Hematologic Cancer after Gene Therapy for Cerebral Adrenoleukodystrophy.

BACKGROUND: Gene therapy with elivaldogene autotemcel (eli-cel) consisting of autologous CD34+ cells transduced with lentiviral vector containing ABCD1 complementary DNA (Lenti-D) has shown efficacy in clinical studies for the treatment of cerebral adrenoleukodystrophy. However, the risk of oncogenesis with eli-cel is unclear. METHODS: We performed integration-site analysis, genetic studies, flow cytometry, and morphologic studies in peripheral-blood and bone marrow samples from patients who received eli-cel therapy in two completed phase 2-3 studies (ALD-102 and ALD-104) and an ongoing follow-up study (LTF-304) involving the patients in both ALD-102 and ALD-104. RESULTS: Hematologic cancer developed in 7 of 67 patients after the receipt of eli-cel (1 of 32 patients in the ALD-102 study and 6 of 35 patients in the ALD-104 study): myelodysplastic syndrome (MDS) with unilineage dysplasia in 2 patients at 14 and 26 months; MDS with excess blasts in 3 patients at 28, 42, and 92 months; MDS in 1 patient at 36 months; and acute myeloid leukemia (AML) in 1 patient at 57 months. In the 6 patients with available data, predominant clones contained lentiviral vector insertions at multiple loci, including at either MECOM-EVI1 (MDS and EVI1 complex protein EVI1 [ecotropic virus integration site 1], in 5 patients) or PRDM16 (positive regulatory domain zinc finger protein 16, in 1 patient). Several patients had cytopenias, and most had vector insertions in multiple genes within the same clone; 6 of the 7 patients also had somatic mutations (KRAS, NRAS, WT1, CDKN2A or CDKN2B, or RUNX1), and 1 of the 7 patients had monosomy 7. Of the 5 patients with MDS with excess blasts or MDS with unilineage dysplasia who underwent allogeneic hematopoietic stem-cell transplantation (HSCT), 4 patients remain free of MDS without recurrence of symptoms of cerebral adrenoleukodystrophy, and 1 patient died from presumed graft-versus-host disease 20 months after HSCT (49 months after receiving eli-cel). The patient with AML is alive and had full donor chimerism after HSCT; the patient with the most recent case of MDS is alive and awaiting HSCT. CONCLUSIONS: Hematologic cancer developed in a subgroup of patients who were treated with eli-cel; the cases are associated with clonal vector insertions within oncogenes and clonal evolution with acquisition of somatic genetic defects. (Funded by Bluebird Bio; ALD-102, ALD-104, and LTF-304 ClinicalTrials.gov numbers, NCT01896102, NCT03852498, and NCT02698579, respectively.).

Early evolutionary branching across spatial domains predisposes to clonal replacement under chemotherapy in neuroblastoma.

Neuroblastoma (NB) is one of the most lethal childhood cancers due to its propensity to become treatment resistant. By spatial mapping of subclone geographies before and after chemotherapy across 89 tumor regions from 12 NBs, we find that densely packed territories of closely related subclones present at diagnosis are replaced under effective treatment by islands of distantly related survivor subclones, originating from a different most recent ancestor compared to lineages dominating before treatment. Conversely, in tumors that progressed under treatment, ancestors of subclones dominating later in disease are present already at diagnosis. Chemotherapy treated xenografts and cell culture models replicate these two contrasting scenarios and show branching evolution to be a constant feature of proliferating NB cells. Phylogenies based on whole genome sequencing of 505 individual NB cells indicate that a rich repertoire of parallel subclones emerges already with the first oncogenic mutations and lays the foundation for clonal replacement under treatment.

Reconstructing tumor clonal heterogeneity and evolutionary relationships based on tumor DNA sequencing data.

The heterogeneity of tumor clones drives the selection and evolution of distinct tumor cell populations, resulting in an intricate and dynamic tumor evolution process. While tumor bulk DNA sequencing helps elucidate intratumor heterogeneity, challenges such as the misidentification of mutation multiplicity due to copy number variations and uncertainties in the reconstruction process hinder the accurate inference of tumor evolution. In this study, we introduce a novel approach, REconstructing Tumor Clonal Heterogeneity and Evolutionary Relationships (RETCHER), which characterizes more realistic cancer cell fractions by accurately identifying mutation multiplicity while considering uncertainty during the reconstruction process and the credibility and reasonableness of subclone clustering. This method comprehensively and accurately infers multiple forms of tumor clonal heterogeneity and phylogenetic relationships. RETCHER outperforms existing methods on simulated data and infers clearer subclone structures and evolutionary relationships in real multisample sequencing data from five tumor types. By precisely analysing the complex clonal heterogeneity within tumors, RETCHER provides a new approach to tumor evolution research and offers scientific evidence for developing precise and personalized treatment strategies. This approach is expected to play a significant role in tumor evolution research, clinical diagnosis, and treatment. RETCHER is available for free at https://github.com/zlsys3/RETCHER.

Cellular origin and clonal evolution of human dedifferentiated liposarcoma.

Dedifferentiated liposarcoma (DDLPS) is the most frequent high-grade soft tissue sarcoma subtype. It is characterized by a component of undifferentiated tumor cells coexisting with a component of well-differentiated adipocytic tumor cells. Both dedifferentiated (DD) and well-differentiated (WD) components exhibit MDM2 amplification, however their cellular origin remains elusive. Using single-cell RNA sequencing, DNA sequencing, in situ multiplex immunofluorescence and functional assays in paired WD and DD components from primary DDLPS tumors, we characterize the cellular heterogeneity of DDLPS tumor and micro-environment. We identify a population of tumor adipocyte stem cells (ASC) showing striking similarities with adipocyte stromal progenitors found in white adipose tissue. We show that tumor ASC harbor the ancestral genomic alterations of WD and DD components, suggesting that both derive from these progenitors following clonal evolution. Last, we show that DD tumor cells keep important biological properties of ASC including pluripotency and that their adipogenic properties are inhibited by a TGF-ß-high immunosuppressive tumor micro-environment.

[Clonal evolution in myeloproliferative neoplasms].

Recent advances in sequencing technologies have clarified the driver gene landscape in Philadelphia chromosomenegative (Ph-) myeloproliferative neoplasms (MPNs) and progressed understanding of MPN pathogenesis. Beyond mutations in the main three drivers of MPN, namely JAK2, MPL and CALR, somatic mutations in the epigenetic regulators and RNA splicing factors have been identified and their association with transformation to myelofibrosis and acute myeloid leukemia have been determined. Clonal expansion of hematopoietic cells with driver mutations (clonal hematopoiesis) has been detected in healthy individuals, especially in elderly people. In MPN patients, however, initial driver mutations such as those in JAK2 and DNMT3A have been shown to be acquired in utero or during childhood. In this review, I will summarize the recent findings about clonal evolution in MPN and the role of driver mutations.

Dynamically adjusted cell fate decisions and resilience to mutant invasion during steady-state hematopoiesis revealed by an experimentally parameterized mathematical model.

A major next step in hematopoietic stem cell (HSC) biology is to enhance our quantitative understanding of cellular and evolutionary dynamics involved in undisturbed hematopoiesis. Mathematical models have been and continue to be key in this respect, and are most powerful when parameterized experimentally and containing sufficient biological complexity. In this paper, we use data from label propagation experiments in mice to parameterize a mathematical model of hematopoiesis that includes homeostatic control mechanisms as well as clonal evolution. We find that nonlinear feedback control can drastically change the interpretation of kinetic estimates at homeostasis. This suggests that short-term HSC and multipotent progenitors can dynamically adjust to sustain themselves temporarily in the absence of long-term HSCs, even if they differentiate more often than they self-renew in undisturbed homeostasis. Additionally, the presence of feedback control in the model renders the system resilient against mutant invasion. Invasion barriers, however, can be overcome by a combination of age-related changes in stem cell differentiation and evolutionary niche construction dynamics based on a mutant-associated inflammatory environment. This helps us understand the evolution of e.g., TET2 or DNMT3A mutants, and how to potentially reduce mutant burden.

The evolutionary theory of cancer: challenges and potential solutions.

The clonal evolution model of cancer was developed in the 1950s-1970s and became central to cancer biology in the twenty-first century, largely through studies of cancer genetics. Although it has proven its worth, its structure has been challenged by observations of phenotypic plasticity, non-genetic forms of inheritance, non-genetic determinants of clone fitness and non-tree-like transmission of genes. There is even confusion about the definition of a clone, which we aim to resolve. The performance and value of the clonal evolution model depends on the empirical extent to which evolutionary processes are involved in cancer, and on its theoretical ability to account for those evolutionary processes. Here, we identify limits in the theoretical performance of the clonal evolution model and provide solutions to overcome those limits. Although we do not claim that clonal evolution can explain everything about cancer, we show how many of the complexities that have been identified in the dynamics of cancer can be integrated into the model to improve our current understanding of cancer.

A Rare Case of X-Linked Four-Way Philadelphia Chromosome Translocation with Therapeutic Challenges and Clonal Evolution.

BACKGROUND: Chronic myeloid leukemia (CML), a myeloproliferative neoplasm defined by the BCR::ABL1 fusion gene arising from the Philadelphia chromosome (Ph) translocation t(9:22)(q34;q11), exhibits diverse clinical courses often influenced by additional chromosomal aberrations (ACAs). This report presents a case of CML har-boring a novel four-way Ph translocation involving the X chromosome, offering insights into the interplay between complex karyotypes and treatment response and emphasizing the need for further research into the role of ACAs in CML management. METHODS: A 42-year-old man diagnosed with CML in the accelerated phase presented a novel four-way Ph translocation involving chromosomes X, 5, 9, and 22: 46,Y,t(X;5;9;22)(q26;q15;q34;q11.2). Despite achieving a major molecular response initially with imatinib and nilotinib, BCR::ABL1 levels (international scale) increased up to 24.0%, which prompted the use of second-line nilotinib. RESULTS: Follow-up bone marrow (BM) studies revealed clonal evolution with trisomy 8 and an unclassified ABL1 mutation (E292V), potentially contributing to resistance. Though a transient major molecular response (MMR) occurred after a switch to third-line dasatinib, this change failed to achieve a deep molecular response, and BCR-ABL1 levels were elevated above the MMR. CONCLUSIONS: This case highlights the challenge of ACAs impacting CML treatment response and prognosis. Limited knowledge exists on complex Ph translocations involving the X chromosome, but this report contributes data for further research. Understanding ACA effects on therapeutic response and prognosis requires a detailed study of such complex chromosomal aberrations.

Evolution from an antecedent chronic myeloid malignancy does not impact survival outcomes in NPM1-mutated AML.

Nucleophosmin-1 (NPM1)-mutated AML is a molecularly defined subtype typically associated with favorable treatment response and prognosis; however, its prognostic significance in AML evolving from an antecedent chronic myeloid malignancy is unknown. This study's primary objective was to determine the impact of mutated NPM1 on the prognosis of AML evolving from an antecedent chronic myeloid malignancy. We conducted a retrospective chart review including patients with NPM1-mutated de novo and sAML. sAML was defined as those with a preceding chronic-phase myeloid malignancy before diagnosis of AML. Of 575 NPM1-mutated patients eligible for inclusion in our study, 51 (8.9%) patients were considered to have sAML. The median time from diagnosis of NPM1-mutated chronic myeloid malignancy to sAML evolution was 3.6 months (0.5-79.3 months). No significant differences in leukemia-free (2-year LKFS 52.0% vs. 51.2%, p = .9922) or overall survival (2-year OS 56.3% vs. 49.4%, p = .4246) were observed between patients with NPM1-mutated de novo versus sAML. Our study suggests that evolution from a preceding myeloid malignancy is not a significant predictor of poor prognosis in the setting of an NPM1 mutation. Our study demonstrated a short time to progression to sAML in most patients, which further supports the consideration of NPM1 as an AML-defining mutation.

Highly parallelized laboratory evolution of wine yeasts for enhanced metabolic phenotypes.

Adaptive Laboratory Evolution (ALE) of microorganisms can improve the efficiency of sustainable industrial processes important to the global economy. However, stochasticity and genetic background effects often lead to suboptimal outcomes during laboratory evolution. Here we report an ALE platform to circumvent these shortcomings through parallelized clonal evolution at an unprecedented scale. Using this platform, we evolved 104 yeast populations in parallel from many strains for eight desired wine fermentation-related traits. Expansions of both ALE replicates and lineage numbers broadened the evolutionary search spectrum leading to improved wine yeasts unencumbered by unwanted side effects. At the genomic level, evolutionary gains in metabolic characteristics often coincided with distinct chromosome amplifications and the emergence of side-effect syndromes that were characteristic of each selection niche. Several high-performing ALE strains exhibited desired wine fermentation kinetics when tested in larger liquid cultures, supporting their suitability for application. More broadly, our high-throughput ALE platform opens opportunities for rapid optimization of microbes which otherwise could take many years to accomplish.

Recurrent adamantinomatous craniopharyngiomas show MAPK pathway activation, clonal evolution and rare TP53-loss-mediated malignant progression.

The two types of craniopharyngioma, adamantinomatous (ACP) and papillary (PCP), are clinically relevant tumours in children and adults. Although the biology of primary craniopharyngioma is starting to be unravelled, little is known about the biology of recurrence. To fill this gap in knowledge, we have analysed through methylation array, RNA sequencing and pERK1/2 immunohistochemistry a cohort of paired primary and recurrent samples (32 samples from 14 cases of ACP and 4 cases of PCP). We show the presence of copy number alterations and clonal evolution across recurrence in 6 cases of ACP, and analysis of additional whole genome sequencing data from the Children's Brain Tumour Network confirms chromosomal arm copy number changes in at least 7/67 ACP cases. The activation of the MAPK/ERK pathway, a feature previously shown in primary ACP, is observed in all but one recurrent cases of ACP. The only ACP without MAPK activation is an aggressive case of recurrent malignant human craniopharyngioma harbouring a CTNNB1 mutation and loss of TP53. Providing support for a functional role of this TP53 mutation, we show that Trp53 loss in a murine model of ACP results in aggressive tumours and reduced mouse survival. Finally, we characterise the tumour immune infiltrate showing differences in the cellular composition and spatial distribution between ACP and PCP. Together, these analyses have revealed novel insights into recurrent craniopharyngioma and provided preclinical evidence supporting the evaluation of MAPK pathway inhibitors and immunomodulatory approaches in clinical trials in against recurrent ACP.

Clonal evolution of the 3D chromatin landscape in patients with relapsed pediatric B-cell acute lymphoblastic leukemia.

Relapsed pediatric B-cell acute lymphoblastic leukemia (B-ALL) remains one of the leading causes of cancer mortality in children. We performed Hi-C, ATAC-seq, and RNA-seq on 12 matched diagnosis/relapse pediatric leukemia specimens to uncover dynamic structural variants (SVs) and 3D chromatin rewiring that may contribute to relapse. While translocations are assumed to occur early in leukemogenesis and be maintained throughout progression, we discovered novel, dynamic translocations and confirmed several fusion transcripts, suggesting functional and therapeutic relevance. Genome-wide chromatin remodeling was observed at all organizational levels: A/B compartments, TAD interactivity, and chromatin loops, including some loci shared by 25% of patients. Shared changes were found to drive the expression of genes/pathways previously implicated in resistance as well as novel therapeutic candidates, two of which (ATXN1 and MN1) we functionally validated. Overall, these results demonstrate chromatin reorganization under the selective pressure of therapy and offer the potential for discovery of novel therapeutic interventions.

Emerging and extensive clonal evolution in the pancreas.

Pancreatic cancer is one of the most lethal malignancies, yet much remains to be learned regarding how its precursors develop. In a recent Nature publication, Braxton and Kiemen et al. found that the normal, adult pancreas harbors hundreds to thousands of pancreatic cancer precursors evolving by a variety of routes.

Relationship between clonal evolution and drug resistance in bladder cancer: A genomic research review.

Bladder cancer stands as a prevalent global malignancy, exhibiting notable sex-based variations in both incidence and prognosis. Despite substantial strides in therapeutic approaches, the formidable challenge of drug resistance persists. The genomic landscape of bladder cancer, characterized by intricate clonal heterogeneity, emerges as a pivotal determinant in fostering this resistance. Clonal evolution, encapsulating the dynamic transformations within subpopulations of tumor cells over time, is implicated in the emergence of drug-resistant traits. Within this review, we illuminate contemporary insights into the role of clonal evolution in bladder cancer, elucidating its influence as a driver in tumor initiation, disease progression, and the formidable obstacle of therapy resistance.

Superior detection of low-allele burden Janus kinase 2 V617F mutation and monitoring clonal evolution in myeloproliferative neoplasms using chip-based digital PCR.

The JAK2 V617F is a prevalent driver mutation in Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPNs), significantly affecting disease progression, immunophenotype, and patient outcomes. The World Health Organization (WHO) guidelines highlight the JAK2 V617F mutation as one of the key diagnostic criterions for Ph-MPNs. In this study, we analyzed 283 MPN samples with the JAK2 V617F mutation to assess the effectiveness of three detection technologies: chip-based digital PCR (cdPCR), real-time quantitative PCR (qPCR), and next-generation sequencing (NGS). Additionally, we investigated the relationship between JAK2 V617F mutant allele burden (% JAK2 V617F) and various laboratory characteristics to elucidate potential implications in MPN diagnosis. Our findings demonstrated high conformance of cdPCR with qPCR/NGS for detecting % JAK2 V617F, but the mutant allele burdens detected by qPCR/NGS were lower than those detected by cdPCR. Moreover, the cdPCR exhibited high sensitivity with a limit of detection (LoD) of 0.08% and a limit of quantification (LoQ) of 0.2% for detecting % JAK2 V617F in MPNs. Clinical implications were explored by correlating % JAK2 V617F with various laboratory characteristics in MPN patients, revealing significant associations with white blood cell counts, lactate dehydrogenase levels, and particularly ß2-microglobulin (ß2-MG) levels. Finally, a case report illustrated the application of cdPCR in detecting low-allele burdens in a de novo chronic myeloid leukemia (CML) patient with a hidden JAK2 V617F subclone, which expanded during tyrosine kinase inhibitor (TKI) treatment. Our findings underscore the superior sensitivity and accuracy of cdPCR, making it a valuable tool for early diagnosis and monitoring clonal evolution.

Comprehensive genomic analysis reveals clonal origin and subtype-specific evolution in a case of sporadic multiple meningiomas.

Meningioma is the most common primary intracranial tumor in adults, with up to 10% manifesting as multiple tumors. Data on the genomic and molecular changes in sporadic multiple meningiomas are scarce, leading to ongoing debates regarding their evolutionary processes. A comprehensive genetic analysis of a large number of lesions, including precursor lesions, is necessary to explore these two possible origins: clonal and independent. In the present study, we performed whole-exome sequencing and analyzed somatic single-nucleotide variants (SNVs), insertions/deletions (INDELs), and copy number alterations (CNAs) in a patient with sporadic multiple meningiomas. These meningiomas included two mass-forming lesions of different histological subtypes (transitional and chordoid) and two small meningothelial nests. Genetic analysis revealed CNAs on chromosomes 22q and Y as common abnormalities in the two largest tumors. Furthermore, we identified SNV/INDELs unique to each focus, with NF2 mutation prevalent in the transitional meningioma and CREBBP mutation in the chordoid meningioma. Loss of chromosome 22 was detected in two small meningothelial nests. Overall, we elucidated the clonal origin and subtype-specific evolution of multiple meningiomas in this case. CNAs may serve as the initial driving event in meningioma development.

Initiating-clone analysis in patients with acute myeloid leukemia secondary to essential thrombocythemia.

Most of essential thrombocythemia (ET) patients have the clone harboring a mutation in one of the JAK2, CALR, or MPL gene, and these clones generally acquire additional mutations at transformation to acute myeloid leukemia (AML). However, the proliferation of triple-negative clones has sometimes been observed at AML transformation. To clarify the clonal evolution of ET to AML, we analyzed paired samples at ET and AML transformation in eight patients. We identified that JAK2-unmutated AML clones proliferated at AML transformation in three patients in whom the JAK2-mutated clone was dominant at ET. In two patients, TET2-mutated, but not JAK2-mutated, clones might be common initiating clones for ET and transformed AML. In a patient with JAK2-mutated ET, SMARCC2, UBR4, and ZNF143, but not JAK2, -mutated clones proliferated at AML transformation. Precise analysis using single-cell sorted CD34+/CD38- fractions suggested that ET clone with JAK2-mutated and AML clone with TP53 mutation was derived from the common clone with these mutations. Although further study is required to clarify the biological significance of SMARCC2, UBR4, and ZNF143 mutations during disease progression of ET and AML transformation, the present results demonstrate the possibility of a common initial clone involved in both ET and transformed AML.