Bergsagel, P. Leif

Abstract Purpose: Naturally occurring tumor-specific T cells derived from peripheral blood (PB) offer a clinically accessible source for adoptive immunotherapy. However, the expansion of these T cells from patients with cancer remains a challenge. We hypothesized that mimicking innate immune activation could optimally stimulate antigen-driven T-cell expansion in vitro, unlocking the therapeutic potential of PB-derived T cells. Patients and Methods: We previously developed an ex vivo culture system that selectively expands tumor antigen–activated T cells from PB mononuclear cells (PBMC), generating multiclonal effector and central memory T cells. In this hypothesis-generating study, we evaluated the therapeutic potential of MUC1-activated T cells in patients with relapsed and/or refractory multiple myeloma (r/rMM) in a phase I clinical trial (NCT05411497). MUC1 is an oncoprotein overexpressed in r/rMM. We translated our small-scale culture into GMP-compliant, large-scale manufacturing, which achieved expansion of T cells from heavily pretreated patients’ PBMCs without exhaustion. Five patients were treated with escalating doses of up to 1 × 1010 T cells. Results: Although the cell infusions were well tolerated, no objective responses occurred. One patient, who received the highest dose, has had stable disease for 2 years after infusion. This patient exhibited transient dermatitis with localized MUC1 and CD3 staining, as potential evidence of on-target, off-tumor T-cell reactivity, perhaps contributing to disease stabilization. T-cell receptor sequencing revealed the T-cell product in four of the patients’ blood, which correlated with the product’s degree of polyfunctionality and MUC1 reactivity. Conclusions: These findings demonstrate the feasibility, safety, and biological activity of PB-derived, MUC1-specific T cells as adoptive immunotherapy. Significance: Expanding tumor antigen–specific T cells for clinical use from the blood of heavily pretreated patients with cancer remains a significant hurdle. We scale up a novel culture method that integrates innate immune signals to stimulate T-cell expansion and demonstrate safety, feasibility, and a case of long-term disease stabilization upon treatment of patients with multiple myeloma in a phase I clinical trial.

Abstract Solitary bone plasmacytoma (SBP) and SBP with minimal marrow involvement (SBPmm) are treated similarly with definitive radiation therapy (RT) followed by observation. However, differences in the natural disease course after RT between the two, are not well described. We performed a multicenter study assessing outcomes after RT in 141 patients with SBP and 102 with SBPmm diagnosed between January 2010 and December 2024. About 92% of patients with SBPmm progressed to multiple myeloma (MM) vs 76% of those with SBP (P = .01). Among patients with SBP, 21% progressed with a second solitary bone lesion vs 4% with SBPmm (P< .01). Patients with longer time to progression (TTP) after initial RT could achieve durable responses with RT to a second solitary plasmacytoma in the absence of other myeloma-defining events (MDE). Multifocal lytic bone lesions were the most common MDE, occurring in 89% and 81% of patients with SBPmm and SBP, respectively. Patients with SBPmm had a median TTP of 15 months (95% confidence interval [CI], 14-21) compared to 43 months (95% CI, 34-55) for those with SBP (P< .0001); 67% with SBPmm progressed within 2 years of diagnosis. Del(17p) and 1q+ (gain or amplification) were associated with shorter TTP to MM in SBPmm, with 2-year progression rates of 100% and 82%, respectively. We report significant differences in risk and modes of progression between SBP and SBPmm. Patients with SBPmm are at particularly high risk for early progression to MM, with most developing bone disease as the MDE. These findings support prospective studies incorporating frontline systemic therapy for SBPmm.

Abstract Immune effector cell–associated enterocolitis (IEC-EC) has been observed after ciltacabtagene autoleucel (cilta-cel [Carvykti]) chimeric antigen receptor T-cell (CAR-T) therapy. It is associated with dismal outcomes and is poorly characterized. Here, we examined its clinical features, risk factors, and proposed management strategies. Among the 229 consecutive patients who received cilta-cel at Mayo Clinic, 9 (3.9%) presented with a nonresolving, nonbloody, grade 3 diarrhea, often requiring prolonged total parenteral nutrition. Gastrointestinal coinfections were seen in most patients. Median time from CAR-T infusion to symptom onset was 85 days (range, 35-166), and there appeared to be a latency from symptom onset to endoscopic evaluation. Histopathology findings resembling a graft-versus-host disease pattern of mucosal injury were most seen on duodenal biopsies, whereas 2 cases had patterns akin to a T-cell lymphoproliferative disorder (1 CD4+ and 1 CD8+). Independent associated factors for IEC-EC included high-risk disease as defined by the International Myeloma Society and International Myeloma Working Group, prolonged cytokine release syndrome, and antecedent delayed neurotoxicity. Response to first-line therapy comprising IV or oral corticosteroids, bile acid sequestrants, IV immunoglobulin, and antimicrobial-directed therapy was poor, with no durable responses. Biologic therapy appeared to induce durable responses in 3 of the 5 patients (2/2 vedolizumab; 1/3 infliximab); none of the responders had gastrointestinal coinfection. Early gastroenterology input for endoscopic evaluation with the inclusion of duodenal biopsy is a key component for diagnosis. Early institution of biologic therapy after failure of a short course of corticosteroids should be considered.

Abstract Intercellular communication between multiple myeloma (MM) cells and osteoclast precursor cells (pre-OCs) contributes extensively to the occurrence and development of myeloma-related bone destruction. However, key interacting modes and the exchanged substances involved in this communication remain unclear. In this study, we discover that tunneling nanotubes (TNTs) directly connect MM and pre-OCs. Using the stable isotope labeling with amino acids in cell culture assay and a positive-negative double selection strategy, we identify filamin A (FLNA) as a major protein transported from MM to pre-OCs. FLNA acts as a molecular clutch linking extracellular matrix–bound MAC1 to the cytoskeleton, activating Rho and MAPK signaling pathways and promoting F-actin polymerization, which subsequently enhances osteoclast differentiation by modulating cellular stiffness, traction force, and deformability. In addition, FLNA directly binds vinculin and promotes its recruitment to podosomes, thereby enhancing the functions of podosomes and the bone resorption capacity of osteoclasts. The conditional depletion of Flna in mice suppresses podosome activity and reduces stiffness, traction force, and deformability in pre-OCs, leading to significantly impaired osteoclast differentiation and increased bone mass. In the Vk∗MYC mouse model of myeloma, the administration of the TNT inhibitor latrunculin B disrupts FLNA transport to osteoclasts and alleviates osteolytic bone disease. These findings highlight the critical role of MM-transferred FLNA in osteoclastogenesis and suggest that targeting TNTs may represent a therapeutic strategy to limit pathological bone resorption associated with MM.

Abstract Multiple myeloma (MM) is an incurable plasma cell neoplasm characterized by diverse and complex genetic abnormalities, including translocations of immunoglobulin (Ig) genes and a hyperdiploid (HRD) karyotype. Much of our current understanding of MM genetics, epigenetics, pathogenesis and response/resistance to myeloma directed therapies originated from studies on human MM-derived cell lines (HMCL). In this study, we conducted an integrated multi-omics analysis in five hyperdiploid cell lines established from serial samples from a single relapsed/refractory hyperdiploid MM patient, enabling detailed characterization of the genetic landscape and molecular alterations through disease progression.

Abstract Multiple myeloma (MM) with t(11;14)(CCND1;IGH) remains the only subset sensitive to the BCL2 inhibitor venetoclax. However, not all patients with t(11;14)(CCND1;IGH) respond to treatment, and some progress early after initial response. To investigate this, we examined 44 whole-genome and whole-exome sequencing data samples from 34 patients with t(11;14) MM treated with venetoclax. The presence of mutations in the RAS pathway was strongly associated with shortened progression-free survival (PFS) and was validated in an independent cohort of 21 patients with MM. The presence of 1q gain was also associated with shorter PFS in patients without RAS mutations. In 10 patients with paired prevenetoclax and postvenetoclax treatment samples, postvenetoclax progression was recurrently driven by the selection of genomic events in the BCL2/MCL1 and RAS pathways and of high-risk features (eg, loss of TP53 and CDKN2C). Overall, our study shows that comprehensive genomic profiling can identify most mechanisms underlying resistance to BCL2 inhibition in t(11;14)(CCND1;IGH) MM.

ABSTRACT Chimeric antigen receptor T-cell (CART) and T-cell engager (TCE) therapies targeting B-cell maturation antigen (BCMA) are transforming the treatment landscape for relapsed multiple myeloma (MM). However, despite impressive initial response rates, most patients eventually relapse. To investigate this unmet medical need, we applied whole-genome sequencing (WGS) to MM cells from cohorts of 102 relapsed patients treated with anti-BCMA CART and TCE therapies. Several genomic alterations were associated with clinical outcomes, particularly primary refractoriness, including high genomic complexity and mutations in genes regulating plasma cell identity, which predicted resistance to therapy. Single-cell RNA sequencing further revealed that MM cells from refractory patients exhibited high proliferation signatures and reduced expression of TNFRSF17 (encoding BCMA), while were less enriched for plasma cell–associated transcriptional programs, a phenomenon we term “plasma cell identity escape.” This profile was strongly associated with immune dysregulation of CD8 T cells including increased activation and exhaustion. This evolution of MM toward a more proliferative and lineage-divergent state, refractory to the anti-BCMA T-cell redirecting therapies, was functionally validated in preclinical MM mouse models. Collectively, our results comprehensively define the cellular and molecular mechanisms underlying primary resistance to anti-BCMA therapies.

Abstract Introduction Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS, mass fix) represents a sensitive and specific alternative to electrophoresis and immunofixation for quantifying monoclonal antibodies in patients with multiple myeloma (MM). Here we report real world data utilizing mass fix and compare its test characteristics to a gold standard of minimal residual disease (MRD) quantification utilizing next generation sequencing (NGS). Results All patients who had obtained mass fix and MRD testing by NGS as part of routine clinical care at Mayo Clinic Arizona were screened for this study. In total, 343 pairs of mass fix and NGS testing within 50 days of each other representing 269 unique patients with secretory multiple myeloma were included. Cases of IgD myeloma and those secreting only free light chains were excluded. The threshold of MRD negativity was set at <1x10-6(less than one malignant cell in one million) and was assessed utilizing the clonoSEQ NGS assay. The median age of our cohort is 67, it is 44% female, and primarily Caucasian (88%) with a median follow-up of 7.3 months from testing date. FISH testing was performed in 96% of patients and at least one high risk cytogenetic abnormality (t(4;14), t(14;16), del(17p)) was found in 23% of patients. The t(11;14) translocation was found in 18%, +1q in 26%, and amp(1q) in 6% of evaluated patients. Notably, patients at all stages of treatment or disease status were included in our study. Induction treatment for newly diagnosed MM (NDMM, 133 patients) was mostly daratumumab-containing quadruplet (63%) or triplet drug regimens (36%). Autologous stem cell transplant (ASCT) was performed in 79% of NDMM and 80% of relapsed refractory MM patients (RRMM, 138 patients). Of the RRMM patients, 94% were triple class exposed and 41% were penta-exposed. Overall agreement between NGS and mass fix results was 65% (Kappa statistic: 0.31, 95% CI: 0.22 – 0.41). Most discordant cases were instances of mass fix negativity and NGS positivity (n = 89, 26% of all testing pairs). Only 7% of cases (n= 30) represented mass fix positivity with NGS negativity. Mass fix was able to define therapeutic monoclonal antibody by its unique mass spectrometric profile in 38 of the total test pairs that otherwise would have been considered disease associated. Of the 30 patients testing mass fix positive and NGS negative, 25 (83%) represent post-treatment evaluations. Thus, ongoing mass fix positivity represents monoclonal protein washout kinetics. All of these cases are noted to have declining mass fix levels at last follow up and 40% had undetectable monoclonal protein. The discordant mass fix negative and NGS positive cases were evenly distributed across NDMM and RRMM representing 32% and 20% of test pairs in each group, respectively. Amongst these 89 total test pairs, the median abundance of malignant clone by NGS was 10 cells per million (range 0.06 to 297,895). This is in contrast to 136 concordantly positive test pairs where the median was 2960 cells per million (range 0.353 to 1,017,360). The median monoclonal protein identified (in those with a quantifiable M-spike) was 0.30 g/dL. Receiver operating characteristic curves using NGS as gold standard found the optimal mass fix threshold to be 0.010g/dL (specificity 95%, sensitivity 51%) for NDMM patients and 0.046g/dL (specificity 84%, sensitivity 55%) in RRMM patients. Conclusions Development of a MRD assay based on peripheral blood sampling could greatly improve the ease and speed of obtaining these assessments. Our experience highlights both the great potential and need to identify appropriate clinical contexts for optimal use of mass fix MRD assessment. A significant limitation of our data is that low level monoclonal proteins without a previous reference above a threshold of 20mg/dL are not reported as positive and may account for a significant fraction of discordant cases reported here. All clonoSEQ NGS samples are referenced to a positive control greatly increasing specificity. The currently reported dataset is biased against mass fix as not all test pairs reported are known to have a positive reference. Further delineation of these cases is forthcoming. Previous reports have also noted that mass fix test characteristics can improve with time from treatment which will be assessed with future follow-up.

Abstract Introduction Cila-cel is associated with a 10-15% risk of delayed neurotoxicity (DNT) with rates of cranial nerve palsies (IEC-NP) and parkinsonism (IEC-PKS) seen at a rate of 10% and 1-4% respectively and can occur as early as 3-4 weeks after infusion. We and others have shown that high post-infusion peak absolute lymphocyte count (ALCpeak) of >3x10^9/L is associated with an increased risk of delayed neurotoxicity (Lim et al., ASCO 2025) whilst the risk was negligible (~1%) when ALCpeak fell below that threshold. Since December 2024, we implemented prophylactic administration of dexamethasone (PPX DEX) when ALC was >3x10^9/L (defined as high ALCpeak). Outcome of these patients are described here. Methods Analysis of clinical outcome included 230 pts treated between Feb 2022 and Nov 2024 as a historical cohort and 102 patients treated between Dec 2024 and May 2025 after the introduction of dexamethasone prophylaxis using an oral dose of 10mg twice a day for at least three days in the high ALCpeak cohort. Results No significant differences were seen between the new and historical cohort in pre-lymphodepletion disease burden (bone marrow plasma cell %, involved light chain or M-spike level), and rates of all/high-grade CRS or ICANS, IEC-HS and rates of tocilizumub use. Median follow-up in the new cohort of 102 pts was 3.6 months (95% CI 3.3-4.2). Eleven (10.8%) developed DNT [3 (2.9%) with IEC-PKS, 8 (7.8%) with IEC-NP]. High ALCpeak of >3x10^9/L was observed in 51 (50%) pts. Among pts with high ALCpeak, DNT was observed in 10 (20%) pts [3 (5.9%) with IEC-PKS, 7 (13.7%) with IEC-NP] whereas among 51 pts with ALCpeak <3x10^9/L, DNT was observed in 1 (2%) pt [1 (2%) with IEC-NP]. In the high ALCpeak cohort, 38 (75%) received DEX [6 (12%) for toxicity management, 32 (63%) PPX DEX]. Of the 38 pts treated with DEX, DNT was observed in 6 (16%) pts (2 (5.3%) IEC-PKS, 4 (10.5%) IEC-CNP). We next compared DNT rates between this new cohort of high ALCpeak pts receiving DEX (n=38) against the historical cohort with high ALCpeak (n=98). Estimated 90-day DNT by Kaplan-Meier method was (19.2% vs 20.5%; p=0.53): IEC-PKS (5.8% vs 7.2%; p=0.60) and IEC-NP (11.7% vs 13.3%; p=0.58). In patients with high ALCpeak, the use of DEX did not appear to significantly reduce the risk of DNT compared to historical controls (HR 0.76, 0.33-1.78), IEC-PKS (HR 0.67, 0.14-3.10) or IEC-NP (0.73, 0.24-2.23). We next compared ALC expansion over the first month post CAR-T infusion (area under the curve, AUC). Among the new cohort of 102 patients, no difference was seen in the AUC of pts who received DEX but did not develop DNT vs those who developed DNT (31.0 vs 57.9, p=0.28); No difference in AUC was seen in the new high ALCpeak cohort receiving DEX vs the historical cohort with high ALCpeak (36.0 vs 43.8, p=0.15) Of the eight cases of IEC-NP, 4 received PPX DEX. No noticeable differences were observed in the severity of presentation, laterality of cranial nerve involvement and rates of multiple cranial nerve involvement between these 4 cases who received PPX DEX and the 16 cases from the historical cohort. At the time of database freeze, 3 of the 4 cases had resolved and 1 was improving. Median time to complete symptom resolution was 38 days vs 69 days in the historical cohort. Of the three cases of IEC-PKS, 2 received PPX DEX. Median time to symptom onset for IEC-PKS was 46 days (vs. 24 days in historical cohort). Both cases had severe symptoms non-responsive to first line high-dose methylprednisolone, second line intrathecal chemotherapy and third line high-dose cyclophosphamide. Both pts subsequently died from sepsis 28 and 67 days post-cyclophosphamide. Conclusion While ALCpeak >3x10^9/L predicts risk of DNT in pts treated with cilta-cel, the use of PPX DEX does not appear to significantly reduce ALC expansion and does not appear to significantly reduce the risk of DNT, IEC-PKS or IEC-NP. Other strategies to reduce ALC or alter T cell functions may need to be considered.