Publications by authors named "Catherine J Wu"

181 Publications

Natural Barcodes for Longitudinal Single Cell Tracking of Leukemic and Immune Cell Dynamics.

Front Immunol 2021 3;12:788891. Epub 2022 Jan 3.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, United States.

Blood malignancies provide unique opportunities for longitudinal tracking of disease evolution following therapeutic bottlenecks and for the monitoring of changes in anti-tumor immunity. The expanding development of multi-modal single-cell sequencing technologies affords newer platforms to elucidate the mechanisms underlying these processes at unprecedented resolution. Furthermore, the identification of molecular events that can serve as barcodes now facilitate the tracking of the trajectories of malignant and of immune cell populations over time within primary human samples, as these permit unambiguous identification of the clonal lineage of cell populations within heterogeneous phenotypes. Here, we provide an overview of the potential for chromosomal copy number changes, somatic nuclear and mitochondrial DNA mutations, single nucleotide polymorphisms, and T and B cell receptor sequences to serve as personal natural barcodes and review technical implementations in single-cell analysis workflows. Applications of these methodologies include the study of acquired therapeutic resistance and the dissection of donor- and host cellular interactions in the context of allogeneic hematopoietic stem cell transplantation.
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http://dx.doi.org/10.3389/fimmu.2021.788891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8761982PMC
January 2022

Durvalumab plus tremelimumab alone or in combination with low-dose or hypofractionated radiotherapy in metastatic non-small-cell lung cancer refractory to previous PD(L)-1 therapy: an open-label, multicentre, randomised, phase 2 trial.

Lancet Oncol 2022 Jan 13. Epub 2022 Jan 13.

Department of Data Science, Dana-Farber Cancer Institute, Boston, MA, USA.

Background: Patients with non-small-cell lung cancer (NSCLC) that is resistant to PD-1 and PD-L1 (PD[L]-1)-targeted therapy have poor outcomes. Studies suggest that radiotherapy could enhance antitumour immunity. Therefore, we investigated the potential benefit of PD-L1 (durvalumab) and CTLA-4 (tremelimumab) inhibition alone or combined with radiotherapy.

Methods: This open-label, multicentre, randomised, phase 2 trial was done by the National Cancer Institute Experimental Therapeutics Clinical Trials Network at 18 US sites. Patients aged 18 years or older with metastatic NSCLC, an Eastern Cooperative Oncology Group performance status of 0 or 1, and progression during previous PD(L)-1 therapy were eligible. They were randomly assigned (1:1:1) in a web-based system by the study statistician using a permuted block scheme (block sizes of three or six) without stratification to receive either durvalumab (1500 mg intravenously every 4 weeks for a maximum of 13 cycles) plus tremelimumab (75 mg intravenously every 4 weeks for a maximum of four cycles) alone or with low-dose (0·5 Gy delivered twice per day, repeated for 2 days during each of the first four cycles of therapy) or hypofractionated radiotherapy (24 Gy total delivered over three 8-Gy fractions during the first cycle only), 1 week after initial durvalumab-tremelimumab administration. Study treatment was continued until 1 year or until progression. The primary endpoint was overall response rate (best locally assessed confirmed response of a partial or complete response) and, along with safety, was analysed in patients who received at least one dose of study therapy. The trial is registered with ClinicalTrials.gov, NCT02888743, and is now complete.

Findings: Between Aug 24, 2017, and March 29, 2019, 90 patients were enrolled and randomly assigned, of whom 78 (26 per group) were treated. This trial was stopped due to futility assessed in an interim analysis. At a median follow-up of 12·4 months (IQR 7·8-15·1), there were no differences in overall response rates between the durvalumab-tremelimumab alone group (three [11·5%, 90% CI 1·2-21·8] of 26 patients) and the low-dose radiotherapy group (two [7·7%, 0·0-16·3] of 26 patients; p=0·64) or the hypofractionated radiotherapy group (three [11·5%, 1·2-21·8] of 26 patients; p=0·99). The most common grade 3-4 adverse events were dyspnoea (two [8%] in the durvalumab-tremelimumab alone group; three [12%] in the low-dose radiotherapy group; and three [12%] in the hypofractionated radiotherapy group) and hyponatraemia (one [4%] in the durvalumab-tremelimumab alone group vs two [8%] in the low-dose radiotherapy group vs three [12%] in the hypofractionated radiotherapy group). Treatment-related serious adverse events occurred in one (4%) patient in the durvalumab-tremelimumab alone group (maculopapular rash), five (19%) patients in the low-dose radiotherapy group (abdominal pain, diarrhoea, dyspnoea, hypokalemia, and respiratory failure), and four (15%) patients in the hypofractionated group (adrenal insufficiency, colitis, diarrhoea, and hyponatremia). In the low-dose radiotherapy group, there was one death from respiratory failure potentially related to study therapy.

Interpretation: Radiotherapy did not increase responses to combined PD-L1 plus CTLA-4 inhibition in patients with NSCLC resistant to PD(L)-1 therapy. However, PD-L1 plus CTLA-4 therapy could be a treatment option for some patients. Future studies should refine predictive biomarkers in this setting.

Funding: The US National Institutes of Health and the Dana-Farber Cancer Institute.
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http://dx.doi.org/10.1016/S1470-2045(21)00658-6DOI Listing
January 2022

Cancer and COVID-19: On the Quest for Effective Vaccines.

Blood Cancer Discov 2021 01 17;2(1):13-18. Epub 2020 Dec 17.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.

Cancer vaccine development has been historically fraught with difficulty, but tremendous progress has been made over the past 5 years. In this article, we reflect on the progress and challenges with vaccine development for cancers in general and for hematologic malignancies in particular, and suggest how our cancer vaccine experience can offer insight into COVID-19 vaccination.
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http://dx.doi.org/10.1158/2643-3230.BCD-20-0205DOI Listing
January 2021

Integrative clinical and molecular characterization of translocation renal cell carcinoma.

Cell Rep 2022 01;38(1):110190

Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA.

Translocation renal cell carcinoma (tRCC) is a poorly characterized subtype of kidney cancer driven by MiT/TFE gene fusions. Here, we define the landmarks of tRCC through an integrative analysis of 152 patients with tRCC identified across genomic, clinical trial, and retrospective cohorts. Most tRCCs harbor few somatic alterations apart from MiT/TFE fusions and homozygous deletions at chromosome 9p21.3 (19.2% of cases). Transcriptionally, tRCCs display a heightened NRF2-driven antioxidant response that is associated with resistance to targeted therapies. Consistently, we find that outcomes for patients with tRCC treated with vascular endothelial growth factor receptor inhibitors (VEGFR-TKIs) are worse than those treated with immune checkpoint inhibitors (ICI). Using multiparametric immunofluorescence, we find that the tumors are infiltrated with CD8 T cells, though the T cells harbor an exhaustion immunophenotype distinct from that of clear cell RCC. Our findings comprehensively define the clinical and molecular features of tRCC and may inspire new therapeutic hypotheses.
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http://dx.doi.org/10.1016/j.celrep.2021.110190DOI Listing
January 2022

Clonal Evolution of High-Risk Chronic Lymphocytic Leukemia: A Contemporary Perspective.

Front Oncol 2021 16;11:790004. Epub 2021 Dec 16.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, United States.

Clonal evolution represents the natural process through which cancer cells continuously search for phenotypic advantages that enable them to develop and expand within microenvironmental constraints. In chronic lymphocytic leukemia (CLL), clonal evolution underpins leukemic progression and therapeutic resistance, with differences in clonal evolutionary dynamics accounting for its characteristically diverse clinical course. The past few years have witnessed profound changes in our understanding of CLL clonal evolution, facilitated by a maturing definition of high-risk CLL and an increasing sophistication of next-generation sequencing technology. In this review, we offer a modern perspective on clonal evolution of high-risk CLL, highlighting recent discoveries, paradigm shifts and unresolved questions. We appraise recent advances in our understanding of the molecular basis of CLL clonal evolution, focusing on the genetic and non-genetic sources of intratumoral heterogeneity, as well as tumor-immune dynamics. We review the technological innovations, particularly in single-cell technology, which have fostered these advances and represent essential tools for future discoveries. In addition, we discuss clonal evolution within several contexts of particular relevance to contemporary clinical practice, including the settings of therapeutic resistance to CLL targeted therapy and immunotherapy, as well as Richter transformation of CLL to high-grade lymphoma.
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http://dx.doi.org/10.3389/fonc.2021.790004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8716560PMC
December 2021

Understanding CLL biology through mouse models of human genetics.

Blood 2021 12;138(25):2621-2631

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA.

Rapid advances in large-scale next-generation sequencing studies of human samples have progressively defined the highly heterogeneous genetic landscape of chronic lymphocytic leukemia (CLL). At the same time, the numerous challenges posed by the difficulties in rapid manipulation of primary B cells and the paucity of CLL cell lines have limited the ability to interrogate the function of the discovered putative disease "drivers," defined in human sequencing studies through statistical inference. Mouse models represent a powerful tool to study mechanisms of normal and malignant B-cell biology and for preclinical testing of novel therapeutics. Advances in genetic engineering technologies, including the introduction of conditional knockin/knockout strategies, have opened new opportunities to model genetic lesions in a B-cell-restricted context. These new studies build on the experience of generating the MDR mice, the first example of a genetically faithful CLL model, which recapitulates the most common genomic aberration of human CLL: del(13q). In this review, we describe the application of mouse models to the studies of CLL pathogenesis and disease transformation from an indolent to a high-grade malignancy (ie, Richter syndrome [RS]) and treatment, with a focus on newly developed genetically inspired mouse lines modeling recurrent CLL genetic events. We discuss how these novel mouse models, analyzed using new genomic technologies, allow the dissection of mechanisms of disease evolution and response to therapy with greater depth than previously possible and provide important insight into human CLL and RS pathogenesis and therapeutic vulnerabilities. These models thereby provide valuable platforms for functional genomic analyses and treatment studies.
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http://dx.doi.org/10.1182/blood.2021011993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8703365PMC
December 2021

Multifunctional barcoding with ClonMapper enables high-resolution study of clonal dynamics during tumor evolution and treatment.

Nat Cancer 2021 Jul 12;2(7):758-772. Epub 2021 Jul 12.

Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.

Lineage-tracing methods have enabled characterization of clonal dynamics in complex populations, but generally lack the ability to integrate genomic, epigenomic and transcriptomic measurements with live-cell manipulation of specific clones of interest. We developed a functionalized lineage-tracing system, ClonMapper, which integrates DNA barcoding with single-cell RNA sequencing and clonal isolation to comprehensively characterize thousands of clones within heterogeneous populations. Using ClonMapper, we identified subpopulations of a chronic lymphocytic leukemia cell line with distinct clonal compositions, transcriptional signatures and chemotherapy survivorship trajectories; patterns that were also observed in primary human chronic lymphocytic leukemia. The ability to retrieve specific clones before, during and after treatment enabled direct measurements of clonal diversification and durable subpopulation transcriptional signatures. ClonMapper is a powerful multifunctional approach to dissect the complex clonal dynamics of tumor progression and therapeutic response.
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http://dx.doi.org/10.1038/s43018-021-00222-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8691751PMC
July 2021

GM-CSF secreting leukemia cell vaccination for MDS/AML after allogeneic HSCT: a randomized double blinded phase 2 trial.

Blood Adv 2021 Nov 22. Epub 2021 Nov 22.

Dana-Farber Cancer Institute, Boston, Massachusetts, United States.

Vaccination using irradiated, adenovirus transduced autologous myeloblasts to secrete GM-CSF (GVAX) early after allogeneic hematopoietic stem cell transplantation (HSCT) can induce potent immune responses. We conducted a randomized phase II trial of GVAX after HSCT for MDS-EB or relapsed/refractory AML. Myeloblasts were harvested before HSCT to generate the vaccine. Randomization to GVAX vs. placebo (1:1) was stratified by disease, transplant center, and conditioning. GVHD prophylaxis included tacrolimus and methotrexate. GVAX or placebo started between day +30-45 if there was engraftment and no GVHD. Vaccines were administered SC/ID weekly x 3, then q2 wks x 3. Tacrolimus taper began after vaccine completion. 123 patients enrolled, 92 proceeded to HSCT, and 57 (GVAX 30, Placebo 27) received at least 1 vaccination. No CTC grade ≥ 3 vaccine related adverse events were reported, but injection site reactions were more common after GVAX (10 vs. 1, p=0.006). With a median follow up of 39 months (range, 9-89), 18-month PFS, OS and relapse incidence were 53% vs 55% (p=0.79), 63% vs. 59% (p= 0.86), and 30% vs. 37% (p=0.51) for GVAX and placebo, respectively. NRM at 18 months was 17% vs. 7.7% (p=0.18), Grade II-IV aGVHD at 12 months 34% vs. 12% (p=0.13), and cGVHD at 3 years 49% vs. 57% for GVAX and placebo, respectively, p=0.26. Reconstitution of T, B, and NK cells were not decreased or enhanced by GVAX. There were no differences in serum MICA/B or other immune biomarkers between GVAX and placebo. GVAX does not improve survival after HSCT for MDS/AML. (Clinicaltrials.gov identifier: NCT01773395).
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http://dx.doi.org/10.1182/bloodadvances.2021006255DOI Listing
November 2021

Venetoclax plus Dose-Adjusted R-EPOCH (VR-EPOCH) for Richter's Syndrome.

Blood 2021 Nov 17. Epub 2021 Nov 17.

DFCI / Harvard Medical School, Boston, Massachusetts, United States.

Richter's Syndrome (RS) of chronic lymphocytic leukemia (CLL) is typically chemoresistant, with a poor prognosis. We hypothesized that the oral Bcl-2 inhibitor venetoclax could sensitize RS to chemoimmunotherapy and improve outcomes. We conducted a single-arm, investigator-sponsored, phase 2 trial of venetoclax plus dose-adjusted rituximab, etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (VR-EPOCH) to determine the rate of complete response (CR). Patients received R-EPOCH for 1 cycle, then after count recovery, accelerated daily venetoclax ramp-up to 400 mg, then VR-EPOCH for up to 5 more 21-day cycles. Responders received venetoclax maintenance or cellular therapy off-study. Twenty-six patients were treated, and 13 of 26 (50%) achieved CR, with 11 achieving undetectable bone marrow minimal residual disease for CLL. Three additional patients achieved partial response (overall response rate 62%). Median progression-free survival was 10.1 months, and median overall survival was 19.6 months. Hematologic toxicity included grade ≥3 neutropenia (65%) and thrombocytopenia (50%), with febrile neutropenia in 38%. No patients experienced tumor lysis syndrome with daily venetoclax ramp-up. VR-EPOCH is active in RS, with deeper, more durable responses than historical regimens. Toxicities from intensive chemoimmunotherapy and venetoclax were observed. Our data suggest that studies comparing venetoclax with chemoimmunotherapy to chemoimmunotherapy alone are warranted. (Funded by Genentech/AbbVie; ClinicalTrials.gov number, NCT03054896).
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http://dx.doi.org/10.1182/blood.2021011386DOI Listing
November 2021

Mapping the evolution of T cell states during response and resistance to adoptive cellular therapy.

Cell Rep 2021 11;37(6):109992

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Harvard Medical School, Boston, MA 02115, USA. Electronic address:

To elucidate mechanisms by which T cells eliminate leukemia, we study donor lymphocyte infusion (DLI), an established immunotherapy for relapsed leukemia. We model T cell dynamics by integrating longitudinal, multimodal data from 94,517 bone marrow-derived single T cell transcriptomes in addition to chromatin accessibility and single T cell receptor sequencing from patients undergoing DLI. We find that responsive tumors are defined by enrichment of late-differentiated T cells before DLI and rapid, durable expansion of early differentiated T cells after treatment, highly similar to "terminal" and "precursor" exhausted subsets, respectively. Resistance, in contrast, is defined by heterogeneous T cell dysfunction. Surprisingly, early differentiated T cells in responders mainly originate from pre-existing and novel clonotypes recruited to the leukemic microenvironment, rather than the infusion. Our work provides a paradigm for analyzing longitudinal single-cell profiling of scenarios beyond adoptive cell therapy and introduces Symphony, a Bayesian approach to infer regulatory circuitry underlying T cell subsets, with broad relevance to exhaustion antagonists across cancers.
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http://dx.doi.org/10.1016/j.celrep.2021.109992DOI Listing
November 2021

Activation of and Signaling via B-cell-Restricted Depletion of Generates a Consistent Murine Model of Chronic Lymphocytic Leukemia.

Cancer Res 2021 12 22;81(24):6117-6130. Epub 2021 Oct 22.

Department of Genome Regulation, Max Planck Institute for Molecular Genetics, Berlin, Germany.

Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase is a key regulator of DNA methylation. Although somatic mutations in CLL are rare, we found that low expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell of origin of disease in this model, and depletion resulted in focal hypomethylation and activation of and signaling. Amplification of chromosome 15 containing the gene was detected in all CLL mice tested, and infiltration of high--expressing CLL cells in the spleen was observed. Notably, hyperactivation of and signaling was exclusively observed in the CLL mice, but not in three other CLL mouse models tested (, , and ), and -depleted CLL were sensitive to pharmacologic inhibition of signaling and . Consistent with these findings, human CLL samples with lower expression were more sensitive to inhibition than those with higher expression. Altogether, these results suggest that depletion induces CLL that is highly dependent on activation of and signaling. SIGNIFICANCE: Loss of expression is a driving event in CLL and is associated with aggressive disease, activation of and signaling, and enhanced sensitivity to inhibition.
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http://dx.doi.org/10.1158/0008-5472.CAN-21-1273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8678341PMC
December 2021

Unannotated proteins expand the MHC-I-restricted immunopeptidome in cancer.

Nat Biotechnol 2021 Oct 18. Epub 2021 Oct 18.

Klarman Cell Observatory, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Tumor-associated epitopes presented on MHC-I that can activate the immune system against cancer cells are typically identified from annotated protein-coding regions of the genome, but whether peptides originating from novel or unannotated open reading frames (nuORFs) can contribute to antitumor immune responses remains unclear. Here we show that peptides originating from nuORFs detected by ribosome profiling of malignant and healthy samples can be displayed on MHC-I of cancer cells, acting as additional sources of cancer antigens. We constructed a high-confidence database of translated nuORFs across tissues (nuORFdb) and used it to detect 3,555 translated nuORFs from MHC-I immunopeptidome mass spectrometry analysis, including peptides that result from somatic mutations in nuORFs of cancer samples as well as tumor-specific nuORFs translated in melanoma, chronic lymphocytic leukemia and glioblastoma. NuORFs are an unexplored pool of MHC-I-presented, tumor-specific peptides with potential as immunotherapy targets.
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http://dx.doi.org/10.1038/s41587-021-01021-3DOI Listing
October 2021

Cancer and COVID-19: On the Quest for Effective Vaccines.

Blood Cancer Discov 2021 Jan 17;2(1):13-18. Epub 2020 Dec 17.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.

Cancer vaccine development has been historically fraught with difficulty, but tremendous progress has been made over the past 5 years. In this article, we reflect on the progress and challenges with vaccine development for cancers in general and for hematologic malignancies in particular, and suggest how our cancer vaccine experience can offer insight into COVID-19 vaccination.
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http://dx.doi.org/10.1158/2643-3230.BCD-20-0205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8500734PMC
January 2021

Impact of cryopreservation and transit times of allogeneic grafts on hematopoietic and immune reconstitution.

Blood Adv 2021 12;5(23):5140-5149

Department of Medical Oncology and.

We sought to evaluate the impact of cryopreservation of unrelated donor (URD) peripheral blood stem cell (PBSC) grafts on engraftment, chimerism, and immune reconstitution in the context of the COVID-19 pandemic. We reviewed stem cell product characteristics and clinical outcomes in 101 patients receiving cryopreserved PBSCs from URDs between January 1, 2019 and 31 December, 2020, compared with 203 patients receiving fresh URD PBSCs. We observed no differences in 6-month overall survival, progression-free survival, or nonrelapse mortality. Patients receiving cryopreserved PBSCs had delayed platelet engraftment and impaired reconstitution of white blood cells and T-cell subsets at day 30. Thirty-four percent of patients receiving cryopreserved grafts had CD3 chimerism <50% at day 30 after transplantation, compared with 14% of patients receiving fresh PBSCs (P = .0002). At day 100, this difference persisted (CD3+ chimerism <50%: 17% of cryopreserved cohort vs 6% of fresh cohort; P = .016). Greater product age at infusion was associated with increased graft failure, independent of cryopreservation. Receipt of grafts >48 hours old at time of cryopreservation or infusion significantly increased the risk of graft failure (subdistribution hazard ratio = 4.57; 95% confidence interval, 1.71-12.3; P = .0025). Our data indicate that cryopreservation is associated with similar overall short-term clinical outcomes compared with fresh PBSC. However, patients must be monitored closely for increased risk of other potentially adverse outcomes, including graft failure and poor immune recovery, particularly for grafts with older overall age at infusion. Longer-term follow-up is needed to determine impact on relapse and survival.
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http://dx.doi.org/10.1182/bloodadvances.2021005139DOI Listing
December 2021

Multi-platform profiling characterizes molecular subgroups and resistance networks in chronic lymphocytic leukemia.

Nat Commun 2021 09 13;12(1):5395. Epub 2021 Sep 13.

Department I for Internal Medicine and Centre for Integrated Oncology, University of Cologne, Cologne, Germany.

Knowledge of the genomic landscape of chronic lymphocytic leukemia (CLL) grows increasingly detailed, providing challenges in contextualizing the accumulated information. To define the underlying networks, we here perform a multi-platform molecular characterization. We identify major subgroups characterized by genomic instability (GI) or activation of epithelial-mesenchymal-transition (EMT)-like programs, which subdivide into non-inflammatory and inflammatory subtypes. GI CLL exhibit disruption of genome integrity, DNA-damage response and are associated with mutagenesis mediated through activation-induced cytidine deaminase or defective mismatch repair. TP53 wild-type and mutated/deleted cases constitute a transcriptionally uniform entity in GI CLL and show similarly poor progression-free survival at relapse. EMT-like CLL exhibit high genomic stability, reduced benefit from the addition of rituximab and EMT-like differentiation is inhibited by induction of DNA damage. This work extends the perspective on CLL biology and risk categories in TP53 wild-type CLL. Furthermore, molecular targets identified within each subgroup provide opportunities for new treatment approaches.
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http://dx.doi.org/10.1038/s41467-021-25403-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8438057PMC
September 2021

Coevolving JAK2V617F+relapsed AML and donor T cells with PD-1 blockade after stem cell transplantation: an index case.

Blood Adv 2021 11;5(22):4701-4709

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA.

Relapse of myeloproliferative neoplasms (MPNs) after allogeneic hematopoietic stem cell transplantation (HSCT) is associated with poor outcomes, as therapeutic approaches to reinstate effective graft-versus-leukemia (GVL) responses remain suboptimal. Immune escape through overexpression of PD-L1 in JAK2V617F-mutated MPN provides a rationale for therapeutic PD-1 blockade, and indeed, clinical activity of nivolumab in relapsed MPN post-HSCT has been observed. Elucidation of the features of response following PD-1 blockade in such patients could inform novel therapeutic concepts that enhance GVL. Here, we report an integrated high-dimensional analysis using single-cell RNA sequencing, T-cell receptor sequencing, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), and assay for transposase-accessible chromatin using sequencing (scATAC-seq), together with mass cytometry, in peripheral blood mononuclear cells collected at 6 timepoints before, during, and after transient response to PD-1 blockade from an index case of relapsed MPN following HSCT. Before nivolumab infusion, acute myeloid leukemia (AML) blasts demonstrated high expression of chemokines, and T cells were characterized by expression of interferon-response genes. This baseline inflammatory signature disappeared after nivolumab infusion. Clinical response was characterized by transient expansion of a polyclonal CD4+ T-cell population and contraction of an AML subpopulation that exhibited megakaryocytic features and elevated PD-L1 expression. At relapse, the proportion of the AML subpopulation with progenitor-like features progressively increased, suggesting coevolution of AML blasts and donor-derived T cells. We thus demonstrate how single-cell technologies can provide complementary insight into cellular mechanisms underlying response to PD-1 blockade, motivating future longitudinal high-dimensional single-cell studies of GVL responses in relapsed myeloid disease.
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http://dx.doi.org/10.1182/bloodadvances.2021004335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8759138PMC
November 2021

Optimized Liquid and Gas Phase Fractionation Increases HLA-Peptidome Coverage for Primary Cell and Tissue Samples.

Mol Cell Proteomics 2021 12;20:100133. Epub 2021 Aug 12.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. Electronic address:

MS is the most effective method to directly identify peptides presented on human leukocyte antigen (HLA) molecules. However, current standard approaches often use 500 million or more cells as input to achieve high coverage of the immunopeptidome, and therefore, these methods are not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples offline followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared with samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8107 distinct peptides starting with as few as 100 million cells. The increased sensitivity obtained using our methods can provide data useful to improve HLA-binding prediction algorithms as well as to enable detection of clinically relevant epitopes such as neoantigens.
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http://dx.doi.org/10.1016/j.mcpro.2021.100133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8724927PMC
August 2021

Clonal hematopoiesis in patients receiving chimeric antigen receptor T-cell therapy.

Blood Adv 2021 08;5(15):2982-2986

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA.

Chimeric antigen receptor (CAR) T-cells have emerged as an efficacious modality in patients with non-Hodgkin lymphoma (NHL) and multiple myeloma (MM). Clonal hematopoiesis of indeterminate potential (CHIP), a state in which mutations in hematopoietic cells give rise to a clonal population of cells, is more common in patients exposed to cytotoxic therapies, has been shown to influence inflammatory immune programs, and is associated with an adverse prognosis in patients with NHL and MM receiving autologous transplantation. We therefore hypothesized that CHIP could influence clinical outcomes in patients receiving CAR T-cell therapy. In a cohort of 154 patients with NHL or MM receiving CAR T-cells, we found that CHIP was present in 48% of patients and associated with increased rates of complete response and cytokine release syndrome severity, but only in patients younger than age 60 years. Despite these differences, CHIP was not associated with a difference in progression-free or overall survival, regardless of age. Our data suggest that CHIP can influence CAR T-cell biology and clinical outcomes, but, in contrast to autologous transplantation, CHIP was not associated with worse survival and should not be a reason to exclude individuals from receiving this potentially life-prolonging treatment.
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http://dx.doi.org/10.1182/bloodadvances.2021004554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8361461PMC
August 2021

Phenotype, specificity and avidity of antitumour CD8 T cells in melanoma.

Nature 2021 08 21;596(7870):119-125. Epub 2021 Jul 21.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses; however, the relationship between phenotypic characteristics and TCR properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8 T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide-human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire.
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http://dx.doi.org/10.1038/s41586-021-03704-yDOI Listing
August 2021

Immune Profiling Mass Cytometry Assay Harmonization: Multicenter Experience from CIMAC-CIDC.

Clin Cancer Res 2021 09 15;27(18):5062-5071. Epub 2021 Jul 15.

Human Immune Monitoring Center, Icahn School of Medicine at Mount Sinai, New York, New York.

Purpose: The Cancer Immune Monitoring and Analysis Centers - Cancer Immunologic Data Commons (CIMAC-CIDC) Network is supported by the NCI to identify biomarkers of response to cancer immunotherapies across clinical trials using state-of-the-art assays. A primary platform for CIMAC-CIDC studies is cytometry by time of flight (CyTOF), performed at all CIMAC laboratories. To ensure the ability to generate comparable CyTOF data across labs, a multistep cross-site harmonization effort was undertaken.

Experimental Design: We first harmonized standard operating procedures (SOPs) across the CIMAC sites. Because of a new acquisition protocol comparing original narrow- or new wide-bore injector introduced by the vendor (Fluidigm), we also tested this protocol across sites before finalizing the harmonized SOP. We then performed cross-site assay harmonization experiments using five shared cryopreserved and one lyophilized internal control peripheral blood mononuclear cell (PBMC) with a shared lyophilized antibody cocktail consisting of 14 isotype-tagged antibodies previously validated, plus additional liquid antibodies. These reagents and samples were distributed to the CIMAC sites and the data were centrally analyzed by manual gating and automated methods (Astrolabe).

Results: Average coefficients of variation (CV) across sites for each cell population were reported and compared with a previous multisite CyTOF study. We reached an intersite CV of under 20% for most cell subsets, very similar to a previously published study.

Conclusions: These results establish the ability to reproduce CyTOF data across sites in multicenter clinical trials, and also highlight the importance of quality control procedures, such as the use of spike-in control samples, for tracking variability in this assay.
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http://dx.doi.org/10.1158/1078-0432.CCR-21-2052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8448982PMC
September 2021

Longitudinal Single-Cell Dynamics of Chromatin Accessibility and Mitochondrial Mutations in Chronic Lymphocytic Leukemia Mirror Disease History.

Cancer Discov 2021 Jun 10. Epub 2021 Jun 10.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.

While cancers evolve during disease progression and in response to therapy, temporal dynamics remain difficult to study in humans due to the lack of consistent barcodes marking individual clones . We employ mitochondrial single-cell assay for transposase-accessible chromatin with sequencing to profile 163,279 cells from 9 patients with chronic lymphocytic leukemia (CLL) collected across disease course and utilize mitochondrial DNA (mtDNA) mutations as natural genetic markers of cancer clones. We observe stable propagation of mtDNA mutations over years in the absence of strong selective pressure, indicating clonal persistence, but dramatic changes following tight bottlenecks, including disease transformation and relapse posttherapy, paralleled by acquisition of copy-number variants and changes in chromatin accessibility and gene expression. Furthermore, we link CLL subclones to distinct chromatin states, providing insight into nongenetic sources of relapse. mtDNA mutations thus mirror disease history and provide naturally occurring genetic barcodes to enable patient-specific study of cancer subclonal dynamics. SIGNIFICANCE: Single-cell multi-omic profiling of CLL reveals the utility of somatic mtDNA mutations as barcodes, which mark subclones that can evolve over time along with changes in accessible chromatin and gene expression profiles to capture dynamics of disease evolution.
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http://dx.doi.org/10.1158/2159-8290.CD-21-0276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8660953PMC
June 2021

60 Years Young: The Evolving Role of Allogeneic Hematopoietic Stem Cell Transplantation in Cancer Immunotherapy.

Cancer Res 2021 09 9;81(17):4373-4384. Epub 2021 Jun 9.

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts.

The year 2020 marked the 30th anniversary of the Nobel Prize in Medicine awarded to E. Donnall Thomas for the development of allogeneic hematopoietic stem cell transplantation (allo-HSCT) to treat hematologic malignancies and other blood disorders. Dr. Thomas, "father of bone marrow transplantation," first developed and reported this technique in 1957, and in the ensuing decades, this seminal study has impacted fundamental work in hematology and cancer research, including advances in hematopoiesis, stem cell biology, tumor immunology, and T-cell biology. As the first example of cancer immunotherapy, understanding the mechanisms of antitumor biology associated with allo-HSCT has given rise to many of the principles used today in the development and implementation of novel transformative immunotherapies. Here we review the historical basis underpinning the development of allo-HSCT as well as advances in knowledge obtained by defining mechanisms of allo-HSCT activity. We review how these principles have been translated to novel immunotherapies currently utilized in clinical practice and describe potential future applications for allo-HSCT in cancer research and development of novel therapeutic strategies.
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http://dx.doi.org/10.1158/0008-5472.CAN-21-0301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8416782PMC
September 2021

Discovery of Candidate DNA Methylation Cancer Driver Genes.

Cancer Discov 2021 Sep 10;11(9):2266-2281. Epub 2021 May 10.

Institute for Computational Biomedicine, Weill Cornell Medicine, New York, New York.

Epigenetic alterations, such as promoter hypermethylation, may drive cancer through tumor suppressor gene inactivation. However, we have limited ability to differentiate driver DNA methylation (DNAme) changes from passenger events. We developed DNAme driver inference-MethSig-accounting for the varying stochastic hypermethylation rate across the genome and between samples. We applied MethSig to bisulfite sequencing data of chronic lymphocytic leukemia (CLL), multiple myeloma, ductal carcinoma , glioblastoma, and to methylation array data across 18 tumor types in TCGA. MethSig resulted in well-calibrated quantile-quantile plots and reproducible inference of likely DNAme drivers with increased sensitivity/specificity compared with benchmarked methods. CRISPR/Cas9 knockout of selected candidate CLL DNAme drivers provided a fitness advantage with and without therapeutic intervention. Notably, DNAme driver risk score was closely associated with adverse outcome in independent CLL cohorts. Collectively, MethSig represents a novel inference framework for DNAme driver discovery to chart the role of aberrant DNAme in cancer. SIGNIFICANCE: MethSig provides a novel statistical framework for the analysis of DNA methylation changes in cancer, to specifically identify candidate DNA methylation driver genes of cancer progression and relapse, empowering the discovery of epigenetic mechanisms that enhance cancer cell fitness..
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http://dx.doi.org/10.1158/2159-8290.CD-20-1334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8419066PMC
September 2021

Splice it up: Atypical transcripts to boost leukemia immunotherapy.

Immunity 2021 04;54(4):608-610

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA; Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA. Electronic address:

Neoantigens are prime targets for cancer immunotherapy, but their identification in low mutational burden malignancies remains challenging. In this issue of Immunity, Ehx et al. show that atypical transcripts, and particularly retained introns, expand the spectrum of leukemia immunotherapy targets.
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http://dx.doi.org/10.1016/j.immuni.2021.03.016DOI Listing
April 2021

Molecular and cellular features of CTLA-4 blockade for relapsed myeloid malignancies after transplantation.

Blood 2021 06;137(23):3212-3217

Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, MD; and.

Relapsed myeloid disease after allogeneic stem cell transplantation (HSCT) remains largely incurable. We previously demonstrated the potent activity of immune checkpoint blockade in this clinical setting with ipilimumab or nivolumab. To define the molecular and cellular pathways by which CTLA-4 blockade with ipilimumab can reinvigorate an effective graft-versus-leukemia (GVL) response, we integrated transcriptomic analysis of leukemic biopsies with immunophenotypic profiling of matched peripheral blood samples collected from patients treated with ipilimumab following HSCT on the Experimental Therapeutics Clinical Trials Network 9204 trial. Response to ipilimumab was associated with transcriptomic evidence of increased local CD8+ T-cell infiltration and activation. Systemically, ipilimumab decreased naïve and increased memory T-cell populations and increased expression of markers of T-cell activation and costimulation such as PD-1, HLA-DR, and ICOS, irrespective of response. However, responding patients were characterized by higher turnover of T-cell receptor sequences in peripheral blood and showed increased expression of proinflammatory chemokines in plasma that was further amplified by ipilimumab. Altogether, these data highlight the compositional T-cell shifts and inflammatory pathways induced by ipilimumab both locally and systemically that associate with successful GVL outcomes. This trial was registered at www.clinicaltrials.gov as #NCT01822509.
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http://dx.doi.org/10.1182/blood.2021010867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8351891PMC
June 2021

Progressive immune dysfunction with advancing disease stage in renal cell carcinoma.

Cancer Cell 2021 05 11;39(5):632-648.e8. Epub 2021 Mar 11.

Section for Bioinformatics, Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark; Center for Genomic Medicine, Rigshospitalet - Copenhagen University Hospital, Copenhagen, Denmark.

The tumor immune microenvironment plays a critical role in cancer progression and response to immunotherapy in clear cell renal cell carcinoma (ccRCC), yet the composition and phenotypic states of immune cells in this tumor are incompletely characterized. We performed single-cell RNA and T cell receptor sequencing on 164,722 individual cells from tumor and adjacent non-tumor tissue in patients with ccRCC across disease stages: early, locally advanced, and advanced/metastatic. Terminally exhausted CD8 T cells were enriched in metastatic disease and were restricted in T cell receptor diversity. Within the myeloid compartment, pro-inflammatory macrophages were decreased, and suppressive M2-like macrophages were increased in advanced disease. Terminally exhausted CD8 T cells and M2-like macrophages co-occurred in advanced disease and expressed ligands and receptors that support T cell dysfunction and M2-like polarization. This immune dysfunction circuit is associated with a worse prognosis in external cohorts and identifies potentially targetable immune inhibitory pathways in ccRCC.
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http://dx.doi.org/10.1016/j.ccell.2021.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138872PMC
May 2021

A hotspot mutation in transcription factor IKZF3 drives B cell neoplasia via transcriptional dysregulation.

Cancer Cell 2021 03;39(3):380-393.e8

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. Electronic address:

Hotspot mutation of IKZF3 (IKZF3-L162R) has been identified as a putative driver of chronic lymphocytic leukemia (CLL), but its function remains unknown. Here, we demonstrate its driving role in CLL through a B cell-restricted conditional knockin mouse model. Mutant Ikzf3 alters DNA binding specificity and target selection, leading to hyperactivation of B cell receptor (BCR) signaling, overexpression of nuclear factor κB (NF-κB) target genes, and development of CLL-like disease in elderly mice with a penetrance of ~40%. Human CLL carrying either IKZF3 mutation or high IKZF3 expression was associated with overexpression of BCR/NF-κB pathway members and reduced sensitivity to BCR signaling inhibition by ibrutinib. Our results thus highlight IKZF3 oncogenic function in CLL via transcriptional dysregulation and demonstrate that this pro-survival function can be achieved by either somatic mutation or overexpression of this CLL driver. This emphasizes the need for combinatorial approaches to overcome IKZF3-mediated BCR inhibitor resistance.
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http://dx.doi.org/10.1016/j.ccell.2021.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034546PMC
March 2021

Preneoplastic Alterations Define CLL DNA Methylome and Persist through Disease Progression and Therapy.

Blood Cancer Discov 2021 Jan 3;2(1):54-69. Epub 2020 Dec 3.

Department of Genome Regulation, Max Planck Institute for Molecular Genetics, Berlin, Germany.

Most human cancers converge to a deregulated methylome with reduced global levels and elevated methylation at select CpG islands. To investigate the emergence and dynamics of the cancer methylome, we characterized genome-wide DNA methylation in pre-neoplastic monoclonal B cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL), including serial samples collected across disease course. We detected the aberrant tumor-associated methylation landscape at CLL diagnosis and found no significantly differentially methylated regions in the high-count MBL-to-CLL transition. Patient methylomes showed remarkable stability with natural disease and post-therapy progression. Single CLL cells were consistently aberrantly methylated, indicating a homogeneous transition to the altered epigenetic state, and a distinct expression profile together with MBL cells compared to normal B cells. Our longitudinal analysis reveals the cancer methylome to emerge early, which may provide a platform for subsequent genetically-driven growth dynamics and together with its persistent presence suggests a central role in the normal-to-cancer transition.
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http://dx.doi.org/10.1158/2643-3230.BCD-19-0058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888194PMC
January 2021
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