Immunoglobulin Variable Gene Region (IGVL) Usage Correlates with Distinct Clinical Presentation in IgM Versus Non-IgM Light Chain Amyloidosis

Introduction: The clinical presentation and frequency of organ involvement are quite distinct in patients with IgM amyloidosis compared to patients with non-IgM amyloidosis, with less heart involvement and more nerve, soft tissue and lung involvement in IgM patients. It has been observed that immunoglobulin light chain variable region (IGVL) gene and gene family of the plasma cell clone impact phenotypic manifestations in AL amyloidosis, particularly organ tropism. (Kourelis et al. Blood. 2017;129). Given the variability in clinical features amongst patients with IgM vs. non IgM AL amyloidosis, we hypothesized that IGVL gene usage would be different between these two groups. Methods: Patients with newly diagnosed IgM and non-IgM amyloidosis seen at our institution from 01/2006 to 12/2015 were identified. IGVL gene usage was assessed by liquid chromatography tandem mass spectrometry (LC/MS/MS) as previously described. (Vrana et al Blood 2009;Dasari et al J of Proteome Res; Kourelis et al. Blood. 2017). Mass spectrometry data for IGVL analysis was obtained from clinically available data. LC/MS/MS was performed on available archival specimens when such data was not available clinically. Results: Patients with newly diagnosed amyloidosis who had sufficient mass spectrometry data on fat aspirate or tissue biopsy for IGVL gene usage identification were included (IgM AL, N=44 and Non-IgM AL, N=391). Clinical Features: As expected, differences were noted in baseline characteristics of patients with IgM vs. non-IgM AL amyloidosis. Patients with IgM amyloidosis were older (68 vs. 64 years, p=0.04) and more likely to have kappa as the involved light chain (34% vs. 25%, p=0.2), though the difference did not reach statistical significance. IgM amyloidosis patients had lower difference in involved and uninvolved free light chains (dFLC) (15.5 vs. 25.8 mg/dL, p=0.01) and lower NTProBNP levels (median: 1987 vs. 2655 pg/mL, p=0.02) compared with non-IgM amyloid patients. Presence of t(11;14) was less common by FISH (30% vs. 48%, p=0.1), though the difference did not reach statistical significance. Patterns of organ involvement were different, with heart involvement being less frequent in IgM patients (64% vs. 76%), while peripheral nerve (30% vs. 18%, p=0.05), soft tissue (41% vs. 21%, p=0.005) and lung involvement (7% vs. 3%, p=0.1) were more common in patients with IgM vs. non-IgM AL amyloidosis. There was no difference in liver (9% vs. 15%), kidney (50% vs. 51%) GI (23% vs. 25%) or autonomic nervous system involvement (18% vs. 14%). IGVL Gene Usage: As shown in Figure 1, IGVL usage differed across the two groups. In the lambda group, LV2-08 (14% vs. 2%, p<0.001) and LV2-14 (36% vs. 10%, p<0.001) were more common in IgM amyloid patients, while LV1-44 (0% vs. 10%, p=0.02) and LV6-57 (2% vs. 18%, p=0.004) were less common in the IgM group. There was also a trend towards less frequent involvement of LV3-01 (2% vs. 12%, p=0.07) in IgM amyloid patients. In the kappa group, KV4-01 (11% vs. 4%, p=0.04) was more common in patients with IgM amyloidosis. Correlation Between IGVL Gene usage and Clinical Features: IGVL gene usage correlated with disease characteristics and differences in the clinical presentation of patients across the two groups. LV2-14 was more commonly seen in IgM amyloid patients (above). It has been previously associated with a higher frequency of peripheral nerve involvement (Kourelis et al. Blood 2017), which was more frequent in IgM group. LV2-14 is also associated with low dFLC levels, which were noted to be lower in the IgM AL cohort. LV1-44 has been associated with higher likelihood of cardiac involvement. LV1-44 was seen less frequently in the IgM amyloid patients, who also had lower rates of cardiac involvement. LV6-57 has been historically associated with higher rates of t(11;14). It was less common in IgM AL patients, who also had lower rates of t(11;14). Conclusions: IGVL gene usage differed significantly in patients with IgM vs. non-IgM amyloidosis, correlating with differences in disease characteristics and organ involvement. This is the first study to our knowledge to report IGVL gene usage differences in IgM vs. non-IgM AL amyloidosis. Differences in IGVL gene usage may explain the distinct clinical presentation seen in IgM amyloidosis, the biology of which has so far remained poorly understood. *SS and SD contributed equally Disclosures Dasari: The Binding Site: Patents & Royalties: US Patent Rights on Mass Spectroscopy Licensing agreement with The Binding Site, Research Funding. Dispenzieri:Alnylam: Research Funding; Celgene: Research Funding; Takeda: Research Funding; Pfizer: Research Funding; Janssen: Consultancy; Akcea: Consultancy; Intellia: Consultancy. Murray:The Binding Site: Patents & Royalties: US Patent Rights on Mass Spectroscopy Licensing agreement with The Binding Site, Research Funding. Kumar:Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Takeda: Research Funding. Gertz:Prothena: Honoraria; Alnylam: Honoraria; Ionis: Honoraria; Janssen: Honoraria; Spectrum: Honoraria, Research Funding; Celgene: Honoraria.

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A Novel Mass Spectrometry Method to Identify the Serum Monoclonal Light Chain Component and Infer Organ Involvement in Systemic Light Chain Amyloidosis

Blood ◽

10.1182/blood-2018-99-114028 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4445-4445 ◽

Cited By ~ 1

Author(s):

Faye Sharpley ◽

Richa Manwani ◽

Shameem Mahmood ◽

Sajitha Sachchithanantham ◽

Helen Lachmann ◽

...

Keyword(s):

Mass Spectrometry ◽

Molecular Mass ◽

Light Chain ◽

Mass Spectra ◽

Amyloid Fibrils ◽

Cardiac Involvement ◽

Small Sample ◽

Organ Involvement ◽

Light Chains ◽

Al Amyloidosis

Abstract Introduction Light chain (AL) amyloidosis is caused by progressive organ dysfunction due to the deposition of structurally abnormal monoclonal light chains (LC) as amyloid fibrils. Serial free light chain measurement is the cornerstone of AL diagnosis but is limited by current methods which measure normal polyclonal and abnormal monoclonal LCs. Since each individual monoclonal LC has a unique amino acid sequence, mass spectrometry (MS) has the ability to detect serum monoclonal LCs. We report a novel MS method for monoclonal LC detection in AL amyloidosis. Methods Twenty patients with systemic AL amyloidosis, diagnosed and treated at the UK National Amyloidosis Centre (UK-NAC), were randomly selected. This included 16 newly diagnosed patients, 2 patients in a complete haematological remission (CR) post-treatment and 2 patients with no amyloidosis (acting as negative controls). All patients had detailed baseline assessments of organ function and serum FLC measurements. Organ involvement was defined according to the international amyloidosis consensus criteria. Magnetic microparticles were covalently coated with modified polyclonal sheep antibodies monospecific for free kappa light chains (anti-free k) and free lambda light chains (anti-free l). The microparticles were incubated with patient sera, washed and treated with acetic acid (5% v/v) containing TCEP (20 mM) in order to elute free light chains in monomeric form. Mass spectra were acquired on a Microflex LT/SH smart matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS; Bruker, GmbH). Results The median number of organs involved was 2 (range 1-4); cardiac involvement was most common in 70% (14/20) patients, followed by renal 40% (8/20), autonomic and soft tissue in 15% (3/20) and peripheral nerve involvement in 5% (1/20). No patients had liver involvement. The median N-terminal pro b-type natriuretic peptide (NT-proBNP) and cardiac troponin T were 3761.5 (range 245-25348 ng/L) and 35.5 (8-170 ng/L) respectively. The median presenting serum albumin was 37 (19-45 g/L) and eGFR 62 (10-100 mls/min). The amyloid deposits were typed as AL lambda in 14(70%), kappa in 2(10%) patients and amyloid of uncertain type in two cases (10%). The median involved FLC was kappa 76 mg/L (range 71-440) and lambda 185 mg/L (44-1023), with difference involved to uninvolved (dFLC) 118 mg/L (33-1015). An intact monoclonal protein was present in 70% (14/20). In all cases, MS correctly identified the presence and type of monoclonal LC, identifying a monoclonal lambda in 14/14 (100%) and a monoclonal kappa in 2/2 (100%); the two patients where a clear monoclonal component could not be identified were patients whose amyloid fibrils failed to be typed by immunohistochemistry or MS. The assay also confirmed normal polyclonal expression of both kappa and lambda LCs in the two control samples. In the two lambda patients in a CR, the MS method identified monoclonal lambda LC expression; in one of these patients next-generation sequencing (NGS) confirmed minimal residual disease (MRD). A clear shift in the LC mass spectra was seen relating to the specific pattern of organ involvement: in patients with renal amyloid the monoclonal LC predominantly displayed a "heavy" molecular mass, (with a mean molecular mass of 11596+/- 436 daltons), whereas in patients with cardiac involvement the monoclonal LC mainly exhibited a "light" mass (with a mean molecular mass of 11443 +/- 102 daltons). Conclusion This small study shows that monoclonal light chains can be accurately detected by MS and be concordant to the tissue amyloid type. A monoclonal LC was detected in 2 patients in serological "CR" - in one case presence of persistent disease was demonstrated by NGS-MRD. Even in this small sample size, there appears to be a marked difference in LC mass for patients with cardiac ("light" LC) vs. renal ("heavy" LC) involvement, raising the interesting possibility that "heavy" LC are trapped by the glomerulus causing renal AL but are unable to penetrate the tight cardiac capillary gap junctions, and vice versa. The unique molecular location of LC on MS offers the possibility of exploiting this technique as a tool to detect amyloidogenic FLC and potentially predict organ involvement in patients with gammopathies. We plan to expand this study to a large cohort of patients to confirm these findings and assess the impact on survival and organ response outcomes. Figure 1 Figure 1. Disclosures Wechalekar: Janssen: Honoraria.

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Phase 2 Trial of Ixazomib, Cyclophosphamide and Dexamethasone for Treatment of Previously Untreated Light Chain Amyloidosis

Blood ◽

10.1182/blood-2020-138814 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 52-53

Author(s):

Eli Muchtar ◽

Morie A Gertz ◽

Betsy Laplant ◽

Francis K. Buadi ◽

Nelson Leung ◽

...

Keyword(s):

Clinical Trials ◽

Board Of Directors ◽

Light Chain ◽

Research Funding ◽

Advisory Board ◽

Organ Involvement ◽

Al Amyloidosis ◽

Advisory Committees ◽

Hematologic Response ◽

Phase 2 Trial

Background: Bortezomib, a proteasome inhibitor, has shown efficacy in the treatment of newly diagnosed and relapsed light chain (AL) amyloidosis, and the combination of bortezomib, cyclophosphamide and dexamethasone is a commonly used regimen in AL. Ixazomib is the first oral proteasome inhibitor to be approved, and the combination of ixazomib with cyclophosphamide and dexamethasone is an all oral effective regimen for the treatment of multiple myeloma. This phase 2 trial was designed to evaluate the efficacy of this regimen in patients with AL, who have not received any therapy. Patients and methods: Newly diagnosed patients with biopsy proven AL amyloidosis, with organ involvement requiring therapy, were enrolled if they had measurable disease (Serum immunoglobulin free light chain ≥5 mg/dL AND abnormal serum free light chain ratio) and adequate organ function. Patients with severe organ involvement were excluded (Alkaline phosphatase >750 U/L, creatinine clearance <30 mL/min or NT-ProBNP ≥ 7500 ng/dL). Treatment consisted of ixazomib 4 mg days 1, 8, 15; cyclophosphamide 500 mg PO weekly and dexamethasone 40 mg, weekly for twelve 28-day cycles, followed by ixazomib maintenance (days 1, 8, 15) at the last tolerated dose till progression. The primary objective was to determine the hematologic response rate of ixazomib, used in combination with cyclophosphamide and dexamethasone in patients with previously untreated AL. A one-stage binomial design was utilized to test the null hypothesis that the hematologic response rate is at most 30% against the alternative hypothesis that it is at least 50%, with 85% power and 9% type I error. Results: Thirty-five patients were enrolled, median age was 67 (range 38-78) years; 69% were male. Organ involvement included cardiac in 23 (65.7%), renal in 19 (54.3%), and nervous system involvement in 5 (14.3%). At data cutoff 8 patients still remain on study with a median follow up of 4.4 months for those who are alive. Across the trial a median of 4 cycles (range 0-23) of treatment have been completed; the most common reason for going off study was institution of alternate therapy in 17 patients (63%). The overall hematologic response was 57% (20/35) and included amyloid CR in 5 (14%), VGPR in 9 (26%) and a PR in 6 (17%) patients. Confirmed organ responses have been observed in 5 patients so far, 2 each for cardiac and renal and 1 hepatic. The median PFS and OS have not been reached; 4 patients had hematological progression; 6 patients (17%) have died. Across 193 cycles of treatment administered, dose modification was required in 5, 3, and 10 patients, respectively, for ixazomib, cyclophosphamide and dexamethasone. A grade 3 or higher adverse event (AE), at least possibly attributed to the study drugs, was observed in 41% of patients. The figure shows the maximum grade of adverse events for individual patients seen in more than one patient across the study. Conclusions: The all-oral regimen of ixazomib, cyclophosphamide, and dexamethasone is active in patients with previously untreated AL amyloidosis with hematologic responses observed in 57% of patients, including complete responses. Organ response has been observed but will likely need longer follow up for accurate assessment, given the delay in organ responses in this disease. Further evaluation of this combination is warranted. Disclosures Gertz: Alnylam: Consultancy; Ionis/Akcea: Consultancy; Amgen: Consultancy; Medscape: Consultancy, Speakers Bureau; Physicians Education Resource: Consultancy; Data Safety Monitoring board from Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Johnson and Johnson: Speakers Bureau; DAVA oncology: Speakers Bureau; Advisory Board for Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Advisory Board for Proclara: Membership on an entity's Board of Directors or advisory committees; i3Health: Consultancy; Springer Publishing: Patents & Royalties; Amyloidosis Foundation: Research Funding; International Waldenstrom Foundation: Research Funding; NCI SPORE MM: Research Funding; Prothena: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; Spectrum: Consultancy, Research Funding; Annexon: Consultancy; Appellis: Consultancy. Kapoor:Celgene: Honoraria; GlaxoSmithKline: Research Funding; Takeda: Honoraria, Research Funding; Amgen: Research Funding; Sanofi: Consultancy, Research Funding; Janssen: Research Funding; Cellectar: Consultancy. Larsen:Janssen Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dingli:Apellis: Consultancy; Janssen: Consultancy; Sanofi-Genzyme: Consultancy; Rigel: Consultancy; Bristol Myers Squibb: Research Funding; Karyopharm Therapeutics: Research Funding; Alexion: Consultancy; Millenium: Consultancy. Dispenzieri:Janssen: Research Funding; Intellia: Research Funding; Alnylam: Research Funding; Celgene: Research Funding; Pfizer: Research Funding; Takeda: Research Funding. Kumar:Adaptive Biotechnologies: Consultancy; Carsgen: Other, Research Funding; AbbVie: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Janssen Oncology: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Genecentrix: Consultancy; Dr. Reddy's Laboratories: Honoraria; Tenebio: Other, Research Funding; Takeda: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; BMS: Consultancy, Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Kite Pharma: Consultancy, Research Funding; Karyopharm: Consultancy; Oncopeptides: Consultancy, Other: Independent Review Committee; IRC member; Merck: Consultancy, Research Funding; Amgen: Consultancy, Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments, Research Funding; Genentech/Roche: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Celgene/BMS: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Cellectar: Other; MedImmune: Research Funding.

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IGVL gene region usage correlates with distinct clinical presentation in IgM vs non-IgM light chain amyloidosis

Blood Advances ◽

10.1182/bloodadvances.2020003671 ◽

2021 ◽

Vol 5 (8) ◽

pp. 2101-2105

Author(s):

Surbhi Sidana ◽

Surendra Dasari ◽

Taxiarchis V. Kourelis ◽

Angela Dispenzieri ◽

David L. Murray ◽

...

Keyword(s):

Light Chain ◽

Clinical Presentation ◽

Variable Region ◽

Immunoglobulin M ◽

Al Amyloidosis ◽

Amyloid Deposits ◽

Peripheral Nerve Involvement ◽

Gene Usage ◽

Region Usage ◽

Organ Tropism

Abstract Patients with immunoglobulin M (IgM) light chain (AL) amyloidosis have a distinct clinical presentation compared with those with non-IgM amyloidosis. We hypothesized that differential immunoglobulin light-chain variable region (IGVL) gene usage may explain the differences in organ involvement, because IGVL usage correlates with organ tropism. IGVL usage was evaluated by mass spectrometry of amyloid deposits (IgM, n = 45; non-IgM, n = 391) and differed across the 2 groups. In the λ family, LV2-08 (13% vs 2%; P < .001) and LV2-14 (36% vs 10%; P < .001) usage was more common in IgM vs non-IgM amyloidosis, whereas LV1-44 (0% vs 10%; P = .02) and LV6-57 (2% vs 18%; P = .004) usage was less common. In the κ family, there was a trend toward higher KV4-01 (11% vs 4%; P = .06) usage in IgM amyloidosis. IGVL usage correlated with disease characteristics/organ tropism. LV2-14 (more common in IgM amyloidosis) has historically been associated with peripheral nerve involvement and lower light chain burden, which were more frequent in IgM amyloidosis. LV1-44 (less common in IgM), associated with cardiac involvement, was less frequent in IgM patients. LV6-57 (less common in IgM) is associated with t(11;14), which was less frequent in IgM patients. In conclusion, IGVL gene usage differs in patients with IgM vs non-IgM amyloidosis and may explain the distinct clinical presentation.

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Quantitative Immunoprecipitation Free Light Chain Mass Spectrometry (QIP-FLC-MS) Simplifies Monoclonal Protein Assessment and Provides Added Clinical Value in Systemic AL Amyloidosis

Blood ◽

10.1182/blood-2019-128175 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4375-4375 ◽

Cited By ~ 1

Author(s):

Faye Amelia Sharpley ◽

Hannah Victoria Giles ◽

Richa Manwani ◽

Shameem Mahmood ◽

Sajitha Sachchithanantham ◽

...

Keyword(s):

Mass Spectrometry ◽

Light Chain ◽

Residual Disease ◽

Renal Involvement ◽

Light Chains ◽

Al Amyloidosis ◽

Free Light Chains ◽

Accurate Identification ◽

Monoclonal Protein ◽

Serum And Urine

Introduction Early diagnosis, effective therapy and precise monitoring are central for improving clinical outcomes in systemic light chain (AL) amyloidosis. Diagnosis and disease response assessment is primarily based on the presence of monoclonal immunoglobulins and free light chains (FLC). The ideal goal of therapy associated with best outcomes is a complete responses (CR), defined by the absence of serological clonal markers. In both instances, detection of the monoclonal component (M-component) is based on serum FLC assessment together with traditional serum and urine electrophoretic approaches, which present inherent limitations and lack sensitivity particularly in AL where the levels are typically low. Novel mass spectrometry methods provide sensitive, accurate identification of the M-component and may prove instrumental in the timely management of patients with low-level amyloidogenic light chain production. Here we assess the performance of quantitative immunoprecipitation FLC mass spectrometry (QIP-FLC-MS) at diagnosis and during monitoring of AL amyloidosis patients treated with bortezomib-based regimens. Methods We included 46 serial patients with systemic AL amyloidosis diagnosed and treated at the UK National Amyloidosis Centre (UK-NAC). All patients had detailed baseline assessments of organ function and serum FLC measurements. Baseline, +6- and +12-month serum samples were retrospectively analysed by QIP-FLC-MS. Briefly, magnetic microparticles were covalently coated with modified polyclonal sheep antibodies monospecific for free kappa light chains (anti-free κ) and free lambda light chains (anti-free λ). The microparticles were incubated with patient sera, washed and treated with acetic acid (5% v/v) containing TCEP (20 mM) in order to elute FLC in monomeric form. Mass spectra were acquired on a MALDI-TOF-MS system (Bruker, GmbH). Results were compared to serum FLC measurements (Freelite®, The Binding Site Group Ltd), as well as electrophoretic assessment of serum and urine proteins (SPE, sIFE, UPE and uIFE). Results Cardiac (37(80%) patients) and renal (31(67%) patients) involvement were most common; 25(54%) patients presented with both. Other organs involved included liver (n=12), soft tissue (n=4), gastrointestinal tract (n=3) and peripheral nervous system (n=2). Baseline Freelite, SPE, sIFE and uIFE measurements identified a monoclonal protein in 42(91%), 22(48%), 34(74%) and 21(46%) patients, respectively. A panel consisting of Freelite + sIFE identified the M-component in 100% of the samples. QIP-FLC-MS alone also identified an M-component in 100% of the samples and was 100% concordant with Freelite for typing the monoclonal FLC (8 kappa, 34 lambda). In 4 patients, QIP-FLC-MS identified an additional M-protein that was not detected by the other techniques. In addition, 4/8(50%) kappa and 4/38(11%) lambda patients showed a glycosylation pattern of monoclonal FLCs at baseline by mass spectrometry. Interestingly, the frequency of renal involvement was significantly lower for patients with non-glycosylated forms (25% vs 76%, p=0.01), while no similar relationship was found for any other organs. During the 1-year follow-up period, 17 patients achieved a CR; QIP-FLC-MS identified serum residual disease in 13(76%) of these patients. Conclusion In our series, QIP-FLC-MS was concordant with current serum methods for identifying the amyloidogenic light chain type and provided, against all other individual tests, improved sensitivity for the detection of the monoclonal protein at diagnosis and during monitoring. The ability to measure the unique molecular mass of each monoclonal protein offers clone-specific tracking over time. Glycosylation of free light chains is over-represented in AL patients which may allow earlier diagnosis and better risk-assessment of organ involvement. Persistence of QIP-FLC-MS positive M component in patients otherwise in CR may allow targeted therapy. Overall, QIP-FLC-MS demonstrates potential to be exploited as a single serum test for precise serial assessment of monoclonal proteins in patients with AL amyloidosis. Disclosures Wechalekar: GSK: Honoraria; Janssen-Cilag: Honoraria; Amgen: Research Funding; Takeda: Honoraria; Celgene: Honoraria.

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Light chain type predicts organ involvement and survival in AL amyloidosis patients receiving stem cell transplantation

Blood Advances ◽

10.1182/bloodadvances.2018016782 ◽

2018 ◽

Vol 2 (7) ◽

pp. 769-776 ◽

Cited By ~ 9

Author(s):

M Hasib Sidiqi ◽

Mohammed A. Aljama ◽

Eli Muchtar ◽

Francis K. Buadi ◽

Rahma Warsame ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Light Chain ◽

Organ Involvement ◽

Al Amyloidosis ◽

Light Chain Type ◽

Key Points ◽

Λ Light Chain ◽

Chain Type

Key Points λ Light chain AL amyloidosis is associated with a shorter PFS and OS compared with κ. Light chain type predicts likelihood of organ involvement in AL amyloidosis.

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Patients with light-chain amyloidosis and low free light-chain burden have distinct clinical features and outcome

Blood ◽

10.1182/blood-2017-02-767467 ◽

2017 ◽

Vol 130 (5) ◽

pp. 625-631 ◽

Cited By ~ 51

Author(s):

Paolo Milani ◽

Marco Basset ◽

Francesca Russo ◽

Andrea Foli ◽

Giampaolo Merlini ◽

...

Keyword(s):

Clinical Features ◽

Light Chain ◽

Free Light Chain ◽

Al Amyloidosis ◽

Renal Survival ◽

Light Chain Amyloidosis ◽

Hematologic Response ◽

Heart Involvement ◽

Key Points

Key PointsPatients with AL amyloidosis and low dFLC burden (<50 mg/L) have less severe heart involvement and better survival. These patients are evaluable for hematologic response with adapted criteria predicting improvement of overall and renal survival.

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Autologous Stem Cell Transplantation In Immunoglobulin Light Chain Amyloidosis With Factor X Deficien

Blood ◽

10.1182/blood.v122.21.2151.2151 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2151-2151

Author(s):

Stefan F Cordes ◽

Morie A Gertz ◽

Francis K Buadi ◽

Yi Lin ◽

Martha Q Lacy ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Stem Cell ◽

Light Chain ◽

Research Funding ◽

Splenic Rupture ◽

Al Amyloidosis ◽

Factor X ◽

Immunoglobulin Light Chain ◽

Hemorrhagic Complications ◽

Transient Improvement

Abstract Background Acquired factor X (FX) deficiency is associated with immunoglobulin light chain (AL) amyloidosis and may be accompanied by hemorrhage. There are limited data on the effects of autologous stem cell transplant (ASCT) on FX deficiency. We reviewed hemorrhagic complications and the effect of high dose melphalan (HDM) and ASCT on FX levels in AL amyloidosis patients with FX deficiency. Methods We conducted a retrospective chart review of patients with AL amyloid with FX levels below 60%, not on chronic anti-coagulation who underwent HDM/ASCT at the Mayo Clinic, Rochester, MN between 1995 and 2011. Results Forty-one of 358 patients (11%) met our study criteria. Median pre-ASCT FX was 45% (range: 2%, 59%). The most common bleeding complication was central line associated n=15 (37%) followed by gastrointestinal n=10 (24%) and genitourinary n=9 (22%). The most frequent and severe bleeding complications occurred in patients with FX levels less than 10%. Four patients required emergent splenectomy owing to splenic rupture; one of these patients died from hemorrhagic shock. Periprocedural prophylaxis included activated recombinant Factor VII (rFVIIa) infusions, fresh frozen plasma (FFP) infusions and platelet transfusions. rFVIIa was efficacious in controlling bleeding during splenectomy (n=5) and, in conjunction with arterial embolization, for retroperitoneal bleed (n=1). Elective splenectomy for FX deficiency (n=1) resulted in only transient improvement in FX level. No relationship between the degree of pre-ASCT FX deficiency and other laboratory values (alkaline phosphatase, AST, total bilirubin, serum albumin, total serum protein, serum creatinine, total urine protein, beta2 microglobulin, troponin T) was found. Post-ASCT FX levels were determined in seventeen patients. In four of these patients, post-ASCT FX levels were determined in the acute/subacute phase of ASCT before steady state FX levels could be achieved; the median change in FX for these patients was -6.5% (range: -19%, 3%). In the remaining thirteen patients, who were between 99 and 1920 days from ASCT, FX improved by median 26% (range: -15%, 92%). Overall post-ASCT FX increased in twelve of thirteen (92%) patients. The improvement in FX correlated with improvement in the degree of proteinuria (p = 0.04) and showed a trend towards significant correlation with improvement in serum alkaline phosphatase (p = 0.06). Conclusions Hemorrhagic complications are most frequent and severe for FX levels below 10%. rFVIIa infusions, FFP and platelets were effective prophylactic agents. In the single patient who underwent elective splenectomy, a transient improvement in FX level was seen. Splenectomy was otherwise reserved for patients with splenic rupture/hematoma. Post-ASCT FX levels increased in twelve (92.3%) of the remaining thirteen patients; five of the patients (38.5%) were no longer FX deficient after ASCT. The degree of improvement in FX levels was correlated with improvement in markers of renal or hepatic involvement by amyloid. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.

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The Acidic Region of Factor VIII Light Chain Contains the Thrombin-Binding Site(s) Responsible for the Cleavage at Arg1689

Blood ◽

10.1182/blood.v126.23.2270.2270 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2270-2270

Author(s):

Hiroaki Minami ◽

Keiji Nogami ◽

Midori Shima

Keyword(s):

Binding Site ◽

Board Of Directors ◽

Light Chain ◽

Research Funding ◽

Coagulation Cascade ◽

Cross Linking ◽

C2 Domain ◽

Advisory Committees ◽

Acidic Residues ◽

Dose Dependent

Abstract Thrombin-catalyzed activation of factor (F)VIII by cleavages at Arg372, Arg740, and Arg1689 is essential for the propagation phase of blood coagulation cascade. Activated FVIII (FVIIIa) forms the tenase complex and markedly amplifies the activation of FX as a cofactor of FIX. We had already demonstrated that thrombin interacts with FVIII through the residues 392-394 and 484-509 in the A2 domain and the C2 domain, and each association regulates cleavage at Arg740, Arg372, and Arg1689, respectively (Nogami K, JBC 2000, 2005; BJH 2008), and recently reported that the A1 acidic clustered region 340-350 involving the sulfated tyrosine regulate the cleavage of Arg372 (Minami et al. 55th ASH). On the other hand, Fay and colleague suggested that recombinant FVIII lacking the C2 domain retains greater than 50% cofactor activity (JBC 2010), supporting the presence of other thrombin-binding region responsible for cleavage at Arg1689 of the light chain. In this study, we attempted to identify this thrombin-binding site(s). We focused on the acidic residues 1659-1669 and 1675-1685 within the light chain, which had similar sequence to the A1 residues 340-350 in terms of involving the clustered acidic residues and sulfated tyrosine as well as hirugen residues 54-65. We prepared four of synthetic peptides corresponding to the residues 1659-1669 and 1675-1685 with sulfated tyrosine, P(1659-69s) and P(1675-85s), and with non-sulfated tyrosine, P(1659-69) and P(1675-85). The inhibitory effect on the thrombin-catalyzed FVIII activation by each peptide was evaluated in a one-stage clotting assay. Each peptide showed a dose-dependent inhibition on thrombin-catalyzed activation. These inhibitory effects were greater in order of P1675-85s, P1659-69s, P1675-85, P1659-69, and the IC50 were 25, 67, 71 and 225 µM, respectively. The peptides with sulfated tyrosine had approximately 3-fold greater inhibition of the FVIII activation by thrombin than with non-sulfated tyrosine. The IC50 in the presence of mixture of P1675-85s and P1659-69s was 30.4 µM, suggesting that these peptides had no an additive effect. The impacts of P1659-69s and P1675-85s on the thrombin-catalyzed cleavage at Arg1689 were examined by SDS-PAGE/western blotting. These peptides blocked the cleavage at Arg1689 in dose-dependent fashions. In timed-course assay, the presence of P1659-69s and P1675-85s decreased the cleavage rate of Arg1689 by 61.3 % and 81.8 %, respectively compared to its absence. The direct binding of P1659-69s and P1675-85s to thrombin was examined by surface resonance plasmon (SPR)-based assay and by the zero-length cross-linking reagent EDC. In SPR-based assay using a Biacore T200TM, thrombin bound to immobilized P1659-69s and P1675-85s directly with high affinity. The Kd values adjusted to 1:1 binding model of global fitting were 203 nM and 94 nM, respectively. EDC cross-linking in fluid-phase assay revealed that formation of EDC cross-linking products between biotinylated P1659-69s or P1675-85s and thrombin were observed in dose-dependent fashions. The products between the biotinylated peptides (800 nM) and thrombin were competitively reduced by the addition of non-biotinylated peptides. Moreover, N-terminal sequence analysis of cross-linking products between both peptides-thrombin indicated that thrombin bound to the residues 1664-1669 and 1683-1684. Taken together, we demonstrated that the A3 residues 1659-1669 (QEEIDYDDTIS) and residues 1675-1685 (EDFDIYDEDEN) contained the thrombin binding-sites responsible for proteolytic cleavage at Arg1689 of the A3 domain. Disclosures Nogami: Bayer, NovoNordisk, Baxalta, Chugai, Kaketsuken, Pfizer, Biogen: Honoraria; Chugai: Membership on an entity's Board of Directors or advisory committees; Bayer, Novo Nordisk, Baxalta. Biogen: Research Funding. Shima:Chugai Pharmaceutical Co., Ltd. and F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Heavy/Light Chain Quantification Identifies Clonal Plasma Cell Disease in Patients with AL Amyloidosis and Normal Serum Free Light Chain Ratio

Blood ◽

10.1182/blood.v126.23.2956.2956 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2956-2956

Author(s):

Tatiana Prokaeva ◽

Brian Spencer ◽

Fangui Sun ◽

Nathaniel McConnell ◽

Richard M O'hara ◽

...

Keyword(s):

Overall Survival ◽

Binding Site ◽

Plasma Cell ◽

Light Chain ◽

Free Light Chain ◽

Al Amyloidosis ◽

Cell Disease ◽

Serum Free ◽

Serum Free Light Chain ◽

Combined Use

Abstract Background: Serum and urine immunofixation electrophoreses (SIFE/UIFE) are routinely used for detection of clonal immunoglobulins (Ig) in AL amyloidosis. Serum free light chain (FLC) assays (Freelite®, The Binding Site Ltd., Birmingham, UK) have significantly improved the management of patients with AL amyloidosis by providing quantitative measure for the detection and monitoring of clonal plasma cell disease. However, up to 20% of patients with AL amyloidosis may have uninformative serum free light chain values. Objective: To assess the quantitative potential of serum Heavy/Light Chain (HLC) pairs (Hevylite®, The Binding Site Ltd., Birmingham, UK) assay in identification of clonal plasma cell disease in AL amyloidosis. Methods: One hundred and ninety-nine untreated patients with AL amyloidosis were included in this study. Patients with multiple myeloma or B cell lymphoproliferative diseases associated AL amyloidosis were excluded. Serum sampleswere obtained at initial evaluation and stored at -20°C. SIFE/UIFE were performed at the time of sample collection. HLC pairs were assessed by the Hevylite® assay. HLC κ/λ normal ratios (HLCR) were: 1.12-3.21 for IgG κ/λ; 0.78-1.94 for IgA κ/λ; and 1.18-2.74 for IgM κ/λ. FLCs were assessed by the Freelite® assay; FLC κ/λ normal ratio (FLCR) was 0.26-1.65. In 103 cases, FLC testing was performed at the time of sample collection; 96 cases were tested at The Binding Site. Vital status of patients was obtained from either medical records or Social Security Death Index. Follow-up ended in June 2014. Results: An abnormal HLCR was found in 74 (37.2%), an abnormal FLCR in 163 (81.9%), and SIFE/UIFE positivity in 187 (94%) of 199 patients with AL amyloidosis. Of 36 patients with a normal FLCR, 23 (63.9%) were noted with an abnormal HLCR compared to 51 (31.3%) patients in an abnormal FLCR group (P = 0.001). In total 186/199 (93.5%) patients with AL amyloidosis had abnormalities in either HLCR or FLCR, compared to 187/199 (94%) of patients who were SIFE/UIFE+ (Table 1). The combined use of both FLCR and HLCR yielded quantifiable information in 93.5% of cases; the use of both tests in combination with SIFE/UIFE identified plasma cell clonality in 100% of patients. Seventy-two cases presented with an abnormal HLCR for a single isotype and 2 in multiple Ig isotypes. In all cases, involved LC type of abnormal HLCR matched LC type identified by SIFE/UIFE. None of 12 cases that were negative on the SIFE/UIFE presented with an abnormal HLCR, however, all showed abnormalities in FLCR. Table 1. Comparative efficiency of FLCR, HLCR and Serum/Urine Immunofixation in AL Amyloidosis patients. SIFE/UIFE+ (n=187) SIFE/UIFE- (n=12) HLCR+/FLCR+ 51 (27.2%) - HLCR+/FLCR- 23 (12.3%) - HLCR-/FLCR+ 100 (53.5%) 12 (100%) HLCR-/FLCR- 13 (7%) - Overall survival was similar in patients with and without abnormal HLCR (Log rank p=0.092; Figure 1), whereas patients with an abnormal FLCR had a significantly inferior overall survival compared to those with a normal FLCR (Log rank p=0.027; Figure 2). Combined use of both HLCR and FLCR demonstrated a trend toward superior overall survival in a group of patients with an abnormal HLCR / normal FLCR (Wilcoxon p=0.037; Log rank p=0.107; Figure 3). Conclusions: The Hevylite® assay provided information in addition to other laboratory tests for clonal plasma cell disease in AL amyloidosis. The combined use of the HLCR and FLCR provided quantifiable information in 93.5% of patients. The use of both assays in combination with SIFE/UIFE detected clonal disease in all patients. HLCR has potential to quantify clonal disease in patients with uninformative FLCR results. An abnormal HLCR was not predictive of overall survival, while an abnormal FLCR was, in this series of patients. Combined use of HLCR and FLCR could be beneficial in prognostication of outcome in AL amyloidosis. Disclosures McConnell: The Binding SIte: Employment. O'hara:The Binding Site: Employment.

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Immunoglobulin M Heavy/Light Chain Pair Measurement Independently Predicts Clinical Outcome and Refines Prognostic Information Provided By Interim PET in Patients with Aggressive Lymphomas

Blood ◽

10.1182/blood.v126.23.3880.3880 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3880-3880

Author(s):

Patricia Johansson ◽

Jan Dürig ◽

Jan Rekowski ◽

Stefan P Müller ◽

Bernd Hertenstein ◽

...

Keyword(s):

Clinical Outcome ◽

B Cell ◽

Binding Site ◽

Light Chain ◽

Research Funding ◽

Prognostic Value ◽

Immunoglobulin M ◽

Prognostic Information ◽

Interim Pet ◽

Aggressive Lymphomas

Abstract Introduction: We recently reported the first results of the large multicenter phase III PETAL trial (EudraCT 2006-001641-33, NCT00554164) showing that 18-fluorodeoxyglucose interim PET (iPET) performed after two cycles of chemotherapy is highly predictive of clinical outcome in patients with aggressive lymphomas. Here, the study`s biobank was utilized to exploratively investigate the prognostic value of immunoglobulin M heavy/light chain pair (HLC-M) abnormalities in this patient cohort. Methods: HLC-M κ and λ were measured in pre-treatment serum samples of a representative subset (N=187, 22%) of patients employing the Hevylite® assay (The Binding Site Ltd, Birmingham, UK). Normal HLC-M ratios and concentrations were defined according to the manufacturer`s recommendations. For statistical analysis standard time-to-event methodology (Kaplan-Meier method, Log Rank test, Cox regression) was used. Results: Median age of the 187 pts was 57 years (range 18-80), whereof 92 (49%) were male, and 95 (51%) were female. 174 pts. had CD20-positive B cell lymphomas (73% diffuse large B cell, 13% other aggressive B cell lymphomas, 7% follicular lymphoma grade 3), 13 had peripheral T cell lymphomas (7%). Normal HLC-M were observed in 150 patients (75.8%). 37/187 (20%) of the patients exhibited a monoclonal IgMκ/IgMλ ratio, where 17 (46%) showed a ratio below and 20 (54%) above the reference range. HLC-M abnormalities were significantly associated with adverse clinical characteristics including advanced Ann Arbor stage (p=0.005), high international prognostic index (IPI, p=0.001) and extranodal disease (p=0.003). Patients with abnormal HLC-M ratios had inferior time to treatment failure (TTTF, Figure 1) and overall survival (OS, Figure 2) as compared to their counterparts with normal HLC-M ratios. Of note, subgroup analyses revealed that the prognostic value of HLC-M abnormalities was limited to patients with a favorable iPET and low-intermediate IPI score. In multivariate analysis with the cox model and controlling for the IPI factors (including age), histology, sex as well as FLC κ and λ, monoclonal HLC-M remained predictive for a shorter TTTF (p=0.0181, covariate-adjusted hazard ratio (aHR) 2.195, 95% CI [1.144;4.214]) and OS (p=0.0034, aHR 3.844, 95% CI [1.559;9.476]). Conclusions: Monoclonal HLC-M is an independent predictor of survival in patients with aggressive lymphoma and refines prognostic information provided by iPET and IPI. Figure 1. Figure 1. Disclosures Dürig: The Binding Site: Research Funding, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy; Novartis: Consultancy; Roche: Consultancy, Other: Travel support, Speakers Bureau; Aicuris: Consultancy; Celgene: Consultancy, Other: Travel support, Speakers Bureau. La Rosée:Roche: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; CTI Lifesciences: Honoraria; Janssen-Cilag: Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers-Squibb: Honoraria, Research Funding; Mundipharma: Other: Travel support; Takaeda: Consultancy, Honoraria, Other: travel support; Celgene: Honoraria. Dührsen:Amgen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria, Research Funding. Hüttmann:Amgen: Consultancy, Research Funding; Gilead: Consultancy; Takeda: Consultancy, Other: Travel support; Roche: Research Funding; Celgene: Other: Travel support, Speakers Bureau.

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