Immunoglobulin Light Research Articles

Widespread amyloidogenicity potential of multiple myeloma patient-derived immunoglobulin light chains

BackgroundIn a range of human disorders such as multiple myeloma (MM), immunoglobulin light chains (IgLCs) can be produced at very high concentrations. This can lead to pathological aggregation and deposition of IgLCs in different tissues, which in turn leads to severe and potentially fatal organ damage. However, IgLCs can also be highly soluble and non-toxic. It is generally thought that the cause for this differential solubility behaviour is solely found within the IgLC amino acid sequences, and a variety of individual sequence-related biophysical properties (e.g. thermal stability, dimerisation) have been proposed in different studies as major determinants of the aggregation in vivo. Here, we investigate biophysical properties underlying IgLC amyloidogenicity.ResultsWe introduce a novel and systematic workflow, Thermodynamic and Aggregation Fingerprinting (ThAgg-Fip), for detailed biophysical characterisation, and apply it to nine different MM patient-derived IgLCs. Our set of pathogenic IgLCs spans the entire range of values in those parameters previously proposed to define in vivo amyloidogenicity; however, none actually forms amyloid in patients. Even more surprisingly, we were able to show that all our IgLCs are able to form amyloid fibrils readily in vitro under the influence of proteolytic cleavage by co-purified cathepsins.ConclusionsWe show that (I) in vivo aggregation behaviour is unlikely to be mechanistically linked to any single biophysical or biochemical parameter and (II) amyloidogenic potential is widespread in IgLC sequences and is not confined to those sequences that form amyloid fibrils in patients. Our findings suggest that protein sequence, environmental conditions and presence and action of proteases all determine the ability of light chains to form amyloid fibrils in patients.

Diagnosis and Treatment of AL Amyloidosis.

Systemic light chain (AL) amyloidosis is caused by an usually small B cell clone that produces a toxic light chain forming amyloid deposits in tissue. The heart and kidney are the major organs affected, but all others, with the exception of the CNS, can be involved. The disease is rapidly progressive, and it is still diagnosed late. Screening programs in patients followed by hematologists for plasma cell dyscrasias should be considered. The diagnosis requires demonstration in a tissue biopsy of amyloid deposits formed by immunoglobulin light chains. The workup of patients with AL amyloidosis requires adequate technology and expertise, and patients should be referred to specialized centers whenever possible. Stagings are based on cardiac and renal biomarkers and guides the choice of treatment. The combination of daratumumab, cyclophosphamide, bortezomib and dexamethasone (dara-CyBorD) is the current standard of care. Autologous stem cell transplant is performed in eligible patients, especially those who do not attain a satisfactory response to dara-CyBorD. Passive immunotherapy targeting the amyloid deposits combined with chemo-/immune-therapy targeting the amyloid clone is currently being tested in controlled clinical trials. Response to therapy is assessed based on validated criteria. Profound hematologic response is the early goal of treatment and should be accompanied over time by deepening organ response. Many relapsed/refractory patients are also treated with daratumumab combination, but novel regimens will be needed to rescue daratumumab-exposed subjects. Immunomodulatory drugs are the current cornerstone of rescue therapy, while immunotherapy targeting B-cell maturation antigen and inhibitors of Bcl-2 are promising alternatives.

Immunoglobulin Light Chain Amyloidosis: Diagnosis and Risk Assessment.

Immunoglobulin light chain (AL) amyloidosis is a clonal plasma cell disorder with multiple clinical presentations. The diagnosis of AL amyloidosis requires a high index of suspicion, making a delay in diagnosis common, which contributes to the high early mortality seen in this disease. Establishing the diagnosis of AL amyloidosis requires the demonstration of tissue deposition of amyloid fibrils. A bone marrow biopsy and fat pad aspirate performed concurrently have a high sensitivity for the diagnosis of AL amyloidosis and negate the need for organ biopsies in most patients. An accurate diagnosis requires amyloid typing via additional testing, including tissue mass spectrometry. Prognostication for AL amyloidosis is largely driven by the organs impacted. Cardiac involvement represents the single most important prognostic marker, and the existing staging systems are driven by cardiac biomarkers. Apart from organ involvement, plasma cell percentage on the bone marrow biopsy, specific fluorescence in situ hybridization findings, age at diagnosis, and performance status are important prognostic markers. This review elaborates on the diagnostic testing and prognostication for patients with newly diagnosed AL amyloidosis.

Case report: Splenic hamartoma: A rare vascular benign tumor of spleen

Splenic hamartoma (or splenoma) is a rare, benign, vascular tumor, often incidentally found at imaging, surgery or autopsy. It is usually asymptomatic. We report a case of 53 yrs old female from Tumkur city which was incidentally found during evaluation for GERD who had a large (9*10*10) splenic mass with small cystic /necrotic areas arising from lower pole of spleen, moderate heterogeneous enhancement on post contrast study, histologically congested sinusoids along with white pulp comprised of lymphoid follicles and immunohistochemically positive for antibodies to T- and B-cells and immunoglobulin light chains, CD 34 and Factor 8.

The Clinical Characteristics of Immunoglobulin Light Chain Amyloidosis in the Chinese Population: A Systematic Scoping Review

Immunoglobulin light chain (AL) amyloidosis is the most common type of systemic amyloidosis in China and is associated with increased morbidity and a poor prognosis. However, the clinical characteristics of Chinese patients with AL amyloidosis have not been systematically investigated. This scoping review aimed to summarize the available literature regarding the clinical characteristics of patients with AL amyloidosis and identify potential knowledge gaps. We searched three electronic databases from inception to 7 February 2021. PICOS (Patient, Intervention, Comparison, Outcome and Study) design structure was used to formulate the data extraction. All statistical calculations and analyses were performed with R (version 3.6.0). Sixty-seven articles with 5022 patients were included. Results suggest Chinese patients were younger (57 years) at the time of diagnosis when compared with other patient populations and were predominantly male (61.2%). The time interval from the onset of symptoms to diagnosis was between 6 and 12 months. It was found that 41.1% of Chinese patients with AL amyloidosis were diagnosed with an advanced stage III disease when diagnosed, and 20.2% had a concurrent disease. The most involved organs were the kidneys (84.3%) and the heart (62.5%). In conclusion, our study shows some similarities and differences with other studies on the clinical characteristics of Chinese patients with AL amyloidosis, including the age at diagnosis, Mayo stage, and organ involvement. However, a nationwide epidemiological investigation is still needed to provide a comprehensive overview of this patient population in China.

Gut mycobiome dysbiosis contributes to the development of hypertension and its response to immunoglobulin light chains.

Human gut microbiome has gained great attention for its proposed roles in the development of hypertension. The fungal microbiome in the human gut (i.e. the mycobiome) is beginning to gain recognition as a fundamental part of our microbiome. However, the existing knowledge of human mycobiome has never revealed the association between gut mycobiome and hypertension. It is known that inflammation and immunity contribute to human hypertension. Here, we sought to investigate whether gut mycobiome could predict the development of hypertension and its association with immunoglobulin light chains. Participants were classified into three cohorts: prehypertension (pre-HTN), hypertension (HTN), and normal-tension (NT) based on their blood pressure. Fresh samples were collected, and the ITS transcribed spacer ribosomal RNA gene sequence was performed. An immunoturbidimetric test was used to examine the serum levels of immunological light chains. Subjects in both of the states of pre-HTN and HTN had different fungal microbiome community compared to the NT group (FDR<0.05). Slightly higher levels of fungal richness and diversity were observed in the groups of pre-HTN and HTN. The relative abundance of Malassezia increased in the HTN group compared to that in the NT group, and the relative abundance of Mortierella enriched in the NT group. For the pre-HTN group, the relative abundance of Malassezia was positively associated with serum the concentration of light chain (LC) κ (r=0.510, P=0.044); for the HTN group, the relative abundance of Mortierella was positively associated with the serum concentration of LC κ (P<0.05), the relative abundance of Malassezia was positively associated with both the serum concentrations of LC κ and LC λ (r>0.30, P<0.05). Our present study demonstrated that gut fungal dysbiosis occurred in the state of prehypertension, and fungal dysbiosis can predict the dysregulation of serum light chains in hypertension patients. Further study on modulating gut fungal community should be focused on balancing the immunological features in hypertension.

880: PULMONARY AMYLOIDOSIS ASSOCIATED WITH B CELL LYMPHOPROLIFERATIVE DISORDER: A CASE REPORT

Introduction: Amyloidosis is caused by the deposition of misfolded autologous proteins in various organs and can be systemic or localized. AL amyloidosis is often associated with plasma cell disorder but here we report a rare case of Isolated Nodular Pulmonary Amyloidosis associated with a B cell Lymphoproliferative Disorder. Description: An 89 year old female with a past medical history of pulmonary arterial hypertension presented to the clinic with chronic stable exertional dyspnea and no associated symptoms. Four years prior, a computed tomography (CT) of the chest showed multiple bilateral calcified and noncalcified lung nodules with no further work-up. Chest X-ray performed at current presentation revealed extensive pulmonary reticulonodular shadowing at the peripheries with extensive fibrotic changes and scattered nodules. Subsequent CT chest showed interval increase of several bilateral nodules. A PET-CT showed multiple mildly hypermetabolic nodules. A CT guided needle biopsy of an FDG avid right upper lobe nodule measuring 1.5 x 1.4 cm was performed. Pathology was significant for homogenous acellular amorphous material with an entrapped plasma cell infiltrate. Congo red stain confirmed the presence of apple green birefringence diagnostic of amyloidosis. Immunohistochemistry showed a mixture of kappa-positive and lambda-positive cells, with kappa-positive cell predominance suggestive of an atypical plasma cell population. Clonal gene rearrangements were detected in both the immunoglobulin heavy chain and light chain gene which highly suggested the presence of a B-cell Lymphoproliferative Disorder. The patient was subsequently referred to oncology for further management. Discussion: Pulmonary Amyloidosis is an incidental radiological finding with patients being asymptomatic. CT chest may show lung nodules with/without calcifications. Biopsy is required for diagnosis with radiology guided fine needle aspiration. Isolated pulmonary amyloidosis has a good prognosis but should trigger evaluation for autoimmune disorders and B cell lymphoproliferative disorders as in this case with early referral and treatment of the underlying disorder.

Diagnosis and management of AL amyloidosis due to B-cell non-Hodgkin lymphoma.

Immunoglobulin light chain (AL) amyloidosis may be caused by a B-cell non-Hodgkin lymphoma (NHL) rather than a plasma cell neoplasm in rare cases, which presents unique diagnostic and management considerations. NHL associated with AL will often have an IgM paraprotein; thus, this disease is termed IgM-related AL amyloidosis (IgM AL). The clinical presentation of IgM AL is more likely to involve the lungs, peripheral nerves, and soft tissue; cardiac involvement is less common. Patients with IgM AL amyloidosis should undergo a lymphoma-directed work-up including evaluation for nodal and extranodal disease. Additionally, patients with an IgM paraproteinemia should be screened for AL amyloidosis through history and physical examination. Treatment regimens active against underlying lymphoma, rather than plasma cell-directed regimens, are recommended. Historical response rates in IgM AL have been poor; prospective studies of novel antineoplastic regimens may improve treatment outcomes.

A rare case of systemic AL-amyloidosis under the mask of myocardial infarction in the absence of myocardial hypertrophy: case report

Systemic amyloidosis is a group of diseases associated with extracellular deposition of fibrillar proteins, resulting in the loss of normal organ structure and function. AL-amyloidosis occurs when amyloid is deposited, consisting of full-length lambda or kappa immunoglobulin light chains, or fragments thereof. This article describes the complexity of timely diagnosis of systemic AL-amyloidosis with a predominant heart lesion in the absence of myocardial hypertrophy on the example of an elderly patient in whom the leading manifestation of the disease was heart failure with a preserved left ventricular ejection fraction.

The patterns and diagnostic significance of the lack of surface immunoglobulin light chain on mature B cells in clinical samples for lymphoma workup.

Surface immunoglobulin (sIg) light chains are not always detected on mature B cells. This may present as a challenge for clonality determination in clinical flow cytometry. To explore the mechanism and diagnostic significance of sIg negative mature B cells, we retrospectively studied 14 cases of sIg negative reactive B-cell lymphocytosis and 89 cases of sIg negative mature B-cell lymphomas. The expression patterns of sIg and cytoplasmic immunoglobulin (cIg) light chains were studied by flow cytometry using both monoclonal and polyclonal antibodies. These 14 cases of sIg negative reactive B-cell lymphocytosis were proven to be polytypic based on cytoplasmic light chain studies. In 89 cases of sIg negative mature B-cell lymphomas, we described four distinct patterns of abnormal light chain expression including partial or complete loss of sIg or cIg, suggesting different underlying mechanisms. This study represents the first reported series of body or cystic fluids where reactive B cells do not have detectable sIg, arguing strongly against making a diagnosis of B-cell lymphoma based on lack of sIg in mature B cells. Since the lack of sIg does not always predict clonal/neoplastic mature B-cell proliferation, further cIg evaluation should be performed when sIg expression is not detected in mature B cells. The lack of both sIg and cIg in mature B cells may serve as a reliable surrogate clonality/neoplastic marker.

T Cell Epitope Prediction within the Clonotypic Immunoglobulin Heavy and Light Chains in Patients with Multiple Myeloma

Despite remarkable therapeutic advances in recent years, multiple myeloma (MM) remains incurable, hence representing an unmet clinical need. Therapeutic approaches based on induced T cell anti-tumor immunity towards cancer eradication show promising results in many hematologic malignancies, including multiple myeloma (MM), highlighting the need for comprehensive understanding of the implicated mechanisms. Similar to other mature B cell malignancies, immunoglobulin (IG) gene rearrangements in MM lead to the expression of unique, novel peptide sequences that can be used as neoantigens, conceivably representing a potential attractive target for cancer-specific immune responses. Here we sought to explore this possibility by performing ad hoc prediction of putative T-cell class I/II neoepitopes contained within the MM clonotypic IG gene rearrangements. To that end, we investigated 16 newly diagnosed patients with MM from whom we isolated the peripheral blood and/or bone marrow mononuclear cell (PBMC/BMMC) fraction as well as CD138+ myeloma cells, CD4+ and CD8+ T cells. Clonotypic IG heavy and light chain gene rearrangements were RT-PCR amplified on RNA extracted from the PBMC/BMMC fraction using subgroup-specific leader primers for the IGHV/IGK/LV gene and universal primers annealing to the constant domain (IGHG, IGHM, IGKC, IGLC), in order to produce the full-length V-(D)-J gene rearrangement sequence, plus the start of the constant domain. The corresponding sequences were determined by bidirectional Sanger sequencing. Moreover, T cell receptor beta (TRB) chain gene rearrangements were RT-PCR amplified on RNA extracted from CD4+ and CD8+ T cells and then subjected to paired-end next generation sequencing (NGS). The deduced amino acid sequences of the IG heavy and light chain variable/constant domains were subsequently parsed in peptides and subjected to bioinformatics analysis for the identification of putative T-cell class I/II neoepitopes using the NetMHCpan and NetMHIICpan softwares. The rank score was calculated, considering the 4-digit HLA-A, -B, -C and DRB1 typing for each individual patient. High- and medium-binding peptides (rank score <2%) were selected. Prediction of the binding specificity of T Cell Receptors (TR) to MHC-peptide complexes (pMHCs) was performed by the ERGO-II tool. The rank score was calculated, considering the peptides resulted by NetMHCpan/ NetMHIICpan, the HLA, and the clonotypes for each patient (AUC>0.8). Exact matches to germline and/or proteome databases were excluded. Overall, 757,263 TRΒ distinct clonotypes were assessed (median= 18,244 /sample). All patients displayed oligoclonal T cell expansions in both CD4+ and CD8+ T cells, albeit these were significantly (p<0.001) more pronounced in the latter. Both T cell subpopulations displayed skewed TRBV and TRBJ gene repertoires (7 TRBV genes and 3 TRBJ genes accounting for about 40% and 59% of the repertoire, respectively). Overall, 1,219 predicted neoepitopes were identified. All patients had predicted CD4+ and CD8 + T-cell epitopes within the MM clonotypic IG, either in the heavy chain (16/16 pts, n=459 epitopes) or the light chain (16/16 pts, n=760 epitopes). There was no statistically significant difference in the rank score of peptides involving the complementarity determining region 3 (CDR3) vs. all other IG regions. Interestingly, 998/1,219 (82%) peptides with strong binding affinity resulted from IG gene sequence motifs outside the CDR3. Most of these peptides (853/1,219, 75%) resulted from somatic hypermutations (SHM) across the IG variable domain. Overall, 51,574 clonotypes of CD4+ T cells and 35,751 clonotypes of CD8+ T cells were in silico predicted by ERGO to have strong binding affinity. Relevant to mention, 528 clonotypes with high binding score were found to be shared between CD4+ T cells of all patients and 209 clonotypes between CD8+ T cells of all patients and, thus, were deemed as public. In conclusion, in silico prediction identified a significant number of putative T-cell class I/II neoepitopes contained within the clonotypic IG of MM patients. Many of the identified peptides derive from SHM, implying that the SHM which shapes the MM BcR IG repertoire may produce immunogenic CD4+/CD8+ T cell epitopes. Their actual immunogenicity has to be tested in ex vivo studies, currently underway by our group.

Density of the Spleen in Low-Dose CT Scans Is Associated with High-Risk Disease Features and Shorter Survival in Newly Diagnosed Multiple Myeloma Patients

Introduction Whole body low-dose computed tomography (WBLDCT) is standard of care in the diagnostic work-up of suspected multiple myeloma. While the focus of clinical routine evaluation lies on the detection of osteolytic lesions or extramedullary myeloma manifestations, the entire informative capacity of WBLDCT scans seems underestimated. Here, the analysis of non-focal signal alterations might provide further diagnostic insights, and a recent study by Terao et al. described tumor-burden dependent loss of the splenic signal in diffusion-weighted MRI in newly diagnosed myeloma (NDMM) patients. However, due to limited availability of MRI, most NDMM patients receive WBLDCT at initial diagnostic work-up. Therefore, we asked if non-focal signal alterations of the spleen in WBLDCT scans (i.e., radiographic tissue density of the organ) in NDMM patients might correlate with disease characteristics, cytogenetics and clinical outcomes. Methods In this retrospective analysis, we screened clinical records of NDMM patients diagnosed and treated at our institution (LMU University Hospital Munich, Germany) between 01/2010 and 12/2020. Clinical and laboratory data from initial diagnosis until first relapse were integrated with segmentation analyses of WBLDCT scans taken within 90 days before diagnosis. Plasma cell dyscrasias others than MM as well as cases with CT scans taken >90 days before diagnosis or CT scans containing contrast media were excluded. Results A total of 231 consecutively treated NDMM patients were included with a median age of 66 years (range 25-89). Cytogenetic analyses were available in 177 (77%) patients of which 38 had high-risk (HR) aberrations including t(4;14), t(14;16) and del(17p). In 44 (19%) of patients a gain(1q) was found. Patients were classified according to ISS and R-ISS: ISS-1 84 (43%), ISS-2 56 (28%), ISS-3 57 (29%) and R-ISS-1 38 (22%), R-ISS-2 119 (70%), R-ISS-3 13 (8%). Induction therapy consisted of a doublet regimen in 61 (26%), a triplet regimen in 167 (72%) and a quadruplet regimen in 3 (1%) patients. From the entire cohort, 141 (61%) patients received high-dose melphalan and autologous stem cell transplantation (auto-SCT), and 46 (20%) patients underwent tandem auto-SCT. Maintenance therapy after auto-SCT was applied to 49 (35% of auto-SCT) patients. Spleen density gradually increased with ISS and R-ISS stages. Thus, ISS3 and R-ISS3 displayed a significantly increased spleen density compared to ISS1 and R-ISS1 patients (ISS3 40.7 HU vs. ISS1 35 HU, p=0.003; R-ISS3 45.4 vs. R-ISS1 32.5 HU, p=0.004, Fig. 1A). We further found spleen density to be significantly higher in patients with HR cytogenetics (HR vs SR HU, p=0.03), in particular with t(4;14) (p=0.04), but not with del(17p), t(14;16) or gain(1q). Moreover, it positively correlated with serum lambda light chain levels in patients with lambda paraproteinemia (r = 0.26, p = 0.02), but not with kappa light chain or immunoglobulin levels. Based on an optimal log-rank test separation algorithm, NDMM patients with a spleen density of > 53.5 HU (Spleenhigh) had a shorter survival compared to Spleenlow patients (median PFS: 18.9 vs 32.1 months, p=0.01, Fig. 1B; median OS: not reached, p=0.02). PFS and OS differences were similar in subgroup analysis of transplant-eligible (PFS: p=0.08; OS: p=0.04) and transplant-ineligible patients (PFS: p=0.02, OS: p=0.04). In multivariate cox regression analyses including Spleenhigh/low and R-ISS stages, spleen density remained an independent factor for shorter survival (HRPFS 2.45, 95%CI 1.21-4.49, p=0.007; HROS 3.24, 95%CI 1.06-8.23, p=0.02). Conclusion Our data suggest that high density of the splenic tissue in WBLDCT is associated with high-risk features and inferior survival of NDMM patients. Increased cell numbers or deposition of light chains within the spleen might represent modes of action. If validated in future prospective studies, spleen WBLDCT density may complement existing risk-stratification tools. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Single Next Generation Sequencing Assay for Detection of Driver Mutations, Translocations and Copy Number Alterations in Patients with Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by several genetic abnormalities that impact risk stratification, outcome, and response to therapy; with fluorescence in situ hybridization (FISH) considered the gold-standard assay to detect the presence of chromosomal translocations and copy-number variations (CNV). Here, we evaluate the performance of a single clinically actionable targeted sequencing (NGS) assay for detecting cytogenetic abnormalities in addition to recurrent driver mutations in MM. Patient-derived bone marrow aspirates were obtained and enriched for CD138+ plasma cells. Targeted sequencing was performed using a 2.3 Mb custom capture panel covering 139 genes and regions surrounding MYC, immunoglobulin heavy chain (IgH), and immunoglobulin light chain kappa (IgK) and lambda (IgL) loci. Chromosomal translocations were identified using an in-house algorithm designed specifically for targeted capture DNA sequencing data analyses. Structural variants were called based on discordant read pairs as well as junction split reads, solving the challenges from both the overlapping paired-end reads due to the longer read lengths (150 bp) and the mapping ambiguities in the Ig regions. CNV were assessed using both on and off target reads, using a 200 kb bin size for segmentation covering the entire genome. Clinical annotations and FISH panel reports were abstracted. We sequenced 264 MM patient samples, 94% of which had available FISH information. A median of two somatic mutated genes per sample (range 0-13) was found. The most commonly mutated genes per sample were: KRAS (35%), NRAS (25%), FAM46C (15%), DIS3 (13%), BRAF (13%), ATM (11%), and TP53 (11%). Comparing NGS with FISH, we found a perfect concordance for the identification of t(4;14), t(6;14), t(14;16), and t(14;20); a 100% specificity and 97% sensitivity for t(11;14); and sensitivity >90% and an average specificity of 85% for trisomies of chromosomes 3, 7, 9, 11, and 15. The NGS approach was also able to detect gain(1q) and mono(13) with a sensitivity and specificity >90%, and del(17p) with a 60% sensitivity and 100% specificity. The reduced sensitivity for del(17p) detection was mostly due to focal deletions and low tumor purity. Because the TP53 gene is included in the custom capture panel, we increased the sensitivity by determining six separate regions of coverage using on-target probes, which enabled the detection of focal del(17p), increasing the sensitivity to 78%. The NGS approach also allowed the characterization of 16 (6%) samples with unavailable FISH. Of the 27 samples with del(17p) detected by FISH, NGS was able to identify TP53 mutations (i.e., biallelic inactivation) in 14 samples (52%). Due to the existence of multiple and promiscuous chromosomal partners involved in IgH translocation, the identification of the chromosomal partners is limited to the clinical FISH probe sets; in fact, 19 study samples presented abnormal IgH without a known partner by FISH. Our NGS assay was able to identify these partners, confirming MYC-IGH translocations in seven (37%) of these cases. Additionally, we detected MYC translocations with immunoglobulin light chains: 14 (5%) cases with IGK, and eight (3%) with IGL. Although IgL translocations, particularly IgL-MYC translocations, have been recently associated to poor prognosis, they are not routinely evaluated in FISH assays. The risk stratification and clinical utility of the NGS assay was demonstrated by the shorter overall survival observed in samples classified as high risk (mSMART 3.0; hazard ratio [HR]= 2.81; CI:1.89-4.17; p<0.0001), which was also observed with risk assessment by FISH (HR = 2.48; CI:1.72-3.58; p<0.0001). Therefore, in addition to the detection of clinically relevant somatic mutations, the targeted NGS approach showed robust detection of recurrent MM specific IgH translocations and CNV. Furthermore, the detection of relevant abnormalities not routinely assessed in most clinical labs, such as TP53 bi-allelic inactivation, IgL and MYC rearrangements, supports the use of this small, targeted panel as a clinically relevant, single molecular assay, for the comprehensive profiling of MM.

Localized light chain amyloidosis: A self-limited plasmacytic B-cell lymphoproliferative disorder.

Immunoglobulin light chain amyloidosis can be either systemic or localized. Although these conditions share a similar name, they are strikingly different. Localized light chain amyloidosis has been challenging to characterize due to its lower incidence and highly heterogeneous clinical presentation. Here, we review the emerging literature, emphasizing recent reports on large cohorts of patients with localized amyloidosis, and provide insights into this condition's pathology and natural history. We find that patients with localized amyloidosis have an excellent prognosis with overall survival similar to that of the general population. Furthermore, the risk of progression to systemic disease is low and likely represents initial mischaracterization as localized disease. Therefore, we argue for the incorporation of more sensitive techniques to rule out systemic disease at diagnosis. Despite increasing mechanistic understanding of this condition, much remains to be discovered regarding the cellular clonal evolution and the molecular processes that give rise to localized amyloid formation. While localized surgical resection of symptomatic disease is typically the treatment of choice, the presentation of this disease across the spectrum of plasmacytic B-cell lymphoproliferative disorders, and the frequent lack of an identifiable neoplastic clone, can make therapy selection a challenge in the uncommon situation that systemic chemotherapy is required.

A Phase 1/2 Multi-Center, Dose-Finding Study to Assess Safety, Tolerability, PK and Efficacy of ZN-d5, a Novel BCL-2 Inhibitor, for Treatment of Subjects with Relapsed/Refractory AL Amyloidosis

Background: Light-chain (AL) amyloidosis arises from a plasma cell clone that overproduces immunoglobulin light chains which aggregate and deposit within tissues and organs, interfering with normal function. Commonly, cardiac, hepatic, and renal involvement produce the major morbidities observed in this rare disease. At present, treatment of AL amyloidosis aims at the elimination of the plasma cell clone to rapidly reduce the level of circulating light chains. Recently, the addition of subcutaneous daratumumab to the standard CyBorD (cyclophosphamide, bortezomib, and dexamethasone) as first-line regimen, was shown to significantly improve complete hematologic and organ response rates. However, the management of patients with relapsed or refractory disease remains challenging. Pre-clinical and clinical data have shown that BCL-2 inhibition is active against malignant plasma cells, and may be particularly effective against cells harboring the t(11;14) translocation, found in about half of AL amyloidosis patients. Interestingly, small, retrospective studies have shown that BCL-2 inhibition may be an effective treatment option for patients with AL amyloidosis, particularly those with t(11;14). ZN-d5 is a novel, highly BCL-2-selective BCL-2 inhibitor in clinical development for hematologic malignancies, which is active against multiple myeloma in preclinical models, and which may be useful in the treatment of AL amyloidosis. Methods: ZN-d5-003 is a multicenter, international Phase 1/2 clinical trial evaluating single-agent oral ZN-d5 in relapsed or refractory AL amyloidosis subjects. In the Phase 1 part, the primary objectives are to determine the safety, tolerability and maximum tolerated dose of ZN-d5, and to determine the recommended Phase 2 dose. Cohorts of increasing doses (200mg; 400mg; 800mg; 1200mg; 1600mg QD) will enroll subjects based on Bayesian Optimum Interval (BOIN) design. In Phase 2, the primary objective is to assess the hematological response rate (HRR) among subjects with or without t(11;14) translocation. Bayesian Optimal Phase 2 (BOP2) design is implemented for interim efficacy monitoring. Key criteria for eligibility include the following: subjects must have a biopsy-confirmed diagnosis of AL amyloidosis, at least 1 and no more than 3 prior lines of therapy (including HSCT), adequate organ function and ECOG performance status of 0-2. All subjects must have measurable disease defined as dFLC of at least 20 mg/L; those considered for the trial will undergo bone marrow aspiration for assessment of t(11;14) status by FISH. Both t(11;14)-positive and -negative subjects are eligible for the study, however they will be separately analyzed for safety and efficacy. Subjects must have a history of organ involvement (current measurable organ disease is not required). Exclusion criteria include non-AL amyloidosis, diagnosis of multiple myeloma per 2014 IMWG criteria, or Mayo 2012 Stage IV disease. The study is expected to enroll up to approximately 140 subject at approximately 25 sites for phase 1, and at least 50 sites for phase 2. Conclusion: This study will evaluate treatment of AL amyloidosis utilizing BCL-2 inhibition and will test the hypothesis of whether t(11;14) is a relevant predictive biomarker. Dose escalation cohorts are currently undergoing evaluation and the study is open to enrollment. Upon determination of a recommended phase 2 dose, the study will commence Phase 2. Clinical trial: NCT05199337.

Gene Expression Sets and Renal Profiling from the Rain (Renal AL Amyloid Involvement and NEOD00) Trial

INTRODUCTION: Despite the high prevalence of kidney involvement in patients with light chain (AL) amyloidosis, a significant knowledge gap exists in understanding how immunoglobulin light chains drive kidney injury. The chief reason is the lack of molecular data from high-quality kidney tissue samples from this patient population. The RAIN trial (NCT03168906) was a phase 2B randomized placebo-controlled, open-label clinical trial that evaluated the efficacy of NEOD001, a monoclonal antibody targeting amyloid fibrils, versus placebo in patients with AL nephropathy. OBJECTIVES: Identifying amyloid-specific molecular signatures in kidney biopsy tissue obtained from AL patients with nephropathy.MATERIALS& METHODS: Renal biopsies were collected on all RAIN participants before randomization to NEOD001 or placebo. Trial clinical data was incorporated to link tissue, molecular, and kidney function data. The histologic assessment included a composite scarring injury score (CSIC) as determined by the degree of glomerular and tubulointerstitial scarring, and a novel amyloid score (AS), as quantified by the extent and distribution of amyloid deposition. Glomerular (G) and tubulointerstitial compartments were micro-dissected and sequenced separately. To identify enriched pathways in AL nephropathy, the expression data was compared to the glomerular and tubular expression profiles of healthy living donor (LD) from the Nephrotic Syndrome Study Network (NEPTUNE) study. Differentially expressed genes (DEGs) were determined with the R limma package based on the voom transformed log2 count data with an FDR of <=0.05. The AL and NEPTUNE datasets were merged and batch-corrected using ComBat. RESULTS: Ten patients were enrolled and randomized in the RAIN trial (5 NEOD001 and five placeboes) before trial closure in April 2018. Kidney tissue was available in nine of the ten patients. Cluster analysis revealed two distinct patient clusters (G1 and G2) within the glomerular and tubulointerstitial compartments. Key data of kidney function and biopsy morphology by compartment and cluster type are shown in Table 1. Despite similar eGFR, there was a higher AS and a non-significant increase in 24-hour urine protein in the glomeruli of individuals in the G2 patient cluster. Subcomponent analysis of AS revealed that the increase in amyloid score in G2 was driven almost entirely by an increase in mesangial and capillary wall amyloid deposition. Pathway enrichment in Amyloid kidney disease versus LD: IPA analysis identified unique activation signatures in the glomeruli and tubules. In the glomeruli of biopsies from the G2 cluster, there was evidence of increased TNF, IFNG, IL33 and VEGFA cytokine activity and a predicted increase in canonical PI3K/Akt signaling and innate immune responses. Tubules showed increased activation of transcriptional regulators TP53, TP73, increased cytokine signaling of IFNG, TGFb1 and VEGF. There was also a predicted activation of major canonical pathways including B cell receptor signaling, PI3K/AKT and Ephrin receptor signaling (Fig 1). Pathway Enrichment in G2 versus G1: Glomeruli showed activation of fibrotic pathways accompanied by a reduction in metabolic processes including gluconeogenesis, lipid transport and xenobiotics. There was an increase in canonical signaling of LPS/IL-1 mediated inhibition of RXR function that drives increased IL1 and TNF release from macrophages. Transcriptomic evidence of TNF activation was also noted in the TI. CONCLUSIONS: The RAIN trial closed prematurely, and thus no conclusions can be reached about the efficacy of NEOD001 in patients with AL nephropathy. However, these limited data revealed the presence of two distinct molecular subtypes of AL nephropathy. Whether these distinct molecular subtypes reflect different stages of the disease along a continuous spectrum or are of unique prognostic entities in themselves remains unclear. Detailed studies, of a larger cohort of patients including investigating diseased tissue at a single cell resolution can help identify the response of individual kidney cell types. Understanding the cellular responses of key cell types to injury from amyloid deposits could provide novel therapeutic targets to mitigate renal injury and improve patient outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

1-Year Results from a Phase 2 Study to Determine Safety and Tolerability of Treating Patients with Light-Chain (AL) Amyloidosis with Cael-101, an Anti-Amyloid Monoclonal Antibody, Combined with Anti-Plasma Cell Dyscrasia

Background: Light-chain (AL) amyloidosis is a rare, systemic disease caused by plasma cell dyscrasia (PCD). Excess immunoglobulin light chains misfold and form insoluble amyloid fibrils that deposit in organs, primarily the heart. Survival depends largely on the extent of heart involvement. Current therapies target PCD to halt fibril formation but do not treat existing fibril deposited in organs. CAEL-101 is a novel, investigational, potentially first-in-class therapy designed to remove amyloid fibrils from organs. Safety, tolerability, and biomarker data after all patients with AL amyloidosis enrolled in the trial received 1 year of treatment with CAEL-101, administered initially with cyclophosphamide-bortezomib-dexamethasone (CyBorD) ± daratumumab, are presented. Methods: Adult patients with AL amyloidosis (European Modification of Mayo Stage I-IIIa) and measurable hematologic disease were eligible for this ongoing, open-label, phase 2 study (NCT04304144). Other forms of amyloidosis and multiple myeloma were excluded. CAEL-101 ≤1000 mg/m2 is administered every other week with anti-PCD therapy as directed by the investigator. In addition to safety assessments, cardiac and renal response were assessed by change over time in N-terminal pro-brain natriuretic peptide (NT-proBNP) and proteinuria, respectively. Results: Patients (n = 25; mean age 65 years; 72% male) with Mayo Stage I (8%), II (76%), and IIIa (16%) AL amyloidosis were treated with CAEL-101 for 1 year at this analysis. Patients presented with cardiac involvement (n = 22), renal involvement (n = 9, reported from one site), and prior anti-PCD therapy (n = 20). All 25 patients experienced treatment-emergent adverse events (TEAEs), and 6 (24%) experienced at least possibly treatment-related TEAEs. Five patients discontinued for non-treatment-related reasons (Table). Fifteen (60%) patients experienced TEAEs of Grade ≥3 severity and 13 (52%) experienced ≥1 serious adverse event (SAE). The most common TEAEs were nausea (n = 10), constipation or fatigue (n = 9 each), anemia, insomnia, or diarrhea (n = 8 each), and dizziness, cough, or rash (n = 7 each). After 1 year of CAEL-101 treatment of the 22 cardiac evaluable patients (baseline NT-proBNP ≥332 pg/mL), 10 (46%) patients experienced ≥30% NT-proBNP decrease from baseline, 4 (18%) were stable (±30% change from baseline), and 3 (14%) showed disease progression (≥30% NT-proBNP increase from baseline); data for 5 patients were missing. Of the 9 renal evaluable patients from a single site as reported by the investigator, 8 showed ≥30% decrease from baseline in proteinuria. Conclusions: The long-term safety evaluation of CAEL-101 continues in this study. All patients currently enrolled have been treated for at least 1 year. At this 1-year time point, CAEL-101 has been generally well tolerated without evidence of organ toxicity. Organ response persisted even after cessation of anti-PCD treatment. Most TEAEs were mild to moderate. A phase 3 program has commenced to elucidate the efficacy and safety of CAEL-101 in cardiac AL amyloidosis European Modification Mayo Stages IIIa and IIIb. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A novel and robust approach to defining population-based reference ranges for a serum free light chain assay in a clinical laboratory

Abstract Serum protein electrophoresis and immunofixation are used to detect the presence of monoclonal heavy and light immunoglobulin chains for the diagnosis of plasma cell neoplasms (PCN). In conjunction, the Freelite® assay has been used to detect excess free kappa and lambda light chains produced by PCN, where the kappa:lambda free light chain ratio (KLR) serves as a surrogate marker of clonality and disease. The manufacturer-defined KLR reference range of 0.26-1.65 is used in internationally recognized criteria for PCN diagnosis and monitoring, and KLR results affect clinical decision making, disease categorization, and response assessment. Our laboratory has verified the manufacturer-defined Freelite® KLR reference range three times using healthy control sera since assay implementation in 2006. Despite this, high percentages of abnormal KLR have consistently been observed among immunofixation-defined monoclonal-negative specimens. We therefore retrospectively interrogated 48,012 serum free light chain clinical results reported at our institution between 2010-2020 by extracting results from our laboratory information system and analyzing population distributions using the R programming language. For all specimens, the distribution of KLR values was shifted towards the towards the upper limit of the manufacturer-defined reference range, indicating that it poorly describes both our monoclonal-negative and monoclonal-positive patient populations. Furthermore, &amp;gt;80% of monoclonal-negative specimens exhibited KLR above the median of the manufacturer-defined reference range (0.26-1.65), while 24% (1,226/5,057) unexpectedly exhibited KLR &amp;gt;1.65. We found that this phenomenon has been present consistently since 2010 and is not affected by patient sex or age, impaired renal function, lot-to-lot variation, or assay drift. We therefore sought to establish an institution-appropriate KLR reference range by defining a reference population from within our existing dataset. We leveraged International Classification of Diseases codes, estimated glomerular function data, and serum/urine immunofixation results to exclude specimens from individuals with PCN- or renal-related diagnoses, impaired renal function, and prior, current, or subsequent monoclonal components. The resultant 1,536 monoclonal-negative reference specimens were then classified according to the manufacturer-defined KLR reference range, where 13% remained abnormally elevated, consistent with the published false positive rate in healthy volunteers. Lastly, we determined a new KLR reference range of 0.66-2.21 using the central 95% of KLR values from these reference specimens. When applied to our dataset, this institution-specific KLR reference range improved the accurate classification of specimens containing monoclonal free light chains by immunofixation, while reducing the false-positive rate in monoclonal-negative specimens. Together, our findings support the use of historical KLR and immunofixation data from routine clinical testing to derive institution-appropriate KLR reference ranges, which can then be used for continuous quality improvement. Implementation of this more-accurate KLR reference range should improve subsequent test utilization by non-specialist providers, reduce concern for/over-diagnosis of kappa-involved disease, and improve the detection and monitoring of lambda-involved disease.

Abstract 14545: Extramedullary Plasmacytoma of Tonsil Presenting as Cardiac Amyloidosis

Introduction: AL amyloidosis is a systemic disease characterized by the deposition of misfolded immunoglobulin light chains produced from clonal plasma cells in multiple organs of the body causing its dysfunction. We present you an unusual case of AL cardiac amyloidosis in a patient with extramedullary plasmacytoma of the tonsils. Case Presentation 61-year-old female presented with dyspnea on exertion with NT-proBNP elevated to 1439 pg/ml. CXR showed increased pulmonary vascular congestion. The patient was started on IV furosemide 40 mg BID with good urine output. Transthoracic echocardiogram revealed left ventricular hypertrophy with an ejection fraction of 60-65% with a global longitudinal strain of -9.0% and regional longitudinal strain reduced in the basal and mid-left ventricular segment but preserved in apical segment consistent with infiltrative cardiomyopathy. The patient underwent a nuclear (nuclear technetium pyrophosphate) scan of the heart consistent with cardiac amyloid deposition. She also was noted to have increased tonsillar mass which was biopsied. In the hospital, the patient had a cardiac arrest with the initial rhythm being ventricular fibrillation. The patient received 6 rounds of shock, and multiple injections of epinephrine, amiodarone, and bicarbonate push. The patient died despite all efforts of resuscitation. Histopathology results of tonsillar mass were consistent with plasmacytoma with amyloid deposition on Congo-red stain. Lambda: Kappa ratio was 70:1. Discussion: In cardiac amyloidosis, light chain deposits between cardiac myocytes disrupt cell-cell interaction. It can present as restrictive cardiomyopathy, conduction system abnormalities, or sudden cardiac death. It is essential to diagnose cardiac amyloidosis earlier. If untreated, the median survival of patients with cardiac AL amyloidosis is 6 months from the onset of heart failure. Conclusion: Our case was unusual in which a rare tumor of tonsils had plasma cell tumor which led to cardiac amyloidosis

ODP034 Circulating Protein Biomarkers for Discriminating Malignant from Benign Adrenal Tumors Using Mass Spectrometry-Based Proteomic Analysis

Abstract Backgrounds Adrenal cortical carcinoma (ACC) is a rare malignant tumor derived from the adrenal gland with a poor prognosis. Since there is no effective medical treatment, early diagnosis and surgical resection are the only way to cure ACC completely. However, early diagnosis of ACC is sometimes tricky because there are no useful diagnostic biomarkers to differentiate ACC from benign adrenal tumors. Therefore, we aimed to identify circulating protein biomarkers to distinguish malignant from benign adrenal tumors using mass spectrometry-based proteomic analysis. Methods Plasma samples were obtained from 27 patients with ACC and 69 age- and sex-matched patients with adrenal adenoma. We performed the liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a data-independent acquisition (DIA) method in plasma samples. Among the quantified 381 proteins, differentially expressed proteins (DEPs) were selected using Student's t-tests for pairwise comparison with a false discovery rate &amp;lt; 0. 05 and |fold-change|&amp;gt; 1.5. In addition, we performed machine learning algorithms such as random forest and XGBoost to select featured DEPs to discriminate ACC. Results The mean age of patients was 52 years, and 38 (39.6%) patients were male. Among 19 DEPs, 8 proteins were overexpressed and 11 were underexpressed in ACC patients. In a further analysis using machine learning algorithms, random forest identified 4 proteins as important features for establishing the classifier. The panels with these four proteins showed a good discrimination performance with an area under the curve (AUC) of 0.94 and an accuracy of 0.96. Eight proteins were identified in the XGBoost algorithm. Four proteins in plasma samples were common DEPs from the traditional methods and machine learning algorithms. Conclusions High plasma levels of two proteins (One is involved in the migration, attachment and organization of cells into tissues. The other is V region of the variable domain of immunoglobulin light chains), and low plasma levels of two proteins (One is involved in cell-cell adhesion and recognition. The other is V region of the variable domain of immunoglobulin heavy chain) might be circulating protein biomarkers of ACCs compared with benign adrenal adenomas. Presentation: No date and time listed