Identification of Clonal Immunoglobulin λ Light-Chain Gene Rearrangements in AL Amyloidosis Using Next Generation Sequencing

[Introduction] AL amyloidosis is caused by the deposition of abnormally folded clonal immunoglobulin (IG) light chains (LCs, λ:κ = 3:1) made by malignant plasma cells in the bone marrow (BM), which leads to multi-organ dysfunction, often involving the heart, kidney, liver, skin, and nerves. However, little is known about what regulates organ tropism of amyloid deposition in this disease. In addition, no study has analyzed the repertoire of IG germlines of plasma cells in the BM in AL amyloidosis using next generation sequencing (NGS). In this study, we aimed to identify the clonal composition of IG λ light-chain variable region (IGLV) genes in BM cells in patients with AL amyloidosis using NGS. [Material and method] BM cells were obtained at diagnosis from 38 patients with AL amyloidosis and those with other plasma cell disorders: multiple myeloma (MM, n = 7), and monoclonal gammopathy of undetermined significance (MGUS, n = 11) with λ-type monoclonal paraprotein. Seven normal control (NC) patients had either immune thrombocytopenia or malignant lymphoma without BM invasion. Genomic DNA was extracted from the BM mononuclear cells preserved in LABO Banker1 or BM clots in O.C.T compound using QIAamp DNA Blood Mini kit. The IGLV1 and IGLV2 genes were amplified by polymerase chain reaction using a 5′ primer for the IGLV1/2 framework 3 (FR3) region and 3′ consensus primers for the IGLJ1/2/3 joining regions. Multiple samples were pooled, and paired-end 2 × 250 base pair sequencing reactions were performed using an Illumina MiSeq sequencer and then analyzed by an open-source program called Vidjil. All subjects provided written informed consent to participate in the study, in accordance with the Declaration of Helsinki. This study was approved by the ethics committee of the Chiba University Graduate School of Medicine and Japanese Red Cross Medical Center. [Results] Clinical and laboratory features of 38 patients with AL amyloidosis were as follows: primary AL amyloidosis (n = 31); 15 and 20 patients had cardiac and renal dysfunctions, respectively, and secondary AL amyloidosis with MM (n = 7); 4 and 1 patient had cardiac and renal dysfunctions, respectively. In patients with AL amyloidosis, the median plasma cell count in BM aspirate smears was 3.3% (0.1%-50.4%), and the median difference in involved and uninvolved light chains (dFLC) was 104.5 mg/L (28.5mg/L -2673.3mg/L). Representative results of the Vidjil analysis in NC, MGUS, AL amyloidosis, and MM are shown in Figure 1. The most abundant IGLV gene accounted for not >1% of the reads, and there was no dominant germline in NC samples. Therefore, we defined the dominant clone as >1% of IG germlines in plasma cells. According to this definition, clonal IG germlines were found in 27 of 31 patients with primary AL amyloidosis (87%), 5 of 7 with secondary AL amyloidosis (71%), 7 of 7 with MM (100%), and 8 of 11 with MGUS (73%). However, the size of clones in AL amyloidosis (median 3.1%, 0.38%-14.3%) was significantly smaller than that in MM (median 17.8%, 2.2%-17.9%) (P<0.001), and similar to that in MGUS (median 3.8%, 0.4%-32.0%). Importantly, in patients with AL amyloidosis, the dFLC and involved/uninvolved FLC ratio was not correlated with the clonal size of plasma cells in our repertoire analysis using NGS, suggesting that small malignant clones of plasma cells may secret FLC and cause LC depositions in AL amyloidosis. Regarding IGLV germline usage, IGLV1-51 was the most frequent repertoire in AL amyloidosis with heart dysfunction (7 of 16 cases) and renal dysfunction (7 of 21 cases). No relationship between the IGLV germlines and organ tropisms was observed. [Conclusion] We successfully identified the clonal composition of IGLV genes in the BM of most patients with AL amyloidosis using NGS, according to the differences in the V and J region recombination and CDR3 sequences using the Vidjil program. In AL amyloidosis, the clonal size of plasma cells in the BM is small and small malignant clones of plasma cells may secret FLC and cause LC depositions in AL amyloidosis. Figure 1 Disclosures Suzuki: Ono: Research Funding; BMS: Honoraria, Research Funding; Takeda: Honoraria; Janssen: Honoraria; Celgene: Honoraria.

Download Full-text

AL Amyloidosis — Pathogenesis and Prognosis Are Determined By the Amyloidogenic Potential of the Light Chain and the Molecular Characteristics of Malignant Plasma Cells

Blood ◽

10.1182/blood-2018-187 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 187-187

Author(s):

Anja Seckinger ◽

Ute Hegenbart ◽

Susanne Beck ◽

Martina Emde ◽

Tilmann Bochtler ◽

...

Keyword(s):

Gene Expression ◽

Plasma Cell ◽

Light Chain ◽

Research Funding ◽

Plasma Cells ◽

Memory B Cells ◽

Light Chains ◽

Molecular Characteristics ◽

Al Amyloidosis ◽

Malignant Plasma Cell

Abstract INTRODUCTION. Systemic light chain amyloidosis (AL) is caused by accumulation of plasma cells producing misfolded monoclonal light chains depositing as amyloid fibrils in different organs, most frequently heart and kidney. AIM of our study is first assessing the molecular characteristics of malignant plasma cells from AL-patients in relation to those from MGUS, asymptomatic, and symptomatic myeloma: Are these plasma cells different, does this difference explain amyloidogenicity? Does AL correspond to a certain developmental stage during evolution of symptomatic myeloma? Secondly, to what extent is prognosis determined by amyloid-deposition (organotropism, amount, amyloidogenicity) vs. number and molecular characteristics of malignant plasma cells? PATIENTS & METHODS . Consecutive patients (n=3023) with AL (n=582), MGUS (n=306), asymptomatic (n=444, AMM), or previously untreated, therapy-requiring multiple myeloma (n=1691, MM) were included. CD138-purified plasma cell samples were subjected to iFISH (n=582/306/444/1691), 1297 to gene expression profiling using Affymetrix U133 2.0 plus arrays (n=196/64/272/765), 712 to RNA- (n=124/52/38/489), and 258 to whole exome sequencing (n=115/53/39/51). Samples of normal bone marrow plasma cells, memory B-cells, and polyclonal plasmablasts were used as comparators. The CoMMpass-cohort (n=647) was used as comparator for the mutational spectrum of myeloma. RESULTS . Prognosis. By AL-factors. Expectedly, organ involvement, i.e. heart only vs. kidney only vs. heart+kidney vs. other (overall survival (OS), P=.001), the amount of free light chains (dFLC ≥18 mg/dL, HR=2.56, P=.01), and the cardiac European Mayo IIIB score (I/II/IIIA/IIIB, median OS 110/55/16/3 months, HR=1/1.94/3.73/7.90, P<.001) strongly determine prognosis (Fig. 1A). By malignant plasma cell factors. High proliferation rate (HR=3.58, P=.001) and expression-based risk factors for MM (GEP70 high, HR=2.38, P=.005; Rs-score high HR=4.63, P<.001) identify patients with very adverse prognosis (Fig. 1A). Tumor load, e.g. plasma cell infiltration >10%/>30% (HR=1.31/1.81, P=.01, P=.002) and M-protein ≥ 30g/l (HR=3.01, P=.005), are likewise prognostic (Fig. 1A). In multivariate analysis, all tested AL-specific (European Mayo IIIB score) and malignant plasma cell factors (proliferation or GEP70 and plasma cell infiltration) are independent. Molecular characteristics.iFISH. As MM (96.2%) and AMM (92.8%) AL-patients (93.1%) carry at least one recurrent myeloma typical aberration. The mean number of progression-associated aberrations in AL (n=0.98) fits between MGUS (n=0.85) and AMM (n=1.45) with significant difference compared to AMM (P<.001) unlike to MGUS. Main differences in frequency are found for t(11;14) and hyperdiploidy with a comparable pattern of non-etiologic aberrations. Gene expression (GEP and RNA-seq). Aberrant plasma cells in AL amyloidosis show the least difference with AMM, followed by MGUS and MM. In principal component analysis, AL overlaps with AMM and MGUS, independent of presence or absence of heart involvement (Fig. 1B). Pairwise assessment of similarity using a multivariate generalization of the squared Pearson correlation coefficient shows closest similarity to AMM and MM followed by MGUS, with comparable differences to normal plasma cells, polyclonal plasmablasts, and memory B-cells. Significantly more AL-patients present with higher proliferation rate vs MGUS (P<.001) and AMM (P<.02). AL and MM differ significantly regarding distinct molecular entities as determined by GEP (e.g. TC-classification; Fig. 1C). Mutation spectrum in AL amyloidosis vs. MM. From the 20 most frequently synonymously mutated non-Ig transcripts (CoMMpass-cohort), 16 could likewise be detected in AL amyloidosis, i.e. KRAS, NRAS, IGLL5, DIS3, FAM46C, MUC16, BRAF, TRAF3, PCLO, RYR2, FATA4, CSMD3, TP53, DNAH5, RYR2A, and FLG. CCND1 mutations were significantly more frequent in AL and AMM compared to MM (P=.02). DISCUSSION & CONCLUSION. Pathogenesis and prognosis of AL amyloidosis are explained both by AL-specific and malignant plasma cell characteristics. Aberrant plasma cells in AL amyloidosis show the same aberration- and expression pattern and a "molecular age" between MGUS and AMM, most closely resembling the latter. AL amyloidosis is thus mostly a rather early plasma cell dyscrasia with an unstable and toxic immunoglobulin light chain. Disclosures Seckinger: Celgene: Research Funding; EngMab: Research Funding; Sanofi: Research Funding. Hose:Celgene: Honoraria, Research Funding; Sanofi: Research Funding; EngMab: Research Funding.

Download Full-text

In Vitro and inVivo Activity of Melflufen in Amyloidosis

Blood ◽

10.1182/blood-2019-125952 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3100-3100 ◽

Cited By ~ 2

Author(s):

Ken Flanagan ◽

Muntasir M Majumder ◽

Romika Kumari ◽

Juho Miettinen ◽

Ana Slipicevic ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Plasma Cell ◽

Light Chain ◽

Research Funding ◽

Plasma Cells ◽

Al Amyloidosis ◽

Advisory Committees

Background: Immunoglobulin light-chain (AL) amyloidosis is a rare disease caused by plasma cell secretion of misfolded light chains that assemble as amyloid fibrils and deposit on vital organs including the heart and kidneys, causing organ dysfunction. Plasma cell directed therapeutics, aimed at preferentially eliminating the clonal population of amyloidogenic cells in bone marrow are expected to reduce production of toxic light chain and alleviate deposition of amyloid thereby restoring healthy organ function. Melphalan flufenamide ethyl ester, melflufen, is a peptidase potentiated alkylating agent with potent toxicity in myeloma cells. Melflufen is highly lipophilic, permitting rapid cellular uptake, and is subsequently enzymatically cleaved by aminopeptidases within cells resulting in augmented intracellular concentrations of toxic molecules, providing a more targeted and localized treatment. Previous data demonstrating multiple myeloma plasma cell sensitivity for melflufen suggests that the drug might be useful to directly eliminate amyloidogenic plasma cells, thereby reducing the amyloid load in patients. Furthermore, the increased intracellular concentrations of melflufen in myeloma cells indicates a potential reduction in systemic toxicity in patients, an important factor in the fragile amyloidosis patient population. To assess potential efficacy in amyloidosis patients and to explore the mechanism of action, we examined effects of melflufen on amyloidogenic plasma cells invitro and invivo. Methods: Cellular toxicity and apoptosis were measured in response to either melflufen or melphalan in multiple malignant human plasma cell lines, including the amyloidosis patient derived light chain secreting ALMC-1 and ALMC-2 cells, as well as primary bone marrow cells from AL amyloidosis patients, using annexin V and live/dead cell staining by multicolor flow cytometry, and measurement of cleaved caspases. Lambda light chain was measured in supernatant by ELISA, and intracellular levels were detected by flow cytometry. To assess efficacy of melflufen in vivo, the light chain secreting human myeloma cell line, JJN3, was transduced with luciferase and adoptively transferred into NSG mice. Cell death in response to melflufen or melphalan was measured by in vivo bioluminescence, and serum light chain was monitored. Results: Melflufen demonstrated increased potency against multiple myeloma cell lines compared to melphalan, inducing malignant plasma cell death at lower doses on established light chain secreting plasma cell lines. While ALMC-1 cells were sensitive to both melphalan and melflufen, the IC50 for melphalan at 960 nM was approximately 3-fold higher than melflufen (334 nM). However, ALMC-2 cells were relatively insensitive to melphalan (12600 nM), but maintained a 100-fold increase in sensitivity to melflufen (121 nM). Furthermore, while 40% of primary CD138+ plasma cells from patients with diagnosed AL amyloidosis responded to melflufen treatment in vitro, only 20% responded to melphalan with consistently superior IC50 values for melflufen (Figure 1). Light chain secreting cell lines and AL amyloidosis patient samples were further analyzed by single cell sequencing. We further examined differential effects on apoptosis and the unfolded protein response in vitro in response to either melflufen or melphalan. This is of particular interest in amyloidosis, where malignant antibody producing plasma cells possess an increased requirement for mechanisms to cope with the amplified load of unfolded protein and associated ER stress. As AL amyloidosis is ultimately a disease mediated by secretion of toxic immunoglobulin, we assessed the effects of melflufen on the production of light chain invitro, measuring a decrease in production of light chain in response to melflufen treatment. Finally, we took advantage of a recently described adoptive transfer mouse model of amyloidosis to assess the efficacy of melflufen and melphalan in eliminating amyloidogenic clones and reducing the levels of toxic serum light chain in vivo. Conclusions: These findings provide evidence that melflufen mediated toxicity, previously described in myeloma cells, extends to amyloidogenic plasma cells and further affects the ability of these cells to produce and secrete toxic light chain. This data supports the rationale for the evaluation of melflufen in patients with AL amyloidosis. Figure 1 Disclosures Flanagan: Oncopeptides AB: Employment. Slipicevic:Oncopeptides AB: Employment. Holstein:Celgene: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees; Sorrento: Consultancy. Lehmann:Oncopeptides AB: Employment. Nupponen:Oncopeptides AB: Employment. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding.

Download Full-text

A Phase I Trial of Pomalidomide, Bortezomib (Velcade), and Dexamethasone (PVD) As Initial Treatment of AL Amyloidosis and Light Chain Deposition Disease

Blood ◽

10.1182/blood.v124.21.4767.4767 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4767-4767

Author(s):

Jeffrey Zonder ◽

Christiane Houde ◽

Sascha Tuchman ◽

Vishal Kukreti ◽

Vaishali Sanchorawala ◽

...

Keyword(s):

Plasma Cell ◽

Light Chain ◽

Research Funding ◽

Median Number ◽

Light Chains ◽

Al Amyloidosis ◽

Light Chain Deposition Disease ◽

Deposition Disease ◽

Plasma Cell Dyscrasias ◽

Light Chain Deposition

Abstract Introduction: AL amyloidosis (AL) and Light Chain Deposition Disease (LCDD) are plasma cell dyscrasias in which misfolded monoclonal light chains form insoluble extracellular protein deposits (fibrillar and amorphous, respectively). In AL particularly, toxic soluble light chain oligomers also play a role in disease pathogenesis. Treatment of AL and LCDD aims at eliminating the abnormal plasma cell clone. Typical agents used include corticosteroids, bortezomib (btz), alkylators, or immunomodulatory drugs (IMiDs) such as lenalidomide (len) or pomalidomide (pom). Len-btz-dexamethasone (dex) is a highly efficacious frontline regimen commonly used for multiple myeloma, a related plasma cell cancer. Despite this, prospective studies using btz-IMiD combos as initial therapy of AL or LCDD are lacking. Here we report our experience with pom-btz-dex(PVD) for pts with AL or LCDD. Methods: This is a prospective Phase I trial using a standard 3+3 dose escalation scheme (described in Table 1). The primary objective is to establish the maximally tolerated dosing (MTD), with assessment for dose limiting toxicity (DLT) extending through cycles 1 and 2 for each pt. Hematologic and organ responses (HR and OR) were assessed using recently updated guidelines. PVD was administered in repeating 28-day cycles until either DLT or progressive disease. Key inclusion/exclusion criteria: biopsy proven AL amyloidosis or LCDD; no more than 1 prior cycle of anti-plasma cell therapy; measurable disease defined as at least a 5 mg/dL difference between the involved (iFLC) and uninvolved (uFLC) serum free light chains, or a serum M-protein of 0.5 g/dL or greater (latter not permissible without measurable sFLCdifference after protocol amendment); ECOG PS of 2 or less; adequate renal, hepatic, and marrow function; no Grade 3 or higher peripheral neuropathy (PN; pts with painful grade 2 PN also excluded). Abnormal left ventricular ejection fraction or cardiac biomarkers allowed, but pts with NYHA class III/IV congestive heart failure or uncontrolled ventricular arrhythmias were excluded. Antithrombotic/antiviral prophylaxis was required for all pts. Results: Six pts have been enrolled thus far (3 each in cohorts 1 and 2, respectively). Three additional pts have already been identified for cohort 3. Five of 6 pts had AL, and 1 had LCDD. Median age was 65.5 yrs (range 49-74 yrs). 5 pts were female. Mayo cardiac stage I/II/III in 1, 2, and 3 pts, respectively. Three pts had one prior cycle of therapy (the others had none). The iFLC was lambda type in all 5 AL pts, and kappa for the pt with LCDD. Median number of organs involved by AL/LCDD was 2 (range, 2-4; 4 with both cardiac and renal, and 1 additional pt with cardiac). The median number of PVD cycles administered was 3 (range 1-6). Two pts are still on therapy. The reasons for stopping PVD in the other 4 pts were: sudden death due to underlying cardiac AL (during cycle 3 of PVD), pt preference after reaching maximal HR (after cycle 6), lack of HR (after cycle 3), and toxicity (after cycle 4). Baseline dex dose adjustment was required for protocol-specified reasons in all pts. One pt required further dex reduction during cycle 4 of PVD. No pts required baseline or subsequent modification of pom or btz. Table 2 summarizes reported adverse events (AEs). No DLTshave been observed. Two pts achieved HR (0 CR, 1 VGPR, 1 PR, 3 SD, 0 PD). Organ responses have not been observed, but the first protocol-specified OR assessment takes place after 4 cycles of PVD and some pts have yet to reach this time point. Conclusions: PVD was well tolerated in this group of pts with AL and LCDD. Importantly, no significant myelosuppression or PN was noted in the first 2 (out of a planned 4) dose cohorts. Most AEs have been related to the ptsÕ underlying AL/LCDD, though dex has posed difficulties for some pts. Hematologic responses have been seen, but organ responses are predictably lagging. Once the MTD is established, an 18-pt expansion cohort dosed at that level willfurther examine the efficacy of PVD as up-front treatment for AL and LCDD. Disclosures Zonder: Celgene: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy. Off Label Use: Pomalidomide and Bortezomib are approved drugs for multiple myeloma; they are used in this trial as treatment for the related plasma cell dyscrasias AL amyloidosis and light chain deposition disease. . Tuchman:Celgene: Honoraria, Research Funding, Speakers Bureau; Millennium: Honoraria, Research Funding, Speakers Bureau. Kukreti:Celgene: Honoraria. Burt:Celgene: Speakers Bureau. Matous:Takeda Pharmaceuticals International Co.: Speakers Bureau; Onyx: Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Seattle Genetics, Inc.: Research Funding, Speakers Bureau.

Download Full-text

The Use of Next Generation Gene Sequencing to Measure Minimal Residual Disease in Patients with AL Amyloidosis and Low Plasma Cell Burden: A Feasibility Study

Blood ◽

10.1182/blood-2019-131863 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4353-4353 ◽

Cited By ~ 2

Author(s):

Shayna Sarosiek ◽

Vaishali Sanchorawala ◽

Mariateresa Fulcinti ◽

Allison P. Jacob ◽

Nikhil C. Munshi ◽

...

Keyword(s):

Bone Marrow ◽

Minimal Residual Disease ◽

Plasma Cell ◽

Peripheral Blood ◽

Research Funding ◽

Plasma Cells ◽

Residual Disease ◽

Al Amyloidosis ◽

Next Generation ◽

Minimal Residual

Background: AL amyloidosis is a bone marrow disorder in which clonal plasma cells produce light chains that misfold and deposit in vital organs, such as the kidneys and heart, leading to organ failure and eventual death. Treatment is directed towards the clonal plasma cell population in an effort to halt the production of toxic light chains and recuperate organ function. Pallidini et al. demonstrated that almost 50% of patients with AL amyloidosis who achieved a complete hematologic response to prior therapy had minimal residual disease (MRD) detectable in their bone marrow by multiparametric flow cytometry (MPF).1. Next generation gene sequencing (NGS) has been a successful tool in measuring MRD among patients with multiple myeloma2 though the data regarding its use in AL amyloidosis are limited. AL amyloidosis is a disease with a much smaller plasma cell burden at baseline (typically 5-10%), making the task of isolating an initial clonal sequence even more challenging. We sought to evaluate NGS as a method of isolating a clonal population of plasma cells among patients with systemic AL amyloidosis in a first-ever feasibility study. Methods: Patients were eligible if they had systemic AL amyloidosis and no clinical evidence of concurrent active multiple myeloma. In this study, feasibility was deemed successful if discovery of a clone could be achieved in 3 out of 10 of patients. Approximately five cc's of peripheral blood and bone marrow aspirate were collected from each patient and processed for CD138 selection and DNA isolation/purification. De-identified samples were sent to Adaptive Biotech Inc. (Seattle, WA) for initial clonal identification using the ClonoSEQ immunoglobulin heavy chain (IGH) assay. Genomic DNA was amplified by implementing consensus primers targeting the IGH complete (IGH-VDJH) locus, IGH incomplete (IGH-DJH) locus, immunoglobulin κ locus (IGK) and immunoglobulin l locus (IGL). The amplified product was sequenced and a clone identified based on frequency. After proof of feasibility in the first 10 patients an additional 27 patients had initial clonal identification via the same process mentioned above. Results: In total, 37 patient samples underwent NGS via the ClonoSEQ IGH assay method. The median patient age was 66 years old (range: 44 to 83), 24% of which were female. All 37 patients had measurable disease based on serum electrophoresis and immunofixation and/or serum free light chain assay (Table 1). Four patients had no monoclonal protein detected on SIFE or UIFE and 13 patients had a normal sFLC ratio. Of the 33 patients with monoclonal disease on immunofixation, 12 patients had only a free lambda monoclonal protein and the remaining 21 patients had a clonal heavy chain with an associated light chain. Bone marrow biopsies demonstrated clonal plasmacytosis of 40% or lower. ClonoSEQ IGH assay identified trackable clones in 31 of 37 patients (84%) (see Table 1). Four patients had at least one trackable sequence (range: 1 to 5 sequences) in the peripheral blood and 29 patients had at least one trackable sequence in the bone marrow aspirate (range: 1 to 7 sequences). No correlation was seen between the detection of a clone and standard measures of plasma cell tumor burden (SIFE, SPEP, UIFE, UPEP, and sFLCs). Conclusion: NGS was successful in identifying an initial clone in 29 of 37 patients with systemic AL amyloidosis, four of which were detectable in the peripheral blood. Due to the low clonal burden in patients with AL amyloidosis, it is often difficult to assess disease status, especially post-treatment. These encouraging results may enhance disease monitoring and improve patient care in this rare disease. We are currently tracking MRD in the patients with identifiable clones as they receive systemic treatment, the results of which will be available for presentation in December 2019. REFERENCES 1. Palladini G, Massa M, Basset M, Russo F, Milani P, Foli A, et al. Persistence of Minimal Residual Disease By Multiparameter Flow Cytometry Can Hinder Recovery of Organ Damage in Patients with AL Amyloidosis Otherwise in Complete Response. Abstr 3261. 2016; 2. Ladetto M, Brüggemann M, Monitillo L, Ferrero S, Pepin F, Drandi D, et al. Next-generation sequencing and real-time quantitative PCR for minimal residual disease detection in B-cell disorders. Leukemia. 2014;28:1299-307. Table 1 Disclosures Sarosiek: Acrotech: Research Funding. Sanchorawala:Proclara: Consultancy, Honoraria; Takeda: Research Funding; Caelum: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Prothena: Research Funding; Celgene: Research Funding. Jacob:Adaptive Biotechnologies: Employment, Other: shareholder. Munshi:Amgen: Consultancy; Adaptive: Consultancy; Celgene: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy.

Download Full-text

Identification of Clonal Immunoglobulin λ Light-Chain Gene Rearrangements in AL Amyloidosis using Next Generation Sequencing

Experimental Hematology ◽

10.1016/j.exphem.2021.08.001 ◽

2021 ◽

Author(s):

Kenji Kimura ◽

Shokichi Tsukamoto ◽

Kanji Miyazaki ◽

Chika Kawajiri-Manako ◽

Arata Ishii ◽

...

Keyword(s):

Next Generation Sequencing ◽

Light Chain ◽

Chain Gene ◽

Al Amyloidosis ◽

Gene Rearrangements ◽

Next Generation ◽

Light Chain Gene ◽

Λ Light Chain ◽

Generation Sequencing

Download Full-text

The Impact of Clonal Hematopoiesis of Indeterminate Potential on Survival in Patients with Newly Diagnosed Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-116240 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4359-4359

Author(s):

Koji Sasaki ◽

Rashmi Kanagal-Shamanna ◽

Guillermo Montalban-Bravo ◽

Rita Assi ◽

Kiran Naqvi ◽

...

Keyword(s):

Next Generation Sequencing ◽

Myeloid Leukemia ◽

Research Funding ◽

Newly Diagnosed ◽

Next Generation ◽

Risk Of Death ◽

Genetics Research ◽

The Impact ◽

Generation Sequencing

Abstract Introduction: Clearance of detected somatic mutations at complete response by next-generation sequencing is a prognostic marker for survival in patients with acute myeloid leukemia (AML). However, the impact of allelic burden and persistence of clonal hematopoiesis of indeterminate potential (CHIP)-associated mutations on survival remains unclear. The aim of this study is to evaluate the prognostic impact of allelic burden of CHIP mutations at diagnosis, and their persistence within 6 months of therapy. Methods: From February 1, 2012 to May 26, 2016, we reviewed 562 patients with newly diagnosed AML. Next-generation sequencing was performed on the bone marrow samples to detect the presence of CHIP-associated mutations defined as DNMT3A, TET2, ASXL1, JAK2 and TP53. Overall survival (OS) was defined as time period from the diagnosis of AML to the date of last follow-up or death. Univariate (UVA) and multivariate Cox proportional hazard regression (MVA) were performed to identify prognostic factors for OS with p value cutoff of 0.020 for the selection of variables for MVA. Landmark analysis at 6 months was performed for the evaluation of the impact of clearance of CHIP, FLT3-ITD, FLT3D835, and NPM1 mutations. Results: We identified 378 patients (74%) with AML with CHIP mutations; 134 patients (26%) with AML without CHIP mutations. The overall median follow-up of 23 months (range, 0.1-49.0). The median age at diagnosis was 70 years (range, 17-92) and 66 years (range, 20-87) in CHIP AML and non-CHIP AML, respectively (p =0.001). Of 371 patients and 127 patients evaluable for cytogenetic in CHIP AML and non-CHIP AML, 124 (33%) and 25 patients (20%) had complex karyotype, respectively (p= 0.004). Of 378 patients with CHIP AML, 183 patients (48%) had TET2 mutations; 113 (30%), TP53; 110 (29%), ASXL1; 109 (29%), DNMT3A; JAK2, 46 (12%). Of 378 patients, single CHIP mutations was observed in 225 patients (60%); double, 33 (9%); triple, 28 (7%); quadruple, 1 (0%). Concurrent FLT3-ITD mutations was detected in 47 patients (13%) and 12 patients (9%) in CHIP AML and non-CHIP AML, respectively (p= 0.287); FLT3-D835, 22 (6%) and 8 (6%), respectively (p= 0.932); NPM1 mutations, 62 (17%) and 13 (10%), respectively (p= 0.057). Of 183 patients with TET2-mutated AML, the median TET2 variant allele frequency (VAF) was 42.9% (range, 2.26-95.32); of 113 with TP53-mutated AML, the median TP53 VAF, 45.9% (range, 1.15-93.74); of 109 with ASXL1-mutated AML, the median ASXL1 VAF was 34.5% (range, 1.17-58.62); of 109 with DNMT3A-mutated AML, the median DNMT3A VAF was 41.8% (range, 1.02-91.66); of 46 with JAK2-mutated AML, the median JAK2 VAF was 54.4% (range, 1.49-98.52). Overall, the median OS was 12 months and 11 months in CHIP AML and non-CHIP AML, respectively (p= 0.564); 16 months and 5 months in TET2-mutated AML and non-TET2-mutated AML, respectively (p <0.001); 4 months and 13 months in TP53-mutated and non-TP53-mutated AML, respectively (p< 0.001); 17 months and 11 months in DNMT3A-mutated and non-DNMT3A-mutated AML, respectively (p= 0.072); 16 months and 11 months in ASXL1-mutated AML and non-ASXL1-mutated AML, respectively (p= 0.067); 11 months and 12 months in JAK2-murated and non-JAK2-mutated AML, respectively (p= 0.123). The presence and number of CHIP mutations were not a prognostic factor for OS by univariate analysis (p=0.565; hazard ratio [HR], 0.929; 95% confidence interval [CI], 0.722-1.194: p= 0.408; hazard ratio, 1.058; 95% confidence interval, 0.926-1.208, respectively). MVA Cox regression identified age (p< 0.001; HR, 1.036; 95% CI, 1.024-1.048), TP53 VAF (p= 0.007; HR, 1.009; 95% CI, 1.002-1.016), NPM1 VAF (p=0.006; HR, 0.980; 95% CI, 0.967-0.994), and complex karyotype (p<0.001; HR, 1.869; 95% CI, 1.332-2.622) as independent prognostic factors for OS. Of 33 patients with CHIP AML who were evaluated for the clearance of VAF by next generation sequencing , landmark analysis at 6 months showed median OS of not reached and 20.3 months in patients with and without CHIP-mutation clearance, respectively (p=0.310). Conclusion: The VAF of TP53 and NPM1 mutations by next generation sequencing can further stratify patients with newly diagnosed AML. Approximately, each increment of TP53 and NPM1 VAF by 1% is independently associated with 1% higher risk of death, and 2% lower risk of death, respectively. The presence of CHIP mutations except TP53 does not affect outcome. Disclosures Sasaki: Otsuka Pharmaceutical: Honoraria. Short:Takeda Oncology: Consultancy. Ravandi:Macrogenix: Honoraria, Research Funding; Seattle Genetics: Research Funding; Sunesis: Honoraria; Xencor: Research Funding; Jazz: Honoraria; Seattle Genetics: Research Funding; Abbvie: Research Funding; Macrogenix: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding; Orsenix: Honoraria; Abbvie: Research Funding; Jazz: Honoraria; Xencor: Research Funding; Orsenix: Honoraria; Sunesis: Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Astellas Pharmaceuticals: Consultancy, Honoraria. Kadia:BMS: Research Funding; Abbvie: Consultancy; Takeda: Consultancy; Jazz: Consultancy, Research Funding; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Celgene: Research Funding; Novartis: Consultancy; Amgen: Consultancy, Research Funding; BMS: Research Funding; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy; Abbvie: Consultancy; Celgene: Research Funding. DiNardo:Karyopharm: Honoraria; Agios: Consultancy; Celgene: Honoraria; Medimmune: Honoraria; Bayer: Honoraria; Abbvie: Honoraria. Cortes:Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Arog: Research Funding.

Download Full-text