Dispenzieri, Angela

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.

Abstract Objective The aim of this study was to evaluate a large cohort of gallbladder amyloid cases to determine clinical and morphologic features. Methods Cholecystectomy specimens (N = 118) typed using proteomics-based techniques between 2008 and 2023 were identified. Clinical and morphologic features were reviewed. Results Six amyloid types were identified: ATTR (n = 63, 53.4%), AL (n = 46, 39.0%), AA (n = 4, 3.4%), AApoA1 (n = 2, 1.7%), ALECT2 (n = 2, 1.7%), and AEFEMP1 (n = 1, 0.8%). Amyloidogenic mutations were detected in 3 ATTR cases and 2 AApoA1 cases. Morphologic review (n = 26) revealed perimuscular vessel involvement in all cases. Amyloidosis was an unexpected diagnosis first made on the cholecystectomy specimen in half of the patients with clinical information (n = 10). All 9 patients with follow-up had evidence of systemic disease. In 2 patients, cholecystic involvement was initially missed and only retrospectively identified after the diagnosis of cardiac amyloidosis. Conclusions In patients with clinical data, amyloidosis was often unexpected, the gallbladder was commonly the first tissue sampled with amyloidosis, and all patients had systemic disease. Thorough review of cholecystectomy specimens with careful inspection of perimuscular vessels, coupled with a low threshold for ordering Congo red stain in elderly individuals and amyloid typing using a robust method such as proteomics, can prevent a delay in amyloid diagnosis and management.

Abstract This retrospective analysis examined 15 years of autologous stem cell transplantation (ASCT) for light chain (AL) amyloidosis at the Mayo Clinic (2010-2024). We aimed to assess ASCT utilization trends, factors influencing practice changes, and current indications amid newer therapies. Four hundred and forty-one ASCTs were divided into cohort 1 (2010-2019) and cohort 2 (2020-2024), revealing a significant ASCT reduction in cohort 2 (385 vs 56, average 71% annual decrease). Cohort 2 patients were older, more likely to have relapsed/refractory disease, and had higher baseline bone marrow plasma cell burden compared to cohort 1. Pre-ASCT induction was more frequent in cohort 2 (89.3% vs 56.4%), with daratumumab (Dara)–cyclophosphamide-bortezomib-dexamethasone (CyBorD) replacing CyBorD as the predominant induction regimen. Lymphoma-based regimens were also more common in cohort 2 (15.1% vs 5.3%, P = .02). Day-100 satisfactory hematological response improved in cohort 2 (91.1% vs 72.7%, P = .001), although hematological complete response rates did not significantly differ (50.9% vs 38.8%, P = .09). In summary, there is a decrease in the utilization of ASCT in AL amyloidosis. This procedure is primarily reserved for patients with suboptimal responses, relapsed/refractory disease, lymphoplasmacytic clones (predominantly immunoglobulin M amyloidosis), or high bone marrow plasma cell burden (myeloma phenotype). This study underscores a significant shift in ASCT practice, driven by novel therapies, emphasizing more personalized management in AL amyloidosis.

ABSTRACTMonoclonal gammopathy of undetermined significance (MGUS) is the term used for a minimal clonal expansion of plasma cells that is most typically detected by the presence of a small serum monoclonal protein. For most—but not all—patients, the presence of a small monoclonal protein will have no clinical significance at the time of recognition or even over time. It is imperative, therefore, that a careful—but sensible—evaluation be performed both at the time of recognition and subsequently because these monoclonal may represent significant pathology including diseases such as multiple myeloma, low grade lymphoma or any of the diseases captured under the moniker of monoclonal gammopathy of clinical significance (MGCS), which includes—but is not limited to—immunoglobulin light chain amyloidosis and POEMS syndrome. Herein is a discussion of the diagnosis and follow‐up MGUS and MGCS.

ABSTRACTCastleman disease is a term that represents a spectrum of diseases whose driving factors are poorly understood. Increased interest prompted by the Castleman's Disease Collaborative Network has led to a better understanding of classification, which will lead to a better understanding of underlying pathology and best practices. It is essential that this entity be recognized and correctly classified by anatomic classification (unicentric, oligocentric, and multicentric), by histopathology (the gradient of hypervascular to plasmacytic), and by clinical phenotype (NOS, TAFRO, IPL, HHV8‐associated, or POEMS‐associated). Most of what we know about therapy is from retrospective studies rather than prospective therapeutic trials, but data are emerging through collaboration. The mainstay of therapy for unicentric disease is surgery. For multicentric disease interleukin‐6 blockade is essential for the non‐POEMS, non‐HHV8 multicentric cases, rituximab‐based (plus or minus antiviral) therapy for HHV8 associated cases, and IL‐6 blockade versus plasma cell directed therapy for the POEMS subtype.

Disease response and progression assessment in multiple myeloma (MM) is based on various measurements of monoclonal protein (serum and urine protein electrophoresis, serum free light chain (FLC, and or quantitative immunoglobulins). Currently, the IMWG consensus response criteria require two sequential assessments of any one marker made at any time before confirmation of disease progression and the institution of any new therapy. However, this can be cumbersome in clinical trials. Herein, we hypothesized that if two markers meet the progression criteria simultaneously, a repeat of either will not be necessary for confirmation. We retrospectively studied all sequential patients with myeloma enrolled in clinical trials at Mayo Clinic. We identified 583 episodes of confirmed progression in our study. Among the 583 progression episodes, nearly 70% (sensitivity of the simultaneous criteria) met the two simultaneous variable criteria at the first testing, indicating progression. Conversely, among 413 patients who met progression criteria by two simultaneous values, 98% (specificity of the simultaneous criteria) of patients subsequently had confirmed progression by sequential values. In summary, for patients with two disease burden markers meeting the simultaneous progression criteria, sequential assessment either one for confirmation may not be necessary to determine disease progression.

11549 Background: Follicular dendritic cell sarcoma (FDCS) is a rare neoplasm with a subset associated with unicentric Castleman disease (UCD). However, the impact of CD association on clinicopathologic features and survival outcomes remains unclear. This study aims to elucidate differences in disease characteristics and outcomes in FDCS with and without CD to guide future research and clinical practice. Methods: We retrospectively analyzed 41 FDCS patients seen at Mayo Clinic between January 2000 and December 2024. Patients were categorized into CD-related (n = 9) and FDCS-only (n = 32) groups. Demographic, clinical, pathological, and survival data were compared. Kaplan-Meier survival analyses were used to evaluate overall survival (OS) and progression-free survival (PFS). Epstein-Barr virus-associated inflammatory FDCS were excluded. Results: The CD-related group was younger at diagnosis (46.0 vs. 58.0 years, p = 0.041). Among those with CD-related FDCS, FDCS was diagnosed concurrently as CD in 6/9 (66.7%) and after the diagnosis of CD in 3/9 (33.3%). The FDCS-only patients were more likely to have extranodal disease (71.9% vs. 22.2%) and to have metastasis (62.5% vs 22.2%). One CD-related FDCS patient had paraneoplastic pemphigus. Among 11 patients with next-generation sequencing (NGS) results, 9 had pathogenic mutations. The most common pathogenic mutations included CDKN2A, CDKN2B, and TRAF3 copy number losses. Surgery was the preferred first-line intervention in FDCS-only and CD-related FDCS groups (19/32, 59.4% vs. 6/9, 66.7%). Eight out of 32 (25.0%) of the FDCS-only group received chemotherapy at first-line, most commonly Gemcitabine/Docetaxel or Adriamycin/Ifosphamide. While Gemcitabine/Docetaxel was the most preferred in the second line, different regimens were pursued in the third and later lines of therapy, including Pembrolizumab, Pazopanib, and stem cell transplant. Five out of 6 patients in the CD-related FDCS group received either adjuvant radiation or chemotherapy, which led to complete remission during the follow-up periods. The median follow-up was 22.4 months for the whole population. There was a trend for better OS and PFS among CD-related FDCS patients. The respective 2-year OS rates were 100% and 66.7%, and 2-year PFS rates were 57.1% and 43.1% for CD-related FDCS and FDCS-only patients, respectively. Conclusions: This study provides novel insights into clinicopathologic differences and survival trends for FDCS. Our data would suggest a less aggressive disease course among CD-related FDCS, which implies the need for different treatment strategies for CD and non-CD-related FDCS. Further research is warranted to elucidate these mechanisms, such as multi-omic analysis on benign FDC proliferation in the microdissected UCD areas and the adjacent FDCS cells to determine factors associated with progression to sarcoma.

e19531 Background: Effective treatment and elimination of clonal plasma cells (PC) in multiple myeloma (MM) are typically associated with recovery of polyclonal (normal) plasma cells (PPC). Studies suggest that robust PPC recovery is associated with better outcomes. However, it is unclear if this remains valid in the context of monoclonal antibodies, such as Daratumumab, that selectively target plasma cells, and in turn, may adversely impact PPC recovery. Methods: We identified patients with MM who underwent MRD testing using Euroflow method at 6-12 months after treatment initiation. Eight patients had disease progression prior to MRD testing and were excluded from further analysis. PPC was quantitated by the proportion of total events acquired during flow examination. Patients were considered MRD-negative if they had <1 clonal PCs in 10 5 cells, as detected in the marrow. We first examined the impact of initial therapy (Daratumumab vs. non-Daratumumab based regimens) and MRD status on PPC recovery. Subsequently, we analyzed impact of PPC recovery on patient outcomes in terms of progression-free survival (PFS) and overall survival (OS). Results: We identified 572 patients who had MRD testing by flow at a median of 9 months (range 6-12) after treatment initiation. The median age was 63 years, 58% were male. Daratumumab was part of initial therapy in 17% of patients; the most common induction therapies were PI-IMiD triplets (73%), and dara-PI-IMiD quads (15%). In our cohort, 74% of patients were MRD-negative. In our practice, only samples with no monotypic PCs identified on initial low-sensitivity flow proceed to MRD testing, hence, the proportion of MRD-negative patients appears higher in our study than previously reported. Median PPC was 0.02% and was similar between MRD-negative and -positive patients. We found the median PPC was significantly low in patients who received Daratumumab compared to the non-Daratumumab treated cohort (0.011% vs. 0.023%; p<0.01). There was a trend towards lower PFS and OS among the lowest PPC quartile group compared to the upper 3 quartiles, although this was not significant (p=0.07 for PFS and OS, Wilcoxon). This trend was more evident among patients who did not receive Daratumumab. Conclusions: Our study demonstrates negative impact of anti-CD38 therapy with Daratumumab on recovery of PPCs after initial treatment, consistent with the persistent hypogammaglobulinemia seen in clinical practice among these patients. However, unlike the observation with the non-Daratumumab treated cohort, the low proportion of PPCs does not significantly impact patient outcomes among patients who received Daratumumab.