Citations

Abstract Background Monoclonal antibody therapies (t-mabs) are known to interfere in serum immunofixation, as they show up as a discrete band. Until recently, most of the t-mabs were IgG kappa monoclonal immunoglobulins, and laboratories were familiar with the clue of a cathodal migrating IgG kappa in a patient sample, suggesting this could be daratumumab or another t-mab in the report. However, with the advent of novel antibody-based medications, identification via traditional laboratory methods has grown more complex. Teclistamab-cqyv is an IgG4 lambda T-cell engager bispecific antibody targeting B-cell maturation antigen (BCMA) and CD3 on T cells, used in relapsed and refractory multiple myeloma. The goal of this study was to determine if teclistamab could be detected on serum immunofixation and mass-fix. Methods Residual waste serum samples with no monoclonal protein via mass-fix (a MALDI-TOF based assay used in place of serum immunofixation) and variable polyclonal background were used to spike teclistamab (n=3). Briefly, one sample had total IgG < 500 mg/dL measured by nephelometry on a Siemens BNII (431 mg/dL, hypo gamma), a second sample had IgG within reference intervals (779 mg/dL, normal gamma) and the third sample had IgG > 1600 mg/dL (1950 mg/dL, polyclonal hypergamma). Each sample was divided into 3 aliquots and spiked with teclistamab (Tecvayli, Johnson &Johnson) obtained from the pharmacy, at final concentrations of 10, 20, and 40 mg/dL in the serum samples. The nine artificial samples were subjected to immunofixation electrophoresis (Sebia) and a new monoclonal protein study using mass-fix, a laboratory developed test. Results In the serum immunofixation experiments, a single discrete IgG lambda band was visible in the hypogamma and normal gamma samples (Table 1) when teclistamab was present at 20 mg/dL or 40 mg/dL. No bands were observed for the 10 mg/dL spike in hypos or normals. No teclistamab was detected at any concentration in the hypergamma sample. When mass-fix was performed, monoclonal protein spikes consistent with the expected mass of the two unique lambda light chains associated with teclistamab (22624Da and 22636Da) were identified in the hypo and normal gamma samples at the 3 concentrations tested: 10, 20, and 40 mg/dL. No mass-fix spikes were seen above the polyclonal spectra in the hypergamma samples. Conclusion The polyclonal background of immunoglobulins in a serum specimen plays a significant role in the identification of any monoclonal protein. Teclistamab is a new IgG lambda t-mab with potential to show up as an interference in both immunofixation and mass-fix, depending on the amount of polyclonal IgG present. Without positive identification, teclistamab presence could complicate interpretation of a patient’s response to treatment. This proof-of-concept study shows that mass-fix can detect smaller concentrations of teclistamab than immunofixation, therefore would be more prone to identifying this t-mab than labs employing the gel-based immunofixation method.

Abstract Background The diagnosis of monoclonal gammopathies relies on the detection of monoclonal immunoglobulins (M-proteins) in serum or urine. Mass-Fix utilizes matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to detect and quantify M-proteins. This method employs immunoaffinity enrichment using antibodies against heavy chains (HC) and light chains (LC), followed by MS analysis to determine M-protein isotypes. Mass-Fix has also revealed novel M-protein characteristics, including LC glycosylation and fragmentation, facilitated by measuring the mass of LC. LC glycosylation is as an independent risk factor for disease progression of monoclonal gammopathy of undetermined significance (MGUS), while light chain fragments have been postulated as the origin of amyloid light chain (AL) plaques. Since Mass-Fix enriches intact immunoglobulins before dissociating LCs, it potentially enhances fragment detection. However, the precise nature and clinical significance of these fragments remain to be fully elucidated. Methods Mass-Fix serum results from Mayo Clinic patients tested from September 10, 2018, to October 1, 2024, were reviewed for the presence of M-protein and LC fragments. The frequency and the isotype of fragment LC fragments was determined in the M-protein positive cohort (n=15410). Patients diagnosed with either MGUS (n=6996), AL amyloidosis (n=315), or both (n=32) who had positive M-protein results on Mass-Fix, either prior to or within one month after the diagnosis, were included for a retrospective clinical association study of LC fragments and disease. To validate that these fragments were not artifacts of MS analysis, PAGE gel analysis and Western blots were performed using clinical residual serum from 9 patients: 2 IgG/kappa and 3 IgG/lambda patients with detectable LC fragments and 2 IgG/kappa and 2 IgG/lambda patients without fragments. Samples were purified using antibodies against gamma heavy chains and reduced with TCEP prior to gel electrophoresis and Western blot. Results Of the 15410 patients with monoclonal proteins detected by Mass-Fix, 2896 (18.7%) had detectable LC fragments; kappa fragments in 1533 and lambda fragments in 1363. Most often, the fragments were observed in pairs, with combined molecular weight typically equaled that of the full-length monoclonal LC present in the same sample. LC fragments were observed more frequently among patients diagnosed with AL amyloidosis (118/347=34%) compared to those without the diagnosis (685/6996=10%), with an odds ratio of 4.75 (95% CI, 3.75-6.01). PAGE with Coomassie blue staining revealed bands with sizes representing LC fragments in the five M-protein samples with detectable LC fragments on Mass-Fix, while no such bands observed in those without LC fragments. Western blot using anti-kappa or anti-lambda antibodies confirmed the identity of the bands with higher molecular weight as a fragment of LC in each of the IgG/kappa and IgG/lambda samples. Conclusion Mass-Fix is able to discriminate LC fragments from intact LC based on their sizes. The detection of LC fragments with Western blot without ionization suggests a biological origin rather than analytical artifacts from MS. The clinical association study suggests that LC fragments may have utility in increasing the suspicion of AL amyloidosis in M-protein positive patients with LC fragments, potentially facilitating earlier intervention and improving patient outcomes.

Abstract Background The Freelite serum free light chain (FLC) assay has been widely used for detecting and monitoring monoclonal gammopathies. Reference intervals (RIs) established by Katzmann et al. in 2002 have served as the standard values for interpreting kappa/lambda FLC ratios. However, multiple reports suggested an assay drift and clinical laboratories started to move towards locally established RIs given the challenge in verifying original RIs. The assay drift over time increased kappa concentration measurements and was attributed to an interaction between the kappa FLC calibrator and serum proteins, including alpha-1-antitrypsin. A stabilized calibrator was developed to improve consistency of results with original RI. The objective of this study was to assess whether the new updated kappa calibrator would warrant a RI update. Methods Healthy adult donors’ serum samples were selected from the institution quality unit. Exclusion criteria included autoimmune diseases, cancers and renal disease. FLC measurements were obtained on the Optilite by turbidimetry using routine reagents for kappa (old lot); the new lot of reformulated kappa calibrator, and a single measurement for lambda FLC. FLC ratios were calculated with the old lot and new lot. All samples were tested by MASS-FIX to confirm absence of a monoclonal protein. Bootstrap resampling (10,000 replicates) estimated standard errors, with statistical significance defined as p < 0.05. Clinical significance was assessed per CLSI EP28-A3 guidelines. A RI was considered for partitioning by sex if the mean difference between males and females exceeded 25% of the RI width or if >4% of either sex had values beyond RI limits. Similar tests were applied to age groups (<60 and =60 years). Results 465 donors (48.2% male, 51.8% female) aged 30-89 years were used. Sixteen donors with incidental monoclonal proteins identified by MASS-FIX were excluded, for a final n=449. No clinically significant relationships of results were found with sex or age. The updated kappa calibrator resulted in a measurable shift down in FLC values with the new lot of reagents. The kappa FLC concentration and corresponding FLC ratio decreased by a median -12.1%, see table 1 for absolute values When reviewing Optilite results of the new lot, 98/449 samples were outside the kappa concentration original RI (21%). Lambda concentration RI was shifted up on Optilite when compared to original RI but passes verification (1.6% of samples, acceptance criteria < or =10%). 19 samples (4.2%) were outside the original RI for the FLC ratio of 0.26 to 1.65 (all >1.65), which was originally established as a 100% diagnostic range. The calibrator reformulation, while decreasing test results by >10%, was not sufficient to verify the original RI for kappa and still left a few cases outside the original diagnostic range for the FLC ratio. Conclusion This study assessed the impact of the stabilized calibrator on RIs for the Optilite Freelite assay. These findings suggest revisiting existing RIs to ensure continued accuracy in clinical interpretation and should be further evaluated with additional lots of reagents for robustness.

Background Telestroke emergency department (ED) workflow emulates that of in-person care, yet limitations of the virtual interaction may require accommodations. The optimal sequence of non-contrast head CT, cerebral CT angiogram, and decision to infuse IV thrombolytic is not defined for telestroke. Methods In this twenty-two-hospital multicenter retrospective study between 1/1/2024-12/31/2024, we characterized timing of ED CT angiograms, either bundled with the non-contrast head CT or non-bundled and performed after the IV-thrombolytic decision. The primary outcome was effect of bundling on door to needle time (DTN) when directed by telestroke. Timing of CT scans was determined by time stamps of scout images. Results In total, 5704 patients were included, with a median age of 65 years (Interquartile range, IQR 58-77) and NIHSS 8 (IQR 6-13). There were 4518 patients in the bundled group (n = 12 hospitals), with time from non-contrast CT to CTA of median 2 min (IQR 0-6 min; vs non-bundled (n = 10 hospitals): 7, 4-15 min; P = .0001). DTN was not statistically different between the groups, bundled vs not: median 47 min (IQR, 35-62 min) vs 54 min (IQR, 41-67 min, P = .21). Door in door out (DIDO) times also were unchanged (169, 117-254 vs 175, 123-247 min; P = .56). These findings, although showing no statistical difference, may have significance for clinical workflows and were consistent with the results of validation analyses conducted on individual patients. Conclusions Bundling non-contrast head CT with the CT angiogram before thrombolytic decision did not delay DTN times, trended to clinically improve times, and may be relevant for telestroke ED workflows.

Importance The Implantable Neurostimulator for the Treatment of Parkinson’s Disease (INTREPID) trial was a randomized, double-blind, sham-controlled study of subthalamic nucleus (STN) deep brain stimulation (DBS) for the treatment of Parkinson disease (PD). Objective To evaluate the long-term (5-year) outcomes and safety of STN-DBS for PD. Design, Setting, and Participants This was a prospective, randomized (3:1), 12-week double-blind sham-controlled study at 23 movement disorder centers across the US with an open-label 5-year follow-up. Patients were implanted and followed up with the Vercise DBS system from May 2013 to December 2022. Eligibility required diagnosis of bilateral idiopathic PD with more than 5 years of motor symptoms, more than 6 hours per day of poor motor function, modified Hoehn and Yahr Scale scores higher than 2, Unified Parkinson’s Disease Rating Scale (UPDRS-III) score of 30 or higher (medication-off state), and 33% or higher improvement in UPDRS-III medication-on score. Intervention Bilateral STN-DBS for moderate to advanced PD. Main Outcomes and Measures Primary outcomes included changes in UPDRS and dyskinesia scores, quality-of-life measures, and safety assessments. Exploratory analyses included medication reduction and DBS association with motor signs. Results A total of 313 patients were enrolled with 191 receiving the DBS system, and 137 participants (72%) completed the study. The study population had a mean (SD) age of 60 (7.9) years, with 139 (73%) male participants. Motor function without medication as measured by UPDRS-III improved from a mean (SD) of 42.8 (9.4) to 21.1 (10.6) at year 1 (51%; 95% CI, 49%-53%; P < .001) and 27.6 (11.6) at year 5 (36%; 95% CI, 33%-38%; P < .001). Activities of daily living without medication as measured by UPDRS-II improved from a mean (SD) of 20.6 (6.0) to 12.4 (6.1) at year 1 (41%; 95% CI, 38%-42%; P < .001) and 16.4 (6.5) at year 5 (22%; 95% CI, 18%-23%; P < .001). Dyskinesia scores decreased from 4.0 (5.1) to 1.0 (2.1) at year 1 (75%; 95% CI, 73%-75%; P < .001) and to 1.2 (2.1) at year 5 (70%; 95% CI, 63%-75%; P < .001). The levodopa equivalent dose was reduced by 28% at year 1, remaining stable at year 5 (28%; 95% CI, 26%-31%; P < .001). The most common serious adverse event was infection (9 participants). Ten deaths were reported, none related to the study. Conclusions and Relevance Although STN-DBS outcomes declined slightly, possibly due to the progressive nature of the disease, patients with PD sustained significant improvement in motor and activities of daily living scores, along with a stable reduction in anti-parkinsonian medication over the 5-year follow-up period.

Abstract Introduction Multiple myeloma (MM) is characterized by the abnormal proliferation of plasma cells, resulting in the overproduction of distinctive monoclonal proteins (M-protein). Suspected MM necessitates screening for M-protein through a combination of serum protein electrophoresis, serum immunofixation (SIFE), and serum free light chain (SFLC) determination. An M-protein appears as a relatively restricted band on agarose gel, where migration in ɑ-2 is rare. Methods A 55-year-old man with pulmonary tuberculosis presented with severe lower back pain. On examination, he appeared chronically ill, with conjunctival pallor. X-rays revealed vertebral compression fractures. The full blood count confirmed anemia; however, serum calcium and creatinine levels did not meet myeloma-defining event criteria. Results The serum protein electrophoresis revealed hypogammaglobulinemia, with the SIFE demonstrating unusual unrestricted κ staining in the ɑ-2 region. A markedly elevated κ SFLC and κ:λ ratio were found. Bone marrow examination demonstrated approximately 90% plasmacytosis. Urine immunofixation revealed a small, restricted κ band disproportionate to the κ SFLC. Notably, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified only polyclonal κ SFLC. Discussion Given the absence of a discernible M-protein on SIFE, a small κ restriction on urine immunofixation, and a polyclonal increase in κ SFLCs, the patient’s condition is being managed as an oligosecretory MM.

Abstract Outcomes in classic Hodgkin lymphoma (cHL) have steadily improved; however, additional therapies are needed for patients who relapse or do not respond to novel agents. Here, we report the efficacy and safety of camidanlumab tesirine (Cami), an anti-CD25 antibody-drug conjugate, in patients with relapsed/refractory cHL after brentuximab vedotin/programmed cell death protein 1 inhibitor therapies from the phase 2 ADCT-301-201 study. Eligible patients were adults with cHL who had received ≥3 previous lines of systemic therapy (or ≥2 if ineligible for hematopoietic stem cell transplant). Patients received 45 μg/kg Cami (IV, once every 3 weeks) in cycles 1 to 2, followed by 30 μg/kg IV once every 3 weeks for ≤1 year. The primary end point was overall response rate (ORR) per 2014 Lugano classification. Secondary end points included complete response rate (CRR), progression-free survival (PFS), and overall survival (OS). In total, 117 patients were enrolled with a median age of 37.0 years (range, 19-87). The ORR was 70.1% (95% confidence interval [CI], 60.9-78.2), with a CRR of 33.3% (95% CI, 24.9-42.6). The median PFS was 9.13 months (95% CI, 5.3-15.0); median OS was not reached. Thirty-three (28.2%) patients discontinued treatment because of treatment-emergent adverse events; the most common reasons were skin and subcutaneous tissue disorders (n = 10 [8.5%] patients), infections and infestations (n = 5 [4.3%]), and nervous systems disorders (n = 5 [4.3%]). Guillain-Barré–type or polyradiculopathy-type events occurred in 8 (6.8%) patients. Cami was efficacious in this heavily pretreated population; however, the efficacy was overshadowed by substantial issues with the safety profile. This trial was registered at www.clinicaltrials.gov as #NCT04052997.

AbstractINTRODUCTIONIncreasing evidence indicates that α‐synuclein (αSYN) oligomers are toxic. We sought to determine whether αSYN oligomers were associated with faster cognitive decline in prospectively‐followed patients with dementia with Lewy bodies (DLB).METHODSEight autopsy‐confirmed patients with DLB were selected based on rapid or slow cognitive decline determined by the rate of change of Mini‐Mental State Examination (MMSE) scores. Quantitative neuropathologic analysis of αSYN oligomers, Lewy‐related pathology, phosphorylated tau, and amyloid‐β was conducted in hippocampal subfields (CA1‐4 and subiculum) and the entorhinal cortex.RESULTSDLB with rapid cognitive decline showed greater CA1 αSYN oligomer burden (p = 0.029) and tau burden (p = 0.029) than DLB with slow decline. The groups showed comparable burden of Lewy‐related pathology and amyloid‐β pathology in the hippocampal formation and entorhinal cortex.DISCUSSIONHippocampal accumulation of αSYN oligomers and phosphorylated tau is associated with rapid cognitive decline in DLB. Therapeutic strategies targeting αSYN oligomers warrant further investigation.Highlights Proximity ligation assay (PLA) and digital pathology for oligomer quantification. Abundant α‐synuclein (αSYN) oligomers in CA1 of patients with dementia with Lewy bodies (DLB) with rapid decline. First human brain study linking αSYN oligomers to cognitive trajectory. Potential therapeutic implications of targeting αSYN oligomers in DLB.