Background: Systemic light chain amyloidosis (AL) is a rare and life-threatening protein-deposition disorder. The diagnosis and especially quantification of the underlying, usually small clonal B ...cell disorder in patients with very low levels of free kappa or lambda light chains in serum (FLC) can be challenging. DFLC (difference of involved minus uninvolved FLC) response to therapy is hardly assessable for initial values below 50 mg/l. Consequently, these patients are frequently excluded from prospective and retrospective studies.
Objective: Characterization of AL amyloidosis patients with dFLC<50.
Methods: We have retrospectively analysedthe clinical features and long-termoutcome of 611 newly diagnosed AL patients with available dFLC and cytogenetic evaluation by iFISH at their first visit to our center between 2003-2014.
Results: Clinical characteristics and detailed results are depicted in table 1. Patients with dFLC<50 significantly showed lower bone marrow plasma cell counts (6% vs. 10%, p<0.001), M-spike (7 g/l vs. 9 g/l, p<0.001) and concentrations of the monoclonal heavy chain (7 g/l vs. 10 g/l, p=0.003), while the mere presence of a monoclonal heavy chain in immunofixation (IF) was more frequent in these patients (55% vs. 38%, p=0.003). All analysed chromosomal aberrations were not associated with dFLC<50 (all p-values >0.05). Patients with cardiac (40% vs. 82%, p<0.001) and soft tissue (26% vs. 42%, p=0.005) involvement, higher Mayo Score and lower Karnofsky Index (KI) were much less frequently found in the group with initial dFLC<50, while kidney involvement was more common (85% vs. 58%, p<0.001). This, however, was not associated with a significantly worse renal function at diagnosis. Median overall survival (OS) was significantly better in patients with dFLC<50 regardless of treatment type (Figure 1): Bortezomib (77 vs. 16 months, p=0.006), Melphalan-Dexamethason (Mdex, 96 vs. 19 months, p=0.001) and high-dose Melphalan (HDM, not reached vs. 99 months, p=0.005).
Conclusion: AL patients with an initial dFLC<50 mg/l represent a distinct clinical entity characterized by infiltration of the marrow with a small plasma cell clone and frequent presence of a monoclonal intact heavy chain, but with a low clonal heavy chain load. Importantly, this group of patients is not associated with any particular chromosomal aberrations as revealed by iFISH. This entity is further associated with a distinct pattern of organ involvement, i.e. a low Mayo Score, more than 80% of patients with renal amyloidosis, and very favourable OS irrespective of primary treatment regimens. Results of prospective clinical trials might be adversely influenced by the exclusion of these patients.
Table 1ParameterAll patientsn=611dFLC < 50 mg/ln=85dFLC ≥ 50 mg/ln=526p valuesAge in years, median range66 38-9065 47-9066 38-90n.s.Sex female, no. of pts (%)235 (39)39 (46)196 (37)n.s.Plasma cell related factorsdFLC in mg/l, median range228 1-12.07829 1-49279 50-12.078-Monoclonal heavy chain in IF, no. of pts (%)248 (41)47 (55)201 (38)0.003M-spike in g/l, median rangeEvaluable pts (%)9 1-41159 (26)7 1-2231 (36)9 1-41128 (24)<0.001Involved heavy chain in g/l,median rangeEvaluable pts (%)9.6 0.5-197243 (40)6.8 1.2-2645 (53)10.2 0.5-197198 (38)0.003Involved light chain λ, no. of pts (%)490 (80)73 (86)417 (79)n.s.BM plasma cell count in %,median range10 1-906 1-4010 1-90<0.001iFISH results, no. of pts (%)t(11;14)350 (58)42 (51)308 (59)n.s.del 13q14201 (33)27 (33)174 (33)n.s.gain 1q21166 (27)22 (26)144 (27)n.s.Hyperdiploidy (Wuilleme Score)98 (16)12 (14)86 (16)n.s.High-risk (del 17p13, t(4;14), t(14;16))47 (8)7 (8)40 (8)n.s.Organ involvementNumber of Organs,median range2 1-62 1-63 1-60.001Heart, no. of pts (%)463 (76)34 (40)429 (82)<0.001Cardiac Mayo Score 2004: I, no. of pts (%)II, no. of pts (%)III, no. of pts (%)101 (18)214 (38)255 (45)35 (46)30 (40)11 (15)66 (13)184 (37)244 (49)<0.001Kidney, no. of pts (%)376 (62)72 (85)304 (58)<0.001MDRD, median range64 2-26468 8-14963 2-264n.s.Soft Tissue, no. of pts (%)243 (40)22 (26)221 (42)0.005KI %, median range80 40-10090 50-10080 40-1000.001Treatment groups, no. of pts / median follow-up in months, median OSBortezomib214 / 272423 / 1977191 / 28160.006Mdex156 / 742721 / 7596135 / 70190.001HDM115 / 7512924 / 75not reached91 / 69990.005
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Hegenbart:Janssen: Honoraria. Bochtler:TEVA: Other: travel support. Goldschmidt:Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Chugai: Honoraria, Research Funding, Speakers Bureau; Millenium: Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Schönland:Janssen, Prothena: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Background
Systemic AL amyloidosis (AL) is associated with an underlying plasma cell dyscrasia and characterised by deposition of abnormally folded monoclonal immunoglobulin light chains. High dose ...melphalan with autologous stem cell transplantation (ASCT) can lead to durable complete haematologic and organ responses. Early studies reported TRM of >20%. Strategies for careful patient selection have made ASCT safer with TRM 2-5% but exclude ~80% of patients with AL from availing this treatment modality. Modern chemotherapy can lead to high clonal and organ responses but durability of response remains unclear. We describe our experience of deferred ASCT in 22 patients treated at two large European amyloidosis centres who were considered ineligible for ASCT at presentation but achieved clonal and organ responses with induction chemotherapy.
Methods
22 patients with AL underwent deferred ASCT from September 2011 to July 2017; 8 were treated at Heidelberg University Amyloidosis Centre and 14 were treated at the UK National Amyloidosis Centre. All underwent serial assessment of organ function, biomarkers and clonal response. Organ involvement and response were defined by international amyloidosis consensus criteria. Survival was calculated by Kaplan-Meier analysis. All analyses are on intention to treat basis (ITT).
Results
Baseline characteristics are presented in Table 1. All patients were diagnosed with AL between January 2011 - November 2016. All had cardiac involvement: Mayo Stage 2, 3A and 3B disease in 4 (18%), 16 (73%) and 2 (9%) patients respectively.
All patients received bortezomib-based induction chemotherapy, median number of cycles was 6 (range 4-9). 6 (27.2%) were treated with a bortezomib-IMID regimen, the remainder had bortezomib-dexamethasone or a bortezomib-alkylator protocol. Haematologic responses after chemotherapy on an ITT basis were: complete haematologic response (CR) - 11 (50%), very good partial response (VGPR) - 8 (36%) partial response (PR) - 3 (14%). At 6 months, 14 (64%) achieved a cardiac response, 5/9 (56%) with renal involvement achieved a renal response and 3/5 (60%) with liver involvement achieved a liver response.
Median time to ASCT from presentation was 14.5 months (range 2-71 months). Indication for ASCT was hematologic progression in 10 (45%) patients and as a consolidation procedure in the remainder. At the time of ASCT, all patients had NYHA class 1-2 symptoms (compared to 86.4% with NYHA Class 1-2 symptoms and 13.6% with NYHA Class 3-4 symptoms at presentation) and ECOG of 0-1 (compared to 95.5% with ECOG 0-2 and 4.5% with ECOG 3-4 at presentation). Median NT-proBNP was 389ng/L (101-2853ng/L)prior to ASCT, having been 2652ng/L (930-28737ng/L) at presentation (p=0.0001) and 1497ng/L (237-5167ng/L) 6 months after diagnosis (p=0.0016). Mean LV wall thickness was 14.5mm, compared to 14.2mm prior to ASCT (p=0.435). Mean LV ejection fraction was 52.5% 6 months after diagnosis, compared to 55.3% prior to ASCT (p=0.478).
Median overall survival has not been reached and 3 patients (13.6%) died (at 6, 11 and 26 months post ASCT) (Fig 1).One patient has yet to reach 3 month follow up. Number of patients in an FLC CR, VGPR, PR and no response on an evaluable basis at 3 months was as follows: 11 (52.4%), 5 (23.8%), 3 (14.3%) and 2 (9.5%). Haematologic response was assessed at 6 months post ASCT in 17 patients (5 have yet to reach 6 month follow up (data to be updated at the meeting) and 1 patient died at 6 months). The number of patients in an FLC CR, VGPR and PR at 6 months was as follows: 10/17 (58.8%), 3/17 (17.6%) and 4/17 (23.5%) respectively (Fig 2). Cardiac response at 6 months was assessed in 14 patients. Of the remainder, one had died and others are awaiting 6 month cardiac assessment. 10/14 (71.4%) were in a cardiac response 6 months post ASCT. Median NT-proBNP 6 months post ASCT was 595ng/L (189-2589 ng/L) (Fig 3).
Conclusion
This study highlights the potential role of deferred ASCT in both a consolidation or relapse setting in selected patients with cardiac AL who have achieved organ responses. No TRM was observed and ASCT was associated with good haematologic and organ responses. Younger patients (<65 years) with AL should be considered for a stem cell sparing regime to potentially avail this treatment modality if clinically significant organ responses are achieved. Longer follow up and larger studies are needed to confirm these findings and assess durability of response.
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Hegenbart:Janssen: Honoraria, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Prothena: Membership on an entity's Board of Directors or advisory committees. Yong:Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Popat:Takeda: Honoraria, Other: Travel support for meetings; Amgen: Honoraria; Celgene: Honoraria, Other: Travel support for meetings; Janssen: Honoraria, Other: Travel support for meetings. Rabin:Janssen: Consultancy, Other: Travel support for meetings, Speakers Bureau; Takeda: Consultancy, Other: Travel support for meetings, Speakers Bureau; Celgene: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Schönland:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; prothena: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Speakers Bureau. Wechalekar:Celgene: Honoraria; Janssen: Honoraria.
We performed a prospective sensitivity analysis to detect amyloid in bone marrow (BM) smears stained with Congo red (CR) and according to Pappenheim of patients with systemic light-chain (AL) ...amyloidosis. Results were directly compared to routine BM histology and fat aspiration. We analysed 198 BM smears from patients with the diagnosis or suspicion of systemic AL amyloidosis. Ultimately, the diagnosis could be established for 168 patients. Amyloid was detected on BM smears with CR in 33% (56/168). All patients suspicious for amyloid on Pappenheim staining (n = 39) showed substantial amyloid infiltration on CR. No patient without systemic AL amyloidosis stained positive. Sensitivity for routine BM histology was 57% (74/129) and for fat aspiration 96% (134/140). Patients with amyloid on BM smears had significantly more hepatic (42 vs. 9%, p < .001), renal (78 vs. 43%, p < .001) and gastrointestinal involvement (40 vs. 22%, p < .01) and less commonly cardiac involvement (58 vs. 76%, p < .03) and consecutively no adverse prognosis. CR staining of BM smears cannot be recommended as a primary screening tool for systemic AL as its overall sensitivity is far inferior to BM histology and fat aspiration. However, we recommend using the technique when suspecting amyloid on Pappenheim staining to establish the diagnosis of systemic AL amyloidosis.
Electron microscopy is the gold standard to characterize centrosomal ultrastructure. However, production of significant morphometrical data is highly limited by acquisition time. We therefore ...developed a generalizable, semi-automated high-throughput electron tomography strategy to study centrosome aberrations in sparse patient-derived cancer cells at nanoscale. As proof of principle, we present electron tomography data on 455 centrioles of CD138
pos
plasma cells from one patient with relapsed/refractory multiple myeloma and CD138
neg
bone marrow mononuclear cells from three healthy donors as a control. Plasma cells from the myeloma patient displayed 122 over-elongated centrioles (48.8%). Particularly mother centrioles also harbored gross structural abnormalities, including fragmentation and disturbed microtubule cylinder formation, while control centrioles were phenotypically unremarkable. These data demonstrate the feasibility of our scalable high-throughput electron tomography strategy to study structural centrosome aberrations in primary tumor cells. Moreover, our electron tomography workflow and data provide a resource for the characterization of cell organelles beyond centrosomes.
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High-throughput electron tomography reveals organelle structure in patient material
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The workflow can be adapted to various cell types and organelles
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We characterize 455 centrioles in human bone marrow cells at nanoscale
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Myeloma cells contain over-elongated centrioles with gross structural aberrations
Visualizing the organelle structure in patient-derived tissues at nanoscale can provide insights into numerous pathologies, but existing tools do not allow for analysis of sufficient numbers of cells at the required resolution. Here, by applying a semi-automated high-throughput electron tomography strategy, we quantify structural abnormalities of centrioles in human bone-marrow-derived cells at high numbers and share our data with the cell biological community to illustrate its utility as a tool for structural organelle analysis.
Dittrich et al. describe a semi-automated high-throughput electron tomography strategy to study organelle structure in patient-derived material at nanoscale. By applying their methodology to centrosomes, they show that plasma cells from a myeloma patient harbor over-elongated centrioles with gross structural abnormalities as the potential cause of chromosomal aberrations in multiple myeloma.
Analysis of intraclonal heterogeneity has yielded insights into the clonal evolution of hematologic malignancies. We compared the clonal and subclonal compositions of the underlying plasma cell ...dyscrasia in 544 systemic light chain amyloidosis (PC-AL) patients with 519 patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), or symptomatic MM; ie, PC–non-AL patients). Using interphase fluorescence in situ hybridization, subclones were stringently defined as clone size below two thirds of the largest clone and an absolute difference of ≥30%. Subclones were found less frequently in the PC-AL group, at 199 (36.6%) of 544 as compared with 267 (51.4%) of 519 in the PC–non-AL group (P < .001), and were not associated with the stage of plasma cell dyscrasia in either entity. In both groups, translocation t(11;14), other immunoglobulin heavy chain translocations, and hyperdiploidy were typically found as main clones, whereas gain of 1q21 and deletions of 8p21, 13q14, and 17p13 were frequently found as subclones. There were no shifts in the subclone/main clone ratio depending on the MGUS, SMM, or MM stage of plasma cell dyscrasia. In multivariate analysis, t(11;14) was associated with lower rates of subclone formation and hyperdiploidy with higher rates. PC-AL itself lost statistical significance, demonstrating that the lower subclone frequency in AL is a reflection of its exceptionally high t(11;14) frequency. In summary, the subclone patterns in PC-AL and PC–non-AL are closely related, implying that subclone formation depends on the main cytogenetic categories and is independent of disease entity and stage.
•Subclone formation is similar in AL and MGUS/MM; t(11;14) is typical main clone; 1q gain and 17p, 8p, or 13q deletion are frequent subclones.•t(11;14) suppresses emergence of subclones, thus accounting for the lower subclone frequency in AL amyloidosis.
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Motivation: With the exponential growth of expression and protein–protein interaction (PPI) data, the frontier of research in systems biology shifts more and more to the integrated analysis of these ...large datasets. Of particular interest is the identification of functional modules in PPI networks, sharing common cellular function beyond the scope of classical pathways, by means of detecting differentially expressed regions in PPI networks. This requires on the one hand an adequate scoring of the nodes in the network to be identified and on the other hand the availability of an effective algorithm to find the maximally scoring network regions. Various heuristic approaches have been proposed in the literature. Results: Here we present the first exact solution for this problem, which is based on integer-linear programming and its connection to the well-known prize-collecting Steiner tree problem from Operations Research. Despite the NP-hardness of the underlying combinatorial problem, our method typically computes provably optimal subnetworks in large PPI networks in a few minutes. An essential ingredient of our approach is a scoring function defined on network nodes. We propose a new additive score with two desirable properties: (i) it is scalable by a statistically interpretable parameter and (ii) it allows a smooth integration of data from various sources. We apply our method to a well-established lymphoma microarray dataset in combination with associated survival data and the large interaction network of HPRD to identify functional modules by computing optimal-scoring subnetworks. In particular, we find a functional interaction module associated with proliferation over-expressed in the aggressive ABC subtype as well as modules derived from non-malignant by-stander cells. Availability: Our software is available freely for non-commercial purposes at http://www.planet-lisa.net. Contact: tobias.mueller@biozentrum.uni-wuerzburg.de
Motivation: Increasing quantity and quality of data in transcriptomics and interactomics create the need for integrative approaches to network analysis. Here, we present a comprehensive R-package for ...the analysis of biological networks including an exact and a heuristic approach to identify functional modules. Results: The BioNet package provides an extensive framework for integrated network analysis in R. This includes the statistics for the integration of transcriptomic and functional data with biological networks, the scoring of nodes as well as methods for network search and visualization. Availability: The BioNet package and a tutorial are available from http://bionet.bioapps.biozentrum.uni-wuerzburg.de Contact: marcus.dittrich@biozentrum.uni-wuerzburg.de; tobias.mueller@biozentrum.uni-wuerzburg.de
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