Multiple Myeloma Plasma Cells Research Articles

Immune escape of multiple myeloma cells results from low miR29b and the ensuing epigenetic silencing of proteasome genes

BackgroundActivation of CD28 on multiple myeloma (MM) plasma cells, by binding to CD80 and CD86 on dendritic cells, decreases proteasome subunit expression in the tumor cells and thereby helps them evade being killed by CD8+ T cells. Understanding how CD28 activation leads to proteasome subunit downregulation is needed to design new MM therapies.MethodsThis study investigates the molecular pathway downstream of CD28 activation, using an in vitro model consisting of myeloma cell lines stimulated with anti-CD28-coated beads.ResultsWe show that CD28 engagement on U266 and RPMI 8226 cells activates the PI3K/AKT pathway, reduces miR29b expression, increases the expression of DNA methyltransferase 3B (DNMT3B, a target of miR29b), and decreases immunoproteasome subunit expression. In vitro transfection of U266 and RPMI 8226 cells with a miR29b mimic downregulates the PI3K/AKT pathway and DNMT3B expression, restores proteasome subunit levels, and promotes myeloma cell killing by bone marrow CD8+ T cells from MM patients. Freshly purified bone marrow plasma cells (CD138+) from MM patients have lower miR29b and higher DNMT3B (mRNA and protein) than do cells from patients with monoclonal gammopathy of undetermined significance. Finally, in MM patients, high DNMT3B levels associate with shorter overall survival.ConclusionsAltogether, this study describes a novel molecular pathway in MM. This pathway starts from CD28 expressed on tumor plasma cells and, through the PI3K-miR29b-DNMT3B axis, leads to epigenetic silencing of immunoproteasome subunits, allowing MM plasma cells to elude immunosurveillance. This discovery has implications for the design of innovative miR29b-based therapies for MM.

A rare case of crystalline deposits over Descemet’s membrane in a suspected case of monoclonal gammopathy of undetermined significance

Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant disorder that can give way to multiple myeloma and related plasma cell disorders. In patients with MGUS, ocular involvement is rare, although several cases of crystalline deposits in the corneal stroma have been reported which may be unilateral or bilateral.[1-4] Along with stromal deposits, corneal involvement may happen with varied presentations making imperative to be differentiated from corneal degenerations and dystrophies. We report a suspected case of MGUS presenting with uniocular involvement in the form of crystalline deposits over Descemet’s membrane, which is an infrequent manifestation of the occult systemic disorder.

Expression of the chemokine receptor CCR1 decreases sensitivity to bortezomib in multiple myeloma cell lines

BackgroundThe proteasome inhibitor bortezomib is one of the primary therapies used for the haematological malignancy multiple myeloma (MM). However, intrinsic or acquired resistance to bortezomib, via mechanisms that are not fully elucidated, is a barrier to successful treatment in many patients. Our previous studies have shown that elevated expression of the chemokine receptor CCR1 in MM plasma cells in newly diagnosed MM patients is associated with poor prognosis. Here, we hypothesised that the poor prognosis conferred by CCR1 expression is, in part, due to a CCR1-mediated decrease in MM plasma cell sensitivity to bortezomib. MethodsIn order to investigate the role of CCR1 in MM cells, CCR1 was knocked out in human myeloma cell lines OPM2 and U266 using CRISPR-Cas9. Additionally, CCR1 was overexpressed in the mouse MM cell line 5TGM1. The effect of bortezomib on CCR1 knockout or CCR1-overexpressing cells was then assessed by WST-1 assay, with or without CCL3 siRNA knockdown or addition of recombinant human CCL3. NSG mice were inoculated intratibially with OPM2-CCR1KO cells and were treated with 0.7 mg/kg bortezomib or vehicle twice per week for 3 weeks and GFP+ tumour cells in the bone marrow were quantitated by flow cytometry. The effect of CCR1 overexpression or knockout on unfolded protein response pathways was assessed using qPCR for ATF4, HSPA5, XBP1, ERN1 and CHOP and Western blot for IRE1α and p-Jnk. ResultsUsing CCR1 overexpression or CRIPSR-Cas9-mediated CCR1 knockout in MM cell lines, we found that CCR1 expression significantly decreases sensitivity to bortezomib in vitro, independent of the CCR1 ligand CCL3. In addition, CCR1 knockout rendered the human MM cell line OPM2 more sensitive to bortezomib in an intratibial MM model in NSG mice in vivo. Moreover, CCR1 expression negatively regulated the expression of the unfolded protein response receptor IRE1 and downstream target gene XBP1, suggesting this pathway may be responsible for the decreased bortezomib sensitivity of CCR1-expressing cells. ConclusionsTaken together, these studies suggest that CCR1 expression may be associated with decreased response to bortezomib in MM cell lines.

Imaging flow cytometric detection of del(17p) in bone marrow and circulating plasma cells in multiple myeloma.

Detection of del(17p) in myeloma is generally performed by fluorescence in situ hybridization (FISH) on a slide with analysis of up to 200 nuclei. The small cell sample analyzed makes this a low precision test. We report the utility of an automated FISH method, called "immuno-flowFISH", to detect plasma cells with adverse prognostic risk del(17p) in bone marrow and blood samples of patients with myeloma. Bone marrow (n = 31) and blood (n = 19) samples from 35 patients with myeloma were analyzed using immuno-flowFISH. Plasma cells were identified by CD38/CD138-immunophenotypic gating and assessed for the 17p locus and centromere of chromosome 17. Cells were acquired on an AMNIS ImageStreamX MkII imaging flow cytometer using INSPIRE software. Chromosome 17 abnormalities were identified in CD38/CD138-positive cells in bone marrow (6/31) and blood (4/19) samples when the percent plasma cell burden ranged from 0.03% to 100% of cells. Abnormalities could be identified in 14.5%-100% of plasma cells. The "immuno-flowFISH" imaging flow cytometric method could detect del(17p) in plasma cells in both bone marrow and blood samples of myeloma patients. This method was also able to detect gains and losses of chromosome 17, which are also of prognostic significance. The lowest levels of 0.009% (bone marrow) and 0.001% (blood) for chromosome 17 abnormalities was below the detection limit of current FISH method. This method offers potential as a new means of identifying these prognostically important chromosomal defects, even when only rare cells are present and for serial disease monitoring.

Serum free light chains among twin siblings: is the kappa/lambda ratio genetically determined?

Background Serum kappa, lambda, the K/λ light chain concentrations are used for screening, diagnosis, and monitoring of patients with multiple myeloma and other plasma cell disorders. Biological variation studies conducted on healthy subjects showed that free light chains have a low within and high between-individual variation. We determined if this variation were genetically linked. Methods We obtained a single serum sample from 16 pairs of identical twins, 8 neonate twins, and 19 presumed directly-related siblings children, measured Κ and λ light chains and computed the Κ/λ ratio. Results As expected, Κ/λ results from each twin neonate were near identical (reflecting maternal/placental transfer). For older children and adult twins, the Κ/λ ratio form a cluster of results that were a subset of the reference range. There was one outlier, a female with a high, different from her twin sister. She likely had a monoclonal gammopathy (no followup was possible). Excluding this pair, results from neonate twins (14.4% ±10.3%) and non-neonate twins (18.0 ± 15.3%) were not significantly different. Results between non-twin siblings were more scattered (53.2%±53.4%) and different from neonate and non-neonate twin adult and children. Conclusion We suggest that the Κ/λ free light chains may be genetically linked.

Circulating Plasma Cells in Multiple Myeloma: Clinical Implications and Prognostic Significance

Circulating Plasma Cells in Multiple Myeloma: Clinical Implications and Prognostic Significance

The Involvement of LAG-3positive Plasma Cells in the Development of Multiple Myeloma.

The Lymphocyte-Activation Protein 3 (LAG-3) inhibitory receptor is expressed on regulatory plasma cells (PCs). Micro-environmental cells that express LAG-3 were found to be increased during the progression of smoldering multiple myeloma (SMM). To assess the possible role of LAG-3 expression on regulatory PCs in patients with plasma cell dyscrasia. Purified Cluster of Differentiation 138 (CD138+) PCs from patients with premalignant conditions, active multiple myeloma (MM), and controls were analyzed for the expression of LAG-3 by flow cytometry. Autologous CD8+T cells were incubated with sorted LAG-3pos or LAG-3neg PCs for 24 h. The expression of granzyme (Grz) in CD8+T cells was assessed by flow cytometry. LAG-3 expression on PCs in active MM (newly diagnosed and relapse refractory MM) was significantly increased compared to monoclonal gammopathy of undetermined significance (MGUS)/ SMM. Grz expression was significantly decreased in CD8+T cells incubated with CD138+LAG-3pos PCs, compared to CD138+LAG-3neg PCs in patients with plasma cell dyscrasia, n = 31, p = 0.0041. LAG-3 expression on malignant PCs can be involved in the development of MM from MGUS by decreasing the expression of Grz in CD8+T cells.

Nicotinamide-Expanded Allogeneic Natural Killer Cells with CD38 Deletion, Expressing an Enhanced CD38 Chimeric Antigen Receptor, Target Multiple Myeloma Cells.

Natural killer (NK) cells are a vital component of cancer immune surveillance. They provide a rapid and potent immune response, including direct cytotoxicity and mobilization of the immune system, without the need for antigen processing and presentation. NK cells may also be better tolerated than T cell therapy approaches and are susceptible to various gene manipulations. Therefore, NK cells have become the focus of extensive translational research. Gamida Cell's nicotinamide (NAM) platform for cultured NK cells provides an opportunity to enhance the therapeutic potential of NK cells. CD38 is an ectoenzyme ubiquitously expressed on the surface of various hematologic cells, including multiple myeloma (MM). It has been selected as a lead target for numerous monoclonal therapeutic antibodies against MM. Monoclonal antibodies target CD38, resulting in the lysis of MM plasma cells through various antibody-mediated mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis, significantly improving the outcomes of patients with relapsed or refractory MM. However, this therapeutic strategy has inherent limitations, such as the anti-CD38-induced depletion of CD38-expressing NK cells, thus hindering ADCC. We have developed genetically engineered NK cells tailored to treat MM, in which CD38 was knocked-out using CRISPR-Cas9 technology and an enhanced chimeric antigen receptor (CAR) targeting CD38 was introduced using mRNA electroporation. This combined genetic approach allows for an improved cytotoxic activity directed against CD38-expressing MM cells without self-inflicted NK-cell-mediated fratricide. Preliminary results show near-complete abolition of fratricide with a 24-fold reduction in self-lysis from 19% in mock-transfected and untreated NK cells to 0.8% of self-lysis in CD38 knock-out CAR NK cells. Furthermore, we have observed significant enhancements in CD38-mediated activity in vitro, resulting in increased lysis of MM target cell lines. CD38 knock-out CAR NK cells also demonstrated significantly higher levels of NK activation markers in co-cultures with both untreated and αCD38-treated MM cell lines. These NAM-cultured NK cells with the combined genetic approach of CD38 knockout and addition of CD38 CAR represent a promising immunotherapeutic tool to target MM.

Monoclonal Gammopathy of Undetermined Cardiovascular Significance; Current Evidence and Novel Insights.

Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant condition characterized by the presence of low levels of a monoclonal protein in the serum and a low percentage of clonal plasma cells in the bone marrow. MGUS may progress to multiple myeloma or other plasma cell disorders at a rate of 1% annually. However, MGUS may also have adverse effects on the cardiovascular system independent of its malignant potential. Emerging data have shown that MGUS is associated with cardiovascular disease. The mechanisms underlying this association are not fully understood but may involve genetic abnormalities, vascular calcification, cryoglobulinemia, cold agglutinin disease, autoantibodies and the direct or indirect effects of the monoclonal protein on the vascular endothelium. Herein, we review current evidence in this field and we suggest that patients with MGUS may benefit from regular cardiovascular risk assessment to prevent severe cardiovascular complications, in parallel with close hematological follow-up to monitor potential disease progression.

Co-Expression of GPRC5D, FcRH5 and BCMA Suggests That Targeting More Than One Cell Surface Marker May be a Viable Strategy in Relapsed/Refractory Multiple Myeloma (RRMM): Biomarker Results from the Phase I Study of Forimtamig, a GPRC5DxCD3 Bispecific Antibody

Background: Forimtamig is a GPRC5DxCD3 T-cell engaging bispecific antibody that induces T-cell mediated multiple myeloma (MM) cell killing and has shown promising clinical activity in a Phase I study (NCT04557150) in patients with RRMM (Carlo-Stella et al. ASH 2022). GPRC5D is known to be overexpressed in MM plasma cells (MMPCs), but little is known about its prevalence in relation to risk status and other identified MM targets like FcRH5 and BCMA. We developed a unique exploratory multiparameter flow cytometry panel to characterize GPRC5D, FcRH5 and BCMA expression on MMPCs (Lelios et al. Cyto 2023). We present data on the prevalence and co-expression of these targets, and their association with baseline patient characteristics and response to forimtamig. Methods: Baseline bone marrow aspirate samples were collected from patients who received intravenous or subcutaneous forimtamig during dose-escalation, and were analyzed with flow cytometry. Target expression was depicted as frequency of positive MMPCs and as receptor density (molecules of equivalent soluble fluorochrome). Patients were grouped by number of prior lines of therapy, ISS score, cytogenetic risk (high risk defined as presence of one or more of the following abnormalities: del(17p), t(4;14), t(14;16), and response to forimtamig (responder [≥PR] or non-responder [<PR]). All patients provided informed consent. Results: At data cut-off (June 15, 2023), 48/163 patients were biomarker evaluable (samples passing pre-defined quality standards and containing ≥100 acquired MMPCs). GPRC5D was detected at baseline in 46/48 (96%) patients, with most MMPCs expressing the target (median 95.6%; min-max: 0-100%). GPRC5D expression was more prevalent on MMPCs than FcRH5 (median 78.6%; min-max: 3.7-100%) and BCMA (median 40.3%; min-max: 0-100%; Figure). Interestingly, GPRC5D receptor density (median 20,612; min-max: 6495-208,072) was 47-100-fold higher and more homogenous than FcRH5 (median 436; min-max: 8-3921) and BCMA (median 206; min-max: 14-6657). Clinical response to forimtamig was not correlated with GPRC5D+ MMPC frequency or GPRC5D receptor density measured by flow cytometry ( Table). Percentages of GPRC5D+, FcRH5+ and BCMA+ MMPCs and receptor densities did not significantly differ between patient groups defined by ISS score, cytogenetic risk and number of prior lines of therapy. Patients previously exposed to BCMA-targeted agents (n=7) tended to have lower BCMA expression levels. GPRC5D and FcRH5 were co-expressed on 75.4% of MMPCs (min-max: 0-100%) followed by MMPCs co-expressing GPRC5D and BCMA (median 31.9%; min-max: 0-100%) or BCMA and FcRH5 (median 25.3%; min- max: 0-94.3%). In the majority of patients, double and triple negative MMPCs were rarely observed (0.5-6% of MMPCs expressed only one or none of the three targets across all analyzed patients). Conclusions: GPRC5D, FcRH5 and BCMA are highly prevalent across MM patient subgroups, including high-risk patients who may warrant targeted therapy approaches. Based on our data and previously published work for FcRH5- and BCMA-targeted T-cell bispecifics (Sumiyoshi et al. EHA 2021; Cortes-Selva et al. ASH 2022), target expression on MMPCs, as measured by flow cytometry, has not been associated with clinical response to the corresponding CD3 bispecifics, likely due to the high efficacy of T-cell mediated tumor cell killing. Co-expression of MM targets suggests that targeting more than one MM surface marker may result in double/triple hits on individual tumor cells. Our data support further testing of dual/triple MMPC targeting as a promising concept to promote fast and deep tumor clearance, and to achieve durable responses, while potentially avoiding clonal selection, in patients with RRMM. Further investigation is needed to understand the relevance of target expression differences measured by flow cytometry compared to other methods like immunohistochemistry, as well as to understand T-cell bispecific resistance mechanisms beyond target expression. Updated data will be presented.

Multiple Myeloma B Cells and Pre-Plasma Cells Are Important Reservoirs for Myeloma Relapse Following Plasma Cell-Directed Therapy and Prevent Cure with Standard Therapies

Multiple Myeloma (MM) is a B cell neoplasm characterized by the accumulation of mature plasma cells (PCs) within the bone marrow (BM). Despite treatment advances, MM remains incurable in the vast majority of patients. Disease relapse typically occurs regardless of treatment with proteasome inhibitors (such as bortezomib and carfilzomib), immunomodulatory drugs (such as lenalidomide and pomalidomide), anti-CD38 monoclonal antibodies (such as daratumumab and isatuximab) or myeloablative melphalan and autologous stem cell transplantation, and also occurs following treatment with immunotherapeutics such as bispecific antibodies and/or CAR-T cells (targeting BCMA, FcRH5 or GPRC5D). This failure to achieve reliable cure in MM, despite the routine attainment of deep treatment responses against PCs, indicates the existence of important intra-tumor heterogeneity and the presence of rare drug-resistant MM cells with full tumorigenic capacity. To identify intra-clonal MM cell subpopulations, we examined BM samples from MM patients using a combination of FACS, single cell resolved immunoflourescence-FISH (IF-FISH), whole exome sequencing (WES), custom-capture targeted deep sequencing (CC-Seq) and single cell RNA sequencing (scRNAseq). We sought to characterize the genomic landscape and to track the dynamic interconnectedness of these intra-clonal subpopulations in patients over time. Using sequential FACS-IF-FISH studies of MM BM samples (n=140) we identified MM clone cellular subpopulations within the BM of MM patients that recapitulate the normal maturation stages between non-malignant post germinal centre B cells and mature PCs. These subpopulations include rare MM cells that resemble CD20 +CD38 -CD138 -Bcma -Irf4 - Xbp1s - B cells, CD20 -CD38 -CD138 - Bcma -Irf4 -Xbp1s - pre-plasmablasts and CD38 +CD138 -Bcma -/lowIrf4 -/+Xbp1s + pre-plasma cells, as well as predominating tumor-bulk CD38 +CD138 +Irf4 +Bcma +Xbp1s + plasma cells. Importantly, MM progenitor cells were typically found to possess all of the chromosomal translocations and copy number variations (CNV) present in MM PCs including whole chromosome ploidy changes, translocations such as t(11;14), t(4;14) and t(14;16), and secondary aberrations such as gain(1q), del(1p) and del(17p). To examine if MM progenitor cells also possess the single nucleotide variations (SNV) present within MM PCs, and are thus fully malignant, and to track MM progenitor subpopulations and their inter-relatedness within patients over time, we performed WES (n=60) plus targeted deep CC-Seq (n=210) on purified cell subpopulations from 17 patients, including 5 patients whom we sampled serially over >3 years. A median of 5 cellular subpopulations were isolated from each BM sample, enriched by factors of up to 1000-fold or more, and each subpopulation was then sequenced to a depth of 4,000-20,000x. Significantly, SNVs that were present in tumor-bulk MM PCs were also regularly detected in MM progenitor cells, although the low frequency of the clonal progenitor cells often prevented capture of complete SNV profiles. At the same time, analyses of serial BM samples from repeat donor patients taken pre- and post-treatment demonstrated that relapsing MM PCs typically did not appear to derive linearly from pre-treatment PCs, as the relapsing PCs commonly lacked a number of SNVs that had been present in pre-treatment PCs. Importantly, we instead observed that relapsed MM plasma cells appeared to consistently derive from MM progenitor cell populations, frequently B cells, as emergent relapse-specific SNVs that were exclusively detected in relapsing post-treatment PCs (but in not pre-treatment PCs) could often also be detected within pre-treatment CD138- CD38- progenitor cells isolated from BM samples that had been collected up to 3 years prior to relapse. We conclude that MM B cells and pre-plasma cell progenitors possess all of the clonal genomic aberrations required for full malignant potential. Tracking of these cells in vivo in patients over time by their SNV profile implicates them as the cellular origin of PC relapse and recurrent MM disease. We propose that cure of MM requires eradication of MM progenitor cells alongside plasma cells. We are currently conducting scRNA-seq and CITE-seq studies of primary MM samples to further characterize MM progenitor cells for gene and protein expression in order to define their optimal therapeutic targets.

A CD38/CD28xCD3 Trispecific T-Cell Engager (TCE) As a Potentially Active Agent in Multiple Myeloma Patients Relapsed and/or Refractory (RRMM) to Anti-CD38 Monoclonal Antibodies (mAbs)

Background: Despite great advances in the treatment of RRMM, including anti-CD38 mAbs, patients become resistant and therapeutic options are limited. Some factors involved in resistance to anti-CD38 mAbs have been described. On the one hand, their efficacy is partially dependent on baseline CD38 expression in MM plasma cells (MMPC), but the treatment induces a rapid and sustained CD38 downregulation. On the other hand, their tumor-killing activity relies on NK cells, but its frequency rapidly decreases following initiation of treatment, and persistent NK cells exhibit a dysfunctional phenotype. Given the reduction of CD38 levels in MMPC and the impaired NK-cell activity in resistance to anti-CD38 mAbs, we hypothesized that RRMM patients previously treated with these agents could benefit from T-cell-based immunotherapeutic strategies that depend less on CD38 antigen density and NK cell function. Aim: Evaluate a CD38/CD28xCD3 trispecific TCE (SAR442257) as a potential therapeutic agent in the RRMM setting. Results: CD38 expression in MMPC and NK-cell frequency in bone marrow were significantly reduced in 29 RRMM patients previously exposed to anti-CD38 mAbs vs 45 newly-diagnosed MM (NDMM) patients ( P <.001 and P =.013, respectively). Moreover, the percentage of CD57+ NK cells was significantly lower in RRMM than NDMM patients ( P =.006), suggesting that both CD38 downregulation and a contraction of cytotoxic NK cells may be associated with resistance to anti-CD38 mAbs. By contrast, T cells did not change significantly between stages, which ignited further interest in the preclinical efficacy of the CD38/CD28xCD3 TCE. First, we observed that, contrary to isatuximab and daratumumab, CD38/CD28xCD3 TCE did not reduce surface CD38 levels in the MOLP 8, RPMI 8226 and KMS 12 BM cell lines. Fluorescence microscopy imaging revealed that CD38 was present in cell membrane up to one week of treatment with CD38/CD28xCD3 TCE, while isatuximab induced an internalization of CD38 at 24h. Next, in a co-culture of the OPM-2 cell line with purified T or NK cells from healthy individuals (E:T ratio 2:1), we confirmed that the activity of CD38/CD28xCD3 TCE relied on T but not NK cells. Namely, no direct NK-cell stimulation was found whereas a significant T-cell activation was observed upon CD38/CD28xCD3 TCE treatment. Surprisingly, when OPM-2 were co-cultured with freshly-isolated peripheral blood mononuclear cells (PBMCs, E:T ratio 10:1), CD38/CD28xCD3 TCE induced activation of T- and NK-cells, suggesting a bystander NK-cell activation. This dual stimulation resulted in a significant reduction of OPM-2 cell viability at 48h. Interestingly, we found a comparable tumor-killing activity and T-cell activation in the presence of the CD38 null trispecific isotype (CD3xCD28 ΔCD38) compared with CD38/CD28xCD3 TCE, suggesting that the latter targets CD28 not only in T-cells, but also in OPM-2 cells, which express high CD28 basal levels. Altogether, these data demonstrate the potential dual CD38/CD28 targeting in RRMM patients previously exposed to anti-CD38 mAbs, particularly in those with double positive MMPC. Additionally, CD38 regulation was analyzed in the co-culture system (measured with a non-competing anti-CD38 antibody), and an unaltered and increased CD38 expression was observed in OPM-2 and T-cells, respectively, indicating again T-cell stimulation. We tested the ex vivo efficacy of CD38/CD28xCD3 TCE (48h, 700 pM) in primary bone marrow samples from eight RRMM patients, which were collected after a median of 10 months since the last anti-CD38 treatment (range, 0.5 - 37). T-cell activation and MMPC lysis were found in six and four samples, respectively, which was accompanied by a reduction in the frequency of CD4 T cells (from 100% to 76%; P =.014). A positive and borderline-significant correlation (r = 0.764, P =.057) was found between the response to CD38/CD28xCD3 TCE and the time since last anti-CD38 treatment, and no differences in CD38 expression were observed between CD38/CD28xCD3 TCE responders and non-responders. Conclusions: We observed a reduction in CD38 levels in MMPC and an impaired cytotoxic activity of NK cells in RRMM patients previously exposed to anti-CD38 mAbs. We propose a second targeting of CD38 using T-cell-based immunotherapeutic agents whose efficacy depends less on CD38 antigen density, especially after longer washout periods, as a potential therapeutic strategy in the RRMM setting.

Targeted Inhibition of Myeloperoxidase (MPO): A New Therapeutic Strategy for the Treatment of Multiple Myeloma

Despite major improvements in therapeutic strategies for patients with multiple myeloma (MM), effective treatment still remains a persistent challenge, as patients ultimately relapse and succumb to the disease. In the last decade, studies have highlighted the reciprocal interaction between MM plasma cells (PC) and the bone marrow (BM) microenvironment in regulating immune evasion, disease progression and persistence. As MM PC rely on BM stromal cells and their secreted factors for their survival and growth, the therapeutic targeting of the BM microenvironment may prove to be a novel and successful strategy for myeloma care in the future. Myeloid-derived suppressor cells (MDSC), a heterogenous population of myeloid cells are described to promote MM progression through immunosuppression and induction of angiogenesis. Myeloperoxidase (MPO), a key inflammatory enzyme important in host defence, is reported to be the most highly upregulated gene in MDSCs in murine cancer models. Recently, the accumulation of MPO within the tumour microenvironment has attracted much attention with a number of studies describing a role for MPO in regulating cancer development due to its potent pro-oxidative and proinflammatory properties. Our most recent findings have revealed new functional roles for myeloid-derived MPO in the BM microenvironment of MM. Specifically, we demonstrate that myeloid cell populations are increased within the BM of 5TGM1 tumour-bearing mice and that MM PC may directly influence Mpo gene expression in BM-derived myeloid cells. Mechanistically, we report that MPO has the capacity to induce the expression of key MM growth factors, and exerts potent immune suppression by inhibiting anti-tumour T-cell responses. Remarkably, in the syngeneic KaLwRij/5TGM1 mouse model of MM, targeted inhibition of MPO with the suicide substrate 4-Aminobenzoic acid hydrazide (4-ABAH) demonstrated a significant reduction in overall MM tumour burden. Here, we investigate for the first time the efficacy of an orally bioavailable irreversible small molecule inhibitor of MPO (MPOi) in the preclinical Vk*MYC murine model of myeloma. Twelve-week-old C57BL/6J mice were intravenously inoculated with Vk*MYC (Vk14451-GFP) cells and tumour progression was monitored by serum paraprotein electrophoresis (SPEP), whilst endpoint GFP+ tumour cells in the bone marrow were quantitated by flow cytometry. To characterise myeloid cell populations and associated Mpo expression in the Vk*MYC tumour landscape, we utilised flow cytometry to quantitate CD11b+ cells and used magnetic activated cell separation to isolate these populations and characterise the expression of Mpo by qPCR. Our studies confirm that CD11b+ myeloid cells are significantly increased in the BM of Vk*MYC tumour-bearing mice (p<0.01), accompanied by an upregulation of MPO mRNA expression (p<0.001). To assess the efficacy of MPOi in MM, mice were treated with MPOi or vehicle alone twice daily by oral gavage, with treatment initiated at the time of Vk*MYC inoculation. Notably, mice receiving MPOi presented with significantly reduced endpoint tumour burden (9 weeks post tumour cell inoculation). We observed a 15.2% and 31.4% reduction in MM tumour as determined by hind limb GFP% (p<0.05) and SPEP (p<0.01) respectively, compared to vehicle control. Additionally, mRNA analysis of complete BM revealed an upregulation of the critical cytokine IFN gamma (p<0.05) and the downregulation of the potent proangiogenic factor VEGF (p<0.05) in mice treated with MPOi, suggesting MPO inhibition in MM may be an advantageous means to regulate the BM microenvironment and impede disease progression. However, when MPOi treatment was initiated at first signs of detectable disease, (as identified by the presence of a monoclonal spike by SPEP; 5 weeks post tumour cell inoculation), no difference in endpoint tumour burden was observed. This suggests that targeted inhibition of MPO using MPOi may be more effective in at early stages of MM development. In conclusion, the findings presented in this study indicate that inhibiting MPO activity with MPOi, as a single agent therapy, attenuates MM tumour growth in the Vk*Myc mouse model. With limited therapies used in the clinic that target the stromal microenvironment, these findings suggest MPOi could be investigated as a potential novel treatment option that may be included in combination with current frontline therapeutic agents.

Lbl-034, a Highly Differentiated T-Cell Engaging Bispecific Antibody Targeting GPRC5D for the Treatment of Relapsed or Refractory Multiple Myeloma

G protein-coupled receptor, class C group 5 member D (GPRC5D) is a type-C 7-pass transmembrane receptor protein that selectively overexpresses on malignant plasma cells in multiple myeloma (MM). The expression of GPRC5D is rarely seen in normal tissues or cells, and is not detectable on other hematopoietic derived cells, therefore indicating that GPRC5D may be a novel target for anti-multiple myeloma therapy. LBL-034, a novel T-cell engaging bispecific antibody targeting GPRC5D, is developed with a specifically designed molecular format and unique 2:1 structure. It showed a potent binding on cell lines with various levels of GPRC5D expression, while its binding to CD3 on Jurkat cells was much lower examined by flow cytometry. Furthermore, LBL-034 re-directed engagement of GPRC5D+ cells and T cells was validated by gating engaged cell population with flow cytometry and confirmed visually with laser scanning confocal microscopy. LBL-034 induced potent T cell dependent cell killing (TDCC) of various types of GPRC5D expressing cells (high to low expression: MM.1R, NCI-H929, MOLP-8 and RPMI 8226 cells), and elevated activation marker (CD25, CD69) expression and cytokine release (IFN-γ, TNF-α, IL-6). LBL-034 demonstrated weak binding to CD3 and minimal effects on cytokine release in the absence of GPRC5D expressing cells and it indicates that LBL-034 activates T cells only in the presence of GPRC5D target cells. LBL-034 was further examined in the MC38-GPRC5D syngeneic model and NCI-H929 xenograft model. Robust antitumor efficacy was observed at the low dose of LBL-034 at 1 mg/kg and 0.3mg/kg respectively. LBL-034 GLP toxicology study with repeated doses was conducted in cynomolgus monkeys. 5, 15 and 50 mg/kg of LBL-034 was given intravenously with Q1W for up to 5 doses. LBL-034 was well tolerated with a good safety profile as determined by pharmacology, pathology and biochemistry analysis. The NOAEL of LBL-034 in cynomolgus monkeys was confirmed at 50 mg/kg. In summary, LBL-034 is a novel T cell engaging bispecific antibody targeting GPRC5D expressing R/R MM with well differentiated binding of GPRC5D and CD3 to enhance anti-tumor activity, while mitigate the risk of CD3-induced CRS, showing a great in vitro potency and in vivo anti-tumor efficacy. LBL-034 IND has been approved by both FDA and NMPA and a FIH study in patients with R/R MM will begin in the 2 nd half of 2023.

CD70/CD27 Signaling Promotes Expansion of Clonal Plasma Cells in Multiple Myeloma and Is a Promising Therapeutic Target in Advanced Disease

Introduction: Multiple Myeloma (MM) is a hematological malignancy characterized by the clonal expansion of malignant plasma cells within the bone marrow (BM). In the last decade, substantial advances in the treatment of MM patients have been made resulting in improved progression-free and overall survival rates. However, to date, MM remains incurable. CD70 and CD27 belong to the TNF receptor family. CD70 is the only known ligand for its receptor CD27. Physiologically, the CD70/CD27 interaction is involved in the regulation of immune cell expansion. However, numerous solid and hematological cancer types overexpress CD70. Aside, CD70 and CD27 co-expression has been described on leukemic stem cells (LSC) in acute myeloid leukemia patients where an autonomous signaling activation drives LSC self-renewal and expansion. While CD70/CD27 signaling plays a key role in the differentiation process of B lymphocytes into plasma cells, only little is known about its impact on myelomagenesis and MM progression. In this study, we analyzed the role of CD70/CD27 signaling in the pathogenesis of MM and studied the possibility to target CD70 by monoclonal antibodies to treat the disease. Materials and Methods: We evaluated CD70 and CD27 gene expression levels and its correlations with overall survival (OS), MM progression and high-risk molecular alterations using different databases (CoMMpass IA15, GSE6477). CD70 and CD27 protein expression patterns were analyzed in a newly established MM tissue microarray (TMA) and a collection of matched BM samples from initial and advanced disease stages (in collaboration with the Institutes of Pathology in Bern, Basel and Tübingen). To assess the functional role of CD70/CD27 signaling on MM initiation and expansion, we generated CD70 knock-out MM cell lines and newly established patient-derived xenografts (PDX) using CRISPR/Cas9 genome editing. CD70-expressing MM cell lines or patient-derived myeloma cells were grown in NOD/SCID (NSG) mice. Moreover, different monoclonal antibodies (Abs) and Ab-derivates were tested including a CD70 blocking antibody (αCD70) and an antibody-dependent cell-mediated cytotoxicity (ADCC) optimized antibody (cusatuzumab, ArgenX) that activates FcγR-expressing effector cells to assess the therapeutic potential of targeting CD70 in MM. Results: High CD70 gene expression levels correlate with increased MKI67 gene expression and elevated lactate dehydrogenase serum levels indicating a more rapid proliferation of CD70-expressing myeloma cells. In addition, high CD70 expression in MM patients correlated with shorter OS. Longitudinal analyses of CD70 gene expression in the precursor stages monoclonal gammopathy of undetermined significance and smoldering myeloma as well as MM and relapsed MM stages showed highest CD70 gene expression levels in relapsed MM patients. Assessment of the molecular background further revealed increased frequencies of the prognostically unfavorable genomic translocations t(4;14) and t(14;16), as well as gain/amplification of the chromosomal region 1q21 (1q+) in CD70 high expressing MM patients. In line with our previous findings, a TMA analysis of over 200 MM core biopsies as well as a longitudinal analysis of matched biopsies from patients at initial diagnosis and MM relapse indicated that CD70 was overexpressed in advanced disease. Functionally, we found that CD70/CD27 signaling promotes plasma cell survival inducing the expansion of MM cell lines and primary malignant plasma cells. CD70 blockade (αCD70) or CD70 knock-out (CD70 KO) resulted in a significant inhibition or complete abrogation of growth of a CD70-expressing MM patient-derived xenograft (PDX) (EMM01 CD70+) compared to controls (Figure 1). Interestingly, in vivo limiting dilution assays revealed that CD70+CD27+ double-positive myeloma subpopulations showed significantly higher cancer stem cell frequencies compared to CD70 negative fractions suggesting that CD70 expression might be related to an immature or stem-cell like phenotype. The ADCC-optimized CD70 specific antibody custatuzumab efficiently eliminated CD70-expressing MM cells in the presence of PBMC-derived natural killer (NK) cells in vitro and in a PDX model in vivo. Conclusion: Our results indicate that CD70-expressing MM cells have a stem-cell like phenotype and propagate the disease. Targeting CD70 is a promising new therapy especially in advanced disease.

Unveiling Novel Therapeutic Targets for CAR Therapy in Multiple Myeloma through Single-Cell RNA Sequencing

Multiple Myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of plasma cells. Among the different therapeutic options in this setting, chimeric antigen receptor (CAR) T cells recently showed unprecedent achievements in term of progression free survival. However, despite advancements in current therapies, the disease's heterogeneity remains a major challenge and all patients unavoidably relapse, rendering innovative approaches to identify precise therapeutic targets eagerly awaited. Along this line, the revolution of single-cell (sc) technologies brings new opportunities to identify precise therapeutic targets, including abundant and unique surface proteins for CAR therapy. In this study, by analyzing individual cancer cells at sc level, we aim to identify novel surface targets, facilitating the development of novel personalized cellular therapies as well as treatment sequences. To this end, we used samples collected from 9 publicly available single-cell RNA sequencing (scRNA-seq) datasets (GEO accession numbers: GSE176131, GSE189460, GSE223060, GSE210079, GSE145977, GSE124310, GSE161801, GSE163278, GSE161722) obtained from 156 patients affected by monoclonal gammopathies (19 monoclonal gammopathy of undetermined significance (MGUS), 10 smoldering multiple myeloma (SMM), 78 MM and 25 relapsed/refractory multiple myeloma (RRMM)) and 24 normal bone marrows (NBM), to conduct an unbiased search for genes showing specific expression in plasma cells (PCs), regardless of disease status. We found 15 genes showing differential expression in PCs (adj.pval<0.01 and logFC>1) and coding for surface proteins: TNFRSF17(BCMA), SDC1 (CD138), FCRL5 (GPRC5D), TNFRSF13B (TACI), CD38, SLAMF7 (CS1), CD59, FCGR2B, FGFR3, SLC44A1 (CTL1), CD320, FCRL2, IL15RA, INSR and SLAMF1. As expected, most of these molecules already represent critical therapeutic targets. After completing our initial analysis, we compared PCs transcriptomes from MM/RRMM patients with NBM, MGUS, and SMM samples. Among previously identified genes, CD320 only was significantly overexpressed in MM PCs. Next, we investigated associations between cytogenetic-related cell subclusters and previously identified surface markers. Interestingly, we found a significant association between the expression levels of TACI/CD59 and the overexpression of CCND2 and MAF, both associated with the presence of t(14:16). Next, we decided to focus on the first five genes (according to expression ranking) that do not currently have an active clinical development program in advanced phases, namely: TNFRSF13B, CD59, FCGR2B, SLC44A1, and CD320 (Fig. 1 A, values dichotomized based on their median) to uncover their (potential) impact/association with patient's outcome. In our analysis of different available expression profiling datasets (GSE4204, GSE2658, GSE57317, GSE4581, GSE4452, GSE9782, the CoMMpass study NCT01454297) involving around 2000 MM patients, we observed TNFRSF13B, CD59, and FCGR2B expression correlated with improved outcome while CD320 and SLC44A1 expression associated with worse outcome (Fig. 1 B). In conclusion, we integrated results from both scRNAseq and bulk-RNAseq/microarray data to identify new targets genes to be used in the context of personalized medicine. Interestingly, CD320 emerges as a potential candidate biomarker useful for monitoring disease evolution and predicting worse outcomes. Additionally, this approach led us to identify a series of potential new targets for MM patients, depicting a new scenario where each patient could be “screened” to identify the best molecule to be targeted. While further investigation is necessary to assess off-target toxicity and confirm clinical relevance, our analyses significantly streamline the search for tumor markers with a method potentially applicable to different malignancies, bringing us closer to identifying the best candidates for effective CAR therapy.

Targeting Mutant KRAS with Novel Mutant-Specific Pharmacological Inhibitors: Activity Against Myeloma and Other Lymphoid Malignant Cells and Molecular Mechanisms of Resistance

Oncogenic RAS mutations are frequent genomic drivers in hematologic neoplasias such as multiple myeloma (MM) but are traditionally considered non-“druggable”. Novel specific inhibitors of G12C and G12D KRAS mutants are clinically active in solid tumors, but their impact on KRAS-mutant lymphoid malignancies has not been explored. Here, we examined the activity of these inhibitors against G12C/D KRAS-mutant cells from MM, B-ALL and T-ALL; the functional sequelae of pharmacological KRAS inhibition; and the potential mechanisms of resistance to these agents. We assessed the activity of novel G12C- (MRTX1257) and G12D- (MRTX1133) specific pharmacological KRAS inhibitors against 2 MM lines with G12C (KHM1B, XG7) and 2 with G12D (KARPAS620, KP6) KRAS mutations; as well as the G12D mutant KOPN-8 (B-ALL) and CCRF-CEM (T-ALL) lines. KRAS inhibitors were tested at concentration ranges achievable in patients with solid tumors. Potent and mutant-specific activity was achieved with both of these KRAS inhibitors in their respective lines, with IC50 in the range of 5-150 nM after 3-7d treatment. This level of in vitro activity was similar to KRAS-mutant lines (e.g., in our in vitro studies and also in the literature) from solid tumors that are clinically responsive to these agents. Furthermore, the KRAS inhibitors effectively abrogated downstream MAPK and AKT pathway activation (e.g. phosphorylation of ERK or AKT by immunoblotting). MRTX1133 led to potent induction of cell death of KARPAS620, KP6 and KOPN8 cells (flow cytometry for Annexin V/PI staining) while MRTX1257 induced cell death in KHM1B and had a predominantly cytostatic effect on XG7 cells. Combinations of MRTX1257 with clinically established anti-MM drugs (incl. melphalan, bortezomib, pomalidomide, trametinib) caused in some cases supra-additive effects, but - more importantly - showed no antagonism with any combination. RNA sequencing revealed that KRAS inhibitor treatment downregulated RAS effector genes (e.g., ETV4, ETV5), and negative regulators of RAS signaling (e.g., DUSP6, SPRY4 reflecting a RAS-driven negative feedback loop controlling expression of these genes), and molecules involved in cell cycle regulation (e.g., D-type cyclin), anti-apoptosis, and DNA replication/repair. Conversely, KRAS inhibition upregulated genes involved in cell cycle arrest and stress responses. Importantly, we performed genome-scale CRISPR activation studies in 4 MM lines with G12C/G12D mutant KRAS to identify genes whose gain-of-function (GOF) enhances or suppresses MM cell response to the respective KRAS inhibitors. Top “hits” from these screens were validated with individual sgRNAs. CRISPR activation of mutant KRAS itself promotes MM cell escape from treatment, likely due to stoichiometric reasons and decreased target saturation at a given drug concentration. Notably, CRISPR activation of positive regulators of RAS-MAPK signaling (e.g., SHOC2), and upstream surface receptors (e.g., EGFR) were among top genes mediating KRAS inhibitor resistance. Beyond KRAS itself, these top “hit” genes are typically not recurrently mutated, amplified, differentially expressed (e.g., compared with normal plasma cells) in MM and are not associated with “high-risk” clinical behavior. Some (e.g., EGFR) of the genes identified from these CRISPR studies are not typically expressed in MM cells at diagnosis, thus these results outline how ectopic expression (e.g., EGFR) or overexpression of these “hit” genes could represent candidate biomarkers for decreased response or early relapse in future clinical studies of KRAS inhibitors in KRAS-mutant MM. Ongoing studies are addressing how these genomic perturbations may affect the in vivo response of KRAS-mutant cells to KRAS inhibitors alone or in combinations with established therapies. Our study documents that novel KRAS G12C and G12D inhibitors exhibit potent and mutant-specific activity against MM and other lymphoid malignant cells at dose levels that are clinically achievable in solid tumor patients. This study also provides functional evidence about genes and molecular pathways that regulate MM cell sensitivity vs. resistance to KRAS inhibitors. These results create a framework for ongoing and future efforts to translate the use of KRAS inhibitors into clinical studies for MM and other lymphoid malignancies.

Single Cell Multi-Omic Analysis of Neoplastic Plasma Cells across the Disease Spectrum Identifies Novel Pathobiologic Mediators and Potential Therapeutic Targets in Multiple Myeloma (MM)

Background: A challenge to developing a full understanding of MM pathobiology using bulk profiling techniques is the large degree of intra-tumoral heterogeneity from minor subclones that, despite their low representation, nonetheless contribute to therapy resistance and disease progression. Moreover, our current therapies tend to be targeted against pathways common to both neoplastic and normal plasma cells (PCs), indicating a need to define novel tumor biology modifiers that could potentially serve to improve risk stratification and as therapeutic targets. Methods: To overcome these limitations, we simultaneously evaluated individual PC single-cell transcriptome and B-cell receptor variable (VDJ) sequences from freshly enriched bone marrow biopsies. These included 33 patients with precursor conditions, 17 each with newly diagnosed symptomatic or relapsed/refractory disease (RRMM), and 3 controls using the 10x Genomics platform. A total of ~320,000 PCs were analyzed, yielding median per sample cell counts of 4,626 and ~300 million total reads. Identical immunoglobulin VDJ sequences defined monoclonal PCs, while diverse VDJ sequences at low frequencies defined polyclonal cells, allowing each patient to serve as their own control. Results: The percentage of monoclonal PCs in active MM was >84%, while precursor stages were more variable (40-100% in smoldering MM; 0.3-82% in monoclonal gammopathy of undetermined significance (MGUS)). Though we could not identify monoclonal PCs in two MGUS cases, we identified them by scRNA-seq in four MGUS patients whose bone marrow showed no clonal cells by standard clinical multiparametric flow cytometry. In differential gene expression analysis where samples were grouped based on the diagnosis, 501 dysregulated genes were identified in monoclonal PCs from symptomatic MM compared to polyclonal PCs. These included genes previously identified as disease modifiers, including Hepatocyte growth factor, Lactate dehydrogenase A, Dickkopf WNT signaling pathway inhibitor 1, CD27, and Lysosomal associated membrane protein family member 5, providing validation for our approach. Next, we identified 279 genes that were commonly dysregulated in monoclonal PCs from symptomatic vs. asymptomatic MM. We also performed unsupervised hierarchical clustering of the top 1000 most variable genes and defined three diagnostic categories. Significant ontology terms characterizing these categories broadly involved antigen processing and recognition in Group 1 (enriched in precursor stages); transcription and translation control in Group 2 (enriched in active MM); and cell division and DNA metabolism in Group 3 (enriched with RRMM) ( A). We focused on dysregulated targets that: showed differential expression in monoclonal vs. polyclonal PCs; expression in most patients within a diagnostic category; a correlation with outcomes in the CoMMpassstudy; and novelty in MM. Among these were Mitotic arrest deficient 2 like 1 (MAD2L1), a component of the mitotic spindle assembly checkpoint, and Methionine adenosyltransferase 2A (MAT2A), which catalyzes the production of S-adenosylmethionine, a key methyl donor in multiple cellular processes, from methionine and ATP. Notably, MAD2L1 inhibition with M2I1, and MAT2A targeting with the allosteric inhibitor AG-270, both produced a dose- and time-dependent inhibition in cell proliferation and reduction in myeloma cell line viability. Importantly, the effects of AG-270 ( B) were associated with induction of markers of apoptosis, including Caspase cleavage, and a reduction of Histone H3 dimethylation. Moreover, they occurred at clinically relevant concentrations based on the known pharmacokinetics of this drug in Phase I testing. Conclusions: Our combined single-cell transcriptomics and VDJ sequencing allowed a greater understanding of heterogeneity among neoplastic PCs compared to each patient's own polyclonal PCs. These analyses can identify putative novel mediators of disease pathobiology that impact prognosis, and that can serve as potential new therapeutic targets using either novel agents, or drug repurposed from other therapeutic areas, setting the stage for their translation to the clinic.

Prevalence, Incidence, and Characterization of LIGHT Chain Amyloidosis in the USA: A Real-World Analysis Utilizing Electronic Health Records (EHR)

Background: Light chain (AL) amyloidosis is a rare disease characterized by the deposition of misfolded immunoglobulin light chains, resulting in organ dysfunction and significant morbidity and mortality. However, population-based studies on the epidemiology of AL are limited. This study aimed to provide an up-to-date assessment of the prevalence and incidence of AL, examine temporal changes in prevalence over a 5-year period, and characterize the AL population in the USA. Methods: A retrospective analysis was conducted using de-identified patient records from Optum's Clinical Electronic Health Record (EHR) database (>140,000 providers and >60 million unique patients). Adults with AL were identified based on ≥2 diagnosis codes (ICD-10-CM code E85.81) or positive mentions about the disease (including clinical features indicative of AL) found in the physician's notes (≥30 days apart). Patients with clinical data at least 12 months pre- index event (defined as the date of the first AL amyloidosis diagnosis or positive mention during the identification period: January 01, 2017 - December 31, 2021) were included in the analysis. Prevalence estimates were calculated for each year between 2017 and 2021, and annual percentage changes were assessed to determine temporal trends. Annual incidence was calculated as the number of newly diagnosed AL cases occurring each year between 2019 and 2021. Additionally, patient demographics, comorbidities, and Mayo 2004 staging with European modification (based on availability of the cardiac biomarkers: NT-proBNP and troponin) were characterized at index date or in the 12-month post index period. Results: A total of 1471 AL patients were included in the study population. The mean age of the prevalent patients was 67.2 years, with 54.5% being male. Most patients were White (78.5%), followed by Black (14.5%), Hispanic (3.3%), Asian (1.2%), and other/unknown (2.6%). The diagnosed prevalence of AL substantially increased from 22.7 to 69.1 cases per million between 2017 and 2021, with an annual growth rate of 32.1%. In 2021, the annual incidence of AL was estimated to be 16.7 patients per million in the general population, with an annual growth rate of 7.7% since 2019, at which time incidence was 14.4 patients per million. The distribution of patients by Mayo stage at diagnosis among those with available biomarker information (n=613, 42% of total sample) was: Stage I, 27%; Stage II, 42%; Stage IIIa, 16%; and Stage IIIb, 15%. The most prevalent comorbidities reported in the year after diagnosis were hypertension (56.6%), dyslipidaemia (51.7%), chronic kidney disease (41.3%), multiple myeloma and malignant plasma cell neoplasms (37.5%), and heart failure (34.5%). Cardiomyopathy was observed in 40.2% of the AL patients. Conclusions: This study provides an updated report of the prevalence and incidence of AL using a large-scale EHR database. The observed rise in AL prevalence and incidence is likely due to a combination of increased disease awareness and improvements in detection and management. The number of comorbidities, especially those related to cardiac function, suggest a continuing unmet need for more therapeutic options. Furthermore, the Mayo stage distribution of patients highlights the need for enhanced efforts to diagnose AL earlier due to the high mortality associated with advanced Mayo stages. These epidemiological findings reinforce the importance of continuous monitoring of prevalence, incidence trends, and the burden of AL in the US.

Frailty Syndrome in Adults Undergoing Autologous Hematopoietic Cell Transplantation. Prospective Study on Behalf of the Grupo Español De Trasplante Hematopoyético y Terapia Celular

INTRODUCTION During the past two years, sixteen institutions members of the Grupo Español de Trasplante Hematopoyético y Terapia Celular (GETH-TC) have undertaken a multicenter and prospective study with the purpose of evaluating the frailty of adults' candidates to autologous HCT (auto-HCT) and of investigating the effect of frailty in transplant outcomes. METHODS All patients consulted for auto-HCT were eligible to be included in the study after providing informed consent. Frailty was evaluated at first consultation, at admission, and after the stem cell infusion, using the HCT Frailty Scale (Salas et al. BMT 2023), designed classify candidates for allogeneic HCT into fit, pre-frail and frail categories ( Table 1). The frailty assessment was made by the hematologists and nurses team as part of the clinical practice and utilizing existing resources. The median time to complete the evaluation ranged from 8 to 10 minutes. As an extension, this study includes the evaluation of patient´s quality of life (QoL) using the EQ-5D-EL questionnaire. QoL was measured at HCT admission and day +100 after the stem cell infusion. This study has no external funding. The results obtained from the frailty assessment were not used to determine HCT eligibility and/or to design the HCT process. RESULTS This study includes 380 consecutive adult candidates for auto-HCT who have been evaluated for frailty in the sixteen participating institutions between February 2021 and May 2023. Overall, the median age was 58 (range, 18-76) and 219 (57.6%) were males. Multiple myeloma or other plasma cell discrasias (PCD) (n=213, 62.1%) and lymphoproliferative disorders (SLP) (n=121, 31.8%) were the most prevalent baseline diagnosis. Prior to auto-HCT, 104 (33.8%) out of 307 adults had an KPS<90% and 35 (12.4%) out of 282 an HCT-CI>3. All patents included underwent their auto-HCT during the study period, and in only 20 (5.3%) of the cases, the standard doses of the conditioning regimen were required to be adjusted secondary to the patient´s condition, age or comorbidities. The first consultation was mostly performed before the stem cell collection. At first consultation, 108 (28.4%) adults were classified as fit, 203 (53.4%) as pre-frail, and 69 (18.2%) as frail. Frail patients were more likely to be older than 60 (OR 5.3, p<0.001), to have a KPS<90% (HR 5.80, p<0.01), and an abnormal Mini-Cog test (<3) (OR 5.81, p=0.0271). The probability of being frail was not affected by sex (HR 1.7, p=0.173), comorbidities (HCT-CI>3) (p=0.196), or underlying diagnosis (multivariate binary regression analysis). Frailty at first consultation was not associated with a more prolonged HCT hospitalization (p=0.663) or higher readmissions (p=0.579). However, a non-significant trend to lower OS was observed in frail patients undergoing auto-HCT than in the rest [ 1-year OS of fit, pre-frail, and frail adults: 98.5%, 95.6%, and 84.4% (p=0.072)] ( Table 1). The impact of frailty in QoL was also investigated. As shown in Table 2, compared with fit patients, pre-frail and frail adults had worse score values throughout all the variables included questionnaire. Since PCD and SLP are the most prevalent indications for auto-HCT, trends on frailty were investigated separately on these subgroups of patients . Trends on frailty were examined and reported in Table 1. At first consultation, the proportions of fit, pre-frail, and frail adults were similar between the two groups (p=0.896). At HCT admission, the proportion of frail patients with PCD tend to be higher than in the other group (16.6% vs.9.9%, p=0.051). Nevertheless, at day +100, frailty stages were again similar between the two study groups. Lastly, when the impact of frailty in OS was investigated in these two subgroups of patients, the negative effect of this syndrome in post-transplant outcomes was only observed in patients with PCD (p=0.012). CONCLUSIONS This study validates the applicability of the HCT Frailty Scale in adult candidates for auto-HCT. At first consultation, frailty had an incidence of 18% being more prevalent in older adults and with worse performance status. Moreover, it´s presence during the post-transplant process was associated with worse QoL. Preliminary data shows that frailty at first consultation correlates with lower OS in patients with PCD. Further analyses will be conducted to better investigate this result.