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Central nervous system involvement in multiple myeloma is a rare complication but carries a very poor prognosis. We provide a review of current literature, including presentation, treatment and survival data, and describe our experience in a regional hematologic malignancy diagnosis center where, over a 15-year period, ten cases were identified. Although the median age of onset, frequently between 50-60 years, is comparatively young, those diagnosed usually have a preceding diagnosis of multiple myeloma and often have had several lines of treatment. We discuss putative underlying factors such as prior treatment and associations including possible risk factors and features suggestive of a distinct biology. Central nervous system involvement may be challenging to diagnose in myeloma, displaying heterogeneous symptoms that can be confounded by neurological symptoms caused by the typical features of myeloma or treatment side-effects. We discuss the clinical features, imaging and laboratory methods used in diagnosis, and highlight the importance of considering this rare complication when neurological symptoms occur at presentation or, more commonly, during the disease pathway. In the absence of clinical trial data to inform an evidence-based approach to treatment, we discuss current and novel treatment options. Finally, we propose the establishment of an International Registry of such cases as the best way to collect and subsequently disseminate presentation, diagnostic and treatment outcome data on this rare complication of multiple myeloma.

Fluorescent in situ hybridization (FISH) techniques can be used to identify a range of chromosome abnormalities that are clinically significant in many cancers. Multicolor FISH can be used to identify multiple targets, which can be simultaneously detected in individual cells using digital imaging microscopy. In an era of precision medicine there is a requirement to make a precise diagnosis and to have a molecular classification of the tumor that can guide therapy. Cancer genomics is now regarded as a sub-specialism in pathology and genomic testing needs to be robustly integrated into the routine diagnostic practice.The FISH techniques described in this chapter have been developed over many years in a busy hematopathology diagnostic laboratory. We describe robust in-house methods for both liquid samples (blood and bone marrow mainly) and formalin-fixed paraffin-embedded (FFPE) tissue biopsies that allow for large numbers of slides to be set up in batches. The techniques described are for interphase cells in tissues where metaphase chromosome techniques are generally not applicable. Some of the FISH tests need to be carried out as an "out-of-hours" emergency test to make a critical diagnosis while others provide prognostic information and are used to guide downstream patient management.

Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation resulting in overexpression of cyclin D1. However, a small subset of cyclin D1− MCL has been recognized, and approximately one-half of them harbor CCND2 translocations while the primary event in cyclin D1−/D2− MCL remains elusive. To identify other potential mechanisms driving MCL pathogenesis, we investigated 56 cyclin D1−/SOX11+ MCL by fluorescence in situ hybridization (FISH), whole-genome/exome sequencing, and gene-expression and copy-number arrays. FISH with break-apart probes identified CCND2 rearrangements in 39 cases (70%) but not CCND3 rearrangements. We analyzed 3 of these negative cases by whole-genome/exome sequencing and identified IGK (n = 2) and IGL (n = 1) enhancer hijackings near CCND3 that were associated with cyclin D3 overexpression. By specific FISH probes, including the IGK enhancer region, we detected 10 additional cryptic IGK juxtapositions to CCND3 (6 cases) and CCND2 (4 cases) in MCL that overexpressed, respectively, these cyclins. A minor subset of 4 cyclin D1− MCL cases lacked cyclin D rearrangements and showed upregulation of CCNE1 and CCNE2. These cases had blastoid morphology, high genomic complexity, and CDKN2A and RB1 deletions. Both genomic and gene-expression profiles of cyclin D1− MCL cases were indistinguishable from cyclin D1+ MCL. In conclusion, virtually all cyclin D1− MCLs carry CCND2/CCND3 rearrangements with immunoglobulin genes, including a novel IGK/L enhancer hijacking mechanism. A subset of cyclin D1−/D2−/D3− MCL with aggressive features has cyclin E dysregulation. Specific FISH probes may allow the molecular identification and diagnosis of cyclin D1− MCL.

Data de publicació electrónica: 29-01-2019 Cytogenomic investigations of haematological neoplasms, including chromosome banding analysis, fluorescence in situ hybridisation (FISH) and microarray analyses have become increasingly important in the clinical management of patients with haematological neoplasms. The widespread implementation of these techniques in genetic diagnostics has highlighted the need for guidance on the essential criteria to follow when providing cytogenomic testing, regardless of choice of methodology. These recommendations provide an updated, practical and easily available document that will assist laboratories in the choice of testing and methodology enabling them to operate within acceptable standards and maintain a quality service.

The Myeloma X trial previously reported improved durability of response (time to disease progression) in patients with relapsed multiple myeloma with salvage autologous stem-cell transplantation (ASCT) compared with oral cyclophosphamide in patients with multiple myeloma relapsing after a first ASCT. We report the final overall survival results of the trial.BSBMT/UKMF Myeloma X was a multicentre, randomised, open-label, phase 3 trial done at 51 centres in the UK. Eligible patients with multiple myeloma relapsing after a previous ASCT were re-induced with intravenous bortezomib (1·3 mg/m(2) on days 1, 4, 8, 11), intravenous doxorubicin (9 mg/m(2) per day on days 1-4), and oral dexamethasone (40 mg/day on days 1-4, 8-11, and 15-18 during cycle 1 and days 1-4 during cycles 2-4), with supportive care as per local institutional protocols before randomisation in a 1:1 ratio to either high-dose melphalan (200 mg/m(2)) and salvage ASCT or weekly oral cyclophosphamide (400 mg/m(2) per week for 12 weeks). Randomisation was by permuted blocks stratified by length of first remission and response to re-induction treatment. The primary endpoint was time to disease progression; the study was also powered to detect a difference in the secondary endpoint, overall survival. Further secondary endpoints were the proportion of patients achieving an objective response, progression-free survival, overall survival, toxic effects and safety, pain, and quality of life. Prespecified exploratory endpoints included time to second objective disease progression (PFS2). Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00747877, and the European Clinical Trials Database, number 2006-005890-24, and is now in long-term follow-up.Between April 16, 2008, and Nov 19, 2012, 297 patients were registered into the study and 174 were randomly assigned to receive either high-dose melphalan and salvage ASCT (n=89) or oral weekly cyclophosphamide (n=85). 173 (58%) of 297 patients relapsed after more than 24 months from first ASCT. 75 (43%) of 174 randomised patients had died at follow-up: salvage ASCT (n=31 [35%]) versus oral weekly cyclophosphamide (n=44 [52%]). Updated time to disease progression shows continued advantage in the salvage ASCT group compared with the weekly cyclophosphamide group (19 months [95% CI 16-26] vs 11 months [9-12]; hazard ratio [HR] 0·45 [95% CI 0·31-0·64] log-rank p0·0001). Median overall survival was superior in the salvage ASCT group compared with weekly cyclophosphamide group (67 months [95% CI 55-not estimable] vs 52 months [42-60]; log-rank p=0·022; HR 0·56 [0·35-0·90], p=0·0169). Time to second objective disease progression was superior in the salvage ASCT group compared with the weekly cyclophosphamide group (67 months [52-not estimable] vs 35 months [31-43]; HR 0·37 [0·24-0·57], log-rank p0·0001). During extended follow-up, no further treatment-related or treatment-unrelated adverse events were reported. 15 second primary malignancies were reported in 12 patients (salvage ASCT [n=7] vs oral weekly cyclophosphamide [n=5]). The cumulative incidence of second primary malignancies at 60 months after trial entry was 5·2% (2·1-8·2).Salvage ASCT increases overall survival during consolidation of re-induction treatment in patients with multiple myeloma at first relapse after a first ASCT. The delay of salvage ASCT to third-line treatment or later might not confer the same degree of advantage as seen with salvage ASCT at first relapse.Cancer Research UK, Janssen-Cilag, and Chugai Pharma UK.

Summary Background Relapsed multiple myeloma has no standard treatment, and the role of autologous stem-cell transplantation (ASCT) has not been fully defined. We aimed to compare high-dose melphalan plus salvage ASCT with cyclophosphamide in patients with relapsed multiple myeloma who had previously undergone ASCT. Methods This multicentre, randomised, open-label, phase 3 study recruited patients aged at least 18 years with multiple myeloma who needed treatment for first progressive or relapsed disease at least 18 months after a previous ASCT from 51 centres across the UK. Before randomisation, eligible patients received bortezomib, doxorubicin, and dexamethasone (PAD) induction therapy and then underwent peripheral blood stem-cell mobilisation and harvesting if applicable. Eligible patients (with adequate stem-cell harvest) were randomly assigned (1:1), using an automated telephone randomisation line, to either high-dose melphalan 200 mg/m 2 plus salvage ASCT or oral cyclophosphamide (400mg/m 2 per week for 12 weeks). Randomisation was stratified by length of first remission or plateau and response to PAD re-induction therapy. The primary endpoint was time to disease progression, analysed by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00747877, and EudraCT, number 2006-005890-24. Findings Between April 16, 2008, and Nov 19, 2012, 297 patients were registered, of whom 293 received PAD re-induction therapy. Between Aug 26, 2008, and Nov 16, 2012, 174 patients with sufficient PBSCs were randomised to salvage ASCT (n=89) or cyclophosphamide (n=85). After a median follow-up of 31 months (IQR 19–42), median time to progression was significantly longer in the salvage ASCT than in the cyclophosphamide group (19 months [95% CI 16–25] vs 11 months [9–12]; hazard ratio 0·36 [95% CI 0·25–0·53]; p 10% of patients) grade 3–4 adverse events with PAD induction, salvage ASCT, and cyclophosphamide were: neutropenia (125 [43%] of 293 patients after PAD, and 63 [76%] of 83 patients in the salvage ASCT group vs 11 [13%] of 84 patients in the cyclophosphamide group), thrombocytopenia (150 [51%] after PAD, and 60 [72%] vs four [5%], respectively), and peripheral neuropathy (35 [12%] after PAD, and none vs none, respectively). Interpretation This study provides evidence for the improved efficacy of high-dose melphalan plus salvage ASCT when compared with cyclophosphamide in patients with relapsed multiple myeloma eligible for intensive therapy, which might help to guide clinical decisions regarding the management of such patients. Funding Cancer Research UK.

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.

Minimal residual disease (MRD) negativity, defined as

A diagnosis of chronic lymphocytic leukemia (CLL) requires a count of over 5000 circulating CLL-phenotype cells per cubic millimeter. Asymptomatic persons with fewer CLL-phenotype cells have monoclonal B-cell lymphocytosis (MBL). The goal of this study was to investigate the relation between MBL and CLL.We investigated 1520 subjects who were 62 to 80 years of age with a normal blood count and 2228 subjects with lymphocytosis (4000 lymphocytes per cubic millimeter) for the presence of MBL, using flow cytometry. Monoclonal B cells were further characterized by means of cytogenetic and molecular analyses. A representative cohort of 185 subjects with CLL-phenotype MBL and lymphocytosis were monitored for a median of 6.7 years (range, 0.2 to 11.8).Monoclonal CLL-phenotype B cells were detected in 5.1% of subjects (78 of 1520) with a normal blood count and 13.9% (309 of 2228) with lymphocytosis. CLL-phenotype MBL had a frequency of 13q14 deletion and trisomy 12 similar to that of CLL and showed a skewed repertoire of the immunoglobulin heavy variable group (IGHV) genes. Among 185 subjects presenting with lymphocytosis, progressive lymphocytosis occurred in 51 (28%), progressive CLL developed in 28 (15%), and chemotherapy was required in 13 (7%). The absolute B-cell count was the only independent prognostic factor associated with progressive lymphocytosis. During follow-up over a median of 6.7 years, 34% of subjects (62 of 185) died, but only 4 of these deaths were due to CLL. Age above 68 years and hemoglobin level below 12.5 g per deciliter were the only independent prognostic factors for death.The CLL-phenotype cells found in the general population and in subjects with lymphocytosis have features in common with CLL cells. CLL requiring treatment develops in subjects with CLL-phenotype MBL and with lymphocytosis at the rate of 1.1% per year.

Chromosome analysis of a patient with intravascular large B-cell lymphoma (IVL) revealed a complex, near-tetraploid karyotype with 83 chromosomes. Abnormalities included a t(11;14)(q13;q32), which was confirmed with both interphase fluorescence in situ hybridization (FISH) using an IGH/cyclin D1 dual-color, dual-fusion probe set and cyclin D1 immunohistochemical analysis. Abnormality of 3q was also evident. Interphase FISH analysis with a dual-color, break-apart probe set confirmed rearrangement of BCL6. To our knowledge, this is the first report of these abnormalities in IVL.

Fluorescence in situ hybridization (FISH) has been used to demonstrate the t(14;18) in up to 100% of follicular lymphoma (FL) cases, however, there is little reproducible data using fixed tissue. The aim was therefore to develop a robust FISH method for the demonstration of translocations in archival tissue. The technique was evaluated by comparison with multiplex polymerase chain reaction (PCR), capable of detecting the majority of known breakpoints. Twenty-eight paired frozen and fixed cases of FL and 20 reactive controls were analyzed. The t(14;18) was detected in 23 of 28 cases using PCR on frozen material and 8 of 20 in paraffin. Using FISH, 24 of 26 frozen and 26 of 28 paraffin cases had a demonstrable translocation. All 20 reactive nodes were negative for the t(14;18) by PCR. Using FISH, one of the reactive cases had occasional cells with a translocation FISH pattern, demonstrable in frozen and paraffin samples. This is consistent with the presence of the t(14;18), which is well described in normal individuals. Both PCR and FISH are highly effective for t(14;18) analysis in unfixed tissue. When only paraffin blocks are available, FISH is the method of choice, and a result was achieved in 100% of cases. The method is applicable to the retrospective analysis of a range of translocations.

Diffuse large B-cell lymphomas (DLBCL) are a heterogeneous group of tumours, varying in clinical features, immunophenotype and cytogenetics. The aim of this study was to investigate the prognostic significance of BCL6 gene rearrangement at the 3q27 locus in patients with primary nodal disease, and to examine interrelationships with immunophenotype and International Prognostic Index (IPI). We have developed a fluorescent in situ hybridization (FISH)-based technique for the retrospective analysis of the effect of BCL6 gene rearrangements on survival, using nuclei extracted from paraffin-embedded tissue. FISH results were obtained in 111 presentation cases of nodal DLBCL. The IPI was calculated and each case was stained immunocytochemically for BCL6, BCL2 and CD10. 3q27 rearrangements were detected in 25% of cases. BCL2 protein and a germinal centre (GC) phenotype (defined as CD10+, BCL6+) were expressed in 56% and 41% of cases respectively. In multivariate analysis, rearrangement of 3q27 and BCL2 expression and the absence of a GC phenotype were associated with a poor prognosis. These factors can be used in conjunction with the IPI to improve risk stratification in nodal DLBCL.

B-cell chronic lymphocytic leukemia (CLL) can be classified as typical or atypical based on morphologic and immunophenotypic features. The relationship between these 2 groups is uncertain, and there is some evidence they may be different entities. We used comparative genomic hybridization (CGH) to explore the cytogenetic relationship between typical and atypical B-cell CLL. Results showed a similar pattern of chromosome gains and losses detected in typical and atypical B-cell CLL, suggesting they are related disorders. Gain on chromosome 12 material occurred in cases that were apparently normal by interphase fluorescence in situ hybridization (FISH), The common region mapped to chromosome 12q21. Gains on chromosome 4 were present in 74% (32) of cases analyzed and were confirmed by interphase FISH in 30% (13) of cases. We previously have shown the strong association between trisomy 12 as detected by FISH and CD11a expression in atypical B-cell CLL. In the present study, CGH demonstrated additional gains on 12p and 12q outside the common amplified region of 12q21 in these patients.

Faith E. Davies, Andrew C. Rawstron, Guy Pratt, Sheila O'Connor, Lela Su'ut, David Blythe, James Fenton, David Claydon, J. Anthony Child, Andrew S. Jack & Gareth J. Morgan

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia resulting from a somatic mutation in a hemopoietic stem cell. In most cases of hemolytic PNH, the majority of the marrow cells are derived from the PNH clone. Recent evidence has indicated, however, that the majority of the most primitive peripheral blood stem cells (PBSCs) in PNH appear to be of normal phenotype. This has led to tentative suggestions that normal PBSCs could be collected and used for autologous transplantation. We have investigated this possibility in four PNH patients by treating them with granulocyte colony-stimulating factor (G-CSF) in an attempt to mobilize normal progenitors. The expression of glycosylphosphatidylinositol (GPI)-linked proteins was analyzed by flow cytometry on mature neutrophils, late stem cells (CD34+/CD38+), and primitive stem cells (CD34+/CD38−). The phenotyping and stem cell quantitation was performed in steady-state blood and post–G-CSF administration. The most primitive PBSCs (CD34+/CD38−) were almost all normal before G-CSF treatment, even when the patients' neutrophils were mainly PNH. However, after G-CSF, the cells that were mobilized into the peripheral blood were of a similar phenotype to the mature neutrophils, ie, mainly PNH. It is possible that PNH-stem cells are preferentially destroyed by complement in the peripheral blood leaving only normal cells in the circulation. After G-CSF, the PNH cells in the marrow are released into the blood. Our findings suggest that it would be difficult to collect sufficient numbers of normal stem cells for autologous transplantation.

Acute promyelocytic leukaemia (APL) is characterized cytogenetically by t(15;17)(q22:q21) which results in the production of a PML/RAR alpha fusion protein. Detection of the translocation or the fusion gene product is required for objective diagnosis of APL. This can be accomplished by conventional cytogenetic methods, fluorescence in situ hybridization or RT-PCR. Such techniques are time consuming and not universally available. The intracellular distribution of the PML protein in promyelocytes is characteristically altered in APL and this can be detected by immunocytochemistry. We have assessed two immunocytochemical methods, immunofluorescence and alkaline phosphatase-anti-alkaline phosphatase staining (APAAP), with regard to sensitivity, specificity and rapidity of diagnosis. 85 patients with AML including 15 cases of APL were studied. Immunofluorescence PML detection was concordant with RT-PCR for t(15:17) in 14/15 (93.3%) cases with no false positives. The negative APL case in our series was a patient with a 5' PML breakpoint who did not express the reciprocal t(17;15) fusion product. APAAP was concordant in only 6/13 (46%) APL cases with one false positive. In conclusion, immunofluorescent localization of PML using 5E10 monoclonal antibody is a rapid, sensitive and specific diagnostic tool for APL.

IgM myeloma is a very rare and poorly defined entity. In a detailed assessment of 10 cases, it was demonstrated that 70% had an aberrant phenotype based on the expression of CD19, CD45, CD27 and Cyclin D1 but all cases lacked CD56 and CD117. Interphase fluorescence in situ hybridization demonstrated deletion 13 in 50% while 5/8 cases assessed had a t(11;14). Despite the high incidence of the t(11;14), CD20 was only expressed in one of nine cases. We conclude that IgM myeloma is a distinctive subset characterized by a CD20-CD56-CD117- phenotype and the t(11;14).

Introduction: Autologous transplantation (ASCT) in myeloma (MM) is standard consolidative therapy in first line therapy in eligible patients. We have shown definitely that a salvage ASCT in relapse setting can induce superior durability of responses (time-to-progression; TTP) over non-transplant consolidation with oral cyclophosphamide after a proteasome inhibitor-based re-induction schedule (ISRCTN601231201). The secondary end point of this multi-centre phase III randomised controlled trial was to evaluate the impact of salvage ASCT on the overall survival (OS_ of patients relapsing after a prior ASCT and delineate patient subgroups that may benefit the most. Patients and Methods: Eligible patients with MM relapsing after a prior ASCT were enrolled. All patients were re-induced with Bortezomib, Doxorubicin and Dexamethasone (PAD) therapy delivered in 2-4 21-day cycles before 1:1 randomization to either a second ASCT (melphalan 200mg/m2 iv; ASCT2 supported by either stored or remobilized stem cells) or low dose consolidation with weekly cyclophosphamide 400mg/m2 PO for 12 weeks (Non-Transplant Consolidation; NTC). Response was assessed (by IMWG criteria) after re-induction and 100 days post-randomization with TTP being determined as the primary end-point. Patients were stratified by β2microglobulin (β2M) at trial entry, ASCT1 TTP and response to re-induction, analyzed according to cytogenetic abnormalities by iFISH (unfavorable: t(4;14), t(14;16) and del17p) with OS was a key secondary endpoint. Results: 297 patients were entered into the study and 174 randomized from April 2008 to November 2012: ASCT2 n=89, NTC n=85. Median age was 61 (range 38-75) with 73.6% of patients relapsing more than 24 months from first ASCT. ORR to re-induction therapy was 79.4% with a 16.0% sCR/CR rate. Post-randomization, sCR/CR was significantly higher after ASCT2 (39.3% [95% CI 29.1,50.3] vs 22.4% [95% CI 14.0,32.7]; p=0á012). The median follow-up is 52 months (IQR range 41, 62) and the up-dated TTP demonstrates continued advantage in ASCT2 cohort compared to NTC (19 months [95% CI 16,26] vs 11 months [95% CI 9,12]; Log Rank p 24m (HR0.60, p=0.089), β2M level Conclusion: This long-term follow-up analysis demonstrates a clear advantage in terms of OS when salvage ASCT consolidates bortezomib-based re-induction therapy in patients with MM at first relapse. The delay of salvage ASCT to third line, though being suggestive of benefit over no salvage ASCT, does not confer the same degree of OS advantage as shown with a salvage transplant in second line. This data is key for patient-centered clinical decision-making. 1. G Cook, et al.. The Lancet Oncology, Vol. 15, No. 8, p874-885 Figure 1. Forest plots showing (a) heterogeneity of effect of randomised treatment on OS and (b) effect on OS of randomised treatment followed by ASCT at second relapse (NTC/ASCT2) Figure 1. Forest plots showing (a) heterogeneity of effect of randomised treatment on OS and (b) effect on OS of randomised treatment followed by ASCT at second relapse (NTC/ASCT2) Figure 1B. Figure 1B. Disclosures Cook: Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Research Funding, Speakers Bureau; BMS: Consultancy; Sanofi: Consultancy, Speakers Bureau; Takeda Oncology: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding, Speakers Bureau. Williams:Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau. Cavenagh:Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau. Snowden:MSD: Consultancy, Other: Educational support, Speakers Bureau; Janssen: Other: Educational support, Speakers Bureau; Celgene: Other: Educational support, Speakers Bureau; Sanofi: Consultancy. Parrish:Janssen: Speakers Bureau; Celgene: Speakers Bureau. Yong:Takeda: Honoraria; Autolous: Consultancy; Janssen: Honoraria; Novartis: Consultancy; BMS: Honoraria; Amgen: Honoraria. Cavet:Celgene: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding, Speakers Bureau. Bird:Janssen: Other: Educational support; Celgene: Speakers Bureau; Amgen: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Heartin:Celgene: Speakers Bureau; Janssen: Consultancy. O'Connor:Celgene: Research Funding. Ashcroft:Janssen: Consultancy, Other: Educational support. Brown:Janssen: Research Funding; Roche: Research Funding; Celgene: Research Funding; Bayer: Research Funding. Morris:Janssen: Other: Meeting support; Celgene: Other: Meeting support.

Introduction: Plasmablastic lymphoma (PBL) is a rare, aggressive lymphoma of differentiated B lymphocytes. PBL sits at the boundary between Diffuse Large B Cell Lymphoma (DLBCL) and plasmablastic myeloma, and is more frequent amongst HIV positive patients. With an increasing divergence between therapy for myeloma and lymphoma and a deficit of prospective studies, the nature of PBL and outcome on current therapy merits further assessment. Methods: We conducted a retrospective analysis of consecutive patients diagnosed between 1st January 2004 and 1st July 2015 with PBL at the HMDS, a regional NHS diagnostic centre serving a population in excess of 3 million and representative of the wider UK population. Using the Haematological Malignancy Research Network (HMRN), a population based epidemiological study overlapping with the population served by HMDS, patient demographics and clinical outcomes were investigated. Results: 53 patients (18 female, 35 male) were diagnosed with PBL. The median age was 67 years (19-95 years). 56.8% were 65 years or older. Six patients were HIV positive and three patients were immunosuppressed following solid organ transplantation. 37/53 (69.8%) patients had extranodal PBL at presentation, including 16/53 (30.2%) with gastrointestinal tract disease. Patient demographics are summarised below. Table 1. Plasmablastic lymphoma Number Male (%) Median age in years (range) Extranodal disease (%) Total 53 35(66.7) 67 (19-95) 37 (69.8) HIV positive 6 4 (66.7) 47 (39-56) 4 (66.7) HIV negative 44 38 (86) 70 (19-95) 33 (75) Post solid organ transplant 3 3 (100) 60 (55-68) 1 (33.3) B cell markers (CD19, CD20, PAX-5) were negative or weakly expressed in all cases. One third demonstrated weak CD45 positivity. The neoplastic cells retained CD79a and expressed plasma cell marker CD138 but were negative for CD56. IRF4/MUM1 and BLIMP1 were expressed in all tumours examined. A high KI67 index was common, with 60.4% of cases having a KI67 index of 80% or greater. Bone marrow involvement was found in eight patients, raising the question of re-assignment to plasmablastic myeloma. Three additional patients had small monoclonal B cell populations detected by flow cytometry. Where suitable material was available, FISH for MYC was performed. MYC/IgH translocation was detected in 23%. The MYC/IgH translocation occurred as a sole abnormality in one third of positive cases and as part of a complex karyotype in15.4%. Extra copies of MYC were found in 15.4% and MYC amplification occurred in a 7.7%. LMP-1 was performed in 17/53 cases, of which three (17.6%) were positive. EBER-ISH was positive in 60% cases tested. HHV8 was detected in a single HIV positive patient. Eight patients were managed with palliative intent, with a median survival of 6 days (95% CI 1-30). Of the patients receiving at least one cycle of chemotherapy, the median was 474 days (95% CI 174-1580). Cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) was used most frequently. The median survival in the overall group is 241 days (95% CI 141-613). The five year overall survival of the overall population was 21.4%. Conclusion: Our unselected cohort represents a large population based series of a rare and challenging disease and describes the experience in a predominantly elderly, HIV negative UK population. The male predominance, propensity for extranodal sites and association with EBV infection mirrors published reports in HIV positive patients. Survival in this population remains dismal. Further investigation is warranted to refine diagnostic criteria and explore molecular pathogenesis of PBL. Innovative therapeutic modalities guided by insights into molecular pathogenesis and exploring options across both lymphoma and myeloma therapeutic strategies are required to improve patient outcome. Given the rarity of PBL, prospective randomised studies are unlikely to be achievable. Our cohort serves as a historical control for future phase II clinical trials. Figure 1. Kaplan Meier survival curve for plasmablastic lymphoma patients Figure 1. Kaplan Meier survival curve for plasmablastic lymphoma patients Disclosures O'Connor: Celgene: Research Funding.

BACKGROUND: The aim was to investigate whether 2 subgroups of Hodgkin lymphoma (Epstein-Barr virus-positive and Epstein-Barr virus-negative) are associated with smoking or alcohol. METHODS: Patients with lymphoma diagnosed between age 16 and 69 years in geographically defined areas of England were recruited between 1998 and 2003. One control, matched to each lymphoma case on sex, date of birth, and area of residence, was randomly selected from population registers. Self-reported histories of tobacco and alcohol use were collected during face-to-face interviews with cases and controls. RESULTS: Compared with lifelong nonsmokers, ever-smokers were at increased risk of Hodgkin lymphoma (odds ratio =1.4; 95% confidence interval = 1.1–1.9). This excess was among current smokers, defined as smoking 2 years before diagnosis (1.7; 1.2–2.3). An increasing trend was observed with rising numbers of years smoked. Risks fell as the number of years stopped smoking increased, becoming equivalent to that of a nonsmoker 10 or more years after quitting. Associations were suggested for Epstein-Barr virus-positive Hodgkin lymphoma, but less so for Epstein-Barr negative Hodgkin lymphoma. No associations between Hodgkin lymphoma and alcohol consumption were observed. CONCLUSIONS: The association between smoking and Hodgkin lymphoma in general, and Epstein-Barr-positive Hodgkin lymphoma in particular, is consistent with previous studies. Further exploration of the relationship between Hodgkin lymphoma and smoking and of the potential mechanisms by which smoking could interact with Epstein-Barr virus status to increase Hodgkin lymphoma risk are required.

Comparatively little is known of the cytogenetics of Waldenstrom macroglobulinemia (WM). This is primarily due to the low proliferation of the clonal B cells, which precludes conventional karyotyping in many cases. Translocations involving the immunoglobulin heavy chain (IGH) gene at 14q32 are characteristic of many B-cell lymphomas and myelomas. Initial reports suggested that the t(9;14) was characteristic of lymphoplasmacytic lymphoma (the underlying pathological diagnosis in WM), but subsequent studies have failed to confirm the uniqueness of the translocation. To clarify this, we examined 69 cases of WM with interphase fluorescence in situ hybridization and failed to demonstrate an IgH translocation in 67 (97%). We conclude that IGH translocations are not a feature of WM, and the implications of this finding are discussed.

Introduction: Composite lymphomas involving Hodgkin lymphoma (HL) and non-Hodgkin lymphomas (CLL, DLBCL, FL, MZL, MCL, T-NHL) are relatively rare but are increasingly frequently diagnosed. This may be a function of the change in diagnostic practice, the more varied and increased treatment of the presenting disease or simply reflect better monitoring post treatment with re-biopsy of lymph nodes. Composite lymphoma is defined as the synchronous and metachronous development of two or more lymphomas in the same patient. The mechanism of pathogenesis underlying its occurrence is not clearly established and in particular the relationship between entities when HL is diagnosed a number of years following successful treatment for follicular lymphoma (FL). Methods: We identified 48 patients with a diagnosis of Hodgkin lymphoma at our centre between January 2005 and June 2014 who had a previous or concurrent diagnosis of another lymphoproliferative disorder. Diagnoses included CLL = 14; follicular lymphoma = 13; DLBCL = 8; mantle cell lymphoma = 1; marginal zone lymphoma = 5; T-cell lymphoma = 3; not specified = 3 and a single patient had CLL, MZL and most recently MCL. The diagnosis of Hodgkin lymphoma was confirmed on the tissue biopsy by using a standard panel of immunohistochemistry markers (CD3, CD20, CD30, IRF4, TARC, CD79, LMP1, BCL6, BOB1, OCT2). This study is focussed on the 13 cases with follicular lymphoma. The aim of the study was to identify a relationship between the Hodgkin lymphoma and the follicular lymphoma using interphase FISH studies to look for identical chromosomal translocations. Results: 8/13 patients had follicular lymphoma diagnosed on an earlier tissue biopsy, range 1-9 years prior to the diagnosis of Hodgkin lymphoma; 7/8 of these FL cases had bone marrow staging carried out at presentation and 5/7 had involvement by FL. 5/13 were newly presenting patients with composite lymphoma; 3/5 had a staging marrow which in each case showed evidence of follicular lymphoma alone with no evidence of Hodgkin lymphoma. In total, 7/13 patients fit the criteria for composite lymphoma with Hodgkin lymphoma and follicular lymphoma occupying distinct zones within the same tissue biopsy. The remaining 6/13 patients show no evidence of follicular lymphoma in the current biopsy and are indistinguishable from de novo presentation of Hodgkin lymphoma. Interphase FISH was used to assess for a genetic relationship between the disease entities. 3µ sections were cut from formalin fixed paraffin processed tissue, using the same block where possible as the H&E and immunohistochemistry. All cases were independently assessed by two experienced scientists with knowledge of histology and FISH reporting on thin sections. 8/13 cases had suitable samples for FISH, the remaining 5 cases had insufficient material remaining in the block. Commercial probes for BCL2 ‘Split-Signal’ (Abbott/Vysis or Dako) were used and FISH results were examined using a Zeiss microscope with MetaSystem image capture. All 8 cases showed BCL2 gene rearrangement in the Reed-Sternberg (RS) cells (5/8 cases were de novo group of Hodgkin lymphoma with no morphological or phenotypic evidence of FL in the tissue and 3/8 were composite lymphoma with distinct zoning). One rare case contained RS cells in a dab/imprint preparation made from the fresh tissue, FICTION technique was carried out on this case combining CD30 immunofluorescent staining with FISH for BCL2 gene rearrangement confirming RS cell type with BCL2 gene rearrangement. CONCLUSIONS The identification of BCL2 gene rearrangement in RS cells, the hallmark cell of Hodgkin lymphoma, in this series of composite lymphomas suggests a relationship between the B cells of follicular lymphoma and the RS cells of HL. The presence of the same chromosomal abnormalities identified in more than one lymphoma cell type indicates the same clonal cell of origin. Disclosures Jack: Roche: Research Funding; Genentech: Collaboration, Collaboration Other.

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.

To establish whether a combination of morphologic and immunophenotypic criteria could be developed to more precisely define Waldenstrom macroglobulinemia (WM) and prognostic factors, we retrospectively assessed the clinical and laboratory features of 111 cases of WM. Bone marrow infiltration by small lymphocytes was documented in each case; and diffuse, interstitial, nodular, and paratrabecular patterns of infiltration were documented in 58%, 32%, 6%, and 4% of cases, respectively. Ninety percent were characterized by a surface immunoglobulin-positive, CD19+CD20+CD5-CD10-CD23- immunophenotype. The median overall survival from diagnosis was 60 months; univariate analysis revealed the following adverse prognostic factors: older than 60 years, performance status more than 1, platelet count less than 100 x 10(3)/microL (< 100 x 10(9)/L), pancytopenia, and diffuse bone marrow infiltration. Associated median survival was 40, 38, 46, 28, and 59 months, respectively. Multivariate analysis revealed age, performance status, and platelet count as prognostically significant, but stratification of patients according to the International Prognostic Index had limited value. We suggest defining WM by the following criteria: IgM monoclonal gammopathy; bone marrow infiltration by small lymphocytes, plasmacytoid cells, and plasma cells in a diffuse, interstitial, or nodular pattern; and a surface immunoglobulin-positive, CD19+CD20+CD5-CD10-CD23- immunophenotype.

Delivering the most effective clinical therapy in acute promyelocytic leukaemia (APL) is dependent on accurately making the diagnosis. The morphological diagnosis can be improved by detecting the presence of a specific chromosome translocation, the t(15;17)(q22;q21). This can be achieved using cytogenetics, RT-PCR, FISH and anti-PML monoclonal antibody. The optimal approach will be rapid, accurate and readily integrated into the routine haematology laboratory. Immunofluorescent detection of microparticulate PML protein fulfils these criteria, however, karyotyping will also detect the variant translocations and remains the 'gold-standard'.

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 Minimal residual disease (MRD) assessment by multicolour flow cytometry (MFC) following upfront therapy is a powerful prognostic tool in multiple myeloma (MM) and there is growing evidence supporting its role as an outcome measure in clinical trials. There is however no published data assessing its utility in the relapse setting. Our aim was to test the utility of MFC at first relapse following a second autologous transplant (ASCT2) or weekly cyclophosphamide (C-weekly) consolidation following re induction chemotherapy in patients entered into the BSBMT/UKMF Myeloma X trial. Patients and Methods Eligible MM patients relapsing after a prior ASCT were enrolled and received a bortezomib-containing re-induction regimen (PAD, Bortezomib, Doxorubicin and Dexamethasone) before being randomized to either ASCT2 or C-weekly. The primary endpoint was time-to-progression (TTP) with secondary endpoints of overall survival and overall response rate (to PAD and randomized intervention). Bone marrow aspirate samples were analyzed by MFC at trial entry to establish neoplastic phenotype and then at specified time-points following treatment. After red cell lysis and washing, samples were labelled with CD27-FITC, CD56-PE, CD19-PerpCPCy5.5, CD38-PE-Cy7, CD138-APC and CD45-APC-Cy7. A minimum of 500,000 events were acquired on a FacsCanto II cytometer and data was analyzed using a standardized gating strategy. Sensitivity of the assay is 0.01% of leucocytes. Response assessment (after re-induction, 100 days post-ASCT2 or 30 days post C-weekly) and disease progression were determined using IMWG criteria. Unfavourable genetic abnormalities were defined as FGFR3/IGH, IGH/MAF and TP53 deletion by interphase FISH. Results 297 patients with a median age of 61 (range 38 -75) were entered into the study and 174 randomized from April 2008 to November 2012: ASCT2 n=89, C-weekly n=85. Of the whole trial cohort, MFC analysis was available in 224 at presentation, 162 at completion of re-induction therapy, 46/89 at D100 post ASCT2 and 50/85 at D30 post C-weekly. In an intention to treat analysis of all available randomized patients, achieving MRD negativity was highly predictive for improved TTP: All patients (MRD+ 12mns 95%CI 10-13 vs. MRD- 24mns 95CI NR; logrank P=0.0003), ASCT arm (MRD+ 15mns 95%CI 11-19 vs. MRD- 24mns 95%CI 16-42; logrank P=0.0227) & C-weekly arm (MRD+ 11mns 95%CI 8-12 vs. MRD- 15mns 95%CI 9-NR) logrank P=0.0107). MRD status was also assessed in conjunction with IMWG conventional categorical response assessment. CR was demonstrable in 39% of patients post ASCT2 and 22% of patients following C-weekly. The presence of MRD predicted outcome in those patients achieving a CR and the most favourable outcome was seen in CR/MRD- patients ( MRD status was also assessed according to cytogenetic risk group as defined by interphase FISH. Adverse cytogenetics were present in 19% of patients. Achieving MRD negativity was associated with an improved outcome in patients with both favourable and adverse cytogenetic profiles: Adverse; MRD+ 10 mns (95%CI 8-11 mns), MRD- 23 mns (95%CI 16-25 mns), Favourable; MRD+ 11 mns (95%CI 9-13 mns), MRD- 16mns (95%CI 10-NR mns); logrank p= 0.0058. Conclusion These data clearly demonstrate that MRD assessment by MFC is a sensitive predictor of outcome in the first relapse setting, irrespective of the consolidation therapy given. MRD assessment predicts outcome in patients achieving CR and is applicable to patients with both favourable and adverse cytogenetics. This data is comparable to that published by us and others in the context of primary therapy and suggests that MRD assessment is an appropriate outcome measure in relapsed MM. Disclosures: Williams: Janssen: Honoraria, Speakers Bureau. Snowden:Janssen: Honoraria, Research Funding, Speakers Bureau.

Introduction Heterogeneity of molecular abnormalities in myeloma plasma cells (PCs) & their impact on clinical outcomes has been clearly demonstrated in newly diagnosed patients & advanced disease stages. Limited analysis at first relapse stage & in particular the impact of molecular risk categories on intensive re-induction/consolidation therapy limits the use of such analysis to predict suitable patient selection. Patients and Methods MM patients relapsing >18 months after a prior ASCT were enrolled into a phase III RCT, receiving a Bortezomib-containing regimen (Bortezomib, Doxorubicin and Dexamethasone) before being randomised to either a second ASCT or Cyclophosphamide weekly. BM aspirate samples at trial entry and at further disease progression were CD138 positively selected (Miltenyi AutoMac) and PC suspensions were fixed in Carnoy’s then stored at -20°C. Interphase FISH analysis (iFISH), was performed with commercial probes, scored and image captured (Zeiss AxioPlan microscopy with MetaSystems software). Depth of response (IMWG criteria) was analysed post-induction and 100 days post-randomisation with durability of response determined as time-to-progression (TTP). Results 222 samples from 297 patients were received & satisfactory CD138+ cell purity and yield was obtained in 149 samples (68% recovery). Of these, only 35 (28%) demonstrated no genetic defects by iFISH at trial entry. A single genetic abnormality was detected in 28 patients (23%): del13q (n=8), IGH rearrangement (n=16), MYC rearrangements (n=2) and del17p (n=2). 2 and ≥3 molecular genetic abnormalities defects were detected in 42 patients (28%) and 32 patients (21%), respectively. The commonest abnormality detected was an IGH rearrangement (n=62, 41%) & there were insufficient CD138+ cells for additional translocation testing in 11/62; Of the 51 IGH rearranged samples, a partner translocation was identified in 32 (63%): CCND1/IGH in n=15 (29%), FGFR3/IGH in n=14 (27%) and IGH/MAF in n=3 (6%) with an additional single genetic defect detected in 31 samples (50%). Del13q was present in 58 patients (39%) of whom 29 patients (50%) demonstrated one additional genetic defect (IGH rearrangement n=22, del17p n=3 and MYC rearrangements n=4), 20 patients (35%) demonstrated 2 additional defects and 2 patients demonstrated >2 additional defects. A rearranged MYC was detected in PCs from 24 patients (16%) with 2 & ≥3 additional defects detected in 13 & 6 patients’ samples, respectively. Del17p was detected in PCs from 22 patients’ samples (15%) with additional copies of Ch17 present in PCs of 8 patients (6%). All Ch17+ patient samples exhibited additional genetic defects. Where del17p clones were detected, 9 and 10 samples had 1 & ≥2 genetic defects. When del17p was present, MYC arrangements were also detected in 16 patients (73%). 20 patients (13%) demonstrated hyperdiploidy with 8 patients demonstrating no other defect. The presence of IGH and MYC rearrangements or del17p did not correlate with the duration of response from ASCT1 nor did it impact the ORR or sCR to bortezomib-based induction. The presence of IGH and MYC rearrangements or del17p did not impact on the TTP in the C-weekly cohort, however both del17p (median TTP - absent: 19 mns, 95%CI, 16-25 mns, vs. present: 10 mns, 95%CI, 2-11 mns; logrank p=0.002) and MYC rearrangements (median TTP absent: 24 mns, 95%CI 12-42 mns vs. present: 11 mns, 95%CI 1-18 mns; logrank p=0.006) impacted on TTP in the ASCT2 cohort. Only the presence of MYC rearrangements were predictive of reduced TTP in the ASCT2 group (Cox regression model interaction test HR 0.08, 95%CI 0.02-0.44; p=0.003) when accounting for treatment response to previous ASCT & response to induction therapy. Conclusion: IGH rearrangements were identified at a similar frequently to analyses reported at presentation though with disproportionately more FGFR3/IGH and MAF/IGH abnormalities. Otherwise, the incidence of genetic abnormalities was similar to rates reported at diagnostic with a frequent association of MYC rearrangements with del17p. Whilst both del17p and MYC rearrangements were associated with inferior durability of response following an ASCT, only a MYC rearrangement was of independent predictive significance. As follow-up is limited, the impact on overall survival currently remains to be determined. Disclosures: Williams: Janssen: Honoraria, Speakers Bureau. Snowden:Janssen: Honoraria, Research Funding, Speakers Bureau.

Numerous studies have shown the prognostic importance of TP53 deletion in CLL. Assessing TP53 deletion prior to initiating therapy is good clinical practice and many laboratories now offer testing. However, there is no harmonisation of methodology and little agreement about ‘Best Practice’. Information about cut-off values is scanty in published series. The best guide to date is the LRF CLL4 trial that showed a cut-off of >20% TP53 deletion as clinically significant. This value relates to a single institution and may not be applicable to other centres using alternative sample preparation methods. We reviewed the TP53 FISH results seen in our institution and looked at the cases with levels of TP53 deletion 10% B cells and had smears/dabs of suitable quality. Minor modification of standard manufacturers protocols were used to reflect the nature of the samples. Slides were examined using a Zeiss microscope and images were captured using ISIS3 (MetaSystems). Deletion analysis was microscopically scored by counting 100 lymphocytes. Normal result is defined as 15% of lymphocytes with one signal. Scores >5% but 20% TP53 deletion at 8 (25% TP53 deletion) months and 18 (>95% TP53 deletion) months. Although the technical aspects of the FISH are straightforward, and results are reproducible, the interpretation can be problematic. A small proportion of these patients show marked expansion of the TP53 clones within short time periods. Rapid clinical progression was observed in these patients. Where repeat testing was possible 2/3 had the same borderline score, these patients continue on a ‘watch & wait’ policy; the remaining 1/3 normalised their TP53 result. This suggests that there are technical quality issues with the sample. CLL cells have a tendency to disintegrate due to their fragility because of this significant subpopulations may not survive manipulation and thus evade scrutiny. A CLL patient in this series who had borderline TP53 deletion on the PB smear was found to have a TP53 level of 40% by the local referring laboratory using their cytogenetic technique. FISH on smears has an advantage over other methodologies as all CLL cells are present. There is a need for improved cross-disciplinary guidelines, and further collaborative harmonisation studies are needed. Our results show that there is no clear cut-off for a TP53 deletion result and that any result over the 5% technical cut-off needs to be reviewed. A proportion of borderline cases reflect technical difficulties of handling CLL samples but in many cases they genuinely detect a small clone of TP53 deleted cells that may expand. Careful follow-up is needed for all cases with borderline TP53 deletion results.

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.

The 2007 IWCLL guidelines indicate that a diagnosis of Chronic Lymphocytic Leukemia (CLL) requires a B-cell count above 5,000/μL in the absence of other features; below this level the diagnosis is Monoclonal B-cell Lymphocytosis (MBL). There is little outcome data for MBL patients and it is not clear whether the detection of low levels of CLL cells, seen in 3% of the general population, is of clinical relevance. We have therefore investigated two hospital populations: the first with normal blood counts and no history of cancer; and the second MBL patients referred for investigation of a current or prior lymphocytosis. Blood samples from 1520 outpatients aged 60–80 with a normal blood count were screened: CLL cells were detected in 78/1520 (5.1%) with a median CLL cell count of 140/μL (range 15 – 1,248). Chromosomal abnormalities were frequently detected in purified CLL-phenotype cells (deletion 13q14 in 15/38, trisomy 12 in 4/22) although poor-risk abnormalities (deletion 11q or 17p) were not detected. The median IgVH mutation was 6.6% (range 0.5 – 13.7%) with 85% of cases showing >2% mutation from germline. The IgVH gene usage was heavily biased with a similar profile to mutated CLL. Detection of CLL cells in individuals with a normal count was not associated with increased mortality (estimated yearly rate 6.2% vs. 8.9% for matched controls, P=0.76) or risk of developing CLL as subsequent lymphocyte counts remained normal in all cases. A diagnosis of MBL was established in 309 of 2228 referrals for investigation of lymphocytosis between 1995 and 2000. A cohort of 185 MBL patients was monitored for a median 6.7 years (range 0.2 – 11.8): the presenting B-cell count was a median 3,100/μL (range 30 – 5,000), age 73 years (range 42 – 96); IgVH mutation rate was 7.1% (range 1.3 – 9.3%) with 96% of cases showing >2% mutation from germline. Progression to a lymphocyte count above 30,000/μL occurred in 15% of cases (28/185) and chemotherapy for progressive CLL was required in 7% (13/185). The absolute B-cell count was the only independent risk factor for an increasing disease levels. Neither IgVH mutation status nor CD38 expression predicted risk of disease progression or requirement for treatment. During follow-up 33% died: age above 70, hemoglobin concentration below 11 g/dL and T-lymphopenia (CD3+ 2,400μL) had significantly longer survival. Development of progressive disease did not predict overall survival: 7/13 patients requiring therapy remain alive at a median 1.9 years (range 0–8.6 years) after initiation of treatment. The total lymphocyte count had no impact on the risk of disease progression, time to treatment or overall survival. CLL-phenotype cells are genetically equivalent to CLL even when detected in the general population but are not associated with increased mortality or risk of progression to CLL when present below 1,500/μL. MBL patients with higher levels of CLL cells show a steady increase in disease levels over time with 1–2% per year requiring chemotherapy for progressive disease. As such, periodic monitoring is indicated but this should have a minimal impact on lifestyle as MBL patients are often elderly with multiple health issues. MBL is a newly described disorder which is related to CLL in a similar way that MGUS is related to myeloma.

A monoclonal B-cell Lymphocytosis (MBL) is detected in the peripheral blood of around 3% of otherwise healthy adults, the majority of these have a CLL immunophenotype. We have previously demonstrated that the cells in MBL are indistinguishable from good risk CLL, sharing the same immunophenotypic profile, genetic aberrations and IgVH gene usage. MBL exerts an increasing burden on haematology clinics with over 100 new patients diagnosed per annum in our regional haemato-oncology laboratory. This is largely as a result of the increased tendency to investigate patients with a mild lymphocyosis although 10,000/μL and included 3 patients who progressed to a clinical stage requiring treatment. VH gene usage was different in each of these 3 patients was with VH3-7, VH4-34 and VH5-51 identified, the levels of SHM were 5.8, 4.0 and 5.3% respectively. Follow-up data shows that a deletion of ATM was detected at progression in one of these patients. In conclusion, we show that MBL is predominantly mutated and that progression to CLL is independent of mutational status. Other prognostic markers, particularly assessment of chromosomal aberrations, may be informative but this would probably require cell selection to increase sample purity. Our data suggests that current prognostic markers are not effective at predicting outcome in MBL and periodic monitoring is required.

Recently published data has identified the type-1 membrane glycoprotein CD200 as a potential therapeutic target in the treatment of CLL. CD200 has been reported to be expressed at significantly higher levels in CLL compared to normal mature B cells. CD200 has an immunosuppressive function and it’s expression by CLL cells may be involved in disease progression by exerting an immunomodulatory effect facilitating escape from host immune responses. In order to further evaluate the clinical and laboratory utility of this antibody the expression of CD200 has been assessed in a range of specimens. We have assessed CD200 expression in 41 CLL patients (36 peripheral blood and 5 bone marrow) and 12 normal samples (5 peripheral blood, 5 bone marrow, 2 lymph node biopsies). Biological features were assessed in 24 known CLL patients (male to female ration 3:1, median age 70 years), 15 untreated and 9 post treatment. Patients were defined as being poor risk if they were ZAP-70+, had deletions of p53, ATM and/or unmutated IgVH genes. Using these criteria 50% of cases were defined as good risk, and 50% as poor risk. Leucocytes were isolated from the specimens by ammonium chloride lysis and an aliquot of 1x106 cells were analysed by 6 colour flow cytometry using the FACS Canto and analysed using FACS Diva software (Becton Dickinson, UK). The level of expression of CD200 above an isotype control was reported for Lymphocytes, Monocytes, Granulocytes, Mature B cells, Germinal centre B cells, Plasma cells and CLL cells. Mean fluorescence intensities were also recorded for each of these groups (table 1). This data shows that CD200 is expressed above control levels by the majority of CLL cells, expression levels were not significantly different between patients with poor and good risk disease (median mfi 1921 and 1677 respectively p=0.42). CD200 was not increased in normal CD5+ B-cells. The very weak expression seen in non-CLL leucocytes may be an artefact of the lower background fluorescence of the control antibody relative to the CD200. Expression levels of CD200 relative to control were to the same magnitude as CD52 and several fold higher than CD20 expression levels, suggesting that CD200 is potentially an excellent target for immunotherapy. In conclusion our data shows that CD200 expression is uniformly higher in CLL than in any other peripheral blood or bone marrow leucocytes. The level of expression appears to be unaffected by prognostic risk factors, suggesting that treatments which target CD200 would be applicable in all patients with CLL. In addition, the lack of CD200 expression on normal leucocytes, particularly on T-cells, is potentially important. The uniformly increased expression of this molecule in CLL also makes this a potential marker for diagnostic and minimal residual disease assays. Table 1. Cell Type Median %CD200+ (range) Mean MFI CD200 Expression in CLL is consistently higher than in normal leucocytes CLL 96.60 (65.2–100) 2773.23 Mature B cells 23.89 (6.06–36.75) 69.29 Germinal Centre B cells 35.60 (2.7–48) 871.86 Plasma Cells 42.50 (2.8–80.9) 1939.5 T cells 0.25 (0.11–0.48) 7.73 Monocytes 3.65 (0.23–6.31) 31.86 Granulocytes 4.47 (0.13–13.25) 34.63

Less than 5% of patients with CLL undergo histological transformation to diffuse large B cell lymphoma (DLBL) while transformation to Classical Hodgkins lymphoma (CHL) is also recognised. It has been assumed that such events reflect genetic changes in the CLL clone but there is now emerging evidence to suggest that at least some transformation events may be Epstein Barr virus (EBV) related neoplasms not clonally related to CLL. In this analysis we have reviewed the clinical and laboratory features of 15 CLL patients with biopsy proven histological transformation to DLBL and 2 patients who developed CHL. Histological sections were assessed for the expression of a wide range of immunophenotypic markers as well as EBV latent membrane protein 1 (LMP-1). Of the 15 patients developing DLBL 5 appeared on phenotypic grounds to be related to the underlying CLL clone. In the remaining patients the tumour cells appeared phenotypically distinct as they lacked CD5 and CD23 and demonstrated expression of germinal centre markers in some instances. LMP1 positivity was demonstrable in 5 patients with DLBL (1 apparently related and 4 unrelated to the underlying CLL). Of the 2 patients who developed CHL 1 was associated with EBV and lacked CD20 expression. The 6 patients with EBV+ tumours were heavily pretreated (median prior therapies 4, range 1–5) while the median time from original diagnosis to histological transformation was 74.5 months (range 17–114). Previous therapies included chlorambucil (n= 4), fludarabine monotherapy (n=3), FC (n=2), FCR (n=1 patient), high dose methyl prednisolone (n= 1) and alemtuzumab (n=2). 2 patient received only one line of therapy for CLL but one of these was heavily immunosuppressed with steroids and azathioprine for autoimmune neutropenia. 4 patients presented with nodal disease and 2 patients presented with extranodal disease (bone marrow and liver). The outcome of the EBV associated tumours in these patients was death in 2 patients (opportunistic infection and complications of intensive chemotherapy), remission with intensive combination chemotherapy in 2 patients one of whom died 10 months later of progressive CLL and spontaneous remission of nodal disease in 1 patient. 1 patient is still undergoing intensive chemotherapy and the outcome is not known yet. We would conclude that not all transformation events in CLL occur within the original clone. The majority appear to be clonally distinct and a significant proportion of these appear to be EBV associated. This presumably occurs as a result of both the underlying immune-deficiency seen in CLL as well the potent immunosuppressive agents given as therapy. This phenomenon may also be a feature of other B-cell disorders as we have recently seen a phenotypically and genotypically distinct EBV associated DLBL in a patient treated with chlorambucil, CHOP and FCR for mantle cell lymphoma. The natural history of these tumours remains uncertain at this stage but it is clear that at least a minority may resolve spontaneously. All biopsies demonstrating histological transformation in CLL patients should be assessed for the presence of EBV.

The t(8:14) is the characteristic cytogenetic abnormality in Burkitt lymphoma and is found as an additional genetic abnormality in some cases of transformed follicular and DLBCL. Rare cases of de novo precursor B-cell lymphoblastic leukaemia (B-ALL) have also been reported to harbour cMYC translocations. We describe 5 cases referred to our regional haematopathology service that presented some diagnostic difficulties that may represent a previously undescribed phenomenon in Germinal Centre (GC)-derived tumours. 2 patients presented with de novo disease whilst of the other 3, 2 had a preceding history of follicular lymphoma and 1 of DLBCL, with all 3 previously demonstrating a t(14:18). Extranodal disease was the primary presenting feature in all 5 patients, with 2 demonstrating bone marrow involvement, 2 demonstrating soft tissue involvement and 1 involving both. The neoplastic cells displayed lymphoblast-type morphology and flow cytometry demonstrated a CD19+ B-cell phenotype with expression of CD10 and CD38, but absence of surface immunoglobulin, surface CD79b, CD20, and weak CD22. This phenotype was suggestive of a precursor B-cell neoplasm but there was no demonstrable CD34 or Tdt. It was therefore unclear whether these cases represented a precursor B-cell neoplasm or atypical forms of DLBCL. To further characterise these cases we performed FISH studies and investigated the expression of transcription factors involved in B-cell differentiation. Immunocytochemistry revealed a common PAX5+MUM-1+BCL-6− expression profile. This transcription factor profile in conjunction with CD10+ is rare and has previously been demonstrated in only 3/224 cases of de novo DLBCL at our institution. Cases were further evaluated for expression of BLIMP-1 using 2 different antibodies. Expression was noted in all cases but this was not accompanied by CD138 expression. FISH studies demonstrated evidence of a t(8:14) in all cases and a t(14:18) in 4 cases, which was pre-existing in 3. These cases are highly unusual and demonstrate loss of a mature B-cell phenotype and partial or abortive plasma cell differentiation in cells of a germinal centre type. Whilst expression of BLIMP-1 provides an explanation for the partial downregulation of CD20, CD22 and CD79b, the failure to repress PAX5 and CD10 indicates that differentiation was abortive in these cases. Rearrangement of cMYC may play a role in this regard as repression of cMYC is a prerequisite for normal plasma cell differentiation. These cases suggest that abortive plasma cell differentiation occurs in a proportion of de novo GC-derived tumours with a t(8:14) and in a proportion of follicular lymphoma/DLBCL cases which acquire a t(8:14) as a transformation or progressive event. This may lead to the erroneous diagnosis of B-ALL.

In the early 1990’s we described a series of patients from Kumasi, Ghana, West Africa who appeared to have a distinctive lymphoproliferative disorder (LPD) characterised by massive splenomegaly and lymphocytosis with characteristic morphology. At the time this was termed tropical splenic lymphoma (TSL). These patients, in contrast to most patients with LPD’s, tended to be female and relatively young and were frequently diagnosed as having hyper-reactive malarial splenomegaly. An aetiological role for chronic malaria infection has been postulated but remains as yet unproven. Only limited phenotypic and genotypic characterisation of such patients has been possible previously. Given the clinical features and potential aetiological role for malaria infection we wanted to determine whether TSL represented a distinct entity or may be more appropriately considered as splenic marginal zone lymphoma (SMZL). We have therefore evaluated the clinical, immunophentypic and genotypic features of 19 pts (median age 63 years, range 40–78) with a clinical diagnosis of TSL. Peripheral blood, bone marrow aspirate and trephine biopsies were obtained in all patients and the laboratory assessment were performed in the HMDS laboratory, Leeds, UK as part of an on-going collaborative project investigating lymphomas in Ghana. 14 of the 19 patients (73%) were female and all had significant splenomegaly (median length below the costophrenic margin of 17cm, range 6–30 cm). 5/19 patients (26%) had lymphadenopathy and 17/19 patients (89%) had a lymphocytosis - median 30.1×09/l (range 5.4×09/l - unrecordable). Bone marrow infiltration was evident in the trephine biopsies of all patients and was extensive in the majority. Immunophenotyping was performed primarily by immunohistochemistry on the trephine sections although flow cytometry was possible in a proportion of patients. The majority of cases were characterised by a CD5− CD10− CD20+ CD23− CD79+ BCL2+ BCL6− MUM1/IRF4- cyclin D1- immunophenotype. The rate of cell proliferation was low in all cases. FISH studies were performed and these demonstrated del7q31 in 4/18 cases and del6q21 in 1/19 cases. There was no evidence of the t(11;14), t(9;14) or MALT1 rearrangements. IgH sequence analysis was also performed in 16 cases and this demonstrated that 9/16 cases (56%) were germline (>98% homology) and 7/16 (44%) were mutated (

A number of balanced translocations have been described in extranodal marginal zone lymphoma (ENMZL). The t(11;18) and variant t(14;18) which deregulate MALT1 are the commonest reported occurring in up to 40% of patients. The t(3;14) which deregulates FOXP1 is reported to occur in up to 10% of cases whilst the t(1;14) and t(14;19) which deregulate BCL10 and BCL3 respectively have been described in a variable proportion of patients. The purpose of this study was to evaluate the incidence of such abnormalities in the routine diagnostic setting in a geographically defined region of Northern England. We have therefore reviewed a series of 56 consecutive cases of presenting ENMZL, diagnosed between November 2003 and July 2006 in the Yorkshire and Humberside Haematology Network (YHHN), which serves a population of approximately 3.6 million. Diagnoses were made using WHO criteria. Presentation tissue included breast (2), gastric (29), colon (3), Conjuctiva/orbit (3), connective tissue/muscle (3), lung (2), parotid (2), salivary gland (2), thyroid (1), prostate (2), and skin (4). In 2 cases the site was unknown. All cases were investigated for MALT1 rearrangements with an alpha satellite 18 control (VYSIS 32-190055/D18Z1) using interphase FISH at the time of diagnosis. Cases with available biopsy material were further investigated for rearrangements of IgH, (VYSIS 32-191019), PAX5, BCL3 (DAKO Y5413 and Y5411) and FOXP1 (in-house probe). FISH was perormed on thin paraffin sections in 54/56 cases and on fresh tissue imprints in 2 cases. 6/56 (11%) cases demonstrated a rearrangement of MALT1, 5/6 were of gastric origin and 1 colonic. 16/56 (29%) cases demonstrated trisomy 18. 6/44 (14%) cases showed rearrangement of IgH (3/6 were unbalanced rearrangement with loss). In 1 of these cases (gastric) BCL3 was also rearranged, suggesting that this may represent a t(14;19)(q32;q13). 1 additional case (parotid) showed an unbalanced rearrangement of BCL3 with loss of the telomeric portion of the gene, giving a total of 2/30 (7%) cases with BCL3 abberrations. In the latter case no other abnormalities were demonstrated. A further 5 cases were suspicious for IgH rearrangements with a low level of cells ( The incidence of MALT1 rearrangements in this study is significicantly lower than most published series. The reasons for this apparrent discrepancy is unclear at present but this study represents a consecutive series of patients presenting in a defined UK cancer network and is therefore free from any referral bias. We similarly failed to demonstrate rearrangements of either FOXp1 or PAX5. Rearrangement of BCL3 was however demonstrated but at low frequency. It is clear that although significant advances have been made in our understanding of the pathobiology of ENMZL the underlying cytogenetic defect remains unknown in the majority of patients.

Cyclooxygenase-2 (COX-2) is the key enzyme involved in prostaglandin synthesis. It appears to support the growth of a number of solid tumours including colon, breast, ovary, lung and uterine cervix and may be an important therapeutic target in at least some of these tumours. COX-2 expression has recently been evaluated (by immunohistochemistry using polyclonal anti-COX-2 antibodies) in multiple myeloma (MM) where expression was documented in 33–57% of patients. COX-2 expression in these studies was strongly associated with an adverse outcome. In addition there is some emerging data to suggest that the use of aspirin in MM may improve survival rates. In order to further evaluate this we have used a monoclonal antibody (Clone SP21, Labvision, Fremont, Ca) to assess COX-2 expression in both normal and neoplastic plasma cells. 52 specimens were assessed using standard streptavidin-biotin immunoperoxidase techniques using a known COX-2+ colon cancer as a positive control. Strong uniform COX-2 expression was seen in 32/33 (97%) of myeloma patients assessed and was also documented in all patients with MGUS (n=10). COX-2 expression was also documented in reactive plasmacytic lesions (oral mucosa, skin and lymph node, n=6) as well as normal bone marrow plasma cells (n=6). Megakaryocytes stained positively in all bone marrow biopsies examined and provided a useful positive internal control while erythroid, myeloid and lymphoid cells were consistently negative. We would conclude that COX-2 is strongly expressed by both normal and neoplastic plasma cells suggesting that COX-2 is a potential therapeutic target in MM. The apparent increase in the proportion of myeloma patients expressing COX-2 in the present study reflects the use of a monoclonal antibody in our immunohistology studies. The fact that polyclonal antibodies identify a lower proportion of patients who appear to have an inferior outcome suggests that the level of expression is of prognostic significance rather than its presence or absence. This is worthy of further study using more appropriate techniques such as RQ-PCR.