Three-dimensional image fusion guidance of percutaneous pulmonary valve implantation to reduce radiation exposure and contrast dose: A comparison with traditional two-dimensional and three-dimensional rotational angiographic guidance.

INTRODUCTION: Three-dimensional rotational angiography (3DRA) has been used in the guidance of various transcatheter therapies including percutaneous pulmonary valve implantation (PPVI). The most recently available 3D image fusion software (VesselNavigator, Philips) extends this technology to use pre-registered computed tomography or magnetic resonance imaging datasets, promising reductions in contrast and radiation exposure along with shorter procedural times. METHODS: In this retrospective review, patients were assigned to three groups according to the mode of imaging guidance: two-dimensional angiography (2DA), 3DRA and VesselNavigator (VN) assisted valve implantation. Patient characteristics and catheterisation data were reviewed with a focus on contrast and radiation exposure, fluoroscopy, and procedural times. RESULTS: Between July 2012 and June 2016, 21 patients underwent PPVI: 8 with 2D guidance, 6 patients with 3DRA and most recently 7 patients with VN assistance. Patents in the VN group received significantly less absolute and weight indexed contrast when compared with those with 2DA or 3DRA guided PPVI. Patients in the 2DA group received a significantly higher total dose area product radiation dose and air kerma in comparison with patients with 3DRA and VN guided intervention. Application of VN resulted in the shortest fluoroscopy time, although not statistically significant, and a significantly shorter study time when compared with 2DA. CONCLUSIONS: Utilisation of pre-intervention image manipulation with VesselNavigator for 3D guidance of PPVI results in a reduction in contrast and radiation exposure and study time as compared with traditional 2D guidance, and contrast usage as compared with 3DRA.

Assessing myeloma bone disease with whole-body diffusion-weighted imaging: comparison with x-ray skeletal survey by region and relationship with laboratory estimates of disease burden.

AIM: To estimate and compare the extent of myeloma bone disease by skeletal region using whole-body diffusion-weighted imaging (WB-DWI) and skeletal survey (SS) and record interobserver agreement, and to investigate differences in imaging assessments of disease extent and apparent diffusion coefficient (ADC) between patients with pathological high versus low disease burden. MATERIALS AND METHODS: Twenty patients with relapsed myeloma underwent WB-DWI and SS. Lesions were scored by number and size for each skeletal region by two independent observers using WB-DWI and SS. Observer scores, ADC, and ADC-defined volume of tumour-infiltrated marrow were compared between patients with high and low disease burden (assessed by serum paraproteins and marrow biopsy). RESULTS: Observer scores were higher on WB-DWI than SS in every region (p<0.05) except the skull, with greater interobserver reliability in rating the whole skeleton (WB-DWI: ICC = 0.74, 95% CI: 0.443-0.886; SS: ICC = 0.44, 95% CI: 0.002-0.730) and individual body regions. WB-DWI scores were not significantly higher in patients with high versus low disease burden (observer 1: mean ± SD: 48.8 ± 7, 38.6 ± 14.5, observer 2: mean ± SD: 37.3 ± 13.5, 30.4 ± 15.5; p = 0.06, p = 0.35). CONCLUSION: WB-DWI demonstrated more lesions than SS in all regions except the skull with greater interobserver agreement. Sensitivity is not a limiting factor when considering WB-DWI in the management pathway of patients with myeloma.

Progression free survival of myeloma patients who become IFE-negative correlates with the detection of residual monoclonal free light chain (FLC) by mass spectrometry.

Deeper responses are associated with improved survival in patients being treated for myeloma. However, the sensitivity of the current blood-based assays is limited. Historical studies suggested that normalisation of the serum free light chain (FLC) ratio in patients who were negative by immunofixation electrophoresis (IFE) was associated with improved outcomes. However, recently this has been called into question. Mass spectrometry (MS)-based FLC assessments may offer a superior methodology for the detection of monoclonal FLC due to greater sensitivity. To test this hypothesis, all available samples from patients who were IFE negative after treatment with carfilzomib and lenalidomide-based induction and autologous stem cell transplantation (ASCT) in the Myeloma XI trial underwent FLC-MS testing. FLC-MS response assessments from post-induction, day+100 post-ASCT and six months post-maintenance randomisation were compared to serum FLC assay results. Almost 40% of patients had discordant results and 28.7% of patients with a normal FLC ratio had residual monoclonal FLC detectable by FLC-MS. FLC-MS positivity was associated with reduced progression-free survival (PFS) but an abnormal FLC ratio was not. This study demonstrates that FLC-MS provides a superior methodology for the detection of residual monoclonal FLC with FLC-MS positivity identifying IFE-negative patients who are at higher risk of early progression.

Targeting bone in myeloma.

Myeloma bone disease (BD) not only impairs quality of life, but is also associated with impaired survival. Studies of the biology underlying BD support the notion that the increased osteoclastogenesis and suppressed osteoblastogenesis is both a consequence and a necessity for tumour growth and clonal expansion. Survival and expansion of the myeloma clone are dependent on its interactions with bone elements; thus, targeting these interactions should have anti-myeloma activities. Indeed, both experimental and clinical findings indicate that bone-targeted therapies, not only improve BD, but also create an inhospitable environment for myeloma cell growth and survival, favouring improved clinical outcome. This chapter summarizes recent progress in our understandings of the biology of myeloma BD, highlighting the role of osteoclasts and osteoblasts in this process and how they can be targeted therapeutically. Unravelling the mechanisms underlying myeloma-bone interactions will facilitate the development of novel therapeutic agents to treat BD, which as a consequence are likely to improve the clinical outcome of myeloma patients.

Cost-effectiveness of zoledronic acid compared with clodronate in multiple myeloma.

BACKGROUND: In the U.K. Medical Research Council Myeloma IX trial (mmix), zoledronic acid 4 mg once every 3-4 weeks, compared with clodronate 1600 mg daily, reduced the incidence of skeletal related events (sres), increased progression-free survival (pfs), and prolonged overall survival (os) in 1970 patients with newly-diagnosed multiple myeloma. The incidence of confirmed osteonecrosis of the jaw was higher with zoledronic acid than with clodronate. The objective of the present study was to evaluate, based on the findings in mmix, the cost-effectiveness of zoledronic acid compared with clodronate in patients with newly-diagnosed multiple myeloma. METHODS: An economic model was used to project pfs, os, the incidence of sres and adverse events, and expected lifetime health care costs for patients with newly diagnosed multiple myeloma who are alternatively assumed to receive zoledronic acid or clodronate. The incremental cost-effectiveness ratio (icer) of zoledronic acid compared with clodronate was calculated as the ratio of the difference in cost to the difference in quality-adjusted life years (qalys). Model inputs were based on results of mmix and published sources. Results were generated under different assumptions regarding the beneficial effects of zoledronic acid on os beyond 5 years after treatment initiation. RESULTS: Assuming lifetime treatment effects of zoledronic acid, treatment with zoledronic acid (compared with clodronate) increased qalys by 0.27 at an additional cost of CA$13,407, yielding an icer of CA$49,829 per qaly gained. If the threshold icer is CA$100,000 per qaly, the estimated probability that zoledronic acid is cost-effective is 80%. Assuming that the benefits of zoledronic acid on pfs and os diminish over 5 years beginning at the end of year 5, the icer is CAN$63,027 per qaly gained. If the benefits of zoledronic acid on pfs and os are assumed to persist for 5 years only, the icer is CAN$76,948 per qaly gained. CONCLUSIONS: Compared with clodronate, zoledronic acid represents a cost-effective treatment alternative in patients with multiple myeloma.

Mycophenolic acid trough level monitoring: relevance in acute and chronic graft versus host disease and its relation with albumin.

Mycophenolate mofetil (MMF) is used to treat acute and chronic graft versus host disease (GvHD). There is scant evidence in the literature about mycophenolic acid (MPA) trough level monitoring in GvHD. We therefore reviewed 32 patients treated with MMF for acute (n = 19) or chronic GvHD (n = 13). Twelve (63%) of 19 patients with acute GvHD and nine (69%) of 13 with chronic GvHD showed a good response. In all 21 patients who responded to MMF, their mean total MPA levels were therapeutic (1-3.5 mg/L), whereas five of 11 patients who did not respond had sub-therapeutic mean MPA levels (p = 0.002). Sixteen (66%) of 24 steroid refractory or dependent patients responded to MMF. Associations between the mean total MPA level for each patient and the corresponding mean serum albumin concentration showed therapeutic mean total MPA levels for all 23 patients with mean albumin ≥ 31 g/L but sub-therapeutic mean total MPA levels in five of nine patients with mean albumin <31 g/L (p = 0.0006). In conclusion, MMF is efficacious in steroid refractory and dependent acute or chronic GvHD with statistically significant correlation between therapeutic plasma total MPA trough levels and clinical response. Serum albumin levels should be taken into account when considering MMF dose adjustments.

Combination of flow cytometry and functional imaging for monitoring of residual disease in myeloma.

The iliac crest is the sampling site for minimal residual disease (MRD) monitoring in multiple myeloma (MM). However, the disease distribution is often heterogeneous, and imaging can be used to complement MRD detection at a single site. We have investigated patients in complete remission (CR) during first-line or salvage therapy for whom MRD flow cytometry and the two imaging modalities positron emission tomography (PET) and diffusion-weighted magnetic resonance imaging (DW-MRI) were performed at the onset of CR. Residual focal lesions (FLs), detectable in 24% of first-line patients, were associated with short progression-free survival (PFS), with DW-MRI detecting disease in more patients. In some patients, FLs were only PET positive, indicating that the two approaches are complementary. Combining MRD and imaging improved prediction of outcome, with double-negative and double-positive features defining groups with excellent and dismal PFS, respectively. FLs were a rare event (12%) in first-line MRD-negative CR patients. In contrast, patients achieving an MRD-negative CR during salvage therapy frequently had FLs (50%). Multi-region sequencing and imaging in an MRD-negative patient showed persistence of spatially separated clones. In conclusion, we show that DW-MRI is a promising tool for monitoring residual disease that complements PET and should be combined with MRD.

Improved reliability of lymphoma diagnostics via PCR-based clonality testing: report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

The diagnosis of malignant lymphoma is a recognized difficult area in histopathology. Therefore, detection of clonality in a suspected lymphoproliferation is a valuable diagnostic criterion. We have developed primer sets for the detection of rearrangements in the B- and T-cell receptor genes as reliable tools for clonality assessment in lymphoproliferations suspected for lymphoma. In this issue of Leukemia, the participants of the BIOMED-2 Concerted Action CT98-3936 report on the validation of the newly developed clonality assays in various disease entities. Clonality was detected in 99% of all B-cell malignancies and in 94% of all T-cell malignancies, whereas the great majority of reactive lesions showed polyclonality. The combined BIOMED-2 results are summarized in a guideline, which can now be implemented in routine lymphoma diagnostics. The use of this standardized approach in patients with a suspect lymphoproliferation will result in improved diagnosis of malignant lymphoma.

Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n=56), mantle cell lymphoma (n=54), marginal zone lymphoma (n=41) and follicular lymphoma (n=109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

Assessing ASDs prior to device closure using 3D echocardiography. Just pretty pictures or a useful clinical tool?

AIMS: To determine the usefulness of three-dimensional transthoracic echocardiography (3D echo) in assessment of secundum atrial septal defects (ASDs) considered for device closure. To compare the findings from 3D echo with those from two-dimensional transoesophageal echocardiography (TOE) regarding dimensions, morphology and suitability for device closure. METHODS AND RESULTS: Twenty-four patients were enrolled in this prospective, crossover study. Three-dimensional echo and TOE data were collected, analysed and compared, assessing quantitative data including maximum defect diameter, area and circumference. Qualitative morphology such as the presence of fenestrations and the defect margins were noted, and an assessment of the suitability for device closure was made using each modality. Eighteen (75%) of the 3D data sets produced usable data for analysis. In each case the maximum diameter of the defect was larger on 3D echo than on TOE (mean difference = 0.34 cm, P < 0.001). On three occasions suitability for device closure could not be determined using 3D echo. On the other 15 occasions there was agreement between the TOE and 3D echo data. CONCLUSIONS: Three-dimensional echo provides comparable data with TOE when attempting to predict suitability for device closure without the need for general anaesthetic or sedation. It also provides useful additional dynamic and morphological information.

Carfilzomib resistance due to ABCB1/MDR1 overexpression is overcome by nelfinavir and lopinavir in multiple myeloma.

Proteasome inhibitor (PI) carfilzomib (CFZ) has activity superior to bortezomib (BTZ) and is increasingly incorporated in multiple myeloma (MM) frontline therapy and relapsed settings. Most MM patients ultimately experience PI-refractory disease, an unmet medical need with poorly understood biology and dismal outcome. Pharmacologic targeting of ABCB1 improved patient outcomes, including MM, but suffered from adverse drug effects and insufficient plasma concentrations. Proteomics analysis identified ABCB1 overexpression as the most significant change in CFZ-resistant MM cells. We addressed the functional role of ABCB1 overexpression in MM and observed significantly upregulated ABCB1 in peripheral blood malignant plasma cells (PCs) vs untreated patients' bone marrow PC. ABCB1 overexpression reduces the proteasome-inhibiting activity of CFZ due to drug efflux, in contrast to BTZ. Likewise, the cytotoxicity of established anti-MM drugs was significantly reduced in ABCB1-expressing MM cells. In search for potential drugs targeting ABCB1 in clinical trials, we identified the HIV protease inhibitors nelfinavir (NFV) and lopinavir (LPV) as potent functional modulators of ABCB1-mediated drug export, most likely via modulation of mitochondria permeability transition pore. NFV and LPV restored CFZ activity at therapeutically relevant drug levels and thus represent ready-to-use drugs to be tested in clinical trials to target ABCB1 and to re-sensitize PC to established myeloma drugs, in particular CFZ.

Prediction of outcome in newly diagnosed myeloma: a meta-analysis of the molecular profiles of 1905 trial patients.

Robust establishment of survival in multiple myeloma (MM) and its relationship to recurrent genetic aberrations is required as outcomes are variable despite apparent similar staging. We assayed copy number alterations (CNA) and translocations in 1036 patients from the NCRI Myeloma XI trial and linked these to overall survival (OS) and progression-free survival. Through a meta-anlysis of these data with data from MRC Myeloma IX trial, totalling 1905 newly diagnosed MM patients (NDMM), we confirm the association of t(4;14), t(14;16), t(14;20), del(17p) and gain(1q21) with poor prognosis with hazard ratios (HRs) for OS of 1.60 (P=4.77 × 10-7), 1.74 (P=0.0005), 1.90 (P=0.0089), 2.10 (P=8.86 × 10-14) and 1.68 (P=2.18 × 10-14), respectively. Patients with 'double-hit' defined by co-occurrence of at least two adverse lesions have an especially poor prognosis with HRs for OS of 2.67 (P=8.13 × 10-27) for all patients and 3.19 (P=1.23 × 10-18) for intensively treated patients. Using comprehensive CNA and translocation profiling in Myeloma XI we also demonstrate a strong association between t(4;14) and BIRC2/BIRC3 deletion (P=8.7 × 10-15), including homozygous deletion. Finally, we define distinct sub-groups of hyperdiploid MM, with either gain(1q21) and CCND2 overexpression (P<0.0001) or gain(11q25) and CCND1 overexpression (P<0.0001). Profiling multiple genetic lesions can identify MM patients likely to relapse early allowing stratification of treatment.

Deletion of chromosome 13 detected by conventional cytogenetics is a critical prognostic factor in myeloma.

In myeloma, the prognostic impact of different strategies used to detect chromosome 13 deletion (Delta13) remains controversial. To address this, we compared conventional cytogenetics and interphase fluorescence in situ hybridization (iFISH) in a large multicenter study (n=794). The ability to obtain abnormal metaphases was associated with a poor prognosis, which was worse if Delta13, p53 deletion or t(4;14) was present, but only Delta13 remained significant on multivariate analysis. Patients with Delta13, by either cytogenetics or iFISH, had a poor prognosis. However, when cases with Delta13 detectable by both cytogenetics and iFISH were separated from those detected by iFISH only, the poor prognosis of iFISH-detectable Delta13 disappeared; their outcome matched that of patients with no detectable Delta13 (P=0.115). Addition of ploidy status to iFISH-Delta13 did not affect the prognostic value of the test. Indeed both cytogenetics and iFISH Delta13 divided both hyperdiploidy and nonhyperdiploidy into two groups with similar prognoses, indicating that the poor prognosis of ploidy is entirely due to its association with Delta13. We conclude that Delta13 detected by metaphase analysis is a critical prognostic factor in myeloma. Absence of Delta13, even in those patients yielding only normal or no metaphases, is associated with a relatively good prognosis.

Hyperhaploidy is a novel high-risk cytogenetic subgroup in multiple myeloma.

Hyperhaploid clones (24-34 chromosomes) were identified in 33 patients with multiple myeloma (MM), demonstrating a novel numerical cytogenetic subgroup. Strikingly, all hyperhaploid karyotypes were found to harbor monosomy 17p, the single most important risk stratification lesion in MM. A catastrophic loss of nearly a haploid set of chromosomes results in disomies of chromosomes 3, 5, 7, 9, 11, 15, 18, 19 and 21, the same basic set of odd-numbered chromosomes found in trisomy in hyperdiploid myeloma. All other autosomes are found in monosomy, resulting in additional clinically relevant monosomies of 1p, 6q, 13q and 16q. Hypotriploid subclones (58-68 chromosomes) were also identified in 11 of the 33 patients and represent a duplication of the hyperhaploid clone. Analysis of clones utilizing interphase fluorescence in situ hybridization (iFISH), metaphase FISH and spectral karyotyping identified either monosomy 17 or del17p in all patients. Amplification of 1q21 was identified in eight patients, demonstrating an additional high-risk marker. Importantly, our findings indicate that current iFISH strategies may be uninformative or ambiguous in the detection of these clones, suggesting this patient subgroup maybe underreported. Overall survival for patients with hyperhaploid clones was poor, with a 5-year survival rate of 23.1%. These findings identify a distinct numerical subgroup with cytogenetically defined high-risk disease.

Overexpression of EZH2 in multiple myeloma is associated with poor prognosis and dysregulation of cell cycle control.

Myeloma is heterogeneous at the molecular level with subgroups of patients characterised by features of epigenetic dysregulation. Outcomes for myeloma patients have improved over the past few decades except for molecularly defined high-risk patients who continue to do badly. Novel therapeutic approaches are, therefore, required. A growing number of epigenetic inhibitors are now available including EZH2 inhibitors that are in early-stage clinical trials for treatment of haematological and other cancers with EZH2 mutations or in which overexpression has been correlated with poor outcomes. For the first time, we have identified and validated a robust and independent deleterious effect of high EZH2 expression on outcomes in myeloma patients. Using two chemically distinct small-molecule inhibitors, we demonstrate a reduction in myeloma cell proliferation with EZH2 inhibition, which leads to cell cycle arrest followed by apoptosis. This is mediated via upregulation of cyclin-dependent kinase inhibitors associated with removal of the inhibitory H3K27me3 mark at their gene loci. Our results suggest that EZH2 inhibition may be a potential therapeutic strategy for the treatment of myeloma and should be investigated in clinical studies.

Bi-allelic inactivation is more prevalent at relapse in multiple myeloma, identifying RB1 as an independent prognostic marker.

The purpose of this study is to identify prognostic markers and treatment targets using a clinically certified sequencing panel in multiple myeloma. We performed targeted sequencing of 578 individuals with plasma cell neoplasms using the FoundationOne Heme panel and identified clinically relevant abnormalities and novel prognostic markers. Mutational burden was associated with maf and proliferation gene expression groups, and a high-mutational burden was associated with a poor prognosis. We identified homozygous deletions that were present in multiple myeloma within key genes, including CDKN2C, RB1, TRAF3, BIRC3 and TP53, and that bi-allelic inactivation was significantly enriched at relapse. Alterations in CDKN2C, TP53, RB1 and the t(4;14) were associated with poor prognosis. Alterations in RB1 were predominantly homozygous deletions and were associated with relapse and a poor prognosis which was independent of other genetic markers, including t(4;14), after multivariate analysis. Bi-allelic inactivation of key tumor suppressor genes in myeloma was enriched at relapse, especially in RB1, CDKN2C and TP53 where they have prognostic significance.

Spatial genomic heterogeneity in multiple myeloma revealed by multi-region sequencing.

In multiple myeloma malignant plasma cells expand within the bone marrow. Since this site is well-perfused, a rapid dissemination of "fitter" clones may be anticipated. However, an imbalanced distribution of multiple myeloma is frequently observed in medical imaging. Here, we perform multi-region sequencing, including iliac crest and radiology-guided focal lesion specimens from 51 patients to gain insight into the spatial clonal architecture. We demonstrate spatial genomic heterogeneity in more than 75% of patients, including inactivation of CDKN2C and TP53, and mutations affecting mitogen-activated protein kinase genes. We show that the extent of spatial heterogeneity is positively associated with the size of biopsied focal lesions consistent with regional outgrowth of advanced clones. The results support a model for multiple myeloma progression with clonal sweeps in the early phase and regional evolution in advanced disease. We suggest that multi-region investigations are critical to understanding intra-patient heterogeneity and the evolutionary processes in multiple myeloma.In multiple myeloma, malignant cells expand within bone marrow. Here, the authors use multi-region sequencing in patient samples to analyse spatial clonal architecture and heterogeneity, providing novel insight into multiple myeloma progression and evolution.

Genome-wide association study of immunoglobulin light chain amyloidosis in three patient cohorts: comparison with myeloma.

Immunoglobulin light chain (AL) amyloidosis is characterized by tissue deposition of amyloid fibers derived from immunoglobulin light chain. AL amyloidosis and multiple myeloma (MM) originate from monoclonal gammopathy of undetermined significance. We wanted to characterize germline susceptibility to AL amyloidosis using a genome-wide association study (GWAS) on 1229 AL amyloidosis patients from Germany, UK and Italy, and 7526 healthy local controls. For comparison with MM, recent GWAS data on 3790 cases were used. For AL amyloidosis, single nucleotide polymorphisms (SNPs) at 10 loci showed evidence of an association at P<10-5 with homogeneity of results from the 3 sample sets; some of these were previously documented to influence MM risk, including the SNP at the IRF4 binding site. In AL amyloidosis, rs9344 at the splice site of cyclin D1, promoting translocation (11;14), reached the highest significance, P=7.80 × 10-11; the SNP was only marginally significant in MM. SNP rs79419269 close to gene SMARCD3 involved in chromatin remodeling was also significant (P=5.2 × 10-8). These data provide evidence for common genetic susceptibility to AL amyloidosis and MM. Cyclin D1 is a more prominent driver in AL amyloidosis than in MM, but the links to aggregation of light chains need to be demonstrated.

Differentiation stage of myeloma plasma cells: biological and clinical significance.

The notion that plasma cells (PCs) are terminally differentiated has prevented intensive research in multiple myeloma (MM) about their phenotypic plasticity and differentiation. Here, we demonstrated in healthy individuals (n=20) that the CD19-CD81 expression axis identifies three bone marrow (BM)PC subsets with distinct age-prevalence, proliferation, replication-history, immunoglobulin-production, and phenotype, consistent with progressively increased differentiation from CD19+CD81+ into CD19-CD81+ and CD19-CD81- BMPCs. Afterwards, we demonstrated in 225 newly diagnosed MM patients that, comparing to normal BMPC counterparts, 59% had fully differentiated (CD19-CD81-) clones, 38% intermediate-differentiated (CD19-CD81+) and 3% less-differentiated (CD19+CD81+) clones. The latter patients had dismal outcome, and PC differentiation emerged as an independent prognostic marker for progression-free (HR: 1.7; P=0.005) and overall survival (HR: 2.1; P=0.006). Longitudinal comparison of diagnostic vs minimal-residual-disease samples (n=40) unraveled that in 20% of patients, less-differentiated PCs subclones become enriched after therapy-induced pressure. We also revealed that CD81 expression is epigenetically regulated, that less-differentiated clonal PCs retain high expression of genes related to preceding B-cell stages (for example: PAX5), and show distinct mutation profile vs fully differentiated PC clones within individual patients. Together, we shed new light into PC plasticity and demonstrated that MM patients harbouring less-differentiated PCs have dismal survival, which might be related to higher chemoresistant potential plus different molecular and genomic profiles.

Fludarabine phosphate and melphalan: a reduced intensity conditioning regimen suitable for allogeneic transplantation that maintains the graft versus malignancy effect.

Reduced intensity conditioning (RIC) for allogeneic stem cell transplantation allows stable donor cell engraftment with the maintenance of a graft versus malignancy effect. Many different regimens exist employing various combinations of chemotherapy, radiotherapy and T-cell depletion. We examined the role of non-T-cell depleted RIC regimens in 56 patients with haematological malignancies. Patients received fludarabine phosphate for 5 days (30 mg/m2 in 35 patients, 25 mg/m2 in 21 patients) and melphalan for 1 day (140 mg/m2 in 36 patients, 100 mg/m2 in 20 patients). Immunosuppression was with CyA alone in 33 patients and CyA/MTX in 23 patients. Twenty-four of the 26 patients with chimerism data showed >95% donor chimerism at 3 months post transplant. aGVHD occurred in 18% of patients receiving CyA/MTX compared to 53% of patients receiving CyA. The 100-day mortality rate was 0.16 (95%CI 0.08-0.28) and 1-year nonrelapse mortality was 0.24 (95%CI 0.13-0.38). Thirty-three patients remained alive and in CR at a median of 19 months post transplant (range 3-38 months). We have shown that patients transplanted with fludarabine phosphate, melphalan 100 mg/m2 and with CyA/MTX as post transplant immunosuppression can achieve good disease control with an acceptable level of toxicity. Further studies are required to confirm these findings.