Dispenzieri, Angela

Background: Venetoclax (Ven), a BCL2 inhibitor, has shown promising responses in multiple myeloma (MM) patients with t(11;14) and/or BCL2 expression. However, consistent benefit in clinical trials remain elusive. This report evaluates the efficacy and safety of Ven combination therapies, including in the non-t(11;14) subgroup, and examines the impact of secondary cytogenetic abnormalities (SGA) on outcomes. Methods: This retrospective study included multiple myeloma (MM) patients without concurrent AL amyloidosis treated with Ven-based combinations at Mayo Clinic sites from January 2015 to December 2023. FISH data was collected at diagnosis and before venetoclax initiation to assess SGAs. Refractoriness was defined by progression on or within 60 days of stopping treatment. Patients refractory to at least one proteasome inhibitor (PI), immunomodulatory drug (ImiD), and anti-CD38 antibody were considered triple-class refractory (TCR). Ven combinations were grouped as: Daratumumab/CD38 antibody (Dara)-Ven based if received Dara ± other drugs, PI-Ven-based without Dara, Ven-dexamethasone (Ven-d), and other combinations (Ven-Misc). BCL2 expression in patients without t(11;14) was determined by immunohistochemistry (IHC). Progression-free survival (PFS) was calculated from treatment initiation to disease progression or last follow-up. Results: We included 232 patients in this study. The median age at diagnosis of MM was 62 years (range 32-87), and the median age at initiation was 68 years (range 35-89). The median time from diagnosis of MM to initiation of Ven was 55 months (95% CI 28, 89 months), and the median follow-up from initiation of Ven-based treatment was 17.2 months (95% CI: 14, 22). Patients had received a median of 3 lines (IQR 4-6); 65% (n=152) of patients were TCR, 71% (n=163) had received autologous stem cell transplant and 7% (n=16) patients had received prior immune effector therapy prior to initiation of Ven-based therapy. The majority (83%, n=190) of the patients harbored a t(11;14); in the patients without t(11;14), a BCL2 expression by IHC was tested in 18 patients and positive in 17 patients. The most common Ven-based combinations included: Dara-Ven-d (19%, n=44), PI-Ven-d (30.2%, n=70), Ven-d (48.3%, n=112), Ven-misc (2.6%, n=6). The overall response rate for the entire cohort was 57% [64% for t(11;14) and 26% for non t(11;14), p<0.0001]. The median PFS for the entire cohort was 9.5 months (95% CI 8.5,13), and the duration of response was 18 months (95% CI 15.2-23.3). The median PFS was 11.8 months (95% CI 9.5-16.7) for patients harboring a t(11;14) compared to 2.9 months (95% CI 1.8, 8.4) for patients without a t(11;14), p<0.0001. Among the non-t(11;14) cohort with BCL2 expression, the median PFS was 1.8 months (95% CI: 1.2-4.2). Among the 190 patients with t(11;14), 18% (34/190) harbored deletion 17p and 32% (60/193) harbored a 1q gain/amplification (referred hereforth as 1q21+) at initiation of Ven-based therapy. In the cohort of patients with t(11;14), the presence of either del17p or 1q21+ (n=85/190) was associated with a significantly inferior PFS [median PFS 8.2 months (95% CI: 5.5,13)] vs. 15.1 months (95% CI: 11.6, 22), p=0.03. At the last follow-up,42 patients had ongoing treatment with ven-based combination. Most common reasons for discontinuation were progression (77% N=140), adverse effect (12%, n=22), patient/provider preference(9%, n=17); 2% (n=4) patients died without progression (3 infection, 1 unclear). The most commonly utilized maximum dose of venetoclax was 400 mg (42%); 15% (n=35) of patients required a dose reduction, and 23% (n=54) received a ramp-up. Tumor lysis syndrome was not noted in any patient. Infectious complications were noted in 23% (n=52). The estimated median OS from initiation of ven-based Rx was 34 months (95%CI 25-47). The estimated median OS for patients without a t(11;14) was 6.2 months (95% CI: 3.3-11.3) compared to 46 months (31.4-54.1), p<0.0001. In the t(11;14) subgroup, the 18-month OS rate was 79% for patients without 1q21+/del17p vs. 50% for patients with a 1q21+/del17p (p=0.002). Conclusion: Venetoclax-based regimens represent an important treatment option for patients harboring a t(11;14). The PFS in patients with BCL2 expression without t(11;14) was poor suggesting lack of efficacy of venetoclax-based regimens for that subgroup. Presence of concurrent del17p and/or 1q21+ with t(11;14) portends an inferior PFS.

Background: There is an increased risk of second primary malignancies including second hematological malignancies (SHM) in multiple myeloma (MM) patients undergoing autologous stem cell transplant (auto-SCT) followed by lenalidomide maintenance. We analyzed the characteristics and outcomes of patients who developed SHM after an auto- SCT for MM. Methods: A retrospective study of all MM patients who underwent auto- SCT at Mayo Clinic, Rochester between January 1, 1990, and 31 August 2023 and later developed SHM. Demographic, clinical and pathologic data were collected retrospectively. Overall survival (OS) was calculated from the date of auto-SCT to the date of last follow up. Statistical analyses were performed using GraphPad prism 9.0. Chi-square test was used to calculate the difference between categorical variables. Kaplan Meier method and log rank test were used to estimate OS. Results: Among 3513 patients who underwent at least 1 auto-SCT, 104 (2.9%) developed a SHM at a median follow up of 59 (range, 0.2-265) months. Patients who developed SHM, more frequently received pretransplant alkylating agents (37.5 vs 24.2 %, odds ratio [OR], 1.87; P=0.003) and topoisomerase II inhibitors (anthracyclines/etoposide) (17.3% vs 10.4%, OR [1.79]; P=0.03) compared to a control group of MM patients without SHM. Further, exposure to lenalidomide (77.8% vs 57.6%, OR[2.59]; P <0.0001) and radiation therapy (32.6% vs 23.1%, [1.54]; P=0.04) were more often seen in the SHM cohort compared to control group. Among the 381 patients who underwent > 1 auto- SCT, 3.1% developed SHM. Therapy related myeloid neoplasms (t-MN) accounted for 90.3% of cases [n=94; t-myelodysplastic syndrome (t-MDS=64), t-acute myeloid leukemia (t-AML=12), t-MDS transformed to AML(n=16), chronic myeloid leukemia (n=1), MDS/myeloproliferative neoplasm overlap syndrome (n=1)]. Eight patients (7.6%) developed therapy related B- cell acute lymphoblastic leukemia (t-ALL); Philadelphia (Ph) negative ALL (n=7) and Ph- like ALL (n=1). One patient each developed mixed phenotype acute leukemia and acute leukemia, not otherwise specified. Sixty patients (57.6%) were males and the median age at diagnosis of SHM was 70 (range, 43-84) years. The median time from auto-SCT to diagnosis of SHM was 58.5 (range, 7-236) months. In a multivariate analysis, receiving an alkylating agent (hazard ratio [95% confidence interval]: 2.51 [1.54 to 4.01]; P = 0.0002) and/or topoisomerase II inhibitor as induction for MM (3.12 [1.59 to 5.88]; P = 0.0006), exposure to lenalidomide (7.08 [3.92 to 13.50]; P = <0.0001) and radiation therapy (1.60 [1.02 to 2.44]; P = 0.03) were independent predictors of developing SHM. At diagnosis, 57% (n=37) of t-MDS and 71% (n=20) with t-AML had high risk disease per R-IPSS and 2022 European Leukemia Net respectively. Clonal abnormality in chromosome 5 and/or 7 was present in 45% (n=42) of patients. Complex and monosomal karyotype was seen in 32% (n=30) with t-MN. The most frequently mutated genes based on next generation sequencing data included TP53 (n=33), DNMT3A (n=9), RUNX1 (n=5), TET2 (n=4) and CEBPA (n=4). In the t-MDS/AML cohort, 3 patients (3.1%) underwent second auto-SCT and 9 (9.5%) received allogenic SCT (allo-SCT) for SHM. After a median follow-up of 27.2 months, the median OS from t-MDS/AML diagnosis was 8.04 months (95%CI, 6.1-11.0 months) and there was no difference in median OS for allo-SCT recipients versus nonrecipients (9.1 vs 6.8 months, P =0.19). In the t-ALL (n=8) cohort, all of them received lenalidomide based induction and five received lenalidomide maintenance. Most of the t-ALL patients had adverse risk cytogenetics [IKZF1 deletion (n=2), complex karyotype (n=2), TP53 mutation (n=1) and P2RY8-CRLF2 fusion (n=1)]. Three patients with t-ALL underwent allo-SCT and the median OS for allo-SCT recipients versus non recipients were 8.3 vs 7.9 months respectively, P=0.50. Among patients with SHM, 92.3% (n = 96) have died with SHM (72%) being the most common cause of death. MM patients who developed SHM had an inferior OS when compared to those without SHM (median OS, 75 vs 90 months, P =0.006). Conclusion: SHM following auto-SCT tends to have aggressive disease biology with poor outcomes. Short term follow up underestimates the risk of SHM and the higher incidence noted in our study may be due to longer follow up. As the OS in MM patients continues to improve, SHM remains an important challenge in improving long term outcomes.

Introduction: For cardiac transthyretin (ATTR) amyloidosis, bone scintigraphy has emerged as a noninvasive diagnostic tool. The use of different tracers, namely Pyrophosphate (PYP), 3,3-diphosphono-1,2-propanodicarboxylic acid (DPD), HydroxymethyleneDiphosphonate (HMDP), allow for the detection of amyloid deposits in the heart. The aim of this systematic review is to evaluate the diagnostic test accuracy of bone scintigraphy in ATTR and light chain (AL) amyloidosis. Methods: As part of the ASH AL Amyloidosis Diagnostic Guideline, we performed systematic reviews for 7 prioritized PICO questions related to the screening, diagnosis and organ involvement evaluation of AL amyloidosis. Electronic searches were conducted on PubMed, Embase, and the Cochrane Central Register of Controlled Trials from inception till January 2024. Two reviewers independently and in duplicate performed title and abstract screening and full text article screening on LASER AI, with conflicts resolved by a third reviewer. Studies were eligible if they included patients that performed both a bone scintigraphy scan and an endomyocardial biopsy for suspicion of ATTR amyloidosis. Test positivity for the visual score was defined to be ≥2, for the heart over contralateral (H/CL) ratio it was > 1.5 at one hour and >1.3 at 3 hours. Diagnostic test accuracy measures of interest were sensitivity, specificity, positive and negative predictive value. Statistical analysis was performed on STATA 18.0. Grading of Recommendations Assessment, Development and Evaluation (GRADE) was used to assess the certainty of evidence. Results: After deduplication, we retrieved 29,237 studies from electronic databases. 12 studies with 1127 patients reported on visual score in patients suspected to have ATTR amyloidosis. The pooled sensitivity for visual score was 96% (95% CI 92.0-98.0) while specificity was 90% (95% CI 84.0-95.0) (Certainty of evidence (COE): Moderate ⨁⨁⨁◯). 3 studies reported on ATTR visual score in combination with evaluating for monoclonal gammopathy, the pooled sensitivity was decreased to 93% (95% CI 65-99) and while specificity increased to 99% (95% CI 80-100) (COE: Moderate ⨁⨁⨁◯). H/CL was evaluated in 5 studies with 189 patients; pooled sensitivity was 97% (95% CI 89.0-99.0) and specificity was 88% (95% CI 64-97) (COE: Low ⨁⨁◯◯). We also performed subgroup analyses for the diagnostic test accuracy of visual score for the different tracers (PYP, DPD, HMDP) in patients suspected to have ATTR amyloidosis. DPD was found to have the highest sensitivity which was found to be 97% (95% CI 91.0-99.0) and a specificity of 95 (95% CI 86.0-98.0) (4 studies, n = 297 COE: Moderate ⨁⨁⨁◯). The sensitivity and specificity for PYP was 95% (95% CI 87.0-98.0) and 90% (95% CI 79.0-96.0) (7 studies, n = 377 COE: Moderate ⨁⨁⨁◯). 1 study reported on HMDP with a sensitivity and specificity of 79% (95% CI 49.0-95.0) and 100% (95% CI 59.0-100.0) (COE: Low ⨁⨁◯◯). Conclusion: Bone scintigraphy has a high sensitivity and specificity for the diagnosis of ATTR amyloidosis, particularly in patients without a monoclonal gammopathy.

Introduction: Although endomyocardial biopsy is the gold standard, echocardiography, and Cardiac Magnetic Resonance (CMR) play a pivotal role in the diagnosis of cardiac amyloidosis. In this systematic review, we aim to estimate the diagnostic test accuracy (DTA) of CMR and echocardiography for cardiac amyloidosis. Methods: As part of the American Society of Hematology guidelines on the diagnosis of Amyloidosis, we searched PubMed, Embase, and the Cochrane Central Register of Controlled Trials from inception till January 2024 for relevant studies. Two reviewers independently performed title and abstract screening and full-text article screening on LASER Al and extracted relevant data using a piloted Excel sheet. We performed statistical pooling using Stata 18.0 software. We evaluated risk of bias using Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) and used the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach to assess the certainty of the evidence. We report diagnostic test accuracy as sensitivity (95% confidence interval) and specificity (95% confidence interval). Results: After screening 29,237, we included 9 studies assessing DTA of late gadolinium enhancement (LGE) on cardiac magnetic resonance (CMR) using an endomyocardial biopsy (EMB) as a reference test in patients with amyloidosis (light chain and ATTR). The majority of studies didn't report on the type of amyloidosis where only 2 out of 9 were in patients with light chain amyloidosis. The pooled estimates for sensitivity and specificity of LGE are 0.95 (0.88. 0.98), and 0.81 (0.60, 0.92), respectively. We did a sensitivity analysis and included only LGE suggestive for amyloidosis which didn't change sensitivity but led to an increase in specificity for amyloidosis to 0.87 (0.76, 0.94), respectively. The pooled estimates were based on moderate certainty in the evidence. For 2-dimensional echocardiography, we addressed DTA for different echocardiographic findings and we did not limit the reference to EMB. A panel of experts judged whether the reference was acceptable or not. Acceptable reference was EMB alone or a combination of extracardiac biopsy plus cardiac imaging (CMR or echocardiography) with or without positive cardiac biomarkers (troponin or BNP or NT-proBNP). Six studies (n=2063) addressed interventricular septum thickness (IVS), sensitivity was 0.77 (0.69, 0.84) and specificity was 0.71 (0.60, 0.81). 4 out of 6 studies used 12 mm as a cutoff while the other 2 studies used 13 mm as a cutoff. Only 2 studies (n=1667) assessed the DTA of posterior wall thickness (PWT), with sensitivity ranging from 0.76 to 1.00, and specificity ranging from 0.84 to 0.89. Six studies (n=438) addressed diastolic dysfunction (grade 2,3), with a sensitivity of 0.71 (0.40, 0.90), and specificity of 0.75 (0.64, 0.84). 7 studies (n=472) reported DTA of only diastolic dysfunction grade 3. When compared to diastolic dysfunction (grade 2,3, sensitivity decreases to 0.42 (0 28. 0.58) and specificity increases to 0.89 (0.83. 0.94). For E/A ratio, only 2 studies (n=127) were included. Sensitivity ranged from 0.45 to 0.65, and specificity ranged from 0.85 to 0.98. Four studies (n=1596) yielded a sensitivity of 0.86 (0.65, 0.95) and specificity of 0.76 (0.55, 0.89) of left ventricular global longitudinal strain (GLS). All except one used -17% as a cutoff for GLS. For apical sparing on GLS, pooled estimates from 8 studies (n=2253) led to a sensitivity of 0.72 (0.64, 0.78) and specificity of 0.78 (0.64, 0.88). There was a variation of cutoff across studies. The certainty of evidence for echocardiographic findings was low to moderate. Conclusion: CMR is a more accurate test with better performance compared to echocardiography.

Introduction: Hyaline-vascular type unicentric Castleman disease (HV-UCD) is a classic variant of Castleman disease, characterized by localized lymph node hyperplasia. Due to its rarity, there have been few systematic reports summarizing its clinicopathological features globally. Also, its pathogenesis remains unelucidated. The present study aims to provide a detailed clinicopathological and transcriptomic analysis of HV-UCD to enhance the understanding of its underlying mechanisms at a molecular level. Methods: We conducted a clinicopathological analysis of 51 HV-UCD cases and performed whole transcriptome analysis on frozen lymph node specimens from four cases. Differentially expressed genes (DEGs) and Gene Ontology (GO) analyses were performed to identify significant pathways and biological processes involved in HV-UCD, with reactive lymphoid hyperplasia as control. Results: The mean age of HV-UCD patients was 42.8 years, with no significant gender differences. The most common sites involved were the abdominal cavity (n = 18, 35.3%), mediastinum (n = 11, 21.6%), and retroperitoneum (n = 9, 17.6%), with an average size of the lesions being 4.8 cm. Histologically, 33.3% of cases exhibited “dysplastic follicular dendritic cells (dFDCs)” characterized by multinucleation or large nuclei within and outside follicles, positive for follicular dendritic cells (FDC) markers. DEGs analysis compared to reactive lymphoid hyperplasia revealed significant upregulation of genes related to FDCs and angiogenesis. Notably, the placental growth factor (PGF), a proangiogenic factor and a member of the VEGF family, demonstrated significant overexpression. GO analysis identified significant enrichment of genes involved in angiogenesis, cell adhesion, tissue architecture, and humoral immunity. Discussion: This large cohort of HV-UCD cohort provides potentially novel clinicopathological insights. Gene expression analysis demonstrated increased expression of FDC-related genes, suggesting a pivotal role of FDCs in HV-UCD pathogenesis. The differences in the gene profiles related to angiogenesis may contribute to the distinct mechanisms and histological features of HV-UCD and iMCD-TAFRO, where HV-UCD is associated with hyalinization of vascular walls and iMCD-TAFRO is characterized by angiogenesis with endothelial cell swelling. Conclusion: The present results suggest the crucial role of FDCs in HV-UCD pathogenesis from the molecular features. Currently, there are no objective diagnostic markers for HV-UCD. Identifying these markers may lead to a clearer understanding of the disease pathophysiology and improve diagnostic accuracy for HV-UCD. Further investigation is warranted to verify the findings.

Background: Beside fibril-forming proteins, amyloid deposits contain other proteins which can influence organ toxicity, disease initiation and clearance. We investigated the association between the amyloid plaque proteomic signature of diagnostic renal biopsies from patients achieving deep and sustained hematologic remissions and renal organ response in AL amyloidosis. Methods: Retrospective study of newly diagnosed AL amyloidosis patients between 2010-2020. Patients were eligible if; (1) they had a baseline renal biopsy which had undergone laser micro-dissection and mass spectrometry, (2) were assessable for renal response [RenR, non-selective proteinuria ≥1g /24 hours, predominantly albumin], (3) were not on renal replacement therapy and did not experience renal progression within 3-months of diagnosis and (4) had RenR measurements, at least twice annually in the first year from treatment and annually thereafter. Patients who did not obtain a renal complete response (RenCR) required a hematologic very good partial response (VGPR) or better within 6 months of treatment initiation, sustained for a minimum of 6 months. Hematological and renal responses were graded according to consensus criteria. Overrepresentation analyses of differentially expressed proteins were performed using the WebGestalt 2019 and the reactome pathway database. A false detection rate corrected p value <0.05 was used to identify significantly overrepresented proteins and pathways. Results: We defined two organ response categories, to capture the extreme ends of the spectrum: renal suboptimal responders (n=24), patients for whom the best renal response was ≤partial response (RenPR), and renal complete responders (n=29), patients who obtained a RenCR at best response. Overall, the median follow-up was 6.7 years (IQR: 5.4-9.2), 6.6 years (IQR: 5.4-8.1) for complete responders, and 8.4 years (IQR: 6.1-11.3) for suboptimal responders. Between renal suboptimal responders and complete responders there was no significant difference in median age (63 vs 63 years, p=0.7), lambda light chain isotype (66% vs 79%, p=0.3), first-line therapy received and renal stage (Stage III: 14% vs 8%, p=0.9). The median time to RenCR was 52-months (IQR: 31-63). Protein pathways over-represented in renal complete responders included 14-3-3 proteins (YWHAG, YWHAH, YWHAQ, YWHAZ), early components of the complement cascade and complement regulatory proteins (C3, C4A, C4B, C8A, complement factor H), keratins, cytoskeletal proteins (tubulin, actin, filamen) and collagen IV. Protein pathways over-represented in renal suboptimal responders included terminal/effector components of the complement cascade (C5, C6, C8B, C9), amyloid signature proteins (ApoA1 and E, vitronectin, clusterin), proteoglycans (heparan sulfate proteoglycan), transgelin and metallopeptidase inhibitor 3. Proteins with the highest-fold increase in renal complete responders included serine protease inhibitor (SERPINA3) and F-box and leucine rich repeat protein (FBXL6), 9.0 x 106 & 7.7-fold, respectively. Proteins with the highest-fold increase in renal suboptimal responders included complement 6 and 8B, 3.5 & 3.6-fold, respectively. Conclusion: This is the first study to identify proteomic determinants of organ response in AL amyloidosis. Molecular chaperones involved in protein aggregate homeostasis, such as the 14-3-3 proteins are upregulated in renal complete responders, similar to what we have shown in our prior work. This suggests that organ responders may have a more favorable microenvironment which “primes” them for organ response after the amyloidogenic light chain has been cleared from the circulation. Renal suboptimal responders (≤RenPR) have elevated levels of terminal complement proteins including membrane attack complex proteins and reduced levels of early components or complement regulatory proteins, which may be responsible for reno-toxic and reno-protective effects, respectively.

Introduction: Light chain (AL) amyloidosis is a clonal plasma cell disorder characterized by tissue deposition of misfolded immunoglobulin light chains in the form of amyloid, leading to organ dysfunction. Halting the production of the amyloidogenic free light chains by achieving a deep hematologic response (HemR) is key to improving organ function, however, variation in organ responses exists. In this work, we investigated variables associated with liver response (LivR) in a large cohort of patients with AL amyloidosis. Methods: Retrospective study of newly diagnosed AL amyloidosis patients with liver involvement seen at Mayo Clinic within 90 days of diagnosis between January 2000 and December 2021. Patients were eligible if they (1) had liver involvement, defined as a baseline alkaline phosphatase (ALP) ≥ 1.5 x upper limit of normal (ULN) and (2) had LivR measurements at least twice annually in the first year from treatment and annually afterward. LivR was defined as a >50% reduction in alkaline phosphatase (ALP) from baseline value or normalization of ALP. LivR was recorded at 6, 12, and 24 months from treatment initiation and at the best LivR achieved. Patients who died prior to landmark assessment were considered non-responders at subsequent milestone assessments. Covariates associated with LivR were identified by univariate and multivariable logistic regression analyses and a proportional subdistribution hazard (Fine-Gray) model for competing risk (treating death as a competing event). Multivariable logistic regression models for LivR were created using best subset selection, with calculation of odds ratio (OR) and 95% confidence interval (CI). Results: A total of 130 patients were included, with a median age of 60 years (IQR 53-66), male predominance (69%), and a median follow-up of 8.8 years. Sixty percent of patients had lambda isotype, the median ALP was 368 IU/mL (IQR 279-589), with 48% of patients having ALP ≥3 fold the upper limit of normal (ULN). Median bilirubin was 0.7 mg/dL (IQR 0.5-1.1). Forty-three percent of patients received front-line autologous stem cell transplant (ASCT) with or without prior induction therapy, and 5% received daratumumab-based therapy as their front-line treatment. Liver response rate increased with time, with 36 patients (28%) achieving a LivR at 6 months, 47 (36%) at 12 months, 63 (48%) at 24 months, and 94 (72%) at best response. The median time to liver response was 14 months (7.22-32.67). At the 24-month landmark, age <60 years, ALP ≥3 fold ULN, and front-line ASCT were significantly associated with achieving a LivR on univariate logistic regression. On univariate logistic regression at best liver response, age <60 years, ALP ≥3 fold the ULN, kappa isotype, ≥HemVGPR within 6 months, and front-line ASCT were significantly associated with obtaining a LivR. On multivariable logistic regression, at 24 months, ALP ≥3 folds of ULN (OR=2.9, 95% CI 1.4-6.3, P=0.004) was significantly associated with an increased likelihood of LivR. Achieving ≥HemVGPR and front-line ASCT were also associated with increased LivR likelihood, although not statistically significant (p=0.094, p=0.078, respectively). At best liver response, achieving ≥HemVGPR within 6 months (OR=5.2, 95% CI 1.9-14.8), kappa isotype (OR=3.2, 95% CI 1.1-10.0), ALP ≥3 folds of ULN (OR=3.3, 95% CI 1.3-9.9), and front-line ASCT (OR=5.3, 95% CI 1.9-16.7) were factors significantly associated with LivR. The Fine-Gray model demonstrated kappa isotype (HR=1.7), baseline ALP ≥3 folds of ULN (HR=2.1), ≥HemVGPR within 6 months (HR=1.9), and age <60 years at diagnosis (HR=1.9) to be positively associated with achieving LivR (P<0.05). Conclusions: In this large cohort of liver AL amyloidosis, LivR was achieved in 72% of patients, and amongst responders, the median time to response was 14 months from therapy initiation. Predictors of LivR included kappa isotype, deep hematological response in the first 6 months of initial therapy, ASCT usage, and higher baseline ALP levels. These findings better inform clinicians on the likelihood of achievement of liver response and may enable ways to improve its achievement.

Background: Systemic light chain (AL) amyloidosis is a clonal plasma cell disorder characterized by the deposition of misfolded immunoglobulin light chain products in vital organs, leading to significant organ dysfunction. The molecular mechanisms underlying its pathogenesis remain incompletely elucidated. Identifying the molecular mechanisms and novel therapeutic targets is critical for improving treatment strategies and patient outcomes. Our previous research work identified that the PI3K/AKT signaling pathway is overexpressed in AL amyloidosis (Zvida et al., Blood, 2022, Fishov et al., Cancer Med., 2023). This pathway plays a critical role in regulating cellular metabolism. Specifically activated AKT can phosphorylate and regulate various target proteins involved in metabolic processes. One of the significant targets is fatty acid synthase (FASN), an enzyme responsible for the synthesis of fatty acids. AKT can enhance FASN expression and activity, thereby promoting lipid biosynthesis. Additionally, elevated FASN levels can drive inflammation through increased fatty acid synthesis, which activates inflammatory signaling pathways and enhances cytokine production (Acosta-Martinez et al.,IJMC, 2022). Elevated levels of FASN have been associated with hematological malignancies and cell survival (Vanauberg, et al., Oncogenesis, 2023). However, its role in AL amyloidosis is yet to be elucidated. Aim: To determine the role of FASN in the pathology and inflammatory response in AL amyloidosis, and to evaluate the potential of targeting FASN and the PI3K/AKT pathway for therapeutic intervention. Methods: We profiled FASN expression in CD138+ bone marrow (BM) samples from patients with AL amyloidosis (n=20), Multiple Myeloma (MM) (n=20), Monoclonal Gammopathy of Undetermined (MGUS) (n=8) and Healthy Controls (HC) (n=6), using qRT-PCR. To further explore the relationship between the PI3K/AKT pathway and FASN, we treated ALMC-1 cells with Capivasertib (an AKT inhibitor) and assessed FASN protein levels, cell proliferation by WST-1 assay, cell cycle distribution by flow cytometry, light chain secretion by ELISA, and expression of AKT and its downstream targets via western blot analysis. Furthermore, TVB-2640 (FASN inhibitor) was used to evaluate the cell response to FASN inhibition. Results: Our analysis revealed a significant upregulation of FASN in AL amyloidosis BM samples compared to MM, MGUS, and HCs. Capivasertib treatment resulted in a dose-dependent reduction in FASN protein levels. Additionally, Capivasertib treatment inhibited cell proliferation, induced G1 phase cell cycle arrest, reduced light chain secretion, and decreased AKT protein expression and its downstream targets p4EBP pS6 and pERK. Elevated levels of proinflammatory cytokines, including TNF and IP-10 (CXCL-10), were observed in AL amyloidosis serum samples, suggesting the involvement of immune mechanisms and the PI3K/AKT pathway, potentially induced by FASN. Finally, treatment with TVB-2640 reduced cell viability and decreased the protein expression of LIPIN-1, which is one of the key target genes regulated by FASN. Conclusions: Our findings highlight the significant upregulation of FASN in AL amyloidosis and its regulation by the PI3K/AKT pathway. The observed effects of Capivasertib on FASN expression and cell proliferation, along with the upregulation of inflammatory cytokines, underscore the potential for targeting the PI3K/AKT signaling pathway and FASN in developing more specific therapeutic strategies for AL amyloidosis.

Background: Multiple myeloma (MM) involves the proliferation of monoclonal plasma cells (PCs) in the bone marrow. The plasma cell labeling index (PCLI), indicating the percentage of PCs in the S-phase, is a key prognostic factor in newly diagnosed multiple myeloma (NDMM) patients. Methods: A retrospective study at Mayo Clinic analyzed bone marrow Affymetrix microarray datasets from 136 NDMM patients (2001-2012). Data normalization and quality assessment were followed by differential gene expression analysis using limma-voom, adjusted for age, BMPC percentage, and disease stage (RISS). Selected genes were dichotomized by median and subjected to logistic regression for high/low PCLI. Data from 594 patients with a Proliferation Index at baseline was pooled from the MMRF (CoMMpass) dataset for validation after normalization across assays. Results: The baseline characteristics of the patients are as follows: 62 (46%) were female and 74 (54%) were male, with a mean age at diagnosis of 61.6 ± 11.5 years. RISS staging included 36 patients (26.5%) at stage 1, 32 (23.5%) at stage 2, 29 (21.32%) at stage 3, and 39 patients (28.67%) with unknown stage. PCLI distribution showed 113 patients (83.7%) with <2%, and 23 (16.3%) ≥2%. Differential Analysis: The differential analyses reveal several critical insights. In the primary continuous differential analysis, 21,367 genes were analyzed, identifying 654 as differentially expressed with an FDR-adjusted p-value ≤0.05. Of these, 481 genes were upregulated (top 5: SPC25, TRAT1, PBK, E2F8, ASPM) and 173 were downregulated (top 5: SPARC, CD24, MYOF, CXCL12, VCAM1). Univariate and multivariate stratified analyses showed significant findings: 668 genes in age stratification, 395 genes in BMPC% stratification, and 536 genes in RISS stratification. In multivariate analysis, 408 genes were significant with an FDR-adjusted p-value <0.05 for PCLI and ≥0.05 for BMPC%, age, and RISS. 3-Fold Internal Validation: 240 significant genes were identified in fold 1, 25 in fold 2, and 167 in fold 3, with 21 genes common across all folds (e.g., MCM10, GTSE1, UBE2S, NCAPG, FOXM1, STMN1, NEK2, CENPF, MND1, HJURP, E2F8, TOP2A, SKA1, CCNB2, CCNF, CHAF1A, PBK, EXO1, AURKB, CDCA2, DHFR). 19 genes were common in the sensitivity analysis by excluding RISS 3. 20 genes were common in categorical analysis (Categories <1%, 1-2%, 2-3%, 3-4%, >4%). 3 Gene-Based Proliferation Score (3GPS): On univariate logistic regression analysis of 21 genes for high/low PCLI, 17 significant genes were identified. In the step-wise multivariate analysis, CDCA2 (OR=11.56), DHFR (OR=9.6), and CENPF (OR=6.11) were significant. Log-odds were used as weights. For the high expression(median and above) of gene as 1 and low expression as 0: 3GPS=2.4*CDCA2+ 2.3*DHFR +1.8*CENPF A cut-off of 2.4 was chosen as CDCA2 had the highest positive concordance rate (90.91%) versus DHFR (86.36%) and CENPF (77.27%). External Validation: In the CoMMpass dataset, the sensitivity and specificity for the presence of high expression of any of the 3 genes with ≥2 Proliferation Index (PI) were 92.4% and 73.8%, respectively (AUC=83.1%). The median 3GPS was 0 for <2 PI (n=238) and 6.5 for ≥2 PI (n=356). The sensitivity and specificity for the 3GPS in predicting ≥2 PI were 82.6% and 93.6%, respectively (AUC=88.1%). Pathway Analysis: Significant pathways using Reactome include polo-like kinase mediated events in G2/M transition, condensation of prometaphase chromosomes in metaphase, resolution of sister chromatid cohesion in anaphase, and APC-Cdc20 mediated degradation of Nek2A for regulation of the cell cycle. p53 signaling, cancer pathways, and microtubule regulation were also mapped. Survival Analysis: In our cohort, for CDCA2 quartiles (Q)3 and Q4, for CENPF Q3 and Q4, and for DHFR Q4 were associated with inferior overall survival. 3GPS ≥2.4 was associated with inferior survival of 60.28 months versus 111.5 months for <2.4 (p<0.01). In the validation dataset, median survival was not reached at 100 months (vs. high 79.7, p<0.01). The multivariate Cox model found 3GPS score ≥2.4 as an independent predictor (HR=1.64, 95% CI: 1.23 - 2.19), along with ECOG status, ISS stage, gender, age, and whether the patient underwent Stem Cell Transplant. Conclusion: CDCA2, DHFR, and CENPF are associated with ≥ 2% PCLI. The 3GPS score can serve as a measure of gene-based proliferation index. Prospective validation is needed for clinical practice.

Background The detection, isotyping and quantitation of over-expressed immunoglobulin(s) (Igs) (M-protein) is key for diagnosing and monitoring patients with monoclonal gammopathies. Traditionally, our lab has used a combination of agarose gel protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE) for these purposes. In 2018, a mass spectrometric method (Mass-Fix) was implemented to replace IFE for M-protein isotyping. This work documents the validation of a quantitative version (Q-Mass-Fix) to replace both SPEP and IFE. Methods 24,645 samples submitted for M-protein testing between 7/2018 and 9/2023 with quantitative IgG, IgA and IgM measurements, Mass-Fix analysis and SPEP results were utilized for this study. A separate cohort of 100,000 M-protein negative samples were utilized to mathematically model the spectral shapes IgG, IgA and IgM polyclonal background. To detect M-proteins, software was developed that fits the polyclonal Ig portion of the spectra and M-proteins are detected as peaks above the polyclonal background. M-proteins isotypes are determined by matching similar peaks in spectra in 5 sperate immuno enrichments (IgG, IgA, IgM, kappa and lambda). M-protein quantitation was obtained by multiplying total M-protein isotype quantity by the fraction area of the M-protein in the spectra. M-protein dilutional studies were used to examine the linearity and level of detection of Q-Mass-Fix. Results SPEP and Q-Mass-Fix M-protein quantitation had the best agreement between the range of 10 to 40 g/L. Below 10 g/L, Q-Mass Fix had on average, lower M-protein values. Above 40 g/dL, Q-Mass-Fix values were on average, higher than SPEP. The automated isotyping assignment was 98.8 percent consistent with the isotype assigned by visual inspection. Dilutional studies revealed that Q-Mass-Fix quantitation was linear over a range of 70 to 0.04 g/L (slope 1.0, r2 0.99) with an analytical limit of quantitation of 0.1 g/L and a limit of detection of 0.04 g/L. Conclusion Q-Mass-Fix represents a significant improvement in comparison to our current SPEP method. Q-Mass-Fix is more capable of removing the interference of polyclonal Igs below 10 g/L and is not subjective to the dye saturation effect at high M-protein levels. The increased analytical measuring range and improved limit of detection of detection could be beneficial in tracking patient's response. Q-Mass Fix is more automated than SPEP and the workflow in our laboratory.