Tarek Mouhieddine | Dana-Farber Cancer Institute (original) (raw)

Papers by Tarek Mouhieddine

Blood, Nov 5, 2021

Our understanding of disease progression in multiple myeloma (MM) and its precursor conditions, m... more Our understanding of disease progression in multiple myeloma (MM) and its precursor conditions, monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), is classically founded on bulk analysis studies. Low disease burden at the precursor stages has precluded comprehensive analyses of the transcriptomic events underlying malignant transformation. Here, we use single-cell RNA sequencing data from 29,387 bone marrow plasma cells from 26 patients with MGUS, SMM, or MM and 9 healthy controls to characterize the transcriptional transformation at each step of progression. Due to varying disease burdens, many samples contained a mixture of healthy and neoplastic plasma cells. We leveraged this impurity to perform a patient-specific characterization of the disease, comparing each patient's neoplastic and healthy plasma cells. This approach isolated the disease phenotype in each patient, which is usually confounded by biological and technical variability when comparing tumors to samples from healthy donors. We found that neoplastic cells from patients with MGUS and SMM already exhibit phenotypic changes similar to those of advanced myeloma. We observed upregulation of genes corresponding to known MM subtypes, such as CCND1 in patients with t(11;14) translocations and other known driver genes such as HIST1H1C. We also found universal downregulation of certain genes such as CD27, a member of the tumor necrosis factor receptor family associated with the differentiation of B cells into plasma cells, which may signify a common loss of a normal plasma phenotype across samples with different driver events and stages. Pathway analysis of differentially expressed genes further revealed that biological pathways related to myeloma were altered as early as MGUS. We observed that SMM patients with hyperdiploidy exhibit upregulation of ribosomal proteins, as reported in advanced disease. Upregulated genes in select MGUS and SMM samples were enriched for the eukaryotic translation initiation factor 3 (eIF3) complex, which plays important roles in translation, as well as proteasome activity, a function central to the survival of MM and targeted by therapies such as bortezomib. We observed enrichment of the E2F family of transcription factors in MGUS and SMM samples; these are master regulators of proliferation that have been suggested as therapeutic targets in myeloma. Five samples were enriched for genes associated with extracellular exosomes, which has been reported to play an important role in cancer cell signaling and to contribute to osteolysis and drug resistance in MM. Pathway enrichment of genes downregulated in neoplastic cell populations revealed weakened response to endoplasmic reticulum and oxidative stress, presumably allowing myeloma cells to tolerate high volumes of abnormal protein production without apoptosing. To further identify shared gene expression patterns across samples, we employed a Bayesian Non-Negative Matrix Factorization method to decompose our data into 31 gene signatures that capture its variability. In addition to recovering signatures corresponding to known MM subtypes, demonstrating that our method captures cohesive transcriptional networks, we find signatures that capture disease biology shared across subtypes. Most notably, we identified a signature that is active in healthy plasma cells across disease stages and dramatically lost in MM and precursor cells. The top genes in this signature include CD27 and CD79A, which are associated with the B cell lineage and whose downregulation may signify dedifferentiation of premalignant cells as early as MGUS, and JSRP1, CTSH, and HCST, genes as of yet unreported to be involved in plasma and MM cell biology. This phenotype would be obscured at early disease stages by bulk analysis. We validated the discovery and behavior of this signature in an external single-cell dataset from Ledergor et al. (Nature Medicine 2018). In summary, using single-cell RNA sequencing, we discovered that canonical MM pathways are altered as early as MGUS and identified a signature of genes which distinguishes healthy and neoplastic cells even at early disease stages. Our identification of patient-specific transcriptional changes as early as MGUS paves the way for future work exploring personalized treatment approaches prior to malignant disease. Haradhvala: Constellation Pharmaceuticals a MorphoSys Company: Consultancy. Zavidij: Constellation Pharmaceuticals: Current Employment. Sontag: curai health: Current holder of individual stocks in a privately-held company; Takeda Pharmaceuticals: Research Funding; Genentech: Research Funding; IBM: Research Funding. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy. Getz: IBM, Pharmacyclics: Research Funding; Scorpion…

Nature Communications, Jun 12, 2020

Journal of Clinical Oncology, Feb 20, 2023

Blood, Nov 5, 2021

Splicing factor mutations are recurrent genetic alterations in blood disorders, highlighting the ... more Splicing factor mutations are recurrent genetic alterations in blood disorders, highlighting the importance of alternative splicing regulation in hematopoiesis. Specifically, mutations in splicing factor 3B subunit 1 (SF3B1) are implicated in the pathogenesis of myelodysplastic syndromes (MDS) and linked to a high-risk of leukemic transformation in clonal hematopoiesis (CH). SF3B1 mutations are associated with aberrant RNA splicing, leading to increased cryptic 3' splice site (ss) usage and MDS with ring sideroblasts phenotype. The study of mutant SF3B1-driven splicing aberrations in humans has been hampered by the inability to distinguish mutant and wildtype single cells in patient samples and the inadequate coverage of short-read sequencing over splice junctions. To overcome these limitations, we developed GoT-Splice by integrating Genotyping of Transcriptomes (GoT; Nam et al. 2019) with Nanopore long-read single-cell transcriptome profiling and CITE-seq (Fig. A). This allowed for the simultaneous single-cell profiling of protein and gene expression, somatic mutation status, and alternative splicing. Our method selectively enriched full-length sequencing reads with the accurate structure, enabling the capture of higher number of junctions per cell and greater coverage uniformity vs. short-read sequencing (10x Genomics; Fig. B, C). We applied GoT-Splice to CD34+ bone marrow progenitor cells from MDS (n = 15,436 cells across 3 patients; VAF: [0.38-0.4]) to study how SF3B1 mutations corrupt human hematopoiesis (Fig. D). High-resolution mapping of SF3B1 mutvs. SF3B1 wt hematopoietic progenitors revealed an increasing fitness advantage of SF3B1 mut cells towards the megakaryocytic-erythroid lineage, resulting in an expansion of SF3B1 muterythroid progenitor (EP) cells (Fig. E, F). Accordingly, SF3B1 mutEP cells displayed higher protein expression of erythroid lineage markers, CD71 and CD36, vs. SF3B1 wt cells (Fig. G). In these SF3B1 mutEP cells, we identified up-regulation of genes involved in regulation of cell cycle and checkpoint controls (e.g., CCNE1, TP53), and mRNA translation (eIFs gene family; Fig. H). Next, while SF3B1 mut cells showed the expected increase of cryptic 3' splicing vs. SF3B1 wt cells (Fig. I), they exhibited distinct cryptic 3' ss usage as a function of hematopoietic progenitor cell identity, displaying stage-specific aberrant splicing during erythroid maturation (Fig. J). In less differentiated EP cells, we observed mis-splicing of genes involved in iron homeostasis, such as the hypoxia-inducible factor HIF1A, and key regulators of erythroid cell growth, such as SEPT2. At later stages, we observed mis-splicing of BAX, a pro-apoptotic member of the Bcl-2 gene family and transcriptional target of p53, and erythroid-specific genes (e.g., PPOX). We further predicted 54% of the aberrantly spliced mRNAs to introduce premature stop codons, promoting RNA degradation through nonsense-mediated decay (NMD). In line with this notion, we observed a significant decrease in expression of NMD-inducing genes in SF3B1 mut vs . SF3B1 wtEP cells (Fig. K). Lastly, splicing factor mutations observed in CH subjects provide an opportunity to interrogate the downstream impact of SF3B1 mutations prior to development of disease. Like MDS, by applying GoT-splice to CD34+ progenitor cells from SF3B1 mut CH subjects (n = 9,007 cells across 2 subjects; VAF: [0.15-0.22]; Fig. L), we revealed increased mutant cell frequency in EP cells (Fig. M) with concomitant increased expression of genes involved in mRNA translation (Fig. N), consistent with SF3B1 mutation causing mis-splicing injury to translational machinery and ineffective erythropoiesis. Notably, CH patients already exhibited cell-type specific cryptic 3' ss usage in SF3B1 mut cells (Fig. O). In summary, we developed a novel multi-omics single-cell toolkit to examine the impact of splicing factor mutations on cellular fitness directly in human samples. With this approach, we showed that, while SF3B1 mutations arise in uncommitted HSCs, their effect on fitness increases with differentiation into committed EPs, in line with the mutant SF3B1-driven dyserythropoiesis phenotype. We revealed that SF3B1 mutations exert cell-type specific mis-splicing that leads to abnormal erythropoiesis. Finally, we demonstrated that the impact of SF3B1 mutations on EP cells begins before disease onset, as observed in CH subjects. Figure 1 Figure 1. Disclosures Dai: Oxford Nanopore Technologies: Current Employment. Beaulaurier: Oxford Nanopore Technologies: Current Employment. Drong: Oxford Nanopore Technologies: Current Employment. Hickey: Oxford Nanopore Technologies: Current Employment. Juul: Oxford Nanopore Technologies: Current Employment. Wiseman: Astex: Research Funding; Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Takeda: Consultancy; StemLine: Consultancy. Harrington: Oxford Nanopore Technologies: Current Employment. Ghobrial: AbbVie, Adaptive, Aptitude…

Prior data have variably implicated the inactivation of the mammalian SWItch/Sucrose Non-Fermenta... more Prior data have variably implicated the inactivation of the mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) complex with increased tumor sensitivity to immune checkpoint inhibitors (ICI). Herein, we examined the association between mSWI/SNF variants and clinical outcomes to ICIs. We correlated somatic loss-of-function (LOF) variants in a predefined set of mSWI/SNF genes (ARID1A, ARID1B, SMARCA4, SMARCB1, PBRM1, and ARID2) with clinical outcomes in patients with cancer treated with systemic ICIs. We identified 676 patients from Dana-Farber Cancer Institute (DFCI, Boston, MA) and 848 patients from a publicly available database from Memorial Sloan Kettering Cancer Center (MSKCC, New York, NY) who met the inclusion criteria. Multivariable analyses were conducted and adjusted for available baseline factors and tumor mutational burden. Median follow-up was 19.6 (17.6–22.0) months and 28.0 (25.0–29.0) months for the DFCI and MSKCC cohorts, respectively. Seven solid tumor subtypes were examined. In the DFCI cohort, LOF variants of mSWI/SNF did not predict improved overall survival (OS), time-to-treatment failure (TTF), or disease control rate. Only patients with renal cell carcinoma with mSWI/SNF LOF showed significantly improved OS and TTF with adjusted HRs (95% confidence interval) of 0.33 (0.16–0.7) and 0.49 (0.27–0.88), respectively, and this was mostly driven by PRBM1. In the MSKCC cohort, where only OS was captured, LOF mSWI/SNF did not correlate with improved outcomes across any tumor subtype. We did not find a consistent association between mSWI/SNF LOF variants and improved clinical outcomes to ICIs, suggesting that mSWI/SNF variants should not be considered as biomarkers of response to ICIs.

Cancers, Jun 12, 2020

Monoclonal Gammopathy of Undetermined Significance (MGUS) is considered to be a benign precursor ... more Monoclonal Gammopathy of Undetermined Significance (MGUS) is considered to be a benign precursor condition that may progress to a lymphoproliferative disease or multiple myeloma. Most patients do not progress to an overt condition, but nevertheless, MGUS is associated with a shortened life expectancy and, in a minority of cases, a number of co-morbid conditions that include an increased fracture risk, renal impairment, peripheral neuropathy, secondary immunodeficiency, and cardiovascular disease. This review aims to consolidate current evidence for the significance of these co-morbidities before considering how best to approach these symptoms and signs, which are often encountered in primary care or within a number of specialties in secondary care.

Nature Genetics, Sep 22, 2022

Somatic mutations in cancer genes have been ubiquitously detected in clonal expansions across hea... more Somatic mutations in cancer genes have been ubiquitously detected in clonal expansions across healthy human tissue, including in clonal hematopoiesis. However, mutated and wildtype cells are morphologically and phenotypically similar, limiting the ability to link genotypes with cellular phenotypes. To overcome this limitation, we leveraged multi-modality single-cell sequencing, capturing the mutation with transcriptomes and methylomes in stem and progenitors from individuals with DNMT3A R882 mutated clonal hematopoiesis. DNMT3A mutations resulted in myeloid over lymphoid bias, and in expansion of immature myeloid progenitors primed toward megakaryocytic-erythroid fate. We observed dysregulated expression of lineage and leukemia stem cell markers. DNMT3A R882 led to preferential hypomethylation of polycomb repressive complex 2 targets and a specific sequence motif. Notably, the hypomethylation motif is enriched in binding motifs of key hematopoietic transcription factors, serving as a potential mechanistic link between DNMT3A R882 mutations and aberrant transcriptional phenotypes. Thus, single-cell multi-omics pave the road to defining the downstream consequences of mutations that drive human clonal mosaicism.

Clinical Lymphoma, Myeloma & Leukemia, Oct 1, 2019

Precursor states of Multiple Myeloma (MM) and its native tumor microenvironment need in-depth mol... more Precursor states of Multiple Myeloma (MM) and its native tumor microenvironment need in-depth molecular characterization to better stratify and treat patients at risk. Using single-cell RNA sequencing of bone marrow cells from precursor stages, MGUS and smoldering myeloma (SMM), to full-blown MM alongside healthy donors, we demonstrate early immune changes during patient

Blood, Dec 7, 2017

Introduction: Multiple myeloma (MM) is an incurable plasma cell malignancy that arises from the p... more Introduction: Multiple myeloma (MM) is an incurable plasma cell malignancy that arises from the precursor states: Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). Some patients rapidly progress from MGUS/SMM to MM, while others remain indolent with minimal progression over their lifetime. The genetic and molecular factors that underlie disease evolution and distinguish progressors from non-progressors are not well elucidated. To address these questions, we examined clinically-annotated samples from SMM patients, including sequential samples at the SMM stage and at time of progression. Methods: We utilized next generation sequencing methods to study 186 SMM patient samples. We performed whole exome sequencing (WES) of 70 tumor-germline matched samples (mean target coverage 50X/100X for germline/tumor) and targeted deep sequencing on 116 samples of progressor and non-progressor SMM (mean target coverage ~500X). We also performed ultra-low pass whole-genome sequencing (ULP-WGS) of 116 SMM samples as well as 20 available cell-free DNA (cfDNA) samples to detect the tumor fraction before proceeding with targeted sequencing. For WES, libraries were constructed with Agilent SureSelect XT2 library prep kit, and hybridized to Agilent9s whole exome V5+UTR capture probes and then sequenced on HiSeq 4000 (Illumina). For targeted sequencing, libraries were enriched by hybridizing to the customized target bait (60 MM related genes and 56 pan-cancer genes). For ULP-WGS, libraries were prepared with Kapa Hyper Prep kit and custom adapters. Sequencing data were analyzed using previously established analytic pipelines including ichorCNA, MuTect, RecapSeg, MutSig, GISTIC, ABSOLUTE, and PHYLOGIC. High-risk SMM (HRSMM) and low-risk SMM (LRSMM) were defined according to the criteria proposed by Rajkumar et al. in Blood 2014. Results: Higher mutation load (average 1.44 mutations/Mb) was observed in HRSMM patients, which is close to the average of 1.6 mutations/Mb in symptomatic MM, compared to LRSMM patients (average 0.73 mutations/Mb, p -value = 0.001). Somatic mutations in known signaling pathways were observed four times more frequently (43.8% vs. 9.5%, p- value = 0.015) in HRSMM patients compared to LRSMM patients. Mutations in MAPK pathway genes (KRAS, NRAS, BRAF) were detected in 21.7% of HRSMM vs. 0% in LRSMM (p- value = 0.004). Mutations in NFkB pathway were present in 10% of patients and were also associated with HRSMM (17.4% vs. 0%, p- value = 0.018). MM somatic copy number aberrations (SCNAs) were detected in 64% of all SMM samples, and were two times more frequently in HRSMM than LRSMM patients (78.1% vs. 42.9%, p- value = 0.020). Phylogenetic studies of sequential patient samples, who progressed from SMM to MM, revealed different patterns of clonal evolution. Furthermore, we have performed ULP-WGS of available cfDNA samples from 20 patients of this cohort. Interestingly, tumor fraction of cfDNA was higher in HRSMM compared to LRSMM patients (p- value = 0.03). Together, this data suggests that specific genomic alterations are associated with HRSMM and could potentially be predictive of the risk to progression in SMM patients. Conclusion: This study demonstrates that next generation sequencing (WES, targeted-sequencing, and ULP-WGS) of SMM patients can identify genomic alterations associated with HRSMM vs LRSMM through different parameters such as mutation load, somatic mutations, and SCNAs. Disclosures Laubach:Novartis, Takeda, Celgene: Consultancy; Novartis, Takeda, Celgene, Onyx: Research Funding. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Oncopeptides AB: Membership on an entity9s Board of Directors or advisory committees; Takeda: Consultancy, Research Funding. Anderson:C4 Therapeutics: Other: scientific founder; MedImmune: Membership on an entity9s Board of Directors or advisory committees; Gilead Sciences: Membership on an entity9s Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity9s Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity9s Board of Directors or advisory committees; Oncopep: Other: scientific founder. Kumar:Celgene, Millennium/Takeda, Onyx, AbbVie, Janssen, Sanofi, Novartis, Amgen, Genentech, Merck, Oncopeptides, Roche, Skyline Diagnostics: Research Funding; Celgene, Millennium, BMS, Onyx, Janssen, Noxxon, AbbVie, Amgen, Merck, Oncopeptides, Skyline Diagnostics, Takeda: Consultancy; Skyline: Honoraria.

JNCI: Journal of the National Cancer Institute

Background Treatment options for penile squamous cell carcinoma are limited. We sought to investi... more Background Treatment options for penile squamous cell carcinoma are limited. We sought to investigate clinical outcomes and safety profiles of patients with penile squamous cell carcinoma receiving immune checkpoint inhibitors. Methods This retrospective study included patients with locally advanced or metastatic penile squamous cell carcinoma receiving immune checkpoint inhibitors between 2015 and 2022 across 24 centers in the United States, Europe, and Asia. Overall survival and progression-free survival were estimated using the Kaplan-Meier method. Objective response rates were determined per Response Evaluation Criteria in Solid Tumours 1.1 criteria. Treatment-related adverse events were graded per the Common Terminology Criteria for Adverse Events, version 5.0. Two-sided statistical tests were used for comparisons. Results Among 92 patients, 8 (8.7%) were Asian, 6 (6.5%) were Black, and 24 (29%) were Hispanic and/or Latinx. Median (interquartile range) age was 62 (53-70) years....

Regular and Young Investigator Award Abstracts, Nov 1, 2022

Figure 2 Kaplan-Meier curves for Progression-Free Survival stratified by HIV status Conclusions I... more Figure 2 Kaplan-Meier curves for Progression-Free Survival stratified by HIV status Conclusions In this matched cohort study, PLWH and metastatic NSCLC had similar toxicity profiles and clinical outcomes to HIV-counterparts receiving ICI supporting their use in PLWH and their inclusion in clinical trials. Larger

Blood, 2021

Introduction: Multiple Myeloma (MM) is an incurable hematologic malignancy characterized by the a... more Introduction: Multiple Myeloma (MM) is an incurable hematologic malignancy characterized by the abnormal growth of clonal plasma cells in the bone marrow (BM). In most cases MM develops from early, asymptomatic disease stages known as Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). Despite effective new therapies, most MM patients inevitably relapse and require further treatment, highlighting the need for better early detection methods for precursor patients and targeted interventions to prevent early disease from progressing. The initial diagnosis of MGUS/SMM remains an incidental process following the identification of increased clonal immunoglobulin in the blood. BM biopsy is the gold standard for diagnosis and monitoring of MM progression, but is intrusive, painful, and comes with possible secondary complications for patients. Consequently, repeated assessment is not a feasible option for MGUS and SMM patients who are asymptomatic...

Cancer Discovery

Multiple myeloma (MM) develops from well-defined precursor stages; however, invasive bone marrow ... more Multiple myeloma (MM) develops from well-defined precursor stages; however, invasive bone marrow (BM) biopsy limits screening and monitoring strategies for patients. We enumerated circulating tumor cells (CTC) from 261 patients (84 monoclonal gammopathy of undetermined significance, 155 smoldering multiple myeloma, and 22 MM), with neoplastic cells detected in 84%. We developed a novel approach, MinimuMM-seq, which enables the detection of translocations and copy-number abnormalities through whole-genome sequencing of highly pure CTCs. Application to CTCs in a cohort of 51 patients, 24 with paired BM, was able to detect 100% of clinically reported BM biopsy events and could replace molecular cytogenetics for diagnostic yield and risk classification. Longitudinal sampling of CTCs in 8 patients revealed major clones could be tracked in the blood, with clonal evolution and shifting dynamics of subclones over time. Our findings provide proof of concept that CTC detection and genomic pro...

Journal of Vascular and Interventional Radiology, 2019

Purpose: To report the results of below-the-elbow (BTE) arterial revascularization in patients wi... more Purpose: To report the results of below-the-elbow (BTE) arterial revascularization in patients with critical hand ischemia (CHI) and End Stage Renal Disease (ESRD). Materials: We retrospectively identified all patients with critical limb ischemia treated with below-the-elbow arterial angioplasty and/or atherectomy between 2013 and 2017. Patient demographics, co-morbidities, and procedural data were reviewed. Rates of hand salvage, technical and clinical success were evaluated. Mean age at treatment was 59.7 (50-72). Average time to follow up was 9 months (2-26 months). Results: 7 ESRD patients (3 female) with a total of 9 hands affected by BTE arterial occlusion and CHI were treated. The technical success rate was 88.9% (8/9). No major or minor complications occurred. 6 hands (66.6%) suffered from non-healing ulcers and gangrene; the remaining 3 (33.3%) presented with rest pain. 1 patient died in the first 30 days of follow up due to preexisting respiratory disease. The mean treated lesion length was 147.5±97.9 mm. Among the successfully treated patients, the hand salvage rate was 87.5% (7/8). One patient did however undergo planned digital amputation following intervention with satisfactory healing of the postoperative stump. Conclusions: Arterial revascularization of the BTE arteries for CHI was a safe and a technically feasible method to improve the hand salvage rate in ESRD patients.

Journal of Clinical Oncology

PURPOSE Compared with people living without HIV (PWOH), people living with HIV (PWH) and cancer h... more PURPOSE Compared with people living without HIV (PWOH), people living with HIV (PWH) and cancer have traditionally been excluded from immune checkpoint inhibitor (ICI) trials. Furthermore, there is a paucity of real-world data on the use of ICIs in PWH and cancer. METHODS This retrospective study included PWH treated with anti–PD-1- or anti–PD-L1-based therapies for advanced cancers. Kaplan-Meier method was used to estimate overall survival (OS) and progression-free survival (PFS). Objective response rates (ORRs) were measured per RECIST 1.1 or other tumor-specific criteria, whenever feasible. Restricted mean survival time (RMST) was used to compare OS and PFS between matched PWH and PWOH with metastatic NSCLC (mNSCLC). RESULTS Among 390 PWH, median age was 58 years, 85% (n = 331) were males, 36% (n = 138) were Black; 70% (n = 274) received anti–PD-1/anti–PD-L1 monotherapy. Most common cancers were NSCLC (28%, n = 111), hepatocellular carcinoma ([HCC]; 11%, n = 44), and head and nec...

Blood, Nov 5, 2021

Our understanding of disease progression in multiple myeloma (MM) and its precursor conditions, m... more Our understanding of disease progression in multiple myeloma (MM) and its precursor conditions, monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), is classically founded on bulk analysis studies. Low disease burden at the precursor stages has precluded comprehensive analyses of the transcriptomic events underlying malignant transformation. Here, we use single-cell RNA sequencing data from 29,387 bone marrow plasma cells from 26 patients with MGUS, SMM, or MM and 9 healthy controls to characterize the transcriptional transformation at each step of progression. Due to varying disease burdens, many samples contained a mixture of healthy and neoplastic plasma cells. We leveraged this impurity to perform a patient-specific characterization of the disease, comparing each patient's neoplastic and healthy plasma cells. This approach isolated the disease phenotype in each patient, which is usually confounded by biological and technical variability when comparing tumors to samples from healthy donors. We found that neoplastic cells from patients with MGUS and SMM already exhibit phenotypic changes similar to those of advanced myeloma. We observed upregulation of genes corresponding to known MM subtypes, such as CCND1 in patients with t(11;14) translocations and other known driver genes such as HIST1H1C. We also found universal downregulation of certain genes such as CD27, a member of the tumor necrosis factor receptor family associated with the differentiation of B cells into plasma cells, which may signify a common loss of a normal plasma phenotype across samples with different driver events and stages. Pathway analysis of differentially expressed genes further revealed that biological pathways related to myeloma were altered as early as MGUS. We observed that SMM patients with hyperdiploidy exhibit upregulation of ribosomal proteins, as reported in advanced disease. Upregulated genes in select MGUS and SMM samples were enriched for the eukaryotic translation initiation factor 3 (eIF3) complex, which plays important roles in translation, as well as proteasome activity, a function central to the survival of MM and targeted by therapies such as bortezomib. We observed enrichment of the E2F family of transcription factors in MGUS and SMM samples; these are master regulators of proliferation that have been suggested as therapeutic targets in myeloma. Five samples were enriched for genes associated with extracellular exosomes, which has been reported to play an important role in cancer cell signaling and to contribute to osteolysis and drug resistance in MM. Pathway enrichment of genes downregulated in neoplastic cell populations revealed weakened response to endoplasmic reticulum and oxidative stress, presumably allowing myeloma cells to tolerate high volumes of abnormal protein production without apoptosing. To further identify shared gene expression patterns across samples, we employed a Bayesian Non-Negative Matrix Factorization method to decompose our data into 31 gene signatures that capture its variability. In addition to recovering signatures corresponding to known MM subtypes, demonstrating that our method captures cohesive transcriptional networks, we find signatures that capture disease biology shared across subtypes. Most notably, we identified a signature that is active in healthy plasma cells across disease stages and dramatically lost in MM and precursor cells. The top genes in this signature include CD27 and CD79A, which are associated with the B cell lineage and whose downregulation may signify dedifferentiation of premalignant cells as early as MGUS, and JSRP1, CTSH, and HCST, genes as of yet unreported to be involved in plasma and MM cell biology. This phenotype would be obscured at early disease stages by bulk analysis. We validated the discovery and behavior of this signature in an external single-cell dataset from Ledergor et al. (Nature Medicine 2018). In summary, using single-cell RNA sequencing, we discovered that canonical MM pathways are altered as early as MGUS and identified a signature of genes which distinguishes healthy and neoplastic cells even at early disease stages. Our identification of patient-specific transcriptional changes as early as MGUS paves the way for future work exploring personalized treatment approaches prior to malignant disease. Haradhvala: Constellation Pharmaceuticals a MorphoSys Company: Consultancy. Zavidij: Constellation Pharmaceuticals: Current Employment. Sontag: curai health: Current holder of individual stocks in a privately-held company; Takeda Pharmaceuticals: Research Funding; Genentech: Research Funding; IBM: Research Funding. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy. Getz: IBM, Pharmacyclics: Research Funding; Scorpion…

Nature Communications, Jun 12, 2020

Journal of Clinical Oncology, Feb 20, 2023

Blood, Nov 5, 2021

Splicing factor mutations are recurrent genetic alterations in blood disorders, highlighting the ... more Splicing factor mutations are recurrent genetic alterations in blood disorders, highlighting the importance of alternative splicing regulation in hematopoiesis. Specifically, mutations in splicing factor 3B subunit 1 (SF3B1) are implicated in the pathogenesis of myelodysplastic syndromes (MDS) and linked to a high-risk of leukemic transformation in clonal hematopoiesis (CH). SF3B1 mutations are associated with aberrant RNA splicing, leading to increased cryptic 3' splice site (ss) usage and MDS with ring sideroblasts phenotype. The study of mutant SF3B1-driven splicing aberrations in humans has been hampered by the inability to distinguish mutant and wildtype single cells in patient samples and the inadequate coverage of short-read sequencing over splice junctions. To overcome these limitations, we developed GoT-Splice by integrating Genotyping of Transcriptomes (GoT; Nam et al. 2019) with Nanopore long-read single-cell transcriptome profiling and CITE-seq (Fig. A). This allowed for the simultaneous single-cell profiling of protein and gene expression, somatic mutation status, and alternative splicing. Our method selectively enriched full-length sequencing reads with the accurate structure, enabling the capture of higher number of junctions per cell and greater coverage uniformity vs. short-read sequencing (10x Genomics; Fig. B, C). We applied GoT-Splice to CD34+ bone marrow progenitor cells from MDS (n = 15,436 cells across 3 patients; VAF: [0.38-0.4]) to study how SF3B1 mutations corrupt human hematopoiesis (Fig. D). High-resolution mapping of SF3B1 mutvs. SF3B1 wt hematopoietic progenitors revealed an increasing fitness advantage of SF3B1 mut cells towards the megakaryocytic-erythroid lineage, resulting in an expansion of SF3B1 muterythroid progenitor (EP) cells (Fig. E, F). Accordingly, SF3B1 mutEP cells displayed higher protein expression of erythroid lineage markers, CD71 and CD36, vs. SF3B1 wt cells (Fig. G). In these SF3B1 mutEP cells, we identified up-regulation of genes involved in regulation of cell cycle and checkpoint controls (e.g., CCNE1, TP53), and mRNA translation (eIFs gene family; Fig. H). Next, while SF3B1 mut cells showed the expected increase of cryptic 3' splicing vs. SF3B1 wt cells (Fig. I), they exhibited distinct cryptic 3' ss usage as a function of hematopoietic progenitor cell identity, displaying stage-specific aberrant splicing during erythroid maturation (Fig. J). In less differentiated EP cells, we observed mis-splicing of genes involved in iron homeostasis, such as the hypoxia-inducible factor HIF1A, and key regulators of erythroid cell growth, such as SEPT2. At later stages, we observed mis-splicing of BAX, a pro-apoptotic member of the Bcl-2 gene family and transcriptional target of p53, and erythroid-specific genes (e.g., PPOX). We further predicted 54% of the aberrantly spliced mRNAs to introduce premature stop codons, promoting RNA degradation through nonsense-mediated decay (NMD). In line with this notion, we observed a significant decrease in expression of NMD-inducing genes in SF3B1 mut vs . SF3B1 wtEP cells (Fig. K). Lastly, splicing factor mutations observed in CH subjects provide an opportunity to interrogate the downstream impact of SF3B1 mutations prior to development of disease. Like MDS, by applying GoT-splice to CD34+ progenitor cells from SF3B1 mut CH subjects (n = 9,007 cells across 2 subjects; VAF: [0.15-0.22]; Fig. L), we revealed increased mutant cell frequency in EP cells (Fig. M) with concomitant increased expression of genes involved in mRNA translation (Fig. N), consistent with SF3B1 mutation causing mis-splicing injury to translational machinery and ineffective erythropoiesis. Notably, CH patients already exhibited cell-type specific cryptic 3' ss usage in SF3B1 mut cells (Fig. O). In summary, we developed a novel multi-omics single-cell toolkit to examine the impact of splicing factor mutations on cellular fitness directly in human samples. With this approach, we showed that, while SF3B1 mutations arise in uncommitted HSCs, their effect on fitness increases with differentiation into committed EPs, in line with the mutant SF3B1-driven dyserythropoiesis phenotype. We revealed that SF3B1 mutations exert cell-type specific mis-splicing that leads to abnormal erythropoiesis. Finally, we demonstrated that the impact of SF3B1 mutations on EP cells begins before disease onset, as observed in CH subjects. Figure 1 Figure 1. Disclosures Dai: Oxford Nanopore Technologies: Current Employment. Beaulaurier: Oxford Nanopore Technologies: Current Employment. Drong: Oxford Nanopore Technologies: Current Employment. Hickey: Oxford Nanopore Technologies: Current Employment. Juul: Oxford Nanopore Technologies: Current Employment. Wiseman: Astex: Research Funding; Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Takeda: Consultancy; StemLine: Consultancy. Harrington: Oxford Nanopore Technologies: Current Employment. Ghobrial: AbbVie, Adaptive, Aptitude…

Prior data have variably implicated the inactivation of the mammalian SWItch/Sucrose Non-Fermenta... more Prior data have variably implicated the inactivation of the mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) complex with increased tumor sensitivity to immune checkpoint inhibitors (ICI). Herein, we examined the association between mSWI/SNF variants and clinical outcomes to ICIs. We correlated somatic loss-of-function (LOF) variants in a predefined set of mSWI/SNF genes (ARID1A, ARID1B, SMARCA4, SMARCB1, PBRM1, and ARID2) with clinical outcomes in patients with cancer treated with systemic ICIs. We identified 676 patients from Dana-Farber Cancer Institute (DFCI, Boston, MA) and 848 patients from a publicly available database from Memorial Sloan Kettering Cancer Center (MSKCC, New York, NY) who met the inclusion criteria. Multivariable analyses were conducted and adjusted for available baseline factors and tumor mutational burden. Median follow-up was 19.6 (17.6–22.0) months and 28.0 (25.0–29.0) months for the DFCI and MSKCC cohorts, respectively. Seven solid tumor subtypes were examined. In the DFCI cohort, LOF variants of mSWI/SNF did not predict improved overall survival (OS), time-to-treatment failure (TTF), or disease control rate. Only patients with renal cell carcinoma with mSWI/SNF LOF showed significantly improved OS and TTF with adjusted HRs (95% confidence interval) of 0.33 (0.16–0.7) and 0.49 (0.27–0.88), respectively, and this was mostly driven by PRBM1. In the MSKCC cohort, where only OS was captured, LOF mSWI/SNF did not correlate with improved outcomes across any tumor subtype. We did not find a consistent association between mSWI/SNF LOF variants and improved clinical outcomes to ICIs, suggesting that mSWI/SNF variants should not be considered as biomarkers of response to ICIs.

Cancers, Jun 12, 2020

Monoclonal Gammopathy of Undetermined Significance (MGUS) is considered to be a benign precursor ... more Monoclonal Gammopathy of Undetermined Significance (MGUS) is considered to be a benign precursor condition that may progress to a lymphoproliferative disease or multiple myeloma. Most patients do not progress to an overt condition, but nevertheless, MGUS is associated with a shortened life expectancy and, in a minority of cases, a number of co-morbid conditions that include an increased fracture risk, renal impairment, peripheral neuropathy, secondary immunodeficiency, and cardiovascular disease. This review aims to consolidate current evidence for the significance of these co-morbidities before considering how best to approach these symptoms and signs, which are often encountered in primary care or within a number of specialties in secondary care.

Nature Genetics, Sep 22, 2022

Somatic mutations in cancer genes have been ubiquitously detected in clonal expansions across hea... more Somatic mutations in cancer genes have been ubiquitously detected in clonal expansions across healthy human tissue, including in clonal hematopoiesis. However, mutated and wildtype cells are morphologically and phenotypically similar, limiting the ability to link genotypes with cellular phenotypes. To overcome this limitation, we leveraged multi-modality single-cell sequencing, capturing the mutation with transcriptomes and methylomes in stem and progenitors from individuals with DNMT3A R882 mutated clonal hematopoiesis. DNMT3A mutations resulted in myeloid over lymphoid bias, and in expansion of immature myeloid progenitors primed toward megakaryocytic-erythroid fate. We observed dysregulated expression of lineage and leukemia stem cell markers. DNMT3A R882 led to preferential hypomethylation of polycomb repressive complex 2 targets and a specific sequence motif. Notably, the hypomethylation motif is enriched in binding motifs of key hematopoietic transcription factors, serving as a potential mechanistic link between DNMT3A R882 mutations and aberrant transcriptional phenotypes. Thus, single-cell multi-omics pave the road to defining the downstream consequences of mutations that drive human clonal mosaicism.

Clinical Lymphoma, Myeloma & Leukemia, Oct 1, 2019

Precursor states of Multiple Myeloma (MM) and its native tumor microenvironment need in-depth mol... more Precursor states of Multiple Myeloma (MM) and its native tumor microenvironment need in-depth molecular characterization to better stratify and treat patients at risk. Using single-cell RNA sequencing of bone marrow cells from precursor stages, MGUS and smoldering myeloma (SMM), to full-blown MM alongside healthy donors, we demonstrate early immune changes during patient

Blood, Dec 7, 2017

Introduction: Multiple myeloma (MM) is an incurable plasma cell malignancy that arises from the p... more Introduction: Multiple myeloma (MM) is an incurable plasma cell malignancy that arises from the precursor states: Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). Some patients rapidly progress from MGUS/SMM to MM, while others remain indolent with minimal progression over their lifetime. The genetic and molecular factors that underlie disease evolution and distinguish progressors from non-progressors are not well elucidated. To address these questions, we examined clinically-annotated samples from SMM patients, including sequential samples at the SMM stage and at time of progression. Methods: We utilized next generation sequencing methods to study 186 SMM patient samples. We performed whole exome sequencing (WES) of 70 tumor-germline matched samples (mean target coverage 50X/100X for germline/tumor) and targeted deep sequencing on 116 samples of progressor and non-progressor SMM (mean target coverage ~500X). We also performed ultra-low pass whole-genome sequencing (ULP-WGS) of 116 SMM samples as well as 20 available cell-free DNA (cfDNA) samples to detect the tumor fraction before proceeding with targeted sequencing. For WES, libraries were constructed with Agilent SureSelect XT2 library prep kit, and hybridized to Agilent9s whole exome V5+UTR capture probes and then sequenced on HiSeq 4000 (Illumina). For targeted sequencing, libraries were enriched by hybridizing to the customized target bait (60 MM related genes and 56 pan-cancer genes). For ULP-WGS, libraries were prepared with Kapa Hyper Prep kit and custom adapters. Sequencing data were analyzed using previously established analytic pipelines including ichorCNA, MuTect, RecapSeg, MutSig, GISTIC, ABSOLUTE, and PHYLOGIC. High-risk SMM (HRSMM) and low-risk SMM (LRSMM) were defined according to the criteria proposed by Rajkumar et al. in Blood 2014. Results: Higher mutation load (average 1.44 mutations/Mb) was observed in HRSMM patients, which is close to the average of 1.6 mutations/Mb in symptomatic MM, compared to LRSMM patients (average 0.73 mutations/Mb, p -value = 0.001). Somatic mutations in known signaling pathways were observed four times more frequently (43.8% vs. 9.5%, p- value = 0.015) in HRSMM patients compared to LRSMM patients. Mutations in MAPK pathway genes (KRAS, NRAS, BRAF) were detected in 21.7% of HRSMM vs. 0% in LRSMM (p- value = 0.004). Mutations in NFkB pathway were present in 10% of patients and were also associated with HRSMM (17.4% vs. 0%, p- value = 0.018). MM somatic copy number aberrations (SCNAs) were detected in 64% of all SMM samples, and were two times more frequently in HRSMM than LRSMM patients (78.1% vs. 42.9%, p- value = 0.020). Phylogenetic studies of sequential patient samples, who progressed from SMM to MM, revealed different patterns of clonal evolution. Furthermore, we have performed ULP-WGS of available cfDNA samples from 20 patients of this cohort. Interestingly, tumor fraction of cfDNA was higher in HRSMM compared to LRSMM patients (p- value = 0.03). Together, this data suggests that specific genomic alterations are associated with HRSMM and could potentially be predictive of the risk to progression in SMM patients. Conclusion: This study demonstrates that next generation sequencing (WES, targeted-sequencing, and ULP-WGS) of SMM patients can identify genomic alterations associated with HRSMM vs LRSMM through different parameters such as mutation load, somatic mutations, and SCNAs. Disclosures Laubach:Novartis, Takeda, Celgene: Consultancy; Novartis, Takeda, Celgene, Onyx: Research Funding. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Oncopeptides AB: Membership on an entity9s Board of Directors or advisory committees; Takeda: Consultancy, Research Funding. Anderson:C4 Therapeutics: Other: scientific founder; MedImmune: Membership on an entity9s Board of Directors or advisory committees; Gilead Sciences: Membership on an entity9s Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity9s Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity9s Board of Directors or advisory committees; Oncopep: Other: scientific founder. Kumar:Celgene, Millennium/Takeda, Onyx, AbbVie, Janssen, Sanofi, Novartis, Amgen, Genentech, Merck, Oncopeptides, Roche, Skyline Diagnostics: Research Funding; Celgene, Millennium, BMS, Onyx, Janssen, Noxxon, AbbVie, Amgen, Merck, Oncopeptides, Skyline Diagnostics, Takeda: Consultancy; Skyline: Honoraria.

JNCI: Journal of the National Cancer Institute

Background Treatment options for penile squamous cell carcinoma are limited. We sought to investi... more Background Treatment options for penile squamous cell carcinoma are limited. We sought to investigate clinical outcomes and safety profiles of patients with penile squamous cell carcinoma receiving immune checkpoint inhibitors. Methods This retrospective study included patients with locally advanced or metastatic penile squamous cell carcinoma receiving immune checkpoint inhibitors between 2015 and 2022 across 24 centers in the United States, Europe, and Asia. Overall survival and progression-free survival were estimated using the Kaplan-Meier method. Objective response rates were determined per Response Evaluation Criteria in Solid Tumours 1.1 criteria. Treatment-related adverse events were graded per the Common Terminology Criteria for Adverse Events, version 5.0. Two-sided statistical tests were used for comparisons. Results Among 92 patients, 8 (8.7%) were Asian, 6 (6.5%) were Black, and 24 (29%) were Hispanic and/or Latinx. Median (interquartile range) age was 62 (53-70) years....

Regular and Young Investigator Award Abstracts, Nov 1, 2022

Figure 2 Kaplan-Meier curves for Progression-Free Survival stratified by HIV status Conclusions I... more Figure 2 Kaplan-Meier curves for Progression-Free Survival stratified by HIV status Conclusions In this matched cohort study, PLWH and metastatic NSCLC had similar toxicity profiles and clinical outcomes to HIV-counterparts receiving ICI supporting their use in PLWH and their inclusion in clinical trials. Larger

Blood, 2021

Introduction: Multiple Myeloma (MM) is an incurable hematologic malignancy characterized by the a... more Introduction: Multiple Myeloma (MM) is an incurable hematologic malignancy characterized by the abnormal growth of clonal plasma cells in the bone marrow (BM). In most cases MM develops from early, asymptomatic disease stages known as Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). Despite effective new therapies, most MM patients inevitably relapse and require further treatment, highlighting the need for better early detection methods for precursor patients and targeted interventions to prevent early disease from progressing. The initial diagnosis of MGUS/SMM remains an incidental process following the identification of increased clonal immunoglobulin in the blood. BM biopsy is the gold standard for diagnosis and monitoring of MM progression, but is intrusive, painful, and comes with possible secondary complications for patients. Consequently, repeated assessment is not a feasible option for MGUS and SMM patients who are asymptomatic...

Cancer Discovery

Multiple myeloma (MM) develops from well-defined precursor stages; however, invasive bone marrow ... more Multiple myeloma (MM) develops from well-defined precursor stages; however, invasive bone marrow (BM) biopsy limits screening and monitoring strategies for patients. We enumerated circulating tumor cells (CTC) from 261 patients (84 monoclonal gammopathy of undetermined significance, 155 smoldering multiple myeloma, and 22 MM), with neoplastic cells detected in 84%. We developed a novel approach, MinimuMM-seq, which enables the detection of translocations and copy-number abnormalities through whole-genome sequencing of highly pure CTCs. Application to CTCs in a cohort of 51 patients, 24 with paired BM, was able to detect 100% of clinically reported BM biopsy events and could replace molecular cytogenetics for diagnostic yield and risk classification. Longitudinal sampling of CTCs in 8 patients revealed major clones could be tracked in the blood, with clonal evolution and shifting dynamics of subclones over time. Our findings provide proof of concept that CTC detection and genomic pro...

Journal of Vascular and Interventional Radiology, 2019

Purpose: To report the results of below-the-elbow (BTE) arterial revascularization in patients wi... more Purpose: To report the results of below-the-elbow (BTE) arterial revascularization in patients with critical hand ischemia (CHI) and End Stage Renal Disease (ESRD). Materials: We retrospectively identified all patients with critical limb ischemia treated with below-the-elbow arterial angioplasty and/or atherectomy between 2013 and 2017. Patient demographics, co-morbidities, and procedural data were reviewed. Rates of hand salvage, technical and clinical success were evaluated. Mean age at treatment was 59.7 (50-72). Average time to follow up was 9 months (2-26 months). Results: 7 ESRD patients (3 female) with a total of 9 hands affected by BTE arterial occlusion and CHI were treated. The technical success rate was 88.9% (8/9). No major or minor complications occurred. 6 hands (66.6%) suffered from non-healing ulcers and gangrene; the remaining 3 (33.3%) presented with rest pain. 1 patient died in the first 30 days of follow up due to preexisting respiratory disease. The mean treated lesion length was 147.5±97.9 mm. Among the successfully treated patients, the hand salvage rate was 87.5% (7/8). One patient did however undergo planned digital amputation following intervention with satisfactory healing of the postoperative stump. Conclusions: Arterial revascularization of the BTE arteries for CHI was a safe and a technically feasible method to improve the hand salvage rate in ESRD patients.

Journal of Clinical Oncology

PURPOSE Compared with people living without HIV (PWOH), people living with HIV (PWH) and cancer h... more PURPOSE Compared with people living without HIV (PWOH), people living with HIV (PWH) and cancer have traditionally been excluded from immune checkpoint inhibitor (ICI) trials. Furthermore, there is a paucity of real-world data on the use of ICIs in PWH and cancer. METHODS This retrospective study included PWH treated with anti–PD-1- or anti–PD-L1-based therapies for advanced cancers. Kaplan-Meier method was used to estimate overall survival (OS) and progression-free survival (PFS). Objective response rates (ORRs) were measured per RECIST 1.1 or other tumor-specific criteria, whenever feasible. Restricted mean survival time (RMST) was used to compare OS and PFS between matched PWH and PWOH with metastatic NSCLC (mNSCLC). RESULTS Among 390 PWH, median age was 58 years, 85% (n = 331) were males, 36% (n = 138) were Black; 70% (n = 274) received anti–PD-1/anti–PD-L1 monotherapy. Most common cancers were NSCLC (28%, n = 111), hepatocellular carcinoma ([HCC]; 11%, n = 44), and head and nec...