Zepeda‐Mendoza, Cinthya

ABSTRACT The clinical relevance of TP53 mutations ( TP53 MUT ) in myeloproliferative neoplasms (MPN) and their prognostic interaction with MPN subtype designation has not been systematically studied. In the current study, 114 patients with MPN harboring TP53 MUT (VAF ≥ 2%) were evaluated for overall survival (OS), calculated from the time of TP53 MUT detection: chronic phase myelofibrosis (MF‐CP; N = 61); blast‐phase (MPN‐BP; N = 31) or accelerated‐phase (MPN‐AP; N = 16) MPN, and polycythemia vera/essential thrombocythemia (PV/ET; N = 6). Sixty‐five (57%) patients harbored International Consensus Classification (ICC)‐defined multihit TP53 MUT and 56 (49%) monosomal/complex karyotype (MK/CK). Majority of MPN‐BP (90%) and MPN‐AP (81%) while 39% of MF‐CP and none of PV/ET patients harbored multihit TP53 MUT . OS in MPN‐BP and MPN‐AP was equally dismal (median 6 vs. 4.5 months, respectively; p = 1.0), regardless of multihit configuration ( p = 0.44), while OS in TP53 MUT MPN‐BP/AP ( N = 47; median 4 months) was inferior to that of a separate cohort ( N = 49) with TP53 wild‐type MPN‐BP/AP (median 11 months; p < 0.01). OS in MF‐CP was significantly shorter with multihit versus non‐multihit TP53 MUT (median 10 vs. 35 months; HR 2.9; p < 0.01), independent of other MF‐relevant genetic risk factors, including ASXL1/SRSF2/U2AF1 mutations. Multihit TP53 MUT was also associated with inferior survival following allogeneic stem cell transplant (ASCT): median 9 months versus “not reached” in patients with ( N = 9) versus without ( N = 8) multihit TP53 MUT ( p < 0.01). The presence of multihit or non‐multihit TP53 MUT in MPN‐BP/AP or multihit TP53 MUT in MF‐CP is associated with exceptionally poor prognosis and justifies inclusion into the ICC category of “myeloid neoplasms with mutated TP53 .” By contrast, detection of non‐multihit TP53 MUT , by itself, might not endanger short‐term survival in MF‐CP, PV, or ET.

Abstract Adult primary leptomeningeal gliomatosis (PLG) is a rare, rapidly progressive and fatal disease characterized by prominent leptomeningeal infiltration by a glial tumor without an identifiable parenchymal mass. The molecular profile of adult PLG has not been well‐characterized. We report the clinical, pathological, and molecular findings of six adult PLG patients (five males and one female), median age 58 years. All cases exhibited pathological leptomeningeal enhancement at presentation. Leptomeningeal biopsy was diagnostic in five (of six) cases, revealing infiltration by an astrocytic glioma with mitotic activity, lacking microvascular proliferation or necrosis. One case was diagnosed at autopsy. All tumors were IDH‐wildtype, with five harboring TERT promoter mutations. Additional mutations identified were PTEN in one case, TP53 in two cases, and NF1 in two cases. A chromosome profile with +7/−10 was found in four cases, whereas the remaining two showed either chromosome 7 or 7p gain only. Four cases showed chromosome 9p loss with CDKN2A/B homozygous deletion, one case showed hemizygous CDKN2A/B loss, and one case showed intact chromosome 9 and CDK4/GLI1 amplification. DNA methylation profiling was performed in four cases and revealed a match to glioblastoma (GBM) family and mesenchymal typical class with high confidence scores in two cases; the other two cases showed only suggestive combined scores for GBM family and mesenchymal atypical class. The molecular profile of all cases closely aligned with that of adult‐type GBM, IDH‐wildtype, CNS WHO grade 4. All patients succumbed to the disease. In five cases with extensive leptomeningeal disease at diagnosis, the course was rapid, with median survival of 24 days following palliative care. Only one case, with relatively localized disease at diagnosis, received chemoradiation therapy and survived 535 days, raising the possibility that early diagnosis and timely treatment could improve outcome. A detailed list of previously reported cases is provided in a supplementary table.

Background: Multiple myeloma (MM) is an incurable plasma cell malignancy with several recurrent primary cytogenetic abnormalities including t(11,14)(q13;q32), resulting in increased expression of CCND1. Although 40% of patients with t(11;14) MM respond favorably to the BCL-2 inhibitor venetoclax (ven), 60% fail to achieve an objective response. Previous studies have suggested upregulation of PMAIP1, encoding the pro-apoptotic NOXA protein, in association with ven sensitivity (Leblay, Blood, 2024). Here we sought to evaluate the role for NOXA in ven response in t(11;14) MM. Methods: We analyzed whole exome, whole genome and RNA sequencing data of CD138+ tumor cells from 1,359 patients with MM in the CoMMpass and Mayo Clinic cohorts. The histone mark H3K27ac was assessed in t(11;14)+ KMS12PE and U266B1 human myeloma cell lines using ChIP-seq. Gene expression and protein levels of PMAIP1 (NOXA) were also detected by qRT-PCR and Western blot, respectively. Live cell detection was evaluated by Annexin V and PI using flow cytometry and by YoYo3 dye using IncuCyte following treatment with ven. Results: The t(11;14) MM subtype was associated with a 1.6-5.5-fold increased expression of PMAIP1 compared to non-t(11,14) MM in the CoMMpass and Mayo Clinic cohorts (p&lt;0.0001). These findings are consistent with a significant correlation between CCND1 and PMAIP1 expression in both cohorts (p&lt;0.001). To evaluate whether cyclin D1 is directly associated with increasing expression of PMAIP1 in t(11;14) MM, we generated a cyclin D1 knock out (KO) version of the U266B1 line using CRISPR/cas9. Two independent U266B1 KO cell lines showed reduced expression of PMAIP1 and NOXA protein compared to the WT U266B1 consistent with a role for cyclin D1 in regulating PMAIP1 expression. In addition, the KO lines had a 4.8-fold reduced H3K27ac signal at the PMAIP1 locus in comparison to the WT control suggesting a role for cyclin D1 in promoting H3K27ac. We previously observed increased expression of CCND1 in association with the G allele compared to the A allele of rs9344 in both the CoMMpass and Mayo Clinic cohorts (Leukemia, in press). We next asked whether the G risk allele of rs9344 within CCND1 impacted gene expression and H3k27ac levels at the PMAIP1 locus. To study the allele specific effect of CCND1, we used the CRISPR/cas9 edited KMS12PE WT (rs9344 AA) and Knock In (KI) (rs9344 GG) cell lines. In the KI cells, the G allele was accompanied by an ~2-fold increased expression of PMAIP1 and NOXA protein detected by western blot (&lt;0.05). Increased PMAIP1 expression in KI lines was also accompanied by a 2.2-fold increased H3K27ac further suggesting a role for the G allele of rs9344 in promoting H3K27ac and gene expression at the PMAIP1 locus. Finally, we evaluated the consequence of PMAIP1 expression levels in relation to ven response. Increased expression of PMAIP1 and elevated levels of NOXA protein in the KMS12PE KI lines was associated with increased sensitivity to ven resulting in ~50% increase in cell death of KI vs. WT lines (p&lt;0.02). Conclusion: We demonstrate a role for cyclin D1 in regulating PMAIP1 expression through H3K27ac resulting in increased NOXA protein. Levels of NOXA are associated with the G allele of rs9344 and increased venetoclax response.

Background Abnormal karyotype is not infrequent in polycythemia vera (PV) and has been associated with shortened survival (BJH 2018;182:437). In the current study, we utilized a large institutional database, in order to describe the spectrum and prevalence of specific cytogenetic abnormalities in PV, during different stages of the disease, and examine their clinical, molecular, and prognostic correlates, in the context of previously established risk factors. Methods After institutional review board approval, we identified 669 cytogenetically-annotated patients with PV, evaluated at the Mayo Clinic between 1973 and 2023, and met the International Consensus Classification diagnostic criteria (Arber et al. Blood 2022;140;1200). Cytogenetic reporting was in accordance with the International System for Human Cytogenomic Nomenclature. Results i) Prevalence of cytogenetic abnormalities at diagnosis and at different stages of the disease 436 of 669 (65%) patients with PV underwent cytogenetic evaluation within one year of diagnosis, 116 (17%) within 1-10 years, 47 (7%) after 10 years, and 70 (10%) at time of fibrotic or leukemic transformation. In patients with cytogenetic information obtained within 1 year of diagnosis, karyotype was normal in 369 (85%), showed loss of Y chromosome (-Y) in 15 (3%), and other abnormalities in 52 (12%). Most common abnormalities included trisomy 9 (n=11; 3%), del(20q) (n=10; 2%), trisomy 8 (n=4; 1%), other sole abnormalities (n=16; 4%), and ≥ 2 abnormalities (n=11; 3%) including complex karyotype (n=4; 0.9%). Frequency of abnormal karyotype was higher with longer disease duration; 15% within a year of diagnosis, 28% in 1-10 years, 49% over 10 years after diagnosis, and 50% at time of fibrotic or leukemic transformation (p&lt;0.01). Sole del (20q), single abnormalities excluding 20q-, +8, +9, or -Y, and ≥ 2 abnormalities including complex karyotype were progressively acquired; respective frequencies within a year of diagnosis, over 10 years after diagnosis, and at time of fibrotic or leukemic transformation were 2%/21%/10% (del (20q), 4%/17%/14% (single abnormality excluding 20q-, +8, +9, or -Y), 3%/11%/19% (≥ 2 abnormalities), 1%/ 2%,/10% (complex karyotype) (all p-values &lt;0.01). In addition, sole +8 displayed highest frequency at fibrotic transformation compared to within a year of diagnosis (5% vs 0.9%; p=0.04). ii) Phenotypic and Molecular Correlates 436 patients with PV (51% males; median age; 64 years) who underwent cytogenetic testing within one year of diagnosis were examined for clinical and molecular correlations. Abnormal karyotype compared to normal karyotype, correlated with older age (median: 70 vs 63 years, p&lt;0.01), male gender (64% vs 48%, p=0.02) and lower platelet count (370 vs 465 × 109/L, p&lt;0.01). Sole del (20q) compared to normal karyotype was associated with older age (median: 76 vs 63 years, p&lt;0.01); sole +8 exclusively occurred in patients ≥ 60 years (p=0.03); sole +9 was associated with venous thrombosis history (45% vs 17%, p=0.03). TET2 and SRSF2 mutations were found to cluster with normal karyotype (18% vs 4%; p=0.04 and 5% vs 0%; p=0.14 in normal vs abnormal karyotype, respectively). iii) Prognostic Impact of Cytogenetic Abnormalities At a median follow-up of 7.4 years (range; 0.04-42.4 years), a total of 163 (37%) patients have died, 50 (11%) and 14 (3%) underwent fibrotic and leukemic transformation, respectively. In univariate analysis for overall survival (OS), abnormal karyotype compared to normal karyotype, was associated with inferior survival (median OS; 10.5 vs 16.3 years, p&lt;0.01). In multivariate analysis, the adverse impact of abnormal karyotype remained significant (p=0.02; HR 2.0, 95% CI 1.2-3.3), along with age ≥60 years, leukocyte count ≥15 × 109/L, and SRSF2 mutations. Abnormal karyotype was also an independent predictor of myelofibrosis-free survival (p&lt;0.01; HR 3.7, 95% CI 1.8-7.8), along with SRSF2 mutations. Analysis of leukemia-free survival revealed abnormal karyotype to be a significant risk factor in univariate (p&lt;0.01), but not multivariate analysis that included SRSF2 mutation (p&gt;0.1). Conclusions The current study provides the distribution of cytogenetic abnormalities at different stages of PV and affirms the prognostic significance of abnormal karyotype at the time of diagnosis, highlighting the importance of incorporating cytogenetic studies in the diagnostic evaluation of PV.

Introduction: Cyclin D1, encoded by the CCND1 gene, is primarily known as a major regulator of cell cycle progression. Emerging studies have demonstrated a role for cyclin D1 in regulating gene transcription. In mantle cell lymphoma with t(11;14), cyclin D1 strongly binds to the promoters of highly transcribed genes and its overexpression led to genome-wide transcriptional downregulation. However, the transcriptional role for cyclin D1 in t(11;14) MM remains undefined. This study aims to elucidate the global chromatin and transcriptional changes induced by cyclin D1 in t(11;14) MM. Methods: Knockout (KO) of CCND1 in U266B1, a MM cell line carrying t(11;14), was performed by the Synthego (Redwood City, CA) using CRISPR/Cas9, which generated a frameshift insertion of base pair (bp) “A” after 69,641,478 bp within exon 1. Two independent KO clones were used for downstream analysis. ChIP-seq and ATAC-seq were performed for KO (n=2) and wild-type clones (WT, n=2). ChIP-seq used antibodies against H3K4me3 (promoter) and H3K4me1 and H3K27ac (enhancer). Libraries were sequenced to 2x51 bp on the Illumina NextSeq 2000 platform. Total RNA libraries (in triplicate) were sequenced to 2x150 bp. ChIP-seq and ATAC-seq reads were mapped to hg38 with BWA, and peaks were identified by MACS2 at FDR 0.01. RNA-seq reads were mapped to hg38 with STAR and gene raw counts were estimated using featureCounts. Differential peaks and differentially expressed genes between KO and WT were identified using edgeR at FDR 0.05 and fold-change 2. Only peaks present in at least 2 samples and genes having reads-per-million value 0.1 in at least 1 sample were included, excluding those from chrY. Pathway analysis was performed with the Enrichr package. Results: Differential expression analysis identified 2-fold more genes that were down-regulated than up-regulated in KO compared to WT (467 vs. 233). Pathway analysis revealed an enrichment of TNF-alpha signaling via NF-kappaB, inflammatory response, and apoptosis in association with down-regulated genes, while up-regulated genes are enriched in myogenesis, KRAS signaling, and epithelial mesenchymal transition pathway. In parallel, differential chromatin analysis identified 2.5% (1,161) of the peaks from H3K27ac and 0.5-1% (359-610) of the peaks for the other 3 marks that showed significant changes of signal levels between KO and WT. For the promoter mark H3K4me3, there are largely a comparable number of peaks showing increased (191 peaks) and decreased (168 peaks) signals, respectively, in KO compared to WT. However, for ATAC-seq and H3K4me1, about 70% of the differential peaks showed an increased signal in KO, in contrast with H3K27ac for which 60% of differential peaks showed decreased signal in KO. The preferential loss of H3K27ac occupancy is consistent with the observation of a higher proportion of genes being down-regulated in KO. These results indicated that knockout of CCND1 led to a global increase of chromatin accessibility, but a reduction of H3K27ac and gene expression. Further annotation revealed that, for H3K27ac and H3K4me1/ATAC-seq, 92% and 97% of the differential peaks were located &gt;2kb away from the transcription start sites (TSSs) of expressed genes. Even for H3K4me3, 88% of the differential peaks are &gt;2kb away from TSSs. The results suggest that the changes of chromatin upon CCND1 knockout predominantly occurred at distal regulatory regions. For up- and down-regulated genes, we estimated ChIP-seq and ATAC-seq signals in the TSS 2kb regions, revealing that up-regulated genes were overall associated with increased signals. Further analysis found that, for ATAC-seq and H3K4me3, 44% and 59% of the differential peaks located in the promoter regions were associated with differentially expressed genes, which dropped to ~20% for the two enhancer marks. These data indicate that changes in chromatin state impact the expression of the associated genes. Conclusion: Knockout of CCND1 impacts local chromatin state, particularly at enhancer regions, and the transcriptional program. In promoter-proximal regulatory regions, the changes of chromatin accessibility and H3K4me3 are strongly correlated with the expression changes of nearby genes. Further studies will identify the genes targeted by the differential enhancers and their possible roles in MM pathogenesis mediated by cyclin D1 following the t(11;14) event.

Background Absolute basophil count (ABC) might be increased in patients with myeloproliferative neoplasms and has been associated with poor prognosis in chronic myeloid leukemia (CML) and, possibly, primary myelofibrosis (PMF, Front. Oncol 2019;9:872). In a Mayo Clinic survey of 382 patients with BCR::ABL1-negative leukocytosis and ABC ≥0.09 x 109/L, 191 (50%) were associated with myeloid neoplasms, including 104 (27%) with CML, 23 (6%) essential thrombocythemia, 21 (6%) polycythemia vera (PV), and 18 (5%) PMF (AJH 2020;95:E216). Herein, we tapped into a well-characterized cohort of patients with PV, in order to describe the prevalence of basophilia, at specific ABC thresholds, and corresponding clinical, molecular, and prognostic correlates. Methods Study patients were retrospectively recruited from Mayo Clinic (USA) databases, after approval from IRB. Diagnostic criteria were according to the International Consensus Classification (Blood 2022;140:1200). Study inclusion criteria included the availability of information on ABC and absolute eosinophil count (AEC). Mayo Clinic institutional reference range for ABC was 0.01 to 0.08 x 109/L and for AEC 0.03 to 0.48 x 109/L. “Eosinophilia” was defined as an AEC of ≥0.6 x 109/L. Basophilia was evaluated both at an ABC of ≥0.1 x 109/L and ABC of ≥0.3 x 109/L, the latter representing 3 x the upper limit of the normal reference range. Cytogenetic information obtained within one year of diagnosis was available in 270 patients. Next-generation sequencing information collected before disease transformation was available in 99 patients. Conventional statistical methods were employed for comparisons of groups and survival analysis (JMP Pro 17.0.0 software SAS Institute, Cary, NC, USA). Results Information on ABC and AEC, obtained within one year of diagnosis, was available in 478 patients (median age 66 years; males 51%). Median values at presentation were 10.6 x 109/L for leukocyte count, 453 x 109/L platelet count, and 17.3 g/dL hemoglobin level. Presenting symptoms included palpable splenomegaly 24%, pruritus 31%, arterial thrombosis 12%, and venous thrombosis 17%. Median and range values for ABC and AEC were 0.12 x 109/L (0-3.2) and 0.27 x 109/L (0-6.5), respectively. ABC was ≥0.3 x 109/L in 80 (17%) patients and ≥0.1 x 109/L in 315 (66%). AEC was ≥0.6 x 109/L in 68 (14.6%) patients. ABC≥0.3 x 109/L was associated with AEC ≥0.6 x 109/L (p&lt;0.01), leukocytosis (p&lt;0.01), and palpable splenomegaly (p=0.02), but not platelet count, pruritus, smoking history, diabetes, or arterial or venous thrombosis; in multivariable analysis, the association with leukocytosis, but not that with splenomegaly, was confirmed. At a median follow-up of 5.7 years (range 0.03-32.8), 132 (28%) deaths, 17 (3.6%) leukemic and 40 (8.4%) fibrotic progressions were documented. Post-diagnosis events also included arterial (7%) and venous (9%) thrombosis. In univariate analysis, neither ABC (p=0.8, 0.7, and 0.96) nor AEC (p=0.7, 0.6, and 0.8), evaluated as continuous variables, affected overall (OS), leukemia-free (LFS), or myelofibrosis-free (MFFS) survival, respectively. Similarly, eosinophilia at AEC ≥0.6 x 109/L did not appear to influence OS (p=0.99), LFS (p=0.6) or MFFS (p=0.5). On the other hand, although basophilia at ABC≥0.3 x 109/L did not affect OS (p=0.27), it was significantly associated with inferior LFS (HR 6.5, 95% CI 2.3-18.0; p&lt;0.01) and MFFS (HR 2.3, 95% CI 1.2-4.5; p=0.01); the corresponding p values for ABC≥0.1 x 109/L were 0.7, 0.1, and 0.17. The detrimental impact of basophilia (ABC≥0.3 x 109/L) on LFS was confirmed (P&lt;0.01) with multivariable analysis that included other clinical variables, including leukocytosis (NS), platelet count (NS), basophil percentage (NS), age (p&lt;0.01), and palpable splenomegaly (p&lt;0.01). Results were similar during multivariable analysis for MFFS with palpable splenomegaly (p&lt;0.01), ABC≥0.3 x 109/L (p=0.03), and age (p=0.05) sustaining independent predictive value. The number of informative cases was too small to evaluate prognostic interaction with mutations and karyotype. Conclusions: Basophilia in PV is associated with palpable splenomegaly and leukocytosis and independently predicts leukemic and myelofibrotic transformation. Additional studies are needed to confirm these observations and examine prognostic interaction with mutations and karyotype.

Abstract Pleomorphic xanthoastrocytomas (PXAs) harbor CDKN2A homozygous deletion in &gt;90% of cases, resulting in loss of p16 expression by immunohistochemistry. Considering the proximity of MTAP to CDKN2A and their frequent concurrent deletions, loss of MTAP expression may be a surrogate for CDKN2A homozygous deletion. We evaluated p16 and MTAP expression in 38 patient PXAs (CNS WHO grade 2: n = 23, 60.5%; grade 3: n = 15, 39.5%) with available chromosomal microarray data to determine whether MTAP can be utilized independently or in combination with p16 to predict CDKN2A status. CDKN2A, CDKN2B, and MTAP homozygous deletion were present in 37 (97.4%), 36 (94.7%), and 25 (65.8%) cases, respectively. Expression of p16 was lost in 35 (92.1%) cases, equivocal in one (2.6%), and failed in 2 (5.3%), while MTAP expression was lost in 27 (71.1%) cases, retained in 10 (26.3%), and equivocal in one (2.6%). This yielded a sensitivity of 94.6% for p16 and 73.0% for MTAP in detecting CDKN2A homozygous deletion through immunohistochemistry. MTAP expression was lost in the 2 cases with failed p16 staining (combined sensitivity of 100%). Our findings demonstrate that combined p16 and MTAP immunostains correctly detect CDKN2A homozygous deletion in PXA, while MTAP expression alone shows reduced sensitivity.

Abstract Multiple myeloma (MM) is the most common blood cancer in African American (AA) individuals. AAs have an increased risk of developing Monoclonal Gammopathy of Undetermined Significance (MGUS) and MM compared to non-Hispanic White (NHW) individuals. While several studies have shown a pivotal role for the immune tumor microenvironment (iTME) in MM pathogenesis, the composition of the iTME in AAs with MM is under investigated. Viably frozen whole bone marrow (BM) aspirates from 128 individuals (53 AA and 75 NHW) with either MM (n=65), MGUS (n=28), smoldering MM (SMM, n=13), or healthy controls (n=22) were evaluated by single cell RNA-sequencing (scRNA-seq) or mass cytometry (CyTOF). We characterized 34 (14 AA and 20 NHW) by scRNA-seq and 94 (39 AA and 55 NHW) by CyTOF. CyTOF samples were stained with Maxpar Direct Immune Profiling Assay Kit (Fluidigm), acquired on a Helios mass cytometer and analyzed in Cytobank. Data were analyzed by logistic equivalent regression model with p&lt;0.05 for significance. ScRNA-seq samples were processed as in Yao, 2022. Data were normalized using scvi-tools and clustered by Uniform manifold approximation and projection (UMAP). Differentially expressed markers between clusters were identified using Model-based Analysis of Single-cell Transcriptomics (MAST) in Seurat R package at FDR &lt; 0.05 and log2 fold change &gt; 0.55. ScRNA-seq of the CD45+ non-tumor lymphocytes revealed 17 immune populations (HSC, erythroblast, MDSC, GMP, CD1c+ DC, CD4+ and CD8+ T cell, CD14+ M1 macrophage, CD16+monocyte, monocyte, pDC, naïve B, pre-B, pro-B, memory B, NK/MAIT). CD3E/D+cells were clustered into naïve/central memory (CCR7+, LEF1+, SELL+), memory (GZMK+), effector (GZMB+, GZMH+, FGFBP2+), g/d (TRDC+), MAIT (SLC4A10+, KLRB1+) and IFN CD8+ T cells (IFIT1+, IFIT2+,IFIT3+). Disease progression was associated with increases in median CD4+ and CD8+ GZMK+ T cells, memory B cells and a reduction in all monocyte clusters. Within the diseased cohort in comparison to NHW patients, AA patients had increased CD3+ T cells (CD4 and CD8+) (AA 57.5% vs. NHW 44.3%) and a reduction in overall monocytes (AA 10.5% vs. 26.5%). These results were validated by CyTOF, which confirmed an increase in CD3+ T cells (AA 53.7% vs. NHW 35.7%, p&lt;0.001) including increases in CD8+ (AA 7.0% vs. NHW 3.2%, p&lt;0.05) and CD4+ terminal effector T cells (AA 5.7% vs. NHW 2.7%, p&lt;0.05) and a reduction in monocytes (AA 18.3% vs. NHW 26.6%) among AAs. After adjusting for age and sex, AAs retained an increase in T cells (OR 1.59 (95%CI 1.2-2.01, p=0.00023)) and a reduction in monocytes (OR 0.88 (95%CI 0.78-1, p=0.045)). Despite the increase in CD8+ T cells in AA patients, we identified reduced GZMK+ T cells and reductions in exhaustion markers (TIGIT, LAG3, TOX) in CD8+ T cells in comparison to NHWs. Nevertheless, the distribution of the immune populations was similar between healthy AA and NHW individuals. Our multiomics approach to characterize the iTME identified significant differences among AA compared to NHW patients with MM. Citation Format: Suganti Shivaram, Hongwei Tang, Huihuang Yan, Shulan Tian, Guyu Qin, Emilie Anderson, Neeraj Sharma, Abiola Bolarinwa, Cinthya Zepeda-Mendoza, Stacey Lehman, Felicia Gomez, Amit Mitra, Surendra Dasari, Taxiarchis Kourelis, Shaji Kumar, Linda Baughn. Characterization of the Immune Tumor Microenvironment in a Diverse Cohort of Patients with Multiple Myeloma [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P06.

Abstract Objectives Fluorescence in situ hybridization (FISH) for plasma cell neoplasms (PCNs) requires plasma cell (PC) identification or purification strategies to optimize results. We compared the efficacy of cytoplasmic immunoglobulin FISH (cIg-FISH) and fluorescence-activated cell sorting FISH (FACS-FISH) in a clinical laboratory setting. Methods The FISH analysis results of 14,855 samples from individuals with a suspected PCN subjected to cytogenetic evaluation between 2019 and 2022 with cIg-FISH (n = 6917) or FACS-FISH (n = 7938) testing were analyzed. Results Fluorescence-activated cell sorting–FISH increased the detection rate of abnormalities in comparison with cIg-FISH, with abnormal results documented in 54% vs 50% of cases, respectively (P &lt; .001). It improved the detection of IGH::CCND1 (P &lt; .001), IGH::MAF (P &lt; .001), IGH::MAFB (P &lt; .001), other IGH rearrangements (P &lt; .001), and gains/amplifications of 1q (P &lt; .001), whereas the detection rates of IGH::FGFR3 fusions (P = .3), loss of 17p (P = .3), and other abnormalities, including hyperdiploidy (P = .5), were similar. Insufficient PC yield for FISH analysis was decreased between cIg-FISH and FACS-FISH (22% and 3% respectively, P &lt; .001). Flow cytometry allowed establishment of ploidy status in 91% of cases. In addition, FACS-FISH decreased analysis times, workload efforts, and operating costs. Conclusions Fluorescence-activated cell sorting–FISH is an efficient PC purification strategy that affords significant improvement in diagnostic yield and decreases workflow requirements in comparison with cIg-FISH.