Developing a membrane-proximal CD33-targeting CAR T cell.

BACKGROUND: CD33 is a tractable target in acute myeloid leukemia (AML) for chimeric antigen receptor (CAR) T cell therapy, but clinical success is lacking. METHODS: We developed 3P14HLh28Z, a novel CD33-directed CD28/CD3Z-based CAR T cell derived from a high-affinity binder obtained through membrane-proximal fragment immunization in humanized mice. RESULTS: We found that immunization exclusively with the membrane-proximal domain of CD33 is necessary for identification of membrane-proximal binders in humanized mice. Compared with clinically validated lintuzumab-based CAR T cells targeting distal CD33 epitopes, 3P14HLh28Z showed enhanced in vitro functionality as well as superior tumor control and increased overall survival in both low antigen density and clinically relevant patient-derived xenograft models. Increased activation and enhanced polyfunctionality led to enhanced efficacy. CONCLUSIONS: Showing for the first time that a membrane-proximal CAR is superior to a membrane-distal one in the setting of CD33 targeting, our results demonstrate the rationale for targeting membrane-proximal epitopes with high-affinity binders. We also demonstrate the importance of optimizing CAR T cells for functionality in settings of both low antigen density and clinically relevant patient-derived models.

Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Jak2V617F Reversible Activation Shows Its Essential Requirement in Myeloproliferative Neoplasms.

Gain-of-function mutations activating JAK/STAT signaling are seen in the majority of patients with myeloproliferative neoplasms (MPN), most commonly JAK2V617F. Although clinically approved JAK inhibitors improve symptoms and outcomes in MPNs, remissions are rare, and mutant allele burden does not substantively change with chronic therapy. We hypothesized this is due to limitations of current JAK inhibitors to potently and specifically abrogate mutant JAK2 signaling. We therefore developed a conditionally inducible mouse model allowing for sequential activation, and then inactivation, of Jak2V617F from its endogenous locus using a combined Dre-rox/Cre-lox dual-recombinase system. Jak2V617F deletion abrogates MPN features, induces depletion of mutant-specific hematopoietic stem/progenitor cells, and extends overall survival to an extent not observed with pharmacologic JAK inhibition, including when cooccurring with somatic Tet2 loss. Our data suggest JAK2V617F represents the best therapeutic target in MPNs and demonstrate the therapeutic relevance of a dual-recombinase system to assess mutant-specific oncogenic dependencies in vivo. SIGNIFICANCE: Current JAK inhibitors to treat myeloproliferative neoplasms are ineffective at eradicating mutant cells. We developed an endogenously expressed Jak2V617F dual-recombinase knock-in/knock-out model to investigate Jak2V617F oncogenic reversion in vivo. Jak2V617F deletion abrogates MPN features and depletes disease-sustaining MPN stem cells, suggesting improved Jak2V617F targeting offers the potential for greater therapeutic efficacy. See related commentary by Celik and Challen, p. 701. This article is featured in Selected Articles from This Issue, p. 695.

A study to assess the efficacy of enasidenib and risk-adapted addition of azacitidine in newly diagnosed IDH2-mutant AML.

ABSTRACT: Enasidenib (ENA) is an inhibitor of isocitrate dehydrogenase 2 (IDH2) approved for the treatment of patients with IDH2-mutant relapsed/refractory acute myeloid leukemia (AML). In this phase 2/1b Beat AML substudy, we applied a risk-adapted approach to assess the efficacy of ENA monotherapy for patients aged ≥60 years with newly diagnosed IDH2-mutant AML in whom genomic profiling demonstrated that mutant IDH2 was in the dominant leukemic clone. Patients for whom ENA monotherapy did not induce a complete remission (CR) or CR with incomplete blood count recovery (CRi) enrolled in a phase 1b cohort with the addition of azacitidine. The phase 2 portion assessing the overall response to ENA alone demonstrated efficacy, with a composite complete response (cCR) rate (CR/CRi) of 46% in 60 evaluable patients. Seventeen patients subsequently transitioned to phase 1b combination therapy, with a cCR rate of 41% and 1 dose-limiting toxicity. Correlative studies highlight mechanisms of clonal elimination with differentiation therapy as well as therapeutic resistance. This study demonstrates both efficacy of ENA monotherapy in the upfront setting and feasibility and applicability of a risk-adapted approach to the upfront treatment of IDH2-mutant AML. This trial is registered at www.clinicaltrials.gov as #NCT03013998.

Development of Resistance to Type II JAK2 Inhibitors in MPN Depends on AXL Kinase and Is Targetable.

PURPOSE: Myeloproliferative neoplasms (MPN) dysregulate JAK2 signaling. Because clinical JAK2 inhibitors have limited disease-modifying effects, type II JAK2 inhibitors such as CHZ868 stabilizing inactive JAK2 and reducing MPN clones, gain interest. We studied whether MPN cells escape from type ll inhibition. EXPERIMENTAL DESIGN: MPN cells were continuously exposed to CHZ868. We used phosphoproteomic analyses and ATAC/RNA sequencing to characterize acquired resistance to type II JAK2 inhibition, and targeted candidate mediators in MPN cells and mice. RESULTS: MPN cells showed increased IC50 and reduced apoptosis upon CHZ868 reflecting acquired resistance to JAK2 inhibition. Among >2,500 differential phospho-sites, MAPK pathway activation was most prominent, while JAK2-STAT3/5 remained suppressed. Altered histone occupancy promoting AP-1/GATA binding motif exposure associated with upregulated AXL kinase and enriched RAS target gene profiles. AXL knockdown resensitized MPN cells and combined JAK2/AXL inhibition using bemcentinib or gilteritinib reduced IC50 to levels of sensitive cells. While resistant cells induced tumor growth in NOD/SCID gamma mice despite JAK2 inhibition, JAK2/AXL inhibition largely prevented tumor progression. Because inhibitors of MAPK pathway kinases such as MEK are clinically used in other malignancies, we evaluated JAK2/MAPK inhibition with trametinib to interfere with AXL/MAPK-induced resistance. Tumor growth was halted similarly to JAK2/AXL inhibition and in a systemic cell line-derived mouse model, marrow infiltration was decreased supporting dependency on AXL/MAPK. CONCLUSIONS: We report on a novel mechanism of AXL/MAPK-driven escape from type II JAK2 inhibition, which is targetable at different nodes. This highlights AXL as mediator of acquired resistance warranting inhibition to enhance sustainability of JAK2 inhibition in MPN.

Non-canonical Hedgehog signaling mediates profibrotic hematopoiesis-stroma crosstalk in myeloproliferative neoplasms.

The role of hematopoietic Hedgehog signaling in myeloproliferative neoplasms (MPNs) remains incompletely understood despite data suggesting that Hedgehog (Hh) pathway inhibitors have therapeutic activity in patients. We aim to systematically interrogate the role of canonical vs. non-canonical Hh signaling in MPNs. We show that Gli1 protein levels in patient peripheral blood mononuclear cells (PBMCs) mark fibrotic progression and that, in murine MPN models, absence of hematopoietic Gli1, but not Gli2 or Smo, significantly reduces MPN phenotype and fibrosis, indicating that GLI1 in the MPN clone can be activated in a non-canonical fashion. Additionally, we establish that hematopoietic Gli1 has a significant effect on stromal cells, mediated through a druggable MIF-CD74 axis. These data highlight the complex interplay between alterations in the MPN clone and activation of stromal cells and indicate that Gli1 represents a promising therapeutic target in MPNs, particularly that Hh signaling is dispensable for normal hematopoiesis.

Interaction between myelodysplasia-related gene mutations and ontogeny in acute myeloid leukemia.

Accurate classification and risk stratification are critical for clinical decision making in patients with acute myeloid leukemia (AML). In the newly proposed World Health Organization and International Consensus classifications of hematolymphoid neoplasms, the presence of myelodysplasia-related (MR) gene mutations is included as 1 of the diagnostic criteria for AML, AML-MR, based largely on the assumption that these mutations are specific for AML with an antecedent myelodysplastic syndrome. ICC also prioritizes MR gene mutations over ontogeny (as defined in the clinical history). Furthermore, European LeukemiaNet (ELN) 2022 stratifies these MR gene mutations into the adverse-risk group. By thoroughly annotating a cohort of 344 newly diagnosed patients with AML treated at the Memorial Sloan Kettering Cancer Center, we show that ontogeny assignments based on the database registry lack accuracy. MR gene mutations are frequently observed in de novo AML. Among the MR gene mutations, only EZH2 and SF3B1 were associated with an inferior outcome in the univariate analysis. In a multivariate analysis, AML ontogeny had independent prognostic values even after adjusting for age, treatment, allo-transplant and genomic classes or ELN risks. Ontogeny also helped stratify the outcome of AML with MR gene mutations. Finally, de novo AML with MR gene mutations did not show an adverse outcome. In summary, our study emphasizes the importance of accurate ontogeny designation in clinical studies, demonstrates the independent prognostic value of AML ontogeny, and questions the current classification and risk stratification of AML with MR gene mutations.

Synthesis and Characterization of Ether Adducts of Thorium Tetrahydroborate Th(BH4)4 and Chemical Vapor Deposition of Thorium Boride Thin Films.

Treating ThCl4 with LiBH4 in various ethereal solvents affords the adducts Th(BH4)4(Et2O)2, Th(BH4)4(thf)2, and Th(BH4)4(dme) (thf = tetrahydrofuran and dme = 1,2-dimethoxyethane). The structures of these three compounds have been established by single-crystal X-ray diffraction: if the tetrahydroborate groups are considered as occupying one coordination site, the Et2O and thf complexes adopt trans-octahedral coordination geometries, whereas the dme complex exhibits a cis-octahedral structure. All four BH4- ligands in each compound are tridentate, rendering each thorium center 14-coordinate. The Th···B distances range from 2.64 to 2.67 Å, and the Th-O bond lengths are 2.47-2.52 Å. We propose that crystals of Th(BH4)4(thf)2 are isomorphous with those of U(BH4)4(thf)2, but owing to pseudosymmetry the latter was reported in a unit cell that was too small by a factor of 2. IR spectra and 1H and 11B NMR data are reported as well. All three adducts are volatile, subliming readily at 60 °C and 10-4 Torr, making them potentially useful as precursors for the chemical vapor deposition (CVD) of thin films of thorium boride. Passage of Th(BH4)4(Et2O)2 over glass, Si(100), and aluminum substrates heated to 350 °C yields amorphous films of approximate stoichiometry ThB2; films deposited from Th(BH4)4(thf)2 have stoichiometries closer to ThB2.5 and contain some oxygen. Auger, XPS, XRD, and SEM studies of these films are reported.

CXCL8/CXCR2 signaling mediates bone marrow fibrosis and is a therapeutic target in myelofibrosis.

Proinflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution are yet to be fully elucidated. Previously, we identified a critical role for combined constitutive JAK/STAT and aberrant NF-κB proinflammatory signaling in MF development. Using single-cell transcriptional and cytokine-secretion studies of primary cells from patients with MF and the human MPLW515L (hMPLW515L) murine model of MF, we extend our previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. Hematopoietic stem/progenitor cells from patients with MF are enriched for a CXCL8/CXCR2 gene signature and display enhanced proliferation and fitness in response to an exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L-adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway among patients with MF.

Molecular predictors of immunophenotypic measurable residual disease clearance in acute myeloid leukemia.

Measurable residual disease (MRD) is a powerful prognostic factor in acute myeloid leukemia (AML). However, pre-treatment molecular predictors of immunophenotypic MRD clearance remain unclear. We analyzed a dataset of 211 patients with pre-treatment next-generation sequencing who received induction chemotherapy and had MRD assessed by serial immunophenotypic monitoring after induction, subsequent therapy, and allogeneic stem cell transplant (allo-SCT). Induction chemotherapy led to MRD- remission, MRD+ remission, and persistent disease in 35%, 27%, and 38% of patients, respectively. With subsequent therapy, 34% of patients with MRD+ and 26% of patients with persistent disease converted to MRD-. Mutations in CEBPA, NRAS, KRAS, and NPM1 predicted high rates of MRD- remission, while mutations in TP53, SF3B1, ASXL1, and RUNX1 and karyotypic abnormalities including inv (3), monosomy 5 or 7 predicted low rates of MRD- remission. Patients with fewer individual clones were more likely to achieve MRD- remission. Among 132 patients who underwent allo-SCT, outcomes were favorable whether patients achieved early MRD- after induction or later MRD- after subsequent therapy prior to allo-SCT. As MRD conversion with chemotherapy prior to allo-SCT is rarely achieved in patients with specific baseline mutational patterns and high clone numbers, upfront inclusion of these patients into clinical trials should be considered.

Myeloproliferative Neoplasms, Version 3.2022, NCCN Clinical Practice Guidelines in Oncology.

The classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) consist of myelofibrosis, polycythemia vera, and essential thrombocythemia and are a heterogeneous group of clonal blood disorders characterized by an overproduction of blood cells. The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for MPN were developed as a result of meetings convened by a multidisciplinary panel with expertise in MPN, with the goal of providing recommendations for the management of MPN in adults. The Guidelines include recommendations for the diagnostic workup, risk stratification, treatment, and supportive care strategies for the management of myelofibrosis, polycythemia vera, and essential thrombocythemia. Assessment of symptoms at baseline and monitoring of symptom status during the course of treatment is recommended for all patients. This article focuses on the recommendations as outlined in the NCCN Guidelines for the diagnosis of MPN and the risk stratification, management, and supportive care relevant to MF.

The CXCR1/CXCR2 Inhibitor Reparixin Alters the Development of Myelofibrosis in the Gata1 low Mice.

A major role for human (h)CXCL8 (interleukin-8) in the pathobiology of myelofibrosis (MF) has been suggested by observations indicating that MF megakaryocytes express increased levels of hCXCL8 and that plasma levels of this cytokine in MF patients are predictive of poor patient outcomes. Here, we demonstrate that, in addition to high levels of TGF-ß, the megakaryocytes from the bone marrow of the Gata1 low mouse model of myelofibrosis express high levels of murine (m)CXCL1, the murine equivalent of hCXCL8, and its receptors CXCR1 and CXCR2. Treatment with the CXCR1/R2 inhibitor, Reparixin in aged-matched Gata1 low mice demonstrated reductions in bone marrow and splenic fibrosis. Of note, the levels of fibrosis detected using two independent methods (Gomori and reticulin staining) were inversely correlated with plasma levels of Reparixin. Immunostaining of marrow sections indicated that the bone marrow from the Reparixin-treated group expressed lower levels of TGF-ß1 than those expressed by the bone marrow from vehicle-treated mice while the levels of mCXCL1, and expression of CXCR1 and CXCR2, were similar to that of vehicle-treated mice. Moreover, immunofluorescence analyses performed on bone marrow sections from Gata1 low mice indicated that treatment with Reparixin induced expression of GATA1 while reducing expression of collagen III in megakaryocytes. These data suggest that in Gata1low mice, Reparixin reduces fibrosis by reducing TGF-ß1 and collagen III expression while increasing GATA1 in megakaryocytes. Our results provide a preclinical rationale for further evaluation of this drug alone and in combination with current JAK inhibitor therapy for the treatment of patients with myelofibrosis.

Comparative Mutational Profiling of Hematopoietic Progenitor Cells and Circulating Endothelial Cells (CECs) in Patients with Primary Myelofibrosis.

A role of endothelial cells (ECs) in Primary Myelofibrosis (PMF) was supposed since JAK2 mutation was found in endothelial precursor cells (EPCs) and in ECs captured by laser microdissection. By Cell Search method, the circulating endothelial cells (CECs) from 14 PMF patients and 5 healthy controls have been isolated and compared by NGS with CD34+Hematopoietic stem and progenitors cells (HSPCs) for panel of 54 myeloid-associated mutations. PMF patients had higher levels of CECs. No mutation was found in HSPCs and CECs from controls, while CECs from PMF patients presented several somatic mutations. 72% of evaluable patients shared at least one mutation between HSPCs and CECs. 2 patients shared the JAK2 mutation, together with ABL1, IDH1, TET2 and ASXL1, KMT2A, respectively. 6 out of 8 shared only NON MPN-driver mutations: TET2 and NOTCH1 in one case; individual paired mutations in TP53, KIT, SRSF2, NOTCH1 and WT1, in the other cases. In conclusion, 70% of PMF patients shared at least one mutation between HSPCs and CECs. These latter harbored several myeloid-associated mutations, besides JAK2V617F mutation. Our results support a primary involvement of EC in PMF and provide a new methodological approach for further studies exploring the role of the "neoplastic" vascular niche.

X-ray Crystal Structure of Thorium Tetrahydroborate, Th(BH4)4, and Computational Studies of An(BH4)4 (An = Th, U).

The crystal structure of Th(BH4)4 is described. Two of the four BH4- ions are terminal and tridentate (κ3), whereas the other two bridge between neighboring ThIV centers in a κ2,κ2 (i.e., bis-bidentate) fashion. Thus, each thorium center is bound to six BH4- groups by 14 Th-H bonds. The six boron atoms describe a distorted octahedron in which the κ3-BH4- ions are mutually cis; the 14 ligating hydrogen atoms define a highly distorted bicapped hexagonal antiprism. The thorium centers are linked into a polymer consisting of interconnected helical chains wound about 4-fold screw axes. The structures of An(BH4)4 (An = Th, U) were also investigated by DFT. The geometries of [An(BH4)6]2-, [An3(BH4)16]4-, and [An5(BH4)26]6- fragments of the polymeric structures were optimized at the B3LYP and/or PBE levels. Most calculated geometries are 14-coordinate and agree with the experimental structures, but isolated [Th(BH4)6]2- units are predicted to feature 16-coordinate ThIV centers.

Epigenetic Dysregulation of Myeloproliferative Neoplasms.

This article reviews the genetic data on epigenetic modifying mutations in myeloproliferative neoplasms and their clinical implications, preclinical studies exploring our current understanding of how mutations in epigenetic modifying proteins cooperate with myeloproliferative neoplasms drivers to promote disease progression, and recent advances in novel therapeutics supporting the role of targeting epigenetic pathways to treat fibrotic progression.

Genomic profiling identifies somatic mutations predicting thromboembolic risk in patients with solid tumors.

Venous thromboembolism (VTE) associated with cancer (CAT) is a well-described complication of cancer and a leading cause of death in patients with cancer. The purpose of this study was to assess potential associations of molecular signatures with CAT, including tumor-specific mutations and the presence of clonal hematopoiesis. We analyzed deep-coverage targeted DNA-sequencing data of >14 000 solid tumor samples using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets platform to identify somatic alterations associated with VTE. End point was defined as the first instance of cancer-associated pulmonary embolism and/or proximal/distal lower extremity deep vein thrombosis. Cause-specific Cox proportional hazards regression was used, adjusting for pertinent clinical covariates. Of 11 695 evaluable individuals, 72% had metastatic disease at time of analysis. Tumor-specific mutations in KRAS (hazard ratio [HR], 1.34; 95% confidence interval (CI), 1.09-1.64; adjusted P = .08), STK11 (HR, 2.12; 95% CI, 1.55-2.89; adjusted P < .001), KEAP1 (HR, 1.84; 95% CI, 1.21-2.79; adjusted P = .07), CTNNB1 (HR, 1.73; 95% CI, 1.15-2.60; adjusted P = .09), CDKN2B (HR, 1.45; 95% CI, 1.13-1.85; adjusted P = .07), and MET (HR, 1.83; 95% CI, 1.15-2.92; adjusted P = .09) were associated with a significantly increased risk of CAT independent of tumor type. Mutations in SETD2 were associated with a decreased risk of CAT (HR, 0.35; 95% CI, 0.16-0.79; adjusted P = .09). The presence of clonal hematopoiesis was not associated with an increased VTE rate. This is the first large-scale analysis to elucidate tumor-specific genomic events associated with CAT. Somatic tumor mutations of STK11, KRAS, CTNNB1, KEAP1, CDKN2B, and MET were associated with an increased risk of VTE in patients with solid tumors. Further analysis is needed to validate these findings and identify additional molecular signatures unique to individual tumor types.

Myeloid/Lymphoid Neoplasms with Eosinophilia and TK Fusion Genes, Version 3.2021, NCCN Clinical Practice Guidelines in Oncology.

Eosinophilic disorders and related syndromes represent a heterogeneous group of neoplastic and nonneoplastic conditions, characterized by more eosinophils in the peripheral blood, and may involve eosinophil-induced organ damage. In the WHO classification of myeloid and lymphoid neoplasms, eosinophilic disorders characterized by dysregulated tyrosine kinase (TK) fusion genes are recognized as a new category termed, myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB or FGFR1 or with PCM1-JAK2. In addition to these aforementioned TK fusion genes, rearrangements involving FLT3 and ABL1 genes have also been described. These new NCCN Guidelines include recommendations for the diagnosis, staging, and treatment of any one of the myeloid/lymphoid neoplasms with eosinophilia (MLN-Eo) and a TK fusion gene included in the 2017 WHO Classification, as well as MLN-Eo and a FLT3 or ABL1 rearrangement.

Leukemia secondary to myeloproliferative neoplasms.

Secondary acute myeloid leukemias (AMLs) evolving from an antecedent myeloproliferative neoplasm (MPN) are characterized by a unique set of cytogenetic and molecular features distinct from de novo AML. Given the high frequency of poor-risk cytogenetic and molecular features, malignant clones are frequently insensitive to traditional AML chemotherapeutic agents. Allogeneic stem cell transplant, the only treatment modality shown to have any beneficial long-term outcome, is often not possible given the advanced age of patients at time of diagnosis and frequent presence of competing comorbidities. Even in this setting, relapse rates remain high. As a result, outcomes are generally poor and there remains a significant unmet need for novel therapeutic strategies. Although advances in cancer genomics have dramatically enhanced our understanding of the molecular events governing clonal evolution in MPNs, the cell-intrinsic and -extrinsic mechanisms driving leukemic transformation at this level remain poorly understood. Here, we review known risk factors for the development of leukemic transformation in MPNs, recent progress made in our understanding of the molecular features associated with leukemic transformation, current treatment strategies, and emerging therapeutic options for this high-risk myeloid malignancy.