Kourelis, Taxiarchis

Abstract Background Extramedullary disease (EMD) in multiple myeloma (MM) represents an aggressive phenotype with poor outcomes. We performed single-cell RNA sequencing (scRNA-seq) on clonal plasma cells (CPCs) from EMD tissue, solitary plasmacytomas (SPM), and bone marrow aspirates (BMAs) from patients with MM to identify transcriptional states unique to EMD. Methods We processed FFPE tumor samples from EMD and SPMs and cryopreserved CD138+ BMAs from MM, using the 10x Genomics FLEX platform. Cells/nuclei were fixed, hybridized with Human WTA probes, pooled, and sequenced (10,000–40,000 cells/library) on NovaSeq X. After removing low quality cells, data was normalized and integrated using RPCA in Seurat. Plasma cell identity was assigned via Cellarium and canonical markers. Clonality was inferred by light chain restrictions and refined via random forest modeling. Cell cycle effects were regressed out to reduce proliferation bias. Results We analyzed 128, 908 CPCs from 16 samples [8 EMD (62, 669 CPCs), 4 SPM (20, 607 CPCs), 4 BMA (45, 632 CPCs)] and identified eight transcriptionally distinct cell clusters. Differential expression and pathway comparisons were performed between EMD/SPM/BMA within each of these clusters. Integration of gene expression and pathway analyses revealed consistent upregulation of cell cycle and E2F pathways in EMD. We identified two highly proliferative cell cycle clusters (Cluster 2 with S-phase genes and Cluster 4 with a G2/M transcriptional program) that were expanded in EMD compared to BMA and SPM. Cluster 0 comprised a quiescent CPC population enriched for RNA processing pathways alongside downregulation of ER stress and autophagy. EMD cells demonstrated suppressed interleukin signaling [fold change (FC) –33.8), and class I MHC antigen processing (FC –23.5) relative to BMA, suggestive of an immune suppressive transcriptional program. Angiogenesis-related genes [VEGFA (log2FC 2.4), ADM2 (log2FC 2.2)] and EZH2 [log2FC 2.1; q<0.0001)] were upregulated in EMD. Compared to SPM, EMD plasma cells in this cluster demonstrated marked downregulation of CD74 (log2FC –2.6; q<0.0001), a key modulator of MHC class II antigen presentation. Cluster 1 comprised of metabolically stressed secretory CPCs and displayed downregulation of early growth response pathways in EMD compared to BMA, concurrent with upregulation of E2F signaling. Suppression of IL-2/STAT5, IL-6/JAK/STAT3, and TGF-β pathways in EMD suggests reduced reliance on bone marrow–derived cytokines and impaired immunoregulatory signaling. CD44 was upregulated in EMD versus BMA (log2FC 3.2), consistent with its role in EMD spread and marrow independence. Cluster 3 was enriched for extracellular matrix–interacting plasma cells (COL1A1, MMP2) with upregulated epithelial–mesenchymal transition (EMT) pathways in EMD compared to both BMA (FC 7.5) and SPM (FC 7.07). Cluster 6 was defined by a migratory, motility-primed plasma cell population (CD44, AHNAK, LMNA and S100A10). Notably, IL32 was overexpressed in EMD compared to BMA (log2FC 5.1; adj. p = 2.02×10⁻¹⁴), a finding linked to hypoxia-driven proliferation. Compared to SPM, EMD cells also had elevated MYC/E2F activity and DNA repair, hallmarks of a proliferative phenotype. Cluster 5 displayed a strong interferon-stimulated gene signature (ISG15, IFIT1, TNFSF10), with enrichment of type I IFN, JAK–STAT, and TNF pathways. Within this inflammatory cluster, EMD exhibited marked suppression of immune signaling, metabolism (TCA, OXPHOS), and TP53 programs, consistent with an immune-evasive, metabolically suppressed, and deregulated state. Finally, within Cluster 7, a smaller subset defined by NFκB–driven anti-apoptotic transcriptional activity, EMD exhibited significant downregulation of KLF2 (log2FC –3.7; adj. p = 1.77×10⁻²⁷), likely reflecting disruption of the KDM3A–KLF2–IRF4 axis, a transcriptional network critical for bone marrow homing and plasma cell survival. We consistently observed downregulation of the MAPK pathway in EMD compared to BMA, aligning with prior bulk RNA-seq data and contrasting with the near-universal presence of MAPK mutations, highlighting a disconnect between mutational status and pathway activation. Conclusion In the largest yet reported single-cell transcriptional atlas of EMD and SPM, we demonstrate EMD to be highly proliferative with metabolic reprogramming, EMT, and downregulation of key immunologic signaling pathways, indicating immune escape and microenvironmental independence.

Abstract Background Measurable residual disease (MRD) negativity post- autologous stem cell transplantation (ASCT) in newly diagnosed multiple myeloma (MM) is associated with favorable outcomes and may overcome the adverse impact of high-risk cytogenetic abnormalities (HRCAs). However, as the definition of high risk evolves and MRD emerges as a key endpoint, understanding its prognostic value within individual HRCAs is critical for treatment guidance. Methods We retrospectively included patients with NDMM that underwent an ASCT between September 19, 2016, and October 19, 2023, and achieved MRD negativity on Day 100 post ASCT. The MRD testing was conducted using multiparameter flow cytometry with a sensitivity of 2x10. HRCAs were defined as del(17p), 1q21+ gain or amplification (1q21+), t(4;14), t(14;16), t(14;20) and del(1p) and rest were considered standard risk (SRCA). Patients were grouped by the number of HRCAs (0, 1, ≥2). An adaptation of the recently proposed International Myeloma Society (IMS)/International Myeloma Working Group (IMWG) classification for high-risk disease (TP53 mutation unavailable) was also used. Progression-free survival (PFS), measured from stem-cell infusion to progression or death, was estimated using Kaplan–Meier methodology and compared using log-rank test. Results We identified 386 patients with MRD negativity post ASCT, of which 381 had cytogenetic data available for analysis. The median follow-up for the study cohort was 47.3 months (95% CI: 43.2-50). The median age at the time of ASCT was 65.7 (25.4-79.2) years and 58.3% patients were males. A total of 49% (n=189) patients had standard-risk cytogenetics, 31.4 % (n=121) harbored 1 HRCA, 15.3% (n=59) patients had two HRCAs and 3.1% (n=12) had 3 HRCAs. The individual HRCA by their prevalence were: 1q21+ in 35.5%(n=137), del(17p) in 14.3% (n=55), t(4;14) in 10.9% (n=42), t(14;16) in 5.4% (n=21), del(1p) in 4.2% (n=16), and t(14;20) in 1.04% (n=4) of patients. The most common double-hit lesions were1q21+ gain and/or amplification co-occurring with t(4;14) (33.9%, n=20) and del(17p) with 1q21+ (18.6%, n=11). Despite universal MRD negativity at Day 100, several HRCAs remained prognostically significant for PFS. Patients with del(17p) had worse 3-year PFS (71%) than those without (81%) [hazard ratio (HR)=1.85, 95% CI 1.11–3.07, p = 0.0172], though 3-year OS did not significantly differ at data cutoff (80% vs. 92%, p=0.1176). Presence of 1q21+ was associated with an inferior 3-year PFS (73%) compared to those without [84%, HR: 1.96 (95% CI: 1.25–3.06), p=0.0026], while 3-year OS was similar (88% vs. 91%, HR:1.33 p=0.36). Isolated 1q21+ had inferior PFS with comparable OS when compared to SRCA [3-year PFS of 82% vs. 88% respectively (HR:1.97, p=0.0177)]; 3-year OS was 91% vs. 94% (HR:1.38, p=0.44)]. Among 1q21+ patients, those with co-occurring HRCAs had a 3-year PFS of 66% vs. 81% for isolated 1q21+ (HR: 1.47, p=0.21). Patients with t(4;14) had worse 3-year PFS (64%) vs. 82% in non t(4;14) [HR 2.11(95% CI: 1.21-3.67) p=0.0064], and 3-year OS of 82% compared to 91% [HR 2.14 (95% CI: 1.02–4.49), p=0.038]. Isolated t(4;14) showed a 3-year PFS of 59% vs. 87% in standard-risk (p=0.06), and 3-year OS of 90% vs. 93% [HR 3.29, 95% CI 0.95–11.33, p = 0.06]. The presence of t(14;16), t(14;20), and del(1p) did not significantly affect 3-year PFS (all p>0.1), likely due to small subsets. When patients were grouped by the number of HRCAs, PFS declined with increasing cytogenetic complexity. Compared to standard risk, the 3-year PFS was 77% for 1 HRCA vs [HR = 2.11 (95% CI: 1.29–3.46), p = 0.0027], 67% for ≥2 HRCA [HR = 2.64 (95% CI: 1.53–4.57), p = 0.0005]. Maintenance therapy was used in 98% of patients. Patients with HRCAs received a doublet maintenance compared to SRCA (p<0.0001), even in MRD negative disease post ASCT. Conclusion In patients with certain HRCA, achievement of MRD negativity post-ASCT does not overcome the adverse impact of HRCA on PFS. In our cohort, patients with del(17p), t(4;14), or 1q21+ continued to exhibit significantly shorter PFS despite achieving MRD-negative status. These findings underscore the need for ongoing risk-adapted strategies even in patients with MM who attain MRD-negative state. The OS estimates are immature at this time.

Abstract Background Primary plasma cell leukemia (pPCL) is a rare and aggressive variant of multiple myeloma (MM), characterized by the presence of more than 5% circulating plasma cells on a peripheral blood smear. Despite advancements in therapy, including novel agents and autologous stem cell transplantation (ASCT), pPCL remains associated with markedly poorer survival outcomes compared to newly diagnosed MM. Although disease progression is common in pPCL, individual courses vary widely upon relapse and thereafter. We aim to describe the natural history of pPCL, focusing on clinical features and survival outcomes at the time of relapse or disease progression. Methods Patients with pPCL who were followed at our institution's cancer center between 2006-2024 were included for retrospective analysis. The electronic health record was reviewed to provide detailed information on patient demographics, disease characteristics, treatment regimens, and clinical outcomes. Time to next treatment (TTNT) and overall survival (OS) analysis were performed using the Kaplan-Meier method. Statistical analyses were performed using the SAS biostatistical software JMP 17.0.1 (SAS Institute Inc). Results We analyzed a cohort of 106 patients with relapsed pPCL after first-line therapy, all of whom received induction with novel agents. To date, 64 patients (60%) have died. Across the entire cohort, median OS is 39 months (mos) (95% CI: 25-68). 59 patients (56%) were diagnosed after 2015. Baseline FISH cytogenetics were available for 100 patients; 57 (57%) had one or more high-risk cytogenetic abnormality. Nearly all patients (96%) had ≥50% clonal plasma cells on initial bone marrow biopsy. While most patients (n = 61; 58%) were planned to undergo upfront ASCT, 20 (33%) experienced disease progression during induction therapy and required a more intensive salvage regimen (often infusional chemotherapy) prior to ASCT. The median TTNT after first-line therapy (TTNT#1) for the entire cohort was 10.4 mos (95% CI: 8–14). The median TTNT after second-line therapy (TTNT#2) for the entire cohort was 5.7 mos (95% CI: 4.3–7.1). Clinical details at first relapse, prompting initiation of second-line therapy, were available for 91 patients. Of these, 26 patients (29%) had symptomatic relapses—due to CRAB features or extramedullary disease—while 65 patients (71%) experienced asymptomatic biochemical progression. Symptomatic relapses were followed by significantly shorter TTNT#2 (3.9 vs. 7.0 mos; p = 0.039) and inferior OS from the start of second-line therapy (7.5 vs. 31.2 mos; p <0.0001) compared to asymptomatic relapses. The median TTNT#2 for patients who relapsed during induction and received salvage ASCT was 6.9 mos (n=20). In contrast, those treated after relapse with non-ASCT regimens (n = 86) fared worse; those receiving novel agent-based combinations, including doublets, triplets, or quadruplets (n=64) had respective median TTNT#2 of 3.3, 6.0, and 3.3 mos. Patients treated with infusional chemotherapy (n = 9) had the poorest outcomes, with a median TTNT#2 of just 1.4 mos. The median TTNT diminished with each subsequent line of therapy: TTNT#3 (n = 73) was 5.8 mos, TTNT#4 (n = 53) was 4.0 mos, and TTNT#5 (n = 35) was 2.6 mos. Patients with pPCL harboring the t(11;14) cytogenetic abnormality who were treated with venetoclax-based regimens (n = 8) during any line of therapy had a longer corresponding median TTNT of 15.0 mos. Additionally, 23 patients received anti-BCMA CAR-T cell therapy as a post-relapse line of therapy. At last follow-up, 15 of these patients (65%) had not relapsed, and after a median 20.7 mos of follow-up, the median TTNT following CAR-T cell therapy had not yet been reached. Patients who received CAR-T after 1-3 prior lines (n=15) had a median TTNT that was not reached; those who had received 4 or more prior lines (n=8) had a median TTNT of 20 mos. ConclusionsDespite the use of novel agent-based combination regimens and ASCT as first-line therapy, most patients with pPCL eventually experience disease progression. Consistent with MM, symptomatic relapse in pPCL is associated with poorer outcomes compared to asymptomatic biochemical progression. Additionally, the duration of response shortens with each successive line of therapy. Notable exceptions include venetoclax-based regimens in patients with t(11;14), and anti-BCMA CAR-T cell therapies: each appear to confer more durable responses than historical treatments in patients with relapsed pPCL.

Abstract Overview: The emergence of effective immune therapies for multiple myeloma has highlighted the importance of understanding the roles of less abundant components within the tumor microenvironment. Single-cell analyses provide snapshots of the functional relationships of individual cells at precise moments in time, as well as insight into the overall cellular composition. By integrating single-cell datasets from multiple studies, we improve our ability to identify trends within rare cell populations, such as macrophages, and explore their clinical implications. We investigate human data here because our prior research of murine models indicated that macrophages expressing C1q, particularly those co-expressing Fcgr4, tend to correlate positively with increased tumor burden. Methods: We combined 56 human bone marrow samples – primarily collected at baseline, post-induction, or post-ASCT – with three publicly available data sets: GSE210079 (post CAR-T bone marrow microenvironment); GSE223060 (single-cell analysis of the MMRF cohort); and GSE271107 (disease progression from healthy donors and MGUS to SMM and NDMM). These data sets were integrated using Seurat to generate robust cellular clusters from over 300,000 cells across 117 samples. We validated our single-cell findings with bulk Affymetrix, whole bone marrow gene expression data (collected at baseline and post-baseline) from the Total Therapy clinical trials (GSE136324). Results: A distinct cluster of C1QA/B/C-expressing macrophages was consistently identified in all single-cell cohorts. These cells co-express FCER1G, CSF1R, and CD68 (markers also expressed in murine C1Q macrophages) and typically cluster adjacent to FCGR3A-expressing monocytes. Within the disease progression cohort, C1Q macrophages were found in all phases of progression: healthy donors, MGUS, SMM, and NDMM. However, C1Q macrophages in progressive disease (SMM and NDMM) have distinct expression features including lower levels of MRC1 and higher levels of APOBEC3A and ISG15 – genes linked to inflammatory responses. Among 11 post-induction samples submitted to lenalidomide maintenance, five with shorter responses displayed elevated expression of APOBEC3A and ISG15 in C1Q-expressing macrophages compared to six samples with longer responses. In a cohort of 22 samples 100 days post-ASCT, seven poor responders showed both an increased frequency of C1Q macrophages and elevated APOBEC3A expression compared to good responders. The MMRF cohort indicated that higher baseline frequencies of C1Q macrophages often preceded rapid disease progression (e.g. MMRF_1720, MMRF_1641). Anecdotal evidence from the CAR-T therapy cohort suggests that expansion of C1Q macrophages could serve as an early warning for tumor recurrence; sample 16 showed retention and expansion of C1Q macrophages 28 days post-CAR-T, that was subsequently followed by increased tumor cell counts at 3 months post-therapy. Analysis of bulk, whole bone marrow gene expression revealed that elevated levels of C1Q genes correlated with poorer outcomes, particularly in post-baseline samples. Multivariate Cox survival analysis revealed that high C1Q expression independently predicted inferior overall survival in post-baseline samples after adjusting for age, ISS stage, high levels of tumor-associated markers (SDC1, JCHAIN, DERL3), and high-risk UAMS7 subtypes (MF, MS, PR). Moreover, post-baseline GEP70 risk scores (from matched bone marrow aspirates) positively correlated with elevated C1Q expression in whole bone marrow samples. Conclusion: Both single-cell and bulk analyses indicate that the persistence of immunosuppressive C1Q macrophages following therapy is associated with inferior clinical outcomes. Although the precise mechanisms are yet to be fully understood, the close proximity of similar macrophages to tumor cells–as observed in other cancers–likely also occurs in myeloma and reflects the crucial relationship between tumor cells and macrophages within the bone marrow. While C1Q macrophages are typically rare in individual samples, analysis of combined cohorts is building evidence of their crucial role in immune cross-talk and therapeutic resistance mechanisms.

ABSTRACT Light chain multiple myeloma (LCMM) is a subtype of multiple myeloma (MM) characterized by the exclusive production of immunoglobulin light chains and accounts for 15%–20% of newly diagnosed MM. A comprehensive comparison of LCMM with MM producing intact immunoglobulin (non‐LCMM) remains limited. In this retrospective study, we described distinct clinical and cytogenetic features and assessed long‐term outcomes in 852 LCMM patients diagnosed between 01/01/2004 and 12/31/2022, compared with non‐LCMM controls matched for age, sex, and year of diagnosis. On univariable analysis, LCMM patients were more likely to present with elevated creatinine (> 2 mg/dL), beta‐2‐microglobulin ≥ 5.5 mg/L, elevated LDH, hypercalcemia, t(11;14), del(13q), and t(6;14). Conversely, IMS‐IMWG high‐risk disease, hyperdiploidy, t(4;14), and serum albumin < 3.5 g/dL were less common. On multivariable analysis, elevated creatinine (OR: 5.1, 95% CI: 1.2–27, p = 0.04), t(11;14) (OR: 2.6, 95% CI: 1.3–5.2, p = 0.006), and del(13q) (OR: 4.1, 95% CI: 2.1–8.2, p < 0.001) were independently associated with LCMM, while IMS‐IMWG high‐risk disease (OR: 0.3, 95% CI: 0.1–0.8, p = 0.02) and hyperdiploidy (OR: 0.3, 95% CI: 0.1–0.7, p = 0.002) were less frequent. Despite these differences, progression‐free survival with frontline treatment (HR: 0.97, 95% CI: 0.84–1.11, p = 0.63) and overall survival (HR: 0.99, 95% CI: 0.87–1.13, p = 0.94) were similar between LCMM and non‐LCMM patients. This study highlights the distinct clinical and cytogenetic features of LCMM, marked by higher rates of renal failure, t(11;14), and del(13q), lower prevalence of IMS‐IMWG high‐risk disease, and comparable survival outcomes.

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 Purpose: R,R-1,2 cyclohexanediamine-pyrophosphato-platinum(II) (PT-112) is a novel immunogenic cell death–inducing small molecule under phase II development in several cancer types. It inhibits ribosome biogenesis and causes organelle stresses, leading to selective immunogenic cell death in cancer cells. The possibility of PT-112’s pyrophosphate moiety driving high drug concentrations to bone sites of disease has led to an interest in PT-112’s use in multiple myeloma. In this study, we present findings from phase I and in vivo studies for PT-112 in relapsed or refractory multiple myeloma. Experimental Design: PT-112 biodistribution was analyzed in mice via laser ablation inductively coupled plasma mass spectrometry. The activity of PT-112 was assessed in de novo and transplantable Tg(Igkv3-5*-MYC)#Plbe (Vk*MYC) multiple myeloma mouse models as monotherapy or combination therapies. M-spike levels and survival were measured. A phase I dose escalation study of PT-112 monotherapy was conducted using a 3 + 3 design in patients with heavily pretreated relapsed or refractory multiple myeloma with exhausted available therapies. Results: In vivo biodistribution imaging revealed high concentrations in the bone, kidney, lung, skin, and liver. PT-112 was active in Vk*MYC multiple myeloma mouse models, both alone and in combination. Phase I data showed that PT-112 monotherapy was safe and well-tolerated, establishing a recommended phase II dose of 360 mg/m2 on days 1, 8, and 15 of a 28-day cycle. Confirmed responses and other signals of activity were observed. Conclusions: These results suggest a lack of cross-resistance with the standard of care and support the translational value of the Vk*MYC model system. Further clinical investigation of PT-112 is warranted in multiple myeloma.