ALBERT

Description: INTRODUCTION: CLL is a disorder with a wide variation in outcome. Patients with adverse cellular features are often refractory to treatment and have a short overall survival. Individuals with CLL-type MBL are unlikely to require treatment and in most cases will eventually die of an unrelated cause. Many factors that predict a poor outcome have been identified, including stage, IGHV mutation status, ZAP-70 expression, and deletions of chromosomes 17p (TP53) and/or 11q23 (ATM). Deletions and mutations in TP53 are generally not presenting features and appear to require clonal evolution. One hypothesis is that the degree of intraclonal variation in genes targeted by the somatic hypermutation machinery, e.g. IGHV and BCL6, may predict the potential for clonal evolution. We have previously tested 66 antigens for their capacity to differentiate proliferating CLL cells, resting CLL cells and normal B-cells and identified 30 potentially relevant markers, including common markers such as CD38 and less frequently used markers such as the Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1). AIM: To compare the expression of relevant cell surface markers with the degree of intraclonal variation in the IGHV and BCL6 genes and to determine if these markers can be used to differentiate CLL-type MBL and CLL with or without adverse biological features. METHODS: The cell surface phenotype was assessed by 6-colour cytometry in 133 patients: 22 CLL with deletion 17p or 11q23, 69 CLL with no adverse prognostic chromosomal abnormalities, and 42 MBL. Surface phenotype was also compared with IGHV mutation status in a cohort of 29 CLL patients (16 ≤2% IGHV mutation, 13 〉2% IGHV mutation). These antigens were also assessed using 4-colour flow cytometry in 20 cases (4 MBL, 16 CLL) and compared with IGHV & BCL6 mutation status and degree of intraclonal variation (defined as the proportion of mutations that were detected in a single clone only), and with ZAP-70 (AF488-1E7.2) expression. RESULTS: CLL cases with ≤2% IGHV mutation showed increased expression of CD38 (6.8 fold, p 0.02), CD49d (4.9-fold, P = 0.04), IgD (2.0-fold, P = 0.05), ZAP-70 (1.5-fold, P=0.04) and decreased expression of LAIR-1 (6.2-fold, P = 0.003) in comparison to CLL cases with 〉2% IGHV mutation. CLL cases with deletions of 17p and 11q23 showed decreased expression of CCR6 (1.7-fold, P = 0.0001), IgD (1.3-fold, P = 0.03) and LAIR-1 (7.1-fold, P2% overall IGHV mutation in both IGHV (median 0.075% vs. 0.049% unique mutations, P〉0.05) and BCL6 (median 0.10% vs. 0.095% unique mutations, P〉0.05). However, there was an inverse relationship between BCL6 and IGHV intraclonal variation and cases with the highest levels of BCL6 intraclonal variation showed significantly decreased expression of CD39 (1.9-fold, P = 0.04) and LAIR1 (4.7-fold, P = 0.019). CONCLUSIONS: There were no markers or marker combinations that could discriminate MBL from CLL. The key differences were decreased expression of markers that are expressed during cell cycle, i.e. CD23, and adhesion markers such as CD62L and CD49d. These markers show sequential changes with disease stage, supporting the hypothesis that cellular interactions are central to the accumulation and expansion of CLL cells. However, the marker most consistently associated with adverse biological features is LAIR1, which is weak or negative in CLL with ≤2% IGHV mutation, high levels of intraclonal variation and TP53 or ATM deletions. LAIR-1 is an inhibitory receptor involved in regulating classs-witching. LAIR1 is strongly expressed in normal circulating peripheral B-cells. As with other prognostic markers, expression is a continuous variable and therefore a suitable cutoff will need to be identified. However, fluorochrome-conjugated antibodies are readily available and expression on CLL cells is stable for several days in EDTA samples which should minimise inter-laboratory analytical variation. LAIR1 expression in CLL is more closely associated with IGHV mutation status than CD38 or ZAP-70 expression. LAIR1 is a promising prognostic marker that appears to be central to the development of aggressive CLL as there is a strong association between downregulation of LAIR1, intraclonal heterogeneity in BCL6 and development of TP53 and ATM deletions.

Description: Abstract 2002 Introduction: Monoclonal B-cells with a Chronic Lymphocytic Leukemia (CLL) phenotype are detectable in more than 10% of adults in the general population using high sensitivity flow cytometry assays designed to detect minimal residual disease after treatment. However, the prevalence of MBL with a phenotype corresponding to other B-lymphoproliferative disorders (B-LPD) is less than 2% of the general population even using the most sensitive assays. No studies have reported CD10+ MBL whereas several studies have demonstrated that the t(14;18) is frequently detectable in the general population at a level which should be detectable by flow cytometry. The lack of CD10+ MBL may indicate that the t(14;18) alone rarely results in the expansion of a clonal B-cell population in the blood, or that currently available assays are inadequate for detecting circulating follicular lymphoma. We have previously investigated 66 markers to determine the best candidates for diagnosis and monitoring B-LPD, which were then tested in over 1500 cases. We have developed a single-tube assay to screen for residual disease that can detect lymphoma cells when they represent as few as 1 in 10,000 leucocytes. The aim of this study was to asses the frequency with which lymphoma-phenotype monoclonal B-cells are detected in the general population using a high sensitivity assay. Methods: Cells from 679 individuals (342 male, 337 female, median age 64, range 40–99) with a normal blood count and no current or prior history of cancer were incubated with antibodies to Kappa, Lambda, CD19, CD20, CD5, CD10, LAIR1, CXCR5 and 0.5 million cells were acquired using a BD FACSCanto II cytometer. In cases with detectable MBL further phenotyping was performed and B-cells were selected and stored for FISH and molecular clonality studies. Results: MBL was detected 129/679 cases (19.0%): CLL-type MBL in 86/679 cases (12.7%), non-CLL MBL in 60/679 cases (8.8%) with both CLL-type and non-CLL MBL were present in 17/679 cases (2.5%). Within the non-CLL MBL group, in 21/60 cases the monoclonal B cells had no additional features to confirm a neoplastic population and it was not possible to ascertain whether these were neoplastic cells or a reactive population with a highly skewed kappa/lambda ratio. Of the remaining 39 specimens: none showed evidence of germinal centre differentiation; 12 (1.7% of total) showed a phenotype most consistent with marginal zone lymphoma/lymphoplasmacytic lymphoma; and 27 cases expressed strong levels of LAIR1 coupled with strong CD19 and CD20 and an extended phenotype that is relatively rare in clinical B-LPD, restricted to hairy cell leukemia and a small proportion (

Description: Solitary plasmacytoma of bone (SPB) typically present as single destructive lesions within the spinal column or long bones. Local radiotherapy is the treatment of choice but approximately 50% of patients progress to myeloma. Many patients have a monoclonal protein detectable in their serum and/or urine at diagnosis and persistence of this for greater than 1 year after radiotherapy predicts for progression to myeloma. Identification of high risk patients at the time of diagnosis is clearly desirable as it would enable risk stratification and more careful monitoring of patients. Although prophylactic adjunctive chemotherapy has not definitively been shown to benefit unselected patients with SPB, future therapeutic advances might be targeted on patients with a high risk of progression. Interestingly it has recently been shown that patients positive for serum free light chains are at greater risk of progression to myeloma. We and others have previously demonstrated that neoplastic bone marrow plasma cells are distinguishable from their normal counterparts by virtue of their lack of CD19 expression and/or their aberrant expression of CD56. We have developed a multiparameter flow cytometry assay which predicts outcome following autologous transplantation in myeloma patients and risk of progression in patients with MGUS. In this study we have applied this assay to assess the staging bone marrow specimens from patients with biopsy proven SPB for the presence of occult disease at sites distant to the primary lesion. 52 patients were included in this analysis (31 male, 21 female; median age 65) and in each case the staging bone marrow aspirate and trephine biopsy (obtained from a site distant from the SPB, typically the right iliac crest) was not indicative of myeloma (30% CD19− and/or CD56+ as per convention) were demonstrable in 35/52 (67%). Neoplastic plasma cells when present comprised a median of 70% (35–100%) of bone marrow plasma cells. 21 patients (40%) developed myeloma with a median time to progression of 476 days (range 18–1632). Progression occurred in 18 of the 35 (51%) patients with neoplastic plasma cells in their staging marrows and in 3/17 (18%) patients with a normal phenotypic profile. The difference was significant using Chi-square analysis with Yates’ correction for continuity (p=0.04). The overall risk of progression was similar in patients with a “myeloma pattern” in which 〉90% of bone marrow plasma cells had a neoplastic phenotype (5/12, 42%) and those with an MGUS or mixed pattern in which distinct populations of both normal and neoplastic cells are demonstrable (13/23, 57%). We would conclude that neoplastic plasma cells are frequently found at bone marrow sites distant to SPB and that their presence predicts for progression to multiple myeloma. Trials of adjuvant systemic therapy are warranted in this group.

Description: Flow cytometry is a powerful tool for diagnosis and detection of minimal residual disease (MRD) in patients with monoclonal gammopathies. Multi-parameter MRD flow analysis is more sensitive than serum immunofixation for the detection of residual disease and is as effective as PCR analysis for predicting outcome after high-dose therapy. Attaining a limit of detection of 0.01% abnormal plasma cells in every patient requires analysis of several neoplastic markers. However, basic immunophenotyping coupled with clonality assessment can be informative in many patients. This approach can also be helpful at diagnosis in MGUS patients to determine whether the primary abnormal component is plasmacytoid or lymphoid. The aim of this study was to develop a single six-colour assay that could be used as a rapid and sensitive screen for the presence of neoplastic plasma cells. Bone marrow aspirate samples were assessed from 83 patients at diagnosis with a suspected plasma cell disorder (median age 69.1 years, 48 male and 35 female) and from 62 patients with myeloma after treatment (median age 61.0 years, 40 male and 22 female). Cells were washed and surface-labelled with CD19 PE, CD38 PE-Cy7, CD138 APC and CD45 APC-Cy7 then fixed, permeabilised and incubated with Kappa FITC and Lambda PE-Cy5. The assay allows assessment of B-cell surface-Ig and plasma cell cytoplasmic-Ig light chain restriction and abnormal plasma cell phenotype according to CD19 and CD45 expression. In presentation cases with more than 10% plasma cells by morphology, neoplastic plasma cells were present in 48/50 samples with one reactive plasmacytosis (32% plasma cells) and one patient with Castlemans disease (11% PC). In the remaining 33 cases with

Description: Bone marrow examination is an established component of the process used to stage patients with diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and classical Hodgkin Lymphoma (CHL); and it is a general requirement of clinical trials. The procedure involves significant discomfort and inconvenience to the patient, as well as expense for the NHS, and this study was designed to evaluate the clinical utility of the information derived from staging bone marrow examinations. All patients presenting with DLBCL (n=1908), CHL (n=604) and FL (n=770) between 1st Sept 2004 and 31st August 2011 in the UK's population-based (3.6M) study area (Haematological Malignancy Research Network - www.hmrn.org) were included. Comprehensive clinical data, including diagnostic, prognostic, outcome and socio-demographic information are available for all patients. All diagnostic and staging specimens are reported in a single specialist haematopathology laboratory (the Haematological Malignancy Diagnostic Service – www.hmds.info). HMDS routinely undertake morphology and flow testing on bone marrow aspirate and trephine biopsies; while further molecular and cytogenetic tests are carried out at clinical discretion. This study aimed to assess the impact of bone marrow examination on the calculation of disease specific prognostic indices. Bone marrow examinations were performed in 85% of patients, providing morphological evidence of disease in 10.2% of DLBCL, 7.2% of CHL and 36.5% of FL. In 18 patients with DLBCL and 16 patients with CHL the bone marrow was the presenting site of disease and this was associated with a very poor clinical outcome. In DLBCL knowledge of the bone marrow result increased the IPI by 1 point in 4.0% and by 2 points in 1.1% of cases. Similarly, the Hasenclaver index increased by 1 point in 3.6% of CHLs and the FLIPI increased by 1 point in 9.0% of FLs. In the case of DLBCL, 70% of these patients had an IPI score of 〉2 before the results of the marrow examination were added. As well as the assessment of prognosis, the results of bone marrow examination are a component of a number of key clinical decisions. Patients with stage 1 FL are routinely treated with radiotherapy with curative intent. In the absence of a bone marrow result, an additional 27 patients would have potentially been treated with radiotherapy. The 5 year relative survival (corrected for underlying population mortality rates) of this group was similar to those with stage 2 disease on watch and wait, and was slightly inferior to that seen in true stage 1 disease. Similarly, patients with stage1A DLBCL may be treated with 3 courses of R-CHOP and radiotherapy in preference to 6 or 8 courses of R-CHOP alone. In our data , lack of knowledge of the bone marrow result would not have affected the classification of this group in. In DLBCL the decision to give prophylaxis to prevent CNS relapse may be based on 2 or more extranodal sites, including bone marrow, involved at presentation. In this group, this decision could have been affected in 51 cases. In the UK the approximate cost of taking and reporting a bone marrow is around $900. When used routinely the cost could approach $25,000 per patient whose IPI score is increased by the results of examination; the figure for CHL is $44,000. It is, therefore, difficult to justify the cost effectiveness of this approach. In newly presenting patients with lymphoma, bone marrow examination should be reserved for those with unexplained cytopenia, and those who may potentially require radiation therapy for early stage FL or CNS prophylaxis in DLBCL. Disclosures: Jack: Roche /Genentech: Research Funding. Off Label Use: Rituximab in Burkitts Lymphoma. Patmore:Roche: Consultancy, Honoraria.

Description: BACKGROUND. The detection of minimal residual disease (MRD) at the level of 0.01%/10-4 or above is a strong independent predictor of reduced progression-free (PFS) and overall survival (OS) in patients with CLL treated with chemoimmunotherapy. Although newer agents such as B-cell receptor pathway inhibitors can result in prolonged survival without achieving complete response, there remains a important role for MRD analysis in assessing therapeutic strategies aimed at disease eradication and cure. This is particularly important in front-line trials for fit patients which now require at least five years of follow-up if PFS is used as an endpoint. The feasibility of using MRD as a surrogate or intermediate endpoint for accelerated approval of new treatments is under review by regulatory agencies but further prospective validation is required. At the same time technology is rapidly evolving and high-throughput sequencing (HTS) technologies now detect MRD at the 0.0001%/10-6 level. It is therefore important to determine the most effective approaches for quantifying MRD that are compatible with previous studies but sufficiently sensitive for current treatments. AIMS. This collaborative project had two objectives. First, to identify the simplest and most flexible flow cytometry panel capable of detecting MRD at the 0.01%/10-4 or lower, that is compatible with published data and independent of instrument/reagent manufacturer. Second, to compare the flow cytometry approach with HTS analysis using the ClonoSEQ assay (Adaptive Biotechnologies, Seattle, WA). METHODS AND RESULTS. A core panel of antibodies for MRD detection was identified by testing an 8-marker combination in 52 samples (27 post-treatment and 25 dilution study) and re-analysing data with serial exclusion of single markers to determine redundancy. A 1-tube core panel of CD19, CD20, CD5, CD43, CD79b, and CD81 was identified and validated against the previously published 2-tube 6-marker and 4-tube 4-marker ERIC panels in 76 samples (19 post-treatment and 57 dilution study). The results showed good concordance (for log-transformed data above the LoQ, linearity=0.977, Pearson correlation co-efficient=0.983, average difference=0.026 log, 95% limit of agreement 0.20log) and a limit of detection of 0.001%/10-5 for the 1-tube core panel. Inter-operator variation was similar to CML MRD monitoring with both experienced operators, or inexperienced cytometrists after ~1 hour of specific education, achieving a 95% limit of agreement less than 0.3log in samples with MRD levels ranging from 0.0001 – 100%. The flow cytometry approach was compared with the ClonoSEQ HTS assay in 109 samples (21 dilution study and 88 post-treatment samples, complete data currently available on 13/88). The assay was applicable to the vast majority CLL patients, often with two clonal markers. There was 94% concordance at the 0.01% (10-4) threshold (15 samples with ≥0.01% CLL by both methods, 14 samples with

Description: Background: Systemic AL amyloidosis is a rare complication of plasma cell dyscrasias. Much progress has occurred in treatment of AL amyloidosis but long term survival remains limited with advanced organ involvement, in particular, cardiac dysfunction determining outcomes. However, controlling the underlying plasma cell clone with chemotherapy or ASCT is the key to improving outcomes. Yet the role of plasma cell clones in determining prognosis remains to be fully explored and understood. The plasma cell burden in patient with AL amyloidosis is generally lower than that of multiple myeloma but reported degree of plasma cell infiltration has varied. A large study from the Mayo group reported markedly poor outcomes for patients with AL amyloidosis who have 〉10% BMPCs, even in the absence of symptomatic myeloma (Kourelis et al, JCO 2013). However, apart from just the number of BMPC, the composition appears to be of importance. Multiparameter flow cytometry (MFC) can identify proportion of normal and clonal plasma cells. Patients with 〉5% normal BMPC (defined as cells expressing CD38+CD138+CD19+) at diagnosis had a better prognosis (Paiva et al. Blood 2011). MFC underestimates the total proportion of BMPCs due to sample dilution effect. We report the impact of normal' plasma cells, as determined by MFC, on the outcome of AL patients in context of the total plasma cell burden as determined by standard morphological techniques in 104 patients with biopsy proven systemic AL amyloidosis, who had both bone marrow trephine and MFC performed at presentation between 2005-2013 assessed at UK National amyloidosis centre and St James's University Hospital. Methods: The bone marrow trephine biopsy (BMTB) plasma cell burden was estimated by morphology supplemented by CD138 immunohistochemistry as required. Patients with 〉10% CD138+ cells were classified as having AL-multiple myeloma (AL-MM) and those with 10% CD138+ PCs on trephine (classed as AL-MM) and 42 (40%) had

Description: Background The benefit of single agent maintenance is largely established from studies in newly diagnosed patients, while extended therapy combination regimens are more commonly used in relapsed disease. Extended therapy on combination protocols may be limited by tolerability and safety, as well as patient compliance. Fixed duration combination treatment followed by single agent maintenance may provide better long term disease control. Such benefit may correlate with depth of response, measured as minimal residual disease (MRD). Prognostic significance of MRD negativity in the relapsed setting is increasingly recognised, but the utility of single agent maintenance in achieving this is unknown. Aims The MUKfive phase 2 trial studied the safety and activity of carfilzomib, cyclophosphamide and dexamethasone (KCd) for patients at first relapse or refractory to one prior line, and compared activity and safety of maintenance K vs observation after fixed duration KCd. Here we provided updated results of the maintenance randomization, including the effect on MRD and interaction with genetic risk. Methods The first part of the study compared KCd with VCd as fixed duration therapy for patients at first relapse, or primary refractory to one line of treatment. Inclusion criteria included Hb〉80g/L, neutrophils〉1.0x109/L, platelets≥50x109/L and GFR〉30ml/min. Participants with ≥SD after KCd were randomised (R2) 1:1 to receive maintenance K (36mg/m2 days 1, 2, 15, 16 for 6m, then days 1, 2 for 12m) or no further treatment, minimization factors: disease response (≥VGPR vs

Description: Background: Ibrutinib inhibits CLL cell proliferation and results in prolonged remission, but MRD responses are rare. Obinutuzumab is a second generation anti-CD20 monoclonal antibody that is effective in CLL and can result in MRD responses. In the IcICLLe study (ISRCTN12695354), 40 participants with CLL requiring treatment (20 treatment-naïve, 20 with relapsed/refractory [R/R] disease) received ibrutinib until complete remission with 1 year prior ibrutinib. Patients: The IcICLLe Extension Study recruited 40 participants with relapsed/refractory CLL requiring treatment. They received continuous ibrutinib (420mg OD) with 6 cycles of obinutuzumab given over 6 months (M). Ten participants had 〉1 year of prior ibrutinib monotherapy in IcICLLe and 30 were ibrutinib-naïve with obinutuzumab started 24 hours after first ibrutinib dose. Patient characteristics and Adverse Events (AEs, collected from registration until 30 days after treatment cessation and reported at 1, 3, and 6M, and 6-monthly thereafter using the Common Terminology Criteria for Adverse Events v4.0) are shown in Table 1. MRD assessment was performed according to ERIC guidelines with a maximum detection limit of 0.001%/10-5. Results: In the 20 R/R patients treated with ibrutinib monotherapy there were no IWCLL CR/CRi responses and no patients achieved 1 year prior ibrutinib monotherapy achieved a higher response rate compared to ibrutinib-naive patients (IWCLL CR/CRi 50% vs. 30%), with a higher proportion of patients achieving 1 year prior ibrutinib treatment had already resolved any lymphadenopathy prior to receiving obinutuzumab. Conclusions: The results suggest that the addition of obinutuzumab to ibrutinib may result in a substantial improvement in the depletion of CLL cells from the PB and BM for ibrutinib-naïve patients. However, a greater impact in MRD response rate and depth of depletion was seen when obinutuzumab was introduced after 〉1 year of ibrutinib treatment and tumour bulk was low. For patients with persistent disease during/ following pathway inhibition treatments, the addition of anti-CD20 antibody therapy may be effective at improving MRD response rates. Disclosures Rawstron: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; BD Bio-sciences: Research Funding; Beckman Coulter: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding. Munir:MorphoSys: Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria; Novartis: Honoraria; Gilead: Honoraria; Janssen: Honoraria; Abbvie: Honoraria. Brock:GlaxoSmithKline: Equity Ownership; AstraZeneca: Equity Ownership; Merck Sharp Dohme: Other: Reimbursement of conference fees; Roche: Other: Reimbursement of expenses; Lilly: Honoraria. Pettitt:AstraZeneca: Research Funding; Celgene: Research Funding; Chugai: Research Funding; Roche: Research Funding; GSK/Novartis: Research Funding; Gilead: Research Funding; Napp: Research Funding. Fox:Celgene: Consultancy, Other: Travel support, Speakers Bureau; Janssen: Consultancy, Other: Personal fees and non-financial support, Speakers Bureau; Gilead: Consultancy, Other: Travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Other: Travel support, Research Funding, Speakers Bureau; Roche: Consultancy, Other: Travel support, Research Funding, Speakers Bureau; Sunesis: Consultancy. Devereux:Janssen: Other: Personal fees; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Fegan:Janssen: Honoraria; Gilead Sciences, Inc.: Honoraria; Abbvie: Honoraria; Roche: Honoraria; Napp: Honoraria. Bloor:Janssen: Research Funding; AbbVie: Research Funding. Hillmen:Alexion Pharmaceuticals, Inc: Consultancy, Honoraria; Acerta: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Novartis: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Gilead Sciences, Inc.: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Key Points Occult marrow disease is demonstrable in 68% of patients with solitary plasmacytoma of bone and is predictive of progression. Trials of adjuvant systemic therapy are warranted in high-risk patients.