phase III trial. The trial demonstrated that oral FCR yields extremely high response rates compared with historical series with intravenous chemotherapy.Future workWe shall compare the results of ARCTIC with those of the ADMIRE (Does the ADdition of Mitoxantrone Improve Response to FCR chemotherapy in patients with CLL?) trial, which compared FCR with FCM-R to assess the efficacy of low- versus standard-dose rituximab, allowing for the toxicity associated with mitoxantrone.Trial registrationCurrent Controlled Trials ISRCTN16544962.FundingThis project was funded by the NIHR Health Technology Assessment programme and will be published in full inHealth Technology Assessment; Vol. 21, No. 28. See the NIHR Journals Library website for further project information.

Purpose Most patients with chronic lymphocytic leukemia (CLL) are elderly and/or have comorbidities that may make them ineligible for fludarabine-based treatment. For this population, chlorambucil monotherapy is an appropriate therapeutic option; however, response rates with chlorambucil are low, and more effective treatments are needed. This trial was designed to assess how the addition of rituximab to chlorambucil (R-chlorambucil) would affect safety and efficacy in patients with CLL. Patients and Methods Patients with first-line CLL were treated with rituximab (375 mg/m2 on day 1, cycle one, and 500 mg/m2 thereafter) plus chlorambucil (10 mg/m2/d all cycles; day 1 through 7) for six 28-day cycles. For patients not achieving complete response (CR), six additional cycles of chlorambucil alone could be administered. The primary end point of the study was safety. Results A total of 100 patients were treated with R-chlorambucil, with a median follow-up of 30 months. Median age of patients was 70 years (range, 43 to 86 years), with patients having a median of seven comorbidities. Hematologic toxicities accounted for most grade 3/4 adverse events reported, with neutropenia and lymphopenia both occurring in 41% of patients and leukopenia in 23%. Overall response rates were 84%, with CR achieved in 10% of patients. Median progression-free survival was 23.5 months; median overall survival was not reached. Conclusion These results compare favorably with previously published results for chlorambucil monotherapy, suggesting that the addition of rituximab to chlorambucil may improve efficacy with no unexpected adverse events. R-chlorambucil may improve outcome for patients who are ineligible for fludarabine-based treatments.

Chronic lymphocytic leukaemia (CLL) is the most common clonal B-cell disorder characterized by clonal diversity, a relapsing and remitting course, and in its aggressive forms remains largely incurable. Current front-line regimes include agents such as fludarabine, which act primarily via the DNA damage response pathway. Key to this is the transcription factor p53. Mutations in the TP53 gene, altering p53 functionality, are associated with genetic instability, and are present in aggressive CLL. Furthermore, the emergence of clonal TP53 mutations in relapsed CLL, refractory to DNA-damaging therapy, suggests that accurate detection of sub-clonal TP53 mutations prior to and during treatment may be indicative of early relapse. In this study, we describe a novel deep sequencing workflow using multiple polymerases to generate sequencing libraries (MuPol-Seq), facilitating accurate detection of TP53 mutations at a frequency as low as 0.3%, in presentation CLL cases tested. As these mutations were mostly clustered within the regions of TP53 encoding DNA-binding domains, essential for DNA contact and structural architecture, they are likely to be of prognostic relevance in disease progression. The workflow described here has the potential to be implemented routinely to identify rare mutations across a range of diseases.

ADMIRE was a multicenter, randomized-controlled, open, phase IIB superiority trial in previously untreated chronic lymphocytic leukemia. Conventional front-line therapy in fit patients is fludarabine, cyclophosphamide and rituximab (FCR). Initial evidence from non-randomized phase II trials suggested that the addition of mitoxantrone to FCR (FCM-R) improved remission rates. Two hundred and fifteen patients were recruited to assess the primary end point of complete remission (CR) rates according to International Workshop on Chronic Lymphocytic Leukemia criteria. Secondary end points were progression-free survival (PFS), overall survival (OS), overall response rate, minimal residual disease (MRD) negativity and safety. At final analysis, CR rates were 69.8 FCR vs 69.3% FCM-R (adjusted odds ratio (OR): 0.97; 95% confidence interval (CI): (0.53-1.79), P=0.932). MRD-negativity rates were 59.3 FCR vs 50.5% FCM-R (adjusted OR: 0.70; 95% CI: (0.39-1.26), P=0.231). During treatment, 60.0% (n=129) of participants received granulocyte colony-stimulating factor as secondary prophylaxis for neutropenia, a lower proportion on FCR compared with FCM-R (56.1 vs 63.9%). The toxicity of both regimens was acceptable. There are no significant differences between the treatment groups for PFS and OS. The trial demonstrated that the addition of mitoxantrone to FCR did not increase the depth of response. Oral FCR was well tolerated and resulted in impressive responses in terms of CR rates and MRD negativity compared with historical series with intravenous chemotherapy.

Minimal residual disease (MRD) negativity, defined as

With immunochemotherapy, remission duration and survival in patients with chronic lymphocytic leukaemia is dependent on the level of minimal residual disease (MRD) after treatment. This phase II trial assessed alemtuzumab consolidation post-chemotherapy in patients who responded with persistent low levels of detectable disease. Blood was screened for MRD using multi-parameter flow cytometry, 6-24 months post-chemotherapy. MRD-positive participants received alemtuzumab 30 mg subcutaneously 3 times weekly for 6 weeks. Following a marrow assessment, MRD-negative participants or non-responders stopped therapy and MRD-positive participants with 1 + log reduction had 6 more weeks of alemtuzumab. Alemtuzumab consolidation was received by 47 participants. One death and 19 of 22 serious adverse events reported from 17 (36%) participants were alemtuzumab-related. MRD eradication from blood and bone marrow was achieved in 39 (83%) participants at the end of consolidation, with 18 (38%) remaining MRD-negative in the blood 6 months later. Of the 18 participants who were MRD-negative at 6 months, the median time to MRD relapse was 46 months, which was similar to patients who were MRD-negative at baseline and were followed up. The 5-year progression-free survival (PFS) and overall survival (OS) of participants who were MRD-negative at 6 months was significantly better than MRD-positive participants [PFS: 78% vs. 39% (P = 0·010), OS: 89% vs. 64% (P = 0·029)].

Histological transformation, typically to diffuse large B-cell lymphoma (DLBCL) is reported to occur in 5%-10% of patients with WM and recent studies have highlighted a possible aetiological role for the nucleoside analogues. It is however becoming increasingly clear that histological transformation is a complex phenomenon and may include clonally unrelated disorders. In order to highlight this pathological heterogeneity we describe 5 patients with diverse histological progression events. These included EBV-associated events namely DLBCL, peripheral T-cell lymphoma and spontaneously resolving mucocutaneous ulcer. A further 2 patients demonstrated a localised plasma cell rich lesion simulating plasmacytoma and a de novo DLBCL arising in an unrelated B-cell clone. It is clear therefore that detailed pathological assessments are required in all suspected cases of transformation and that the pathological heterogeneity demonstrated by this study needs to be taken into account when potential aetiological factors are being assessed.

P>Combination fludarabine (F), cyclophosphamide (C) and rituximab (R) is the standard front-line therapy in chronic lymphocytic leukaemia (CLL), but appropriate treatment of relapsed/refractory CLL is less clear. Combined FC and mitoxantrone (M) has been reported to be effective in a single arm study, and rituximab when added to chemotherapy in CLL is synergistic. A randomized, two-stage, Phase II trial of FCM and FCM-R was conducted in relapsed CLL. The primary endpoint was response rate 2 months after therapy, assessed according to the 2008 International Workshop CLL criteria. In addition, minimal residual disease (MRD) in the marrow was studied 2 months after therapy, with MRD negativity defined as < 0 center dot 01% CLL cells. Fifty-two patients were entered, 26 in each arm. The overall response rates to FCM and FCM-R were 58% and 65% respectively. Combined complete response (CR) and CR with incomplete marrow recovery [CR(i)] was 15% (95% confidence interval [CI]:4-35%) for FCM and 42% (95%CI:23-63%) for FCM-R, with eight patients achieving MRD negativity (3 FCM; 5 FCM-R). The toxicity of both regimens was acceptable. In conclusion, the addition of rituximab to FCM improves the response rates in relapsed CLL, resulting in more complete remissions and without additional safety concerns. Efficacy and safety should be fully tested in a randomized Phase III trial.

Even though chronic lymphocytic leukemia (CLL) is the most prevalent leukemia of the Western world, the development of treatment approaches for CLL has lagged behind the development of approaches to various other hematologic malignancies for a variety of reasons. In recent years, the treatment approach to patients with CLL has evolved rapidly, with the addition of several new prognostic markers, highly effective immunochemotherapy combinations, and attainment of remission up to the point of the eradication of minimal residual disease (MRD). Highly sensitive methods now available to detect MRD can detect a single CLL cell in 10(4) leukocytes using either allele-specific oligonucleotide polymerase chain reaction or four-color or six-color flow cytometry. Over the past decade, several studies have examined the possible advantage of MRD eradication in CLL. This article reviews our current understanding of MRD eradication and analyzes whether it is a desirable goal in the routine clinical treatment of CLL, which will optimize the management of individual patients.

Introduction FCR improves PFS and OS in CLL. Previous non-randomised Phase II trials suggest adding mitoxantrone to FCR (FCM-R) improves outcomes. The rituximab dose in CLL was inferred from lymphoma but in CLL even low doses of rituximab lead to the loss of CD20 expression (“shaving”) suggesting that low dose rituximab with chemotherapy may be effective. Aims The primary objective was to assess whether the CR rate to FCM-miniR was not inferior to FCR. Secondary objectives were MRD eradication, overall response rates and safety. Late endpoints (not reported) were PFS, OS and MRD relapse. Methods ARCTIC was a phase IIB, randomised, controlled, parallel group trial in previously untreated CLL performed in 34 UK centres. 206 patients were to be randomised to FCR or FCM-miniR with an 80% power to show that FCM-miniR did not lead to 10% worse CR rates compared to FCR. A formal interim analysis on the primary endpoint was planned after 103 patients. The schedule for oral FCR was equivalent to iv FCR with iv rituximab given on Day 1 (375mg/m2 Cycle 1; 500mg/m2 Cycles 2-6), oral fludarabine (24mg/m2/day for 5 days; Days 1-5) and oral cyclophosphamide (150mg/m2/day for 5 days; Days 1-5). FCM-miniR included intravenous mitoxantrone (6mg/m2/day) and 100mg rituximab on Day 1 of 6 cycles given 28 days apart. Patients with neutropenia delaying therapy received G-CSF (lenograstim 263mcg/day; Days 7-13) on all remaining cycles. Prophylaxis with co-trimoxazole and acyclovir was used. Results 200 patients were recruited, 100 to each arm. The interim analysis after 103 patients indicated that although the CR rate to FCM-miniR was similar to predicted it was inferior to FCR. The independent DMC advised that the trial would not show non-inferiority for FCM-miniR and recruitment was stopped. A further 97 patients were recruited up to the interim analysis. 21 patients still receiving FCM-miniR were advised to cross-over to FCR. 100 patients received FCR, 79 FCM-miniR and 21 patients randomised to FCM-miniR crossed over to FCR. The treatment arms were well balanced for the following: median age 63 (36-80) and 20% (n=40) over 70; 67.5% male; 17% prog. stage A, 47% stage B and 35.5% stage C. 33.5% had a creatinine clearance under 70ml/min. 60% (87/146) had unmutated Ig genes; 4% (6/164) 17p deletion; and 15% (26/168) 11q deletion. More 11q del patients were randomised to FCM-miniR (15 vs 7). 29.5% (59/200) discontinued therapy early including 30% (n=30) FCR and 35.4% (n=28) FCM-miniR. 141 (70.5%) completed 6 cycles. 93 (49%) of 189 received G-CSF - more with FCM-miniR compared with FCR (54% vs 44%). 117 (58.5%) patients experienced a dose modification; 57% for FCR and 62% for FCM-miniR; the most common being dose delay in 47% of patients. 181 SAE's were reported from 104 patients; 79 events from 49% (49/100) receiving FCR, 80 from 58% (46/79) receiving FCM-miniR and 47% (9/22) cross-over patients. 62.5% of related SAE's were infectious. 3 patients died due to an SAE, 1 FCR and 2 FCM-miniR. We report only the 179 intention-to-treat patients who did not cross-over. To date 146 of 179 patients (82%) have undergone independent central review. 70% (102/146) achieved a CR or CRi; 78% (62/79) FCR and 60% (40/67) FCM-miniR. 13/22 (59%) patients with a baseline creatinine clearance of Conclusion ARCTIC was stopped prematurely as it was clear that the arm containing mitoxantrone and low dose rituximab would not be non-inferior to FCR. However both FCR and FCM-miniR performed well with CR rates of 78% and 60% respectively. This compares favourably to CR rates of 38% for FC in LRF CLL4 Trial and 44% for FCR in the GCLLSG CLL8 Trial. MRD was undetectable in 55% of patients receiving FCR and 46% for FCM-miniR. Oral FCR in front-line CLL results in extremely high CR rates and the eradication of detectable MRD in the majority. Low dose rituximab (100mg per cycle; 600mg total dose compared to 2875mg/m2 for standard FCR) in combination with FCM appears to be effective although FCM-miniR is inferior to full dose FCR. However there is more toxicity associated with the addition of mitoxantrone confounding the comparison of rituximab doses. Disclosures: Hillmen: Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Roche Pharmaceuticals: Honoraria, Research Funding; GlaxoSmithKline: Honoraria, Research Funding; Celgene: Honoraria. Off Label Use: The use of ofatumumab in combination with chlorambucil in previously untreated CLL. The reported trial will support the extension of the ofatumumab licence. Schuh:GSK: Honoraria; Celgene: Honoraria; Mundipharma: Honoraria.

In this study we used bone marrow flow cytometry and immunohistochemistry to evaluate response to fludarabine therapy in patients with Waldenström's macroglobulinemia (WM)/lymphoplasmacytic lymphoma. Responses in serum M protein were typically delayed with a median time to maximum response of 6 months following the completion of therapy (range, 0-18 months). In contrast, bone marrow responses occurred promptly in responding patients such that there were no detectable clonal B cells at the end of therapy in 55% of patients assessed. Persistent monoclonal plasma cells were, however, readily identified by CD138 immunohistochemistry, explaining the persistence of serum M protein in these patients. This simple observation has significant implications for the assessment of responses in WM as well as the design of future therapeutic strategies.

Background: The detection of minimal residual disease (MRD) in CLL using a 0.01% cut-off is an independent predictor of progression-free (PFS) and overall survival (OS) after chemo-immunotherapy. There are several new treatments available and using MRD as a trial endpoint could greatly speed up the identification of the best treatment approaches. MRD assessments can be made in the peripheral blood (PB) or bone marrow (BM) but there is substantial variation in the extent to which different treatments deplete disease in the PB, BM, and nodal compartments. Aims: To compare MRD levels in the PB & BM during/after different treatment approaches to determine differential compartment effects and evaluate the impact on predicting PFS/OS. Methods: The level of residual disease was determined using multi-parameter flow cytometry according to ERIC consensus protocols with a limit of quantification of 10-4 / 0.01% or better; "+" and "-" denotes ≥0.01% and Results: The MRD level in PB & BM samples taken at the same time, either during or within 6 months of treatment, was available in 556 cases of which 85 (15%) showed discordant results at the 0.01% threshold. The frequency of discrepant PB & BM MRD levels varied across treatment and patient groups (table 1). In 322 evaluable cases from the ADMIRE/ARCTIC trials at 3 months after the end FCR-based therapy, 66/322 were BM+PB-, representing 54% of the total MRD+ cases. Discrepant results with >1 log difference between PB & BM MRD levels were most common in refractory patients treated with alemtuzumab. There were no discrepant results in patients undergoing idelalisib or ibrutinib monotherapy although all patients have ≥0.01% MRD to date. A small compartment effect was apparent in patients treated with a combination of ibrutinib and obinutuzumab but the relative difference in PB vs. BM disease level appears to be approximately half that of rituximab in combination with chemotherapy. To determine whether the discrepant results affect the ability of MRD status to predict outcome, we compared the PFS according to BM & PB MRD status. In the CLL202 (fludarabine + alemtuzumab) trial the PFS for BM+PB- cases was similar to BM+PB+ cases (median PFS was 1.6 and 6.5 months respectively) and both were substantially shorter than for BM-PB- cases (median PFS 44.7 months, Log-rank P66 months vs. 54 months vs. 33 months for BM-PB- vs. BM+PB- vs. BM+PB+ respectively, Log-rank P1% MRD in the PB also had >1% disease in the BM. Summary/Conclusion: The compartment effect varies greatly according to patient group and treatment. Rituximab typically results in 0.7log lower disease in the PB compared to BM. Patients on BCR-pathway inhibitors have similar levels of disease in PB & BM even in combination with obinutuzumab. This suggests either that BCR-pathway inhibitors counteract the compartment effects of anti-CD20 antibody treatment, or that obinutuzumab is more effective in depleting bone marrow disease than rituximab. Peripheral blood MRD status may be appropriate for predicting outcome in some settings but underestimates MRD level in a substantial proportion of patients. Bone marrow remains the most sensitive and reliable source for MRD detection but is not required if there is >1% MRD in the blood. Evaluating the compartment effect of specific treatment combinations will help to identify approaches to achieve maximal MRD response using peripheral blood monitoring. Table 1 Compartment effect - difference in MRD detection in PB vs. BM samples across different trials. * median log difference only calculated in cases with >0.01% MRD in both PB & BM so may underestimate compartment effect. Table 1. Compartment effect - difference in MRD detection in PB vs. BM samples across different trials. / * median log difference only calculated in cases with >0.01% MRD in both PB & BM so may underestimate compartment effect. Disclosures Rawstron: AbbVie: Honoraria; Roche: Honoraria; Celegene: Honoraria; Janssen: Research Funding; BD Biosciences: Other: Remuneration; Gilead: Consultancy, Honoraria, Research Funding; Genzyme: Honoraria; GlaxoSmithKline: Honoraria. Munir:Alexion pharmaceuticals: Honoraria. Brock:AstraZeneca: Equity Ownership; GlaxoSmithKline: Equity Ownership. Hillmen:Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Research Funding.

Chronic lymphocytic leukaemia (CLL) is a low grade lymphoproliferative disorder (LPD) characterized by accumulation of monomorphic small mature lymphocytes in peripheral blood, bone marrow and lymphoid tissues. CLL was recognized as a separate clinical disorder towards the beginning of the twentieth century only, although leukaemia was first described in 1845. Even though in the early part of the century there were several case summaries and some mention about CLL in the text book published by Sir William Osler, The Principles and Practice of Medicine, the first comprehensive clinical reports on 80 patients with CLL were published by Minot and Isaacs in 1924 [1–3]. As with any other types of leukaemia, the diagnosis was initially purely based on morphology and over many years several diagnostic tests were introduced which helped to distinguish the various low grade LPDs with somewhat similar morphological appearance, which were originally all considered to be CLL. Currently the diagnosis of CLL can be made in a majority of patients by morphology and immunophenotyping of peripheral blood, but a minority of patients need an integrated approach including tissue histology and the analysis of chromosome deletions, duplications and translocations to make an accurate diagnosis. The distinction of CLL from other similar LPDs is important prognostically as well as increasingly in targeting specific therapies.

Combination fludarabine (F), cyclophosphamide (C) and rituximab (R) is the standard front-line therapy in chronic lymphocytic leukaemia (CLL), but appropriate treatment of relapsed/refractory CLL is less clear. Combined FC and mitoxantrone (M) has been reported to be effective in a single arm study, and rituximab when added to chemotherapy in CLL is synergistic. A randomized, two-stage, Phase II trial of FCM and FCM-R was conducted in relapsed CLL. The primary endpoint was response rate 2 months after therapy, assessed according to the 2008 International Workshop CLL criteria. In addition, minimal residual disease (MRD) in the marrow was studied 2 months after therapy, with MRD negativity defined as0·01% CLL cells. Fifty-two patients were entered, 26 in each arm. The overall response rates to FCM and FCM-R were 58% and 65% respectively. Combined complete response (CR) and CR with incomplete marrow recovery [CR(i)] was 15% (95% confidence interval [CI]:4-35%) for FCM and 42% (95%CI:23-63%) for FCM-R, with eight patients achieving MRD negativity (3 FCM; 5 FCM-R). The toxicity of both regimens was acceptable. In conclusion, the addition of rituximab to FCM improves the response rates in relapsed CLL, resulting in more complete remissions and without additional safety concerns. Efficacy and safety should be fully tested in a randomized Phase III trial.

BACKGROUND: Minimal residual disease (MRD) in CLL is an independent predictor of progression-free and overall survival after chemo-immunotherapy. Data from the DCLLSG trials indicate that a high, intermediate and low risk of disease progression is seen in patients with >1%, 0.01-1%, or AIM: To apply the ERIC consensus 1-tube multiparameter MRD strategy prospectively in two UK clinical trials of FCR-based treatment (ADMIRE and ARCTIC) to determine the survival benefit per log depletion. METHODS: The level of residual disease was determined using multi-parameter flow cytometry according to the ERIC consensus protocol with a limit of quantification of 10-4 / 0.01% or better on 415 patients at 3 months after end of treatment. RESULTS: The level of MRD at end of treatment was a powerful predictor of PFS and OS independent of age, stage, IWCLL response, FISH and IGHV mutation status. Per log reduction in CLL level, the hazard of disease progression decreased by 33% (95%CI 27-38%) and the hazard of dying decreased by 22% (95%CI 13-29%). Although there were statistically significant improvements per log reduction, the DCLLSG 2-log model showed more meaningful differences in outcome over the period of follow-up. The median PFS for patients with >1% vs. 0.01-1% vs 1% MRD predicts equivalent or worse outcome than a clinical PR. PB MRD analysis at 18 months after randomisation (~1 year after treatment) was also strongly predictive of outcome: 98% of patients with CONCLUSIONS: Prospective enumeration of MRD using the ERIC consensus 1-tube multiparameter protocol confirms that MRD at end of treatment is a powerful independent predictor of progression-free and overall survival. Post-treatment follow-up using peripheral blood MRD could be more informative than clinic assessments because patients with 1% MRD at end of treatment in the peripheral blood have a similar outcome to those with a clinical PR and a bone marrow assessment is not informative in these cases. The level of MRD is highly informative with sequential improvements in outcome per log depletion. The DCLLSG 2-log categorisation (>1%, 0.01-1%, or 6yrs. Figure 1. the level of MRD in the bone marrow at 3 months after treatment is highly predictive of outcome with >1% MRD equating to 5yrs median PFS Figure 1. the level of MRD in the bone marrow at 3 months after treatment is highly predictive of outcome with >1% MRD equating to 5yrs median PFS Figure 2. sequential PB MRD analysis identifies patients with negligible risk of requiring treatment within the following 12 months. Figure 2. sequential PB MRD analysis identifies patients with negligible risk of requiring treatment within the following 12 months. Disclosures Rawstron: BD Biosciences: Patents & Royalties; Roche: Honoraria; Celgene: Honoraria; Abbvie: Honoraria; Gilead: Honoraria, Research Funding; Pharmacyclics: Research Funding. Gregory:Celgene: Honoraria; Janssen: Honoraria. Hillmen:Roche: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Celgene: Research Funding.

Background Eradication of minimal residual disease (MRD) is an independent predictor of survival outcome in patients with chronic lymphocytic leukaemia (CLL) receiving fludarabine and cyclophosphamide (FC) or fludarabine, cyclophosphamide, and rituximab (FCR) as first-line treatment. However, the independent prognostic relevance of MRD status in other therapeutic settings is not clear. The goal of this study was to investigate the independent importance of achieving MRD negativity in CLL on progression-free survival (PFS) and overall survival (OS) with different treatments in frontline and relapsed or refractory settings compared with known prognostic markers. Methods We included all patients at our centre in Leeds and associated hospitals in West and North Yorkshire who had completed treatment for CLL from 1996 to 2007, achieved at least a partial response, and received an MRD assessment from a bone-marrow specimen after treatment. MRD assessments were done by multiparametric flow cytometry using CD5–CD19 in combination with CD20, CD22, CD32, CD38, CD79b, and CD81 in accordance with the international harmonised approach. 133 patients (17 receiving fludarabine, 65 fludarabine-based combination therapies, 26 alemtuzumab, and 25 other treatment including chlorambucil and autologous stem-cell transplantation) were followed up for a median of 5·2 years (IQR 3·0–7·3) to assess PFS and OS. Findings MRD negativity (defined as less than one CLL cell in 10 000 leucocytes) at the end of therapy independently correlated with both PFS (hazard ratio [HR] 3·22 [95% CI 2·09–4·97], Cox proportional hazards model p vs 31) and OS (78 vs 64) than did MRD-positive individuals without such adverse cytogenetic abnormalities. Interpretation MRD status is a powerful independent predictor of survival outcome in CLL across a range of therapeutic approaches including in frontline and relapse settings. MRD negativity is the most appropriate therapeutic goal for CLL patients who are fit enough for such an approach. Funding None.

Large cell transformation is a well-recognised late event in follicular lymphoma, mantle cell lymphoma (MCL), Waldenstrom macroglobulinemia (WM) and chronic lymphocytic leukemia (CLL). This is trad...

Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired bone marrow disorder characterised by intravascular hemolysis and hemoglobinuria, potentially life-threatening thrombosis and an association with aplastic anemia. Most of the clinical features and complications of PNH are due to the unopposed activity of complement due to the absence of CD59 and CD55, two key regulators of complement. Eculizumab prevents the cleavage of C5 complement thereby preventing terminal complement activity and protecting PNH cells from lysis. The inhibition of C5 preserves the early part of complement pathway and leads to the build up of C3 on the PNH red cells, perhaps in part due to their lack of CD55. The majority of PNH patients receiving eculizumab have evidence of extravascular haemolysis that can be clinically significant, including with anemia, hyperbilirubinemia and in some a continued requirement for transfusions. This extravascular hemolysis in thought to be due to the C3 loading of PNH red cells. Methods We report the C3-loading of the PNH red cells from 119 patients treated with eculizumab and correlate this with hemoglobin, LDH, bilirubin, reticulocytes and transfusions. We have studied genetic polymorphisms that affect both C3 and FCγR. We have genotyped 46 eculizumab patients for a functional mutation in the C3 gene (rs2230199). The two alleles of this gene can be distinguished by the presence or absence of a HindIII restriction site that distinguishes the electophoretically slow (arg80) from the electrophoretically fast (gly80) allotype. The fast (C3F) allotype allele of this snp is associated with a range of disorders including age-related macular degeneration, IgA nephropathy, systemic vasculitis and partial lipodystrophy. APL-1 is a small cyclic peptide that binds to and inhibits the activation of complement C3. APL-2 is a large conjugate of APL-1 with enhanced bioactivity and a long systemic half-life. APL-1 and APL-2 molecules as well as other complement inhibitors were studied for lysis of red cells and C3 loading in vitro in a modified Ham test in which flow cytometry was used to identify non-lysed cells. Results Out of the 119 Eculizumab treated patients, 55 (46.2%) required at least one transfusion on treatment. 110 patients had C3 detectable by flow cytometry on their PNH red cells (mean of 19.8%; range: We studied one functional polymorphism in the Fcγ receptor but this showed no correlation with haemolytic parameters. Conversely, for the C3 polymorphism eculizumab-treated patients homozygous for the slow (C3S) allele had a significantly higher degree of C3 loading on PNH red blood cells with C3S/C3S having a mean of 33.7% C3 loaded PNH red cells (n=26), C3S/C3F 19.0% (n=19) and C3F/C3F 12.8% (n=3)(all P In vitro experiments show that inhibitors of C5, such as eculizumab, protect the PNH red cells from lysis but lead to a very rapid deposition of C3 on the PNH red cells. However both APL1 and APL2 demonstrated similar protection of PNH red cell lysis with virtually no C3 loading on PNH red cells. Conclusion A significant proportion of patients on eculizumab experience extravascular haemolysis with the majority of patients developing C3 loading. We show for the first time that a functional polymorphism in the C3 gene is associated parameters of hemolysis. The S(low) allele alters the function of C3 protein and appears to be associated with extravascular haemolysis in patients with PNH. The C3 inhibitors, APL-1 and APL-2, protect PNH red cells and prevent C3 loading in vitro and if safe to be given chronically would be expected to reduce extravascular hemolysis significantly. Disclosures: Hill: Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Kelly:Alexion: Honoraria. Richards:Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Hillmen:Alexion: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Background The advent of anti-CD20 therapy has significantly improved the management of various autoimmune conditions including rheumatoid arthritis (RA). The persistence of B-cells and/or plasmablasts in the immediate post-therapeutic phase (2 weeks) by highly sensitive flow cytometry has been shown to predict poor clinical response (1). However in patients with complete depletion, the return of clinical symptoms post B-cell reconstitution is variable, although clinical relapse is rare in the absence of return of B-lineage cells. Objectives To establish the reconstitution pattern of B-cells post anti-CD20 therapy in the phase prior to clinical relapse. Methods 79 samples from autoimmune patients and 36 disease controls were evaluated by 10-parameter flow cytometry. Scatter properties and surface expression of CD19, CD20, CD27 and CD38 was used to classify the B-lineage cells into transitional, naive, memory B-cells and plasmablasts. Further analysis of 34 different surface markers on selected RA patients and controls was used to identify the 4 most informative markers that might help to predict disease relapse. Results We were able to classify the memory B-cells into 16 different subsets using the 4 markers identified as above. We identified a subset with plasmablast like phenotype (CD24 Lo CD84 Lo CD95 Hi similar to plasmablasts and CD19, CD20 & CD38 expression levels similar to memory B-cells), which constituted a higher proportion of memory B-cells and was the dominant subtype in the majority of RA patients relapsing post anti-CD20 therapy (14/18) compared to controls (2/10). The dominance of this subset was not seen in patients with Rituximab-naive activeRA (0/22) and only in 1 patient during remission (1/9) with different biologics/DMARDs. This abnormal reconstitution pattern was not seen in patients treated with Rituximab for B-cell lymphomas (0/4) or following pre-renal transplant Campath (Anti-CD52) conditioning (0/4). The samples from lymphoma patients in remission were collected 12-18 months since the last Rituximab dose and 3-6 months post-therapy in case of Campath. Conclusions The abnormal reconstitution of memory B-cell subsets is associated with clinical relapse following anti-CD20 therapy for RA and is likely to be related to the disease process rather than the direct effect of B-cell depletion. The patients with such altered memory B-cell reconstitution might benefit from early re-treatment. References Dass et al Arthritis Rheum. 2008 Oct;58(10):2993-9. Disclosure of Interest None Declared

Abstract 3899 Introduction: Combination chemo-immunotherapy has improved the survival in previously untreated CLL, but the treatment of relapsed and fludarabine refractory CLL continues to be challenging. Inhibiting the B-cell receptor (BCR) signalling pathway appears to be beneficial in the majority of patients but is not sufficient to eradicate disease and combination approaches are already being investigated. We have previously undertaken large scale protein expression profiling in CLL and identified several molecules involved in neurotransmission that are highly expressed in CLL. Aim: To explore signalling mechanisms in neuronal markers that are aberrantly expressed in CLL, and to determine whether they have therapeutic potential in combination with BCR inhibitors. Methods: RNA expression data (Haslinger et al; Journal of Clinical Oncology. 2004) was used to identify candidate antigens that could be screened for protein level expression. Screening was restricted to antigens expressed on plasma membrane with an extracellular epitope and a commercially available antibody suitable for testing in combination with mouse monoclonal gating antibodies (i.e. fluorochrome-conjugated monoclonal, non-conjugated mouse monoclonal labelled using the zenon technique, or rabbit polyclonal detected using fluorochrome-conjugated secondary goat F(ab')2 anti-rabbit antibody). In vitro viability experiments were performed using either mononuclear cells, separated using lymphoprep, or purified CLL cells isolated with a B-cell isolation kit (Miltenyi Biotec), cultured with or without the presence of CD40L transfected fibroblasts as a supporting stromal layer. Viability was assessed using Annexin-V, 7-AAD and counting beads. To study pathway interactions, the B-cell receptor was stimulated by crosslinking using goat F(ab')2anti-human IgM or IgD and signalling responses were assessed by Syk phosphorylation status (Syk pY348 PE, BD Phosflow) and calcium flux (Fluo-3/Fura red, Invitrogen). Results: 84 target antigens were assessed of which 11 showed binding on all mononuclear cells and 9/84 showed binding on CLL cells and/or normal B-cells but not other leucocytes. Of the B-cell targets, 4/9 molecules have a role in neurotransmission, such as the nicotinic acetyl choline receptor subunit β4 (CHRNB4) and dopamine receptor D4 (DRD4). CHRNB4 had the highest level of expression on B-cell and CLL surface membranes and therefore other subunits of nicotinic acetyl choline receptor were further evaluated using RT-PCR. Of the 15 subunits tested 10 were expressed on CLL cells suggesting the possibility of an important role in the pathophysiology of CLL. However, in vitro experiments did not show a significant effect on viability of CLL cells with either acetyl choline or its antagonist mecamylamine. Unlike acetyl choline, dopamine was found to be effective in reducing CLL cell survival in vitro, in a dose dependent manner with an LC50 of 8.2μM (95%CI- 5.2 to 12.9, n=6). Assessment of dopamine receptor agonists and agonists demonstrated that the D2-like family specific antagonist domperidone enhances the activity of dopamine and also has single agent activity in reducing in vitro CLL cell viability. To determine the effect of dopamine on BCR pathway signalling, syk phosphorylation and calcium flux after IgD crosslinking were measured. Stimulation of the BCR pathway by crosslinking using goat F(ab')2 anti human IgD increased the mean fluorescent intensity of syk phosphorylation by 1.9 fold (p=0.0024) compared to 1.5 fold (p=0.0186) when pre-incubated with dopamine and 1.2 fold (p=0.0059) with a syk inhibitor. Similarly the mean percentage rise in CLL cells fluxing calcium has fallen from 20.1% to 8.2% (p=< 0.0001) when treated with dopamine and to 5.6% (p=< 0.0001) when treated with syk inhibitor. In vitro testing demonstrated a synergistic effect in blocking of the D2 and the BCR pathways as a combination of PI3 kinase δ inhibitor, GS-1101, along with domperidone reduced CLL cell viability more efficiently than either agent alone. Conclusion: Systematic screening has identified several new targets in CLL, in particular several neuronal markers which are highly expressed and functionally active. Biological investigations suggest that these neuronal receptors could play an important role in the pathophysiology of CLL and show potential synergies with therapeutically active BCR inhibitors. Disclosures: Hillmen: Alexion Pharmaceuticals, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 60 Background: The length of remission in patients with chronic lymphocytic leukemia (CLL) is dependent on the level of minimal residual disease (MRD) at the end of therapy, regardless of the therapy received. The conversion of remissions from MRD positive to negative should prolong remissions and survival. Several small studies have used alemtuzumab as consolidation therapy following conventional chemotherapy but with concerns over toxicity; primarily due to immune suppression and infections. The dose and timing (i.e. interval between prior chemotherapy and alemtuzumab) seem to be critical. Trial Design: The Phase II NCRN CLL207 Trial assessed alemtuzumab consolidation post-chemotherapy. Between 6 and 24 months from completing treatment (1 to 3 prior therapies) blood was screened for MRD using multi-parameter flow cytometry with a sensitivity of less than a single CLL cell in 10,000 leucocytes (0.01%). The marrow of MRD positive patients was assessed to quantify the CLL before treatment with alemtuzumab at a dose of 30mg given subcutaneously 3 times a week for 6 weeks, at which time the marrow was repeated. MRD negative patients and non-responders stopped therapy; MRD positive patients with at least one log reduction in MRD continued therapy for a further 6 weeks. All patients received prophylaxis with co-trimoxazole and aciclovir as well as weekly cytomegalovirus (CMV) monitoring by PCR. It was pre-determined that at least 14 of up to 54 (26%) patients would need to be converted from MRD positivity to negativity to justify further studies of this treatment strategy. Results: 47 patients received alemtuzumab in NCRN CLL207 with median age of 58 yrs (40-77) and 35 (74.5%) males. There was a median of 2 prior therapies (range 1 to 4) with 46 pts receiving fludarabine combinations as the latest therapy and 9 receiving rituximab-containing combinations. There were a total of 21 SAE's in 17 (36.2%) pts with 2 (4.3%) treatment related deaths (EBV-LPD and a parainfluenza infection). Alemtuzumab was stopped in 6 patients before week 6 mainly due to toxicity, 32 patients received 6–8 weeks of treatment and 9 patients received a 12-week course. G-CSF was given when the neutrophil count fell below 1 × 10^9/l and 14 (30%) patients required G-CSF during alemtuzumab with an additional 13 (28%) receiving G-CSF after completion of alemtuzumab. Positive CMV PCRs were detected in 21 (45%) patients, all of whom were successfully treated with pre-emptive antiviral therapy. Prior to alemtuzumab 24 patients were in complete remission (CR) and 23 were in partial remission (PR). Three months after alemtuzumab 13/23 (56%) PR converted to CR. 39/47 (83%) patients had MRD negative marrows at the end of alemtuzumab, 7 (15%) remained MRD positive and 1 (2%) was not evaluable. Blood MRD assessment 6 months after completing alemtuzumab showed that 15/31 (48%) MRD negative patients became MRD positive, although all except 2 had low CLL levels (below 0.1×10^9/l) at this time-point. Therefore overall 16/39 (41%) patients were MRD negative 6 months after completing alemtuzumab and of these 8/9 (89%) remained MRD negative in the blood at 12 months. Therefore MRD negativity in the blood at 6 months seems to better predict for persistent MRD negativity than the marrow at the end of therapy and appears to be the most appropriate assessment for the outcome of consolidation. Patients who are MRD negative at this time-point usually have durable remissions. 6 of the 9 patients receiving 12 weeks of alemtuzumab were MRD negative at the end of treatment but only 1 (11%) remained MRD negative in the blood at 6 months. In contrast 33/38 patients receiving up to 8 weeks of alemtuzumab were MRD negative at the end of treatment and 15 (39%) remained MRD negative at 6 months suggesting that the patients who benefit most from alemtuzumab consolidation are those who are MRD negative at 6 weeks. Conclusion: Alemtuzumab consolidation results in the eradication of detectable MRD in 83% of patients and 41% remain MRD negative 6 months later. Consolidation with alemtuzumab is associated with mainly infective toxicities, which are largely manageable with prophylaxis and close monitoring. These results justify the continued investigation of this approach in CLL within a clinical trial setting and with appropriate monitoring of patients. To this end we are now commencing a randomized Phase III trial of consolidation with alemtuzumab compared to observation (the CLARET study). Disclosures: Pocock: F.Hoffmann-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees. Rawstron:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; BD Bioscience: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Genzyme: Honoraria. Dearden:Roche Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hillmen:Genzyme: Honoraria, Research Funding, Speakers Bureau; Roche Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau; Glaxo Smith Kline: Honoraria, Research Funding.

Abstract 922 Background AND TRIAL DESIGN: Patients with fludarabine refractory chronic lymphocytic leukemia (CLL) have a very poor prognosis with conventional chemotherapy with a median survival of 10 months and Results: 53 patients were enrolled but 2 patients died before receiving alemtuzumab and 1 withdrew consent. 50 patients have completed therapy (median duration of alemtuzumab was 18.8 months with a median dose of 1370mg [106-2323mg]). Response assessment was possible in 49 patients of which 8 patients achieved a complete remission (CR) and 16 patients achieved a partial remission (PR) giving an overall response rate of 49%. 17 patients had fludarabine added to the alemtuzumab (6 PR, 11 non-responders [NR]) and 3 had an improvement in response (1 from PR to CR and 2 from NR to PR). Median overall survival (OS) was 20.2 mo (CI 95%, 13.2–27.1) and progression free survival (PFS) was 13.0 mo (CI95%, 10.3–15.7). The 5-year OS and PFS rates were 24% and 9% respectively. Patients who achieved a CR, PR and less than a PR had a median OS of 63.6 mo, 20.2 mo and 18.2 mo, respectively and a median PFS of 47.1 mo, 13.8 mo and 7.5 mo respectively. Of 18 patients evaluable for MRD status, 7/18 achieved an MRD negative response and 11 were MRD positive with OS of not reached vs 13 mo (p=.02) and PFS of 47.1 vs 22.3 mo (p=.02), respectively. Of the 7 patients achieving MRD negativity, 5/7 (71%) were alive at 5 years. The IGHV gene was unmutated (>98% germ line homology) in 23/31 evaluable patients. Comparing unmutated with mutated for OS gave 18.2 and 13.1 mo (p=0.3) and PFS of 16.6 and 12.3 mo (p=0.08). FISH revealed poor risk deletions (11q and/or 17p) in 21/45 patients (17p- in 10, 11q- in 8, both in 3). 25/38 patients had dysfunction p53 in a functional assay previously reported by Pettitt et al (Blood, 2001; 98: 814–822). The table shows the lack of impact on PFS and OS. In addition to prospective FISH evaluation we have performed retrospective SNP microarray using Illumina human CytoSNP-12 BeadChip on stored bone marrow and peripheral blood slides. Each BeadChip includes a complete panel of genome-wide tag SNPs and markers (>330,000 in total) targeting all regions of known cytogenetic importance. 36 patients were tested, SNP analysis identified all of the deletions present in more than 20% of CLL cells detected by FISH except in 1 patient due to technical issues. SNP analysis identified 2 additional patients with abnormalities in 17p (one deletion and one copy neutral loss of heterozygosity) and 2 more with deletions in 11q. A number of other chromosomal aberrations (average number per patient =2.6, with a maximum of 12 in one patient) were also detected although no recurrent abnormalities were identified. Summary: We report that subcutaneous alemtuzumab is effective in poor-risk fludarabine-refractory CLL. In this group, patients with poor risk deletions havenÕt got a survival disadvantage over the non deleted group presumably because all of the patients were resistant to conventional chemotherapy regardless of whether a p53 pathway abnormality could be detected. SNP microarray is a useful research tool in studying genomic aberration from archived bone marrow and blood samples including in stored blood and marrow slides. The long term survival data shows that subcutaneous alemtuzumab improves the OS and PFS in fludarabine refractory CLL particularly in patients achieving MRD negative remissions. Disclosures: Rawstron: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; BD Bioscience: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Genzyme: Honoraria. Dearden:Roche Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees. Pettitt:Glaxo Smith Kline: Research Funding. Kennedy:Roche Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Abstract 540 The depth of remission in CLL correlates with survival in a large number of trials regardless of the therapy used and the depletion of minimal residual disease (MRD), when reported, is usually associated with improved progression free and overall survival. However it is not clear whether this improved outcome is due to the attainment of MRD negativity or whether MRD eradication is a surrogate for other variables that predict for good response. In order to address the true impact of achieving MRD negativity we present data from 137 patients with CLL who were treated between 1996 and 2007, achieved a good clinical response and had bone marrow examined post-therapy to assess remission status including MRD. The MRD assessment was performed in a single laboratory (HMDS, Leeds, UK) using multicolor flow cytometry capable of detecting minimal residual disease (MRD) to a level of one CLL cell in 10000 leukocytes as recently recommended in the IWCLL Guidelines. Patients were followed for a median duration of 3.1 years (range 0.2 - 12.7) after treatment with chlorambucil (n=13), fludarabine (n=17), fludarabine and cyclophosphamide (n=58), fludarabine and cyclophosphamide with mitoxantrone and/or rituxumab (n=8), alemtuzumab (n=29), autologous stem cell transplantation (n=7) and various other treatments (n=5). Of these, 48 achieved a complete response (CR), 24 achieved a CR with incomplete marrow recovery (CRi), 27 achieved a nodular partial response (nPR) and 38 achieved a partial response (PR). Altogether 58 individuals (42.3%) were MRD negative at the end of treatment, including 28 CR, 20 CRi, 3 nPR and 7 PR patients. Results of the univariate and multivariate analyses are summarized below: Progression Free Survival Overall Survival Parameter (Cut-off points) Univariate (Log-Rank) P Value Multivariate (Cox) P Value Hazard Ratio (95% CI) Univariate (Log-Rank) P Value Multivariate (Cox) P Value Hazard Ratio (95% CI) Age pre-treatment (55,70) 0.122 0.001 0.002 2.20 (1.32-3.64) Hemoglobin pre-treatment (10g/dL) 0.435 0.028 0.921 1.03 (0.60-1.78) Leukocytes pre-treatment (30×10 9/L) 0.235 0.092 Platelets pre-treatment (100×10 9/L) 0.002 0.432 0.82 (0.50-1.34) 0.028 0.465 0.81 (0.46-1.43) Prior treatment (Yes/No) 0.004 0.014 1.94 (1.14-3.29) 0.035 2.29 (1.06-4.94) Prior fludarabine (Yes/No) 0.611 0.042 0.078 1.85 (0.93-3.66) Type of treatment 0.017 0.209 0.89 (0.75-1.06) 0.214 Response (CR vs nPR vs PR) 0.023 1.45 (1.05-2.00) 0.015 0.624 1.10 (0.75-1.62) MRD Status ( 5.32 (2.70-10.5) 0.002 0.012 2.40 (1.21-4.76) Age, number of prior therapies and MRD negativity were independently correlated with overall survival. MRD negativity in the marrow at the end of therapy was independently significant in multi-variate analysis including when analysed against age, stage, prior therapy, IWCLL response assessment and cytopenia. One of the most striking findings was in patients having their first CLL treatment. Of the 58 patients in this series who had achieved a clinical response to first line therapy 24 patients (21 patients following FC, 1 FCR, 1 chlorambucil and 1 fludarabine a autologous SCT) achieved an MRD-negative remission and 34 were MRD-positive. With a median follow-up of 38 months (range 7-153) the 5 year PFS for MRD negative patients was 89% (95% CI 55-97%) compared to 0% for the MRD positive patient (95% CI In conclusion, we demonstrate that achieving MRD negativity in CLL is an independent predictor of survival in multivariate even when a variety of different treatment approaches are considered and regardless of the line of therapy.. This is the strongest evidence yet that achieving MRD negativity is the most appropriate aim of therapy in CLL for patients who are fit enough for such an approach. Furthermore patients who achieve MRD negativity after their first therapy have a 5 year PFS of 89% and a 5 year overall survival of 95% suggesting that the optimal time to attempt to achieve MRD negativity is with first line therapy. Disclosures: Hillmen: Roche Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees.