Search

Follicular lymphoma (FL) is morphologically and clinically diverse, with mutations in epigenetic regulators alongside t(14;18) identified as disease-initiating events. Identification of additional mutational entities confirms this cancer’s heterogeneity, but whether mutational data can be resolved into mechanistically distinct subsets remains an open question. Targeted sequencing was applied to an unselected population-based FL cohort (n = 548) with full clinical follow-up (n = 538), which included 96 diffuse large B-cell lymphoma (DLBCL) transformations. We investigated whether molecular subclusters of FL can be identified and whether mutational data provide predictive information relating to transformation. DNA extracted from FL samples was sequenced with a 293-gene panel representing genes frequently mutated in DLBCL and FL. Three clusters were resolved using mutational data alone, independent of translocation status: FL_aSHM, with high burden of aberrant somatic hypermutation (aSHM) targets; FL_STAT6, with high STAT6 & CREBBP mutation and low aSHM; and FL_Com, with the absence of features of other subtypes and enriched KMT2D mutation. Analysis of mutation signatures demonstrated differential enrichment of predicted mutation signatures between subgroups and a dominant preference in the FL_aSHM subgroup for G(C>T)T and G(C>T)C transitions consistent with previously defined aSHM-like patterns. Of transformed cases with paired samples, 17 of 26 had evidence of branching evolution. Poorer overall survival (OS) in the aSHM group (P = .04) was associated with older age; however, overall tumor genetics provided limited information to predict individual patient risk. Our approach identifies 3 molecular subclusters of FL linked to differences in underlying mechanistic pathways. These clusters, which may be further resolved by the inclusion of translocation status and wider mutation profiles, have implications for understanding pathogenesis as well as improving treatment strategies in the future.

Cancer Biomarkers are the key to unlocking the promise of precision oncology, selecting which patients will respond to a more personalised treatment while sparing non-responders the therapy-related toxicity. In this paper, we highlight the primacy of cancer biomarkers, but focus on their importance to patients and to health systems. We also highlight how cancer biomarkers represent value for money. We emphasise the need for cancer biomarkers infrastructure to be embedded into European health systems. We also highlight the need to deploy multiple biomarker testing to deliver the optimal benefit for patients and health systems and consider cancer biomarkers from the perspective of cost, value and regulation. Cancer biomarkers must also be situated in the context of the upcoming In Vitro Diagnostics Regulation, which may pose certain challenges (e.g. non-compliance of laboratory developed tests, leading to cancer biomarker shortages and increased costs) that need to be overcome. Cancer biomarkers must be embedded in the real world of oncology delivery and testing must be implemented across Europe, with the intended aim of narrowing, not widening the inequity gap for patients. Cancer patients must be placed firmly at the centre of a cancer biomarker informed precision oncology care agenda.

Supplementary Tables

Supplementary Figures

Purpose:Inhibition of histone deacetylase 6 (HDAC6) is predicted to deliver both direct antitumor activity and modulation of the antitumor immune response. This study describes the development of a novel HDAC6 inhibitor.Patients and Methods:KA2507 was characterized in HDAC biochemical and cellular target engagement assays and in preclinical efficacy models of melanoma and colorectal cancer. In a phase I study, KA2507 was administered orally using a 3+3 dose-escalation design (NCT03008018).Results:KA2507 is a potent and selective inhibitor of HDAC6 (biochemical IC50 = 2.5 nmol/L). Preclinical models demonstrated antitumor efficacy in syngeneic tumor-bearing mice, with translational studies highlighting modulation of the antitumor immune response. Twenty patients were treated in a phase I study. KA2507 was well tolerated; dose-limiting toxicity was not observed up to the maximum dose administered. Pharmacokinetic profiling supported twice-daily oral dosing. Pharmacodynamic analysis demonstrated selective HDAC6 target engagement in peripheral blood cells, free from off-target class I HDAC activity. Stable disease was the best clinical response (7 patients). Three of these patients (adenoid cystic carcinoma, n = 2; rectal adenocarcinoma, n = 1) had prolonged disease stabilization that lasted for 16.4, 12.6, and 9.0 months, respectively.Conclusions:KA2507 is a potent and selective inhibitor of HDAC6 showing antitumor efficacy and immune modulatory effects in preclinical models. In a phase I study, KA2507 showed selective target engagement, no significant toxicities, and prolonged disease stabilization in a subset of patients. Further clinical studies of KA2507 are warranted, as a single agent or, preferably, combined with other immuno-oncology drugs.

Purpose: Inhibition of histone deacetylase 6 (HDAC6) is predicted to deliver both direct antitumor activity and modulation of the antitumor immune response. This study describes the development of a novel HDAC6 inhibitor. Patients and Methods: KA2507 was characterized in HDAC biochemical and cellular target engagement assays and in preclinical efficacy models of melanoma and colorectal cancer. In a phase I study, KA2507 was administered orally using a 3+3 dose-escalation design (NCT03008018). Results: KA2507 is a potent and selective inhibitor of HDAC6 (biochemical IC50 = 2.5 nmol/L). Preclinical models demonstrated antitumor efficacy in syngeneic tumor-bearing mice, with translational studies highlighting modulation of the antitumor immune response. Twenty patients were treated in a phase I study. KA2507 was well tolerated; dose-limiting toxicity was not observed up to the maximum dose administered. Pharmacokinetic profiling supported twice-daily oral dosing. Pharmacodynamic analysis demonstrated selective HDAC6 target engagement in peripheral blood cells, free from off-target class I HDAC activity. Stable disease was the best clinical response (7 patients). Three of these patients (adenoid cystic carcinoma, n = 2; rectal adenocarcinoma, n = 1) had prolonged disease stabilization that lasted for 16.4, 12.6, and 9.0 months, respectively. Conclusions: KA2507 is a potent and selective inhibitor of HDAC6 showing antitumor efficacy and immune modulatory effects in preclinical models. In a phase I study, KA2507 showed selective target engagement, no significant toxicities, and prolonged disease stabilization in a subset of patients. Further clinical studies of KA2507 are warranted, as a single agent or, preferably, combined with other immuno-oncology drugs.

Summary Hepatoid tumors (HT) are rare neoplasms morphologically resembling hepatocellular carcinoma, which arise in several organs other than the liver. A comprehensive molecular profile of this group of neoplasms is still lacking. Genomic characterization of 19 HTs from different organs (three colon HTs, four esophagogastric HTs, four biliary HTs, six genitourinary HTs, two lung HTs) was performed using a multigene next-generation sequencing panel. NGS unraveled a composite molecular profile of HT. Their genetic alterations were clearly clustered by tumor site: (i) colorectal HT displayed microsatellite instability, high tumor mutational burden, mutations in ARID1A/B genes and NCOA4-RET gene fusion (2/3 cases); (ii) gastric HT had TP53 mutations (2/4); (iii) biliary HT displayed loss of CDKN2A (3/4) and loss of chromosome 18 (2/4); (iv) genital HT showed gain of chromosome 12 (3/6); (v) lung HT had STK11 somatic mutations (2/2). The only commonly mutated gene occurring in HT of different sites was TP53 (8/19 cases: colon 2, esophagogastric 2, biliary 2, genital 1, lungs 1). This study shows that most genetic alterations of HT were clustered by site, indicating that context matters. The novel potential targets for HT precision oncology are also clustered based on the anatomic origin. This study shed light on the biology of these rare cancers and may have important consequences for treatment decisions and clinical trial selection for HT patients.

For learnings from academic research to impact health outcomes, they must be embedded into routine clinical practice. Research innovations such as the human genome project, next-generation sequenci...

PURPOSE To analyze the prevalence of homologous recombination deficiency (HRD) in patients with pancreatic ductal adenocarcinoma (PDAC). MATERIALS AND METHODS We conducted a systematic review and meta-analysis of the prevalence of HRD in PDAC from PubMed, Scopus, and Cochrane Library databases, and online cancer genomic data sets. The main outcome was pooled prevalence of somatic and germline mutations in the better characterized HRD genes ( BRCA1, BRCA2, PALB2, ATM, ATR, CHEK2, RAD51, and the FANC genes). The secondary outcomes were prevalence of germline mutations overall, and in sporadic and familial cases; prevalence of germline BRCA1/2 mutations in Ashkenazi Jewish (AJ); and prevalence of HRD based on other definitions (ie, alterations in other genes, genomic scars, and mutational signatures). Random-effects modeling with the Freeman-Tukey transformation was used for the analyses. PROSPERO registration number: (CRD42020190813). RESULTS Sixty studies with 21,842 participants were included in the systematic review and 57 in the meta-analysis. Prevalence of germline and somatic mutations was BRCA1: 0.9%, BRCA2: 3.5%, PALB2: 0.2%, ATM: 2.2%, CHEK2: 0.3%, FANC: 0.5%, RAD51: 0.0%, and ATR: 0.1%. Prevalence of germline mutations was BRCA1: 0.9% (2.4% in AJ), BRCA2: 3.8% (8.2% in AJ), PALB2: 0.2%, ATM: 2%, CHEK2: 0.3%, and FANC: 0.4%. No significant differences between sporadic and familial cases were identified. HRD prevalence ranged between 14.5%-16.5% through targeted next-generation sequencing and 24%-44% through whole-genome or whole-exome sequencing allowing complementary genomic analysis, including genomic scars and other signatures (surrogate markers of HRD). CONCLUSION Surrogate readouts of HRD identify a greater proportion of patients with HRD than analyses limited to gene-level approaches. There is a clear need to harmonize HRD definitions and to validate the optimal biomarker for treatment selection. Universal HRD screening including integrated somatic and germline analysis should be offered to all patients with PDAC.

PI3-kinase p110δ is mainly expressed in lymphocytes and is an attractive therapeutic target in B cell lymphomas. Targeting p110β may further suppress tumor growth and overcome escape mechanisms. KA2237 is an oral, potent, dual p110β/p110δ inhibitor. In preclinical studies, KA2237 inhibited p110β- and p110δ-dependent AKT activation and suppressed proliferation of diverse hematological and epithelial tumors. Twenty-one patients received KA2237 in a first-in-human phase I study (NCT02679196; diffuse large B cell, n = 8; follicular, n = 5; mantle cell, n = 3; chronic lymphocytic leukemia/small lymphocytic lymphoma, n = 3; marginal zone, n = 1; Waldenstrom's, n = 1). Median age 69; median prior therapies 3. Eighty-six percent of patients experienced treatment-related adverse events (TRAEs). Forty-three percent of patients experienced grade ≥3 TRAEs, with rash (n = 3), pneumonia (n = 3), transaminitis (n = 2), and pneumonitis (n = 2) being most common. Thirty-three percent discontinued treatment due to adverse events. KA2237 induced objective responses in indolent and aggressive lymphoma (overall response rate 37%; complete response n = 4, partial response n = 3).

Through the delivery of large international projects including ICGC and TCGA, knowledge of cancer genomics is reaching saturation point. Enabling this to improve patient outcomes now requires embedding comprehensive genomic profiling into routine oncology practice. Towards this goal, this study defined the biologically and clinically relevant genomic features of adult cancer through detailed curation and analysis of large genomic datasets, accumulated literature and biomarker-driven therapeutics in clinic and development. The characteristics and prevalence of these features were then interrogated in 2348 whole genome sequences, covering 21 solid tumour types, generated by the PCAWG project. This analysis highlights the predominant contribution of copy number alterations and identifies a critical role for disruptive structural variants in the inactivation of clinically important tumour suppressor genes, including PTEN and RB1, which are not currently captured by diagnostic assays. This study defines a set of essential genomic features for the characterisation of common adult cancers., Highlights • Genomic profiling requires coverage of all variant classes to unlock full utility. • Small variants comprise just under a half of all clinically useful information. • Appropriate analysis of copy number and structural alteration is essential. • Available assays are frequently suboptimal in gene and variant class selection. • Comprehensive profiling is within the economic reach of socialised healthcare.

We recently published results of targeted sequencing applied to 928 unselected cases of DLBCL registered in the Haematological Malignancy Research Network (HMRN) registry (1). Clustering allowed us to resolve five genomic subtypes. These subtypes shared considerable overlap with those proposed in two independent genomic studies(2, 3), suggesting the potential to use genetics to stratify patients by both risk and biology. In the original studies, clustering techniques were applied to sample cohorts to reveal molecular substructure, but left open the challenge of how to classify an individual patient. This was addressed by the LymphGen classification tool (4). LymphGen assigns an individual case to one of six molecular subtypes. The tool accommodates data from exome or targeted sequencing, either with or without copy number variant (CNV) data. Separate gene expression data allows classification of a seventh, MYC-driven subtype defined by a double hit (DHL) or molecular high-grade (MHG) gene expression signature(5-7).

Recent advances in single-cell molecular analytical methods and clonal growth assays are enabling more refined models of human hematopoietic lineage restriction processes to be conceptualized. Here, we report the results of integrating single-cell proteome measurements with clonally determined lymphoid, neutrophilic/monocytic, and/or erythroid progeny outputs from >1000 index-sorted CD34+ human cord blood cells in short-term cultures with and without stromal cells. Surface phenotypes of functionally examined cells were individually mapped onto a molecular landscape of the entire CD34+ compartment constructed from single-cell mass cytometric measurements of 14 cell surface markers, 20 signaling/cell cycle proteins, and 6 transcription factors in ∼300 000 cells. This analysis showed that conventionally defined subsets of CD34+ cord blood cells are heterogeneous in their functional properties, transcription factor content, and signaling activities. Importantly, this molecular heterogeneity was reduced but not eliminated in phenotypes that were found to display highly restricted lineage outputs. Integration of the complete proteomic and functional data sets obtained revealed a continuous probabilistic topology of change that includes a multiplicity of lineage restriction trajectories. Each of these reflects progressive but variable changes in the levels of specific signaling intermediates and transcription factors but shared features of decreasing quiescence. Taken together, our results suggest a model in which increasingly narrowed hematopoietic output capabilities in neonatal CD34+ cord blood cells are determined by a history of external stimulation in combination with innately programmed cell state changes.

Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models., A dearth of adequate preclinical models to faithfully mimic diffuse large B-cell lymphoma has hampered the identification of driver genes. Here, the authors present a co-culture system that enables ex vivo expansion, viral transduction and transformation of primary human germinal center B cells.

Hematopoietic clones with leukemogenic mutations arise in healthy people as they age, but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may play an important role in modulating human AML population dynamics. To investigate this concept further, we examined the combined and separate effects of an oncogene (c-MYC) and exposure to interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized, rapidly fatal, and serially transplantable blast population, phenotypically and transcriptionally similar to human AML cells, but only in mice producing IL-3, GM-CSF, and SCF transgenically or in regular mice in which the cells were exposed to IL-3 or GM-CSF delivered using a cotransduction strategy. In their absence, the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months, but, on transfer to secondary mice producing the human cytokines, the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38−) and late granulocyte-macrophage progenitor (GMP) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them.

Distinguishing genomic events that drive cancer (drivers) from inconsequential damage (passengers) is key to matching patients with biomarker-associated therapies and clinical trials. Whilst population level genomic analyses can estimate the proportion of driver events within a given gene, they cannot categorize individual variants. Reliable annotation remains a significant barrier to unlocking the full utility of cancer genomics. GENIE (Genomics Evidence Neoplasia Information Exchange) data was used to generate codon recurrence scores (CRS) for known cancer genes. GENIE was parsed to remove duplicates, sequencing artefact (recurrent variants reported by one institution, invariably associated with amplicon-based sequencing) and hypermutated samples (>15 mutations per megabase). For missense mutations, a codon recurrence of ≥10 was used to classify driver mutations. The table shows proportion of missense mutations classified as drivers by gene and cancer type, comparing population-based non-synonymous to synonymous mutation ratio; dN/dS (Martincorena 2017, PMID 29056346) with assessment of individual variants by CRS. This comparison demonstrates a high degree of concordance. For a few genes, CRS under called driver mutations (ie VHL in renal cancer) likely due to reduced power with small sample numbers. Importantly, CRS is able to assign driver status to individual missense mutations. Comparison with informatic analyses (PMIDs 28115009, 29247016, 30365005, 31034466) showed equivalent or superior performance of the GENIE approach. These findings demonstrate how a large (and expanding) real-world dataset can be used to predict the driver status of somatic missense mutations at the n=1 variant level. This process is amenable to implementation as a rules-based classification process for somatic missense mutations as part of an automated annotation pipeline. +corrected dN/dS ratio not significantly different from one. Pan- cancer Breast cancer Renal cancer Lung adenocarcinoma Gene dN/dS CRS dN/dS CRS dN/dS CRS dN/dS CRS BRAF 91% 76% Citation Format: Philip A. Beer, Susanna L. Cooke, Xuan Shirley Li, Andrew V. Biankin. Leveraging the GENIE dataset to distinguish somatic cancer drivers from passenger events in routine oncology practice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1176.

Elucidation of the identity and diversity of mechanisms that sustain long-term human blood cell production remains an important challenge. Previous studies indicate that, in adult mice, this property is vested in cells identified uniquely by their ability to clonally regenerate detectable, albeit highly variable levels and types, of mature blood cells in serially transplanted recipients. From a multi-parameter analysis of the molecular features of very primitive human cord blood cells that display long-term cell outputs in vitro and in immunodeficient mice, we identified a prospectively separable CD33+CD34+CD38-CD45RA-CD90+CD49f+ phenotype with serially transplantable, but diverse, cell output profiles. Single-cell measurements of the mitogenic response, and the transcriptional, DNA methylation and 40-protein content of this and closely related phenotypes revealed subtle but consistent differences both within and between each subset. These results suggest that multiple regulatory mechanisms combine to maintain different cell output activities of human blood cell precursors with high regenerative potential.

Summary DDX3X is a ubiquitously expressed RNA helicase involved in multiple stages of RNA biogenesis. DDX3X is frequently mutated in Burkitt lymphoma, but the functional basis for this is unknown. Here, we show that loss-of-function DDX3X mutations are also enriched in MYC-translocated diffuse large B cell lymphoma and reveal functional cooperation between mutant DDX3X and MYC. DDX3X promotes the translation of mRNA encoding components of the core translational machinery, thereby driving global protein synthesis. Loss-of-function DDX3X mutations moderate MYC-driven global protein synthesis, thereby buffering MYC-induced proteotoxic stress during early lymphomagenesis. Established lymphoma cells restore full protein synthetic capacity by aberrant expression of DDX3Y, a Y chromosome homolog, the expression of which is normally restricted to the testis. These findings show that DDX3X loss of function can buffer MYC-driven proteotoxic stress and highlight the capacity of male B cell lymphomas to then compensate for this loss by ectopic DDX3Y expression.

Xenograft models are transforming our understanding of the output capabilities of primitive human hematopoietic cells in vivo . However, many variables that affect posttransplantation reconstitution dynamics remain poorly understood. Here, we show that an equivalent level of human chimerism can be regenerated from human CD34 + cord blood cells transplanted intravenously either with or without additional radiation-inactivated cells into 2- to 6-month-old NOD-Rag1 –/– -IL2Rγc –/– (NRG) mice given a more radioprotective conditioning regimen than is possible in conventionally used, repair-deficient NOD- Prkdc scid/scid - IL2Rγc –/– (NSG) hosts. Comparison of sublethally irradiated and non-irradiated NRG mice and W 41 /W 41 derivatives showed superior chimerism in the W 41 -deficient recipients, with some differential effects on different lineage outputs. Consistently superior outputs were observed in female recipients regardless of their genotype, age, or pretransplantation conditioning, with greater differences apparent later after transplantation. These results define key parameters for optimizing the sensitivity and minimizing the intraexperimental variability of human hematopoietic xenografts generated in increasingly supportive immunodeficient host mice.

Based on the profile of genetic alterations occurring in tumor samples from selected diffuse large B-cell lymphoma (DLBCL) patients, 2 recent whole-exome sequencing studies proposed partially overlapping classification systems. Using clustering techniques applied to targeted sequencing data derived from a large unselected population-based patient cohort with full clinical follow-up (n = 928), we investigated whether molecular subtypes can be robustly identified using methods potentially applicable in routine clinical practice. DNA extracted from DLBCL tumors diagnosed in patients residing in a catchment population of ∼4 million (14 centers) were sequenced with a targeted 293-gene hematological-malignancy panel. Bernoulli mixture-model clustering was applied and the resulting subtypes analyzed in relation to their clinical characteristics and outcomes. Five molecular subtypes were resolved, termed MYD88, BCL2, SOCS1/SGK1, TET2/SGK1, and NOTCH2, along with an unclassified group. The subtypes characterized by genetic alterations of BCL2, NOTCH2, and MYD88 recapitulated recent studies showing good, intermediate, and poor prognosis, respectively. The SOCS1/SGK1 subtype showed biological overlap with primary mediastinal B-cell lymphoma and conferred excellent prognosis. Although not identified as a distinct cluster, NOTCH1 mutation was associated with poor prognosis. The impact of TP53 mutation varied with genomic subtypes, conferring no effect in the NOTCH2 subtype and poor prognosis in the MYD88 subtype. Our findings confirm the existence of molecular subtypes of DLBCL, providing evidence that genomic tests have prognostic significance in non-selected DLBCL patients. The identification of both good and poor risk subtypes in patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) clearly show the clinical value of the approach, confirming the need for a consensus classification.

DDX3X is a ubiquitously expressed RNA helicase involved in multiple stages of RNA biogenesis. DDX3X is frequently mutated in Burkitt lymphoma but the functional basis for this is unknown. Here, we show that loss-of-function DDX3X mutations are also commonly found in MYC-translocated diffuse large B cell lymphoma and reveal functional co-operation between mutant DDX3X and MYC. DDX3X promotes translation of mRNAs encoding components of the core translational machinery, thereby driving global protein synthesis. Loss-of-function DDX3X mutations moderate MYC-driven global protein synthesis, thereby buffering MYC-induced proteotoxic stress during early lymphomagenesis. Established lymphoma cells subsequently restore full protein synthetic capacity by aberrant expression of DDX3Y, a Y-chromosome homologue that is normally expressed exclusively in testis. These findings show that DDX3X loss-of-function can buffer MYC-driven proteotoxic stress, and highlight the capacity of male B cell lymphomas to later compensate for this loss by ectopic DDX3Y expression.

e18055 Background: Entrectinib, a TRK kinase inhibitor, has been approved for the treatment of tissue agnostic rare tumors positive for TRK fusions. A very low frequency molecular subset of TRK fusion tumors dubbed as secretory carcinoma (SC) are characterized by organ-agnostic epithelial origin, ETV6-NTRK3 (EN) fusion and distinguishable secretory-type tumor cells. These tumors have been frequently miscategorized as acinic cell carcinoma or adenocarcinoma. A previous mouse study identified EGF dependent epithelial progenitors as putative cell-of-origin for SC. Methods: To test the role of EGFR signaling in EN mediated transformation and therapy resistance, we expressed EN, kinase-dead EN-K380M, and drug-resistant EN-G623R in human epithelial MCF10A cells with EGF-dependent primitive function and investigated their ability to grow in the presence and absence of EGF and/or entrectinib. To understand the significance of findings based on our model, we analyzed a total of 22 ‘rare’ patients from Mayo Clinic Tissue Registry and analyzed TCGA PanCan datasets. Results: We report herein that EGF signaling is essential for normal growth but dispensable during EN driven transformation. Our findings suggest that levels equivalent to circulating EGF (0.5-1ng/ml) is sufficient to drive 100% resistance to entrectinib in vitro. Three different strategies to blockade EGF/EGFR axis including depletion of EGF in culture system, genetic depletion of EGFR using shRNA as well as cetuximab antibody-based EGFR neutralization potentiated oncogene-addiction and hypersensitivity to entrectinib in our models. As predicted, models with G623R mutation in EN was refractory to entrectinib under all experimental conditions. Further omics analysis of TCGA PanCan suggested that EN and EGFR mutations are mutually exclusive and entrectinib-resistant G623R mutation were uncommon. Interestingly, nearly all EN tumors from Mayo Clinic Tissue Registry immunostained weakly or strongly for EGFR and showed perfect concordance with pEGFR suggesting pathway activation. Conclusions: Together, these findings raise an important question whether blockade of ‘wildtype’ EGFR signaling could improve medical intervention in SC patients presenting with wildtype EGFR and no drug-resistant mutation in entrectinib, by improving oncogene-addiction and attendant hypersensitization of transformed cells to entrectinib.

Background: KA2507 is a potent and selective small molecule inhibitor of HDAC6. Inhibition of HDAC6 elicits both cell intrinsic and extrinsic anti-cancer activity. HDAC6 is also involved in the regulation of the co-inhibitory molecule programmed death ligand 1 (PD-L1). We conducted a first-in-human (FIH) study to assess the pharmacokinetics, safety and tolerability of KA2507 monotherapy in patients with advanced cancer with preliminary assessment of any antitumor activity. Methods: In this FIH, dose-escalation phase I study, patients (pts) with advanced metastatic cancer who had failed standard therapy were enrolled. KA2507 was administered orally at escalating doses of 50 mg QD, 100 mg QD, 200 mg QD, 200 mg BID, 400 mg BID, and 800 mg BID (1 cycle = 28 days). A “3+3” design was used. PD assessment was performed measuring changes in acetylated tubulin and histone in peripheral blood cells post dosing. Response was assessed by RECIST 1.1 criteria. (www.clinicaltrials.gov, NCT03008018). Results: Twenty pts with various tumor types commenced treatment from Aug 2017 to Aug 2019. The median age was 56 years (27-78 years) and 13 were males. The most common tumor types were adenoid cystic carcinoma (ACC, n=6) and colorectal cancer (n=4). Seventeen of 20 (85%) pts reported at least one treatment emergent adverse event (AE). Five (25%) pts across all dose cohorts reported treatment related AEs, considered to be at least possibly related to treatment (all Grade 1). No treatment related hematologic toxicities were noted. No dose-limiting toxicity was observed up to the maximum dose level tested. PK analysis demonstrated that the PK parameters of KA2507 were similar across all doses tested and after single and repeated oral dosing, with rapid absorption following oral administration, Tmax ~2 h; t½ ~3 h; dose-proportional exposure; low accumulation. PD assessment demonstrated that the fold-increase in acetylation of tubulin in T-cells increased with exposure (P=0.02), with no significant effect on acetylated histone (P=0.4). Seven (35%) of these heavily pre-treated pts with advanced cancer had stable disease (SD). One pt with ACC had SD and remained on study for 18 cycles, and another pt for 12+ cycles. No objective response was noted. Discussion: KA2507 was well tolerated at all doses tested (up to 800 mg BID) with evidence of selective target engagement at all exposure levels. The dose was not limited by the AEs previously reported in clinical trials of other, less selective HDAC6 inhibitors. A dose of 800 mg BID has been identified for further development and is considered the maximum feasible dose, representing a dose that is well tolerated, achieving exposure that is invariably above the biologically active level identified in pre-clinical syngeneic efficacy models, yet not exceeding the human equivalent dose of the NOAEL in GLP-toxicology and maintaining selectivity for HDAC6. A dose expansion cohort to further investigate this dose is planned. Citation Format: Apostolia M. Tsimberidou, Philip Beer, Jennifer Bendall, James Dow, Justine King, Hilary McElwaine-Johnn, Ignacio I. Wistuba. Phase I study of KA2507, a selective HDAC6 inhibitor, in patients with relapsed or refractory solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT151.

TPS4652 Background: The ABC-02 trial provided Level A evidence supporting the use of cisplatin plus gemcitabine as first-line chemotherapy for advanced biliary tract cancer (ABC) [Valle, 2010]. In second line therapy oxaliplatin and 5FU and ivosedinib for IDH1 mutated cancers are options [Lamarca, 2019; Abou-Alfa, 2019] however there remains significant unmet need for patients without actionable alterations. Histone deacetylase 6 (HDAC6) is over-expressed in cholangiocarcinoma, reducing primary cilia. This is mediated through increased resorption in normal human cholangiocytes via tubulin deacetylation in the ciliary axoneme. Inhibition of HDAC6 elicits both cell intrinsic and extrinsic anti-cancer activity. HDAC6 inhibition reversed oncogenic loss of ciliation and demonstrated preclinical efficacy in a syngeneic rat orthotopic biliary cancer model [Gradilone, 2013]. KA2507 is a potent and selective small molecule inhibitor of HDAC6. Phase I dose escalation study identified an oral dose of 800mg bid for further development, being well tolerated and showing evidence of selective target engagement. Methods: ABC-11 is a Phase II multi-centre, open-label study of KA2507 in 40 evaluable patients with advanced biliary tract cancer previously treated with standard of care chemotherapy. The study follows a single-arm single-stage design using A’Hern’s methodology. Eligible patients receive continuous 28-day cycles of fixed daily oral dose of KA2507 until death, disease progression or other pre-defined reason for study drug discontinuation. Tumour assessment is made at baseline and at 8-weekly intervals using RECIST 1.1 criteria until disease progression; primary endpoint is PFS at 4 months. Independent Data Monitoring Committee will review 4 month PFS and other data after first six patients, after a total of 17 patients (futility analysis, corresponding to cut-off of the Simon’s minimax 2-stage design; 33% was set as the target 4-month PFS rate expected with KA2507) and at least annually thereafter. Subject to availability of adequate tissue, mandatory pre-treatment and on-study tumour biopsy samples will undergo multiparameter flow cytometry of immune cell subsets, immunofluorescence analysis of immune cell subsets (activation status and topology) and T cell repertoire studies. The study received regulatory and ethical approval to proceed in January 2020 and enrolment is in progress. Clinical trial information: NCT04186156 .

The discovery of rare, heterogeneous self-renewing stem cells with shared developmental and molecular features within epithelial components of mammary gland and breast cancers has provided a conceptual framework to understand cellular composition of these tissues and mechanisms that control their number. These normal mammary epithelial stem cells (MaSCs) and breast cancer stem cells (BCSCs) were identified and analyzed using transplant assays (namely mammary repopulating unit (MRU) assay, mammary tumor-initiating cell (TIC) assay), which reveal their latent ability to regenerate respective normal and malignant epithelial tissues with self-renewing units displaying hierarchical cellular differentiation over multiple generations in recipient mice. “Next-generation” methods using “barcoded” normal and malignant mammary cells, with the help of next-generation sequencing (NGS) technology, have revealed hidden complexity and heterogeneous growth potential of MaSCs and BCSCs. Several single markers or combinations of markers have been reported to prospectively enrich MaSCs and BCSCs. Such markers and the extent to which they enrich for MaSCs and BCSCs activity require a critical appraisal. Also, knowledge of the functional assays and their limitations and harmonious reporting of results is a prerequisite to improve our understanding of MaSCs and BCSCs. This chapter describes evolution of the concept of MaSCs and BCSCs, and specific methodologies to investigate them.

Recent advances in single-cell molecular analytical methods and clonal growth assays are enabling more refined models of human hematopoietic lineage restriction processes to be conceptualized. Here, we report the results of integrating single-cell proteome measurements with clonally determined lymphoid, neutrophilic/monocytic, and/or erythroid progeny outputs from >1000 index-sorted CD34(+) human cord blood cells in short-term cultures with and without stromal cells. Surface phenotypes of functionally examined cells were individually mapped onto a molecular landscape of the entire CD34(+) compartment constructed from single-cell mass cytometric measurements of 14 cell surface markers, 20 signaling/cell cycle proteins, and 6 transcription factors in ∼300 000 cells. This analysis showed that conventionally defined subsets of CD34(+) cord blood cells are heterogeneous in their functional properties, transcription factor content, and signaling activities. Importantly, this molecular heterogeneity was reduced but not eliminated in phenotypes that were found to display highly restricted lineage outputs. Integration of the complete proteomic and functional data sets obtained revealed a continuous probabilistic topology of change that includes a multiplicity of lineage restriction trajectories. Each of these reflects progressive but variable changes in the levels of specific signaling intermediates and transcription factors but shared features of decreasing quiescence. Taken together, our results suggest a model in which increasingly narrowed hematopoietic output capabilities in neonatal CD34(+) cord blood cells are determined by a history of external stimulation in combination with innately programmed cell state changes.

The incidence of acute myeloid leukaemia (AML) increases with age and mortality exceeds 90% when diagnosed after age 65. Most cases arise without any detectable early symptoms and patients usually present with the acute complications of bone marrow failure(1). The onset of such de novo AML cases is typically preceded by the accumulation of somatic mutations in preleukaemic haematopoietic stem and progenitor cells (HSPCs) that undergo clonal expansion(2,3). However, recurrent AML mutations also accumulate in HSPCs during ageing of healthy individuals who do not develop AML, a phenomenon referred to as age-related clonal haematopoiesis (ARCH)(4-8). Here we use deep sequencing to analyse genes that are recurrently mutated in AML to distinguish between individuals who have a high risk of developing AML and those with benign ARCH. We analysed peripheral blood cells from 95 individuals that were obtained on average 6.3 years before AML diagnosis (pre-AML group), together with 414 unselected age- and gender-matched individuals (control group). Pre-AML cases were distinct from controls and had more mutations per sample, higher variant allele frequencies, indicating greater clonal expansion, and showed enrichment of mutations in specific genes. Genetic parameters were used to derive a model that accurately predicted AML-free survival; this model was validated in an independent cohort of 29 pre-AML cases and 262 controls. Because AML is rare, we also developed an AML predictive model using a large electronic health record database that identified individuals at greater risk. Collectively our findings provide proof-of-concept that it is possible to discriminate ARCH from pre-AML many years before malignant transformation. This could in future enable earlier detection and monitoring, and may help to inform intervention.

Without effective therapy, chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized, but biologically poorly characterized, accelerated phase (AP). Here, we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts, which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML, we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML, including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients.

Elucidation of the identity and diversity of mechanisms that sustain long-term human blood cell production remains an important challenge. Previous studies indicate that, in adult mice, this property is vested in cells identified uniquely by their ability to clonally regenerate detectable, albeit highly variable levels and types, of mature blood cells in serially transplanted recipients. From a multi-parameter analysis of the molecular features of very primitive human cord blood cells that display long-term cell outputs in vitro and in immunodeficient mice, we identified a prospectively separable CD33

Purpose: We sought to identify the genomic abnormalities in squamous cell carcinomas (SCC) arising in ovarian mature cystic teratoma (MCT), a rare gynecological malignancy of poor prognosis. Experimental design: We performed copy number, mutational state, and zygosity analysis of 151 genes in SCC arising in MCT (n = 25) using next-generation sequencing. The presence of high-/intermediate-risk HPV genotypes was assessed by quantitative PCR. Genomic events were correlated with clinical features and outcome. Results: MCT had a low mutation burden with a mean of only one mutation per case. Zygosity analyses of MCT indicated four separate patterns, suggesting that MCT can arise from errors at various stages of oogenesis. A total of 244 abnormalities were identified in 79 genes in MCT-associated SCC, and the overall mutational burden was high (mean 10.2 mutations per megabase). No SCC was positive for HPV. The most frequently altered genes in SCC were TP53 (20/25 cases, 80%), PIK3CA (13/25 cases, 52%), and CDKN2A (11/25 cases, 44%). Mutation in TP53 was associated with improved overall survival. In 8 of 20 cases with TP53 mutations, two or more variants were identified, which were bi-allelic. Conclusions: Ovarian SCC arising in MCT has a high mutational burden, with TP53 mutation the most common abnormality. The presence of TP53 mutation is a good prognostic factor. SCC arising in MCT share similar mutation profiles to other SCC. Given their rarity, they should be included in basket studies that recruit patients with SCC of other organs. Clin Cancer Res; 23(24); 7633–40. ©2017 AACR.

The past years have witnessed significant success for cancer immunotherapies that activate a patient's immune system against their cancer cells. At the same time our understanding of the genetic changes driving tumor evolution have progressed dramatically. The study of cancer genomes has shown that tumors are best understood as cell populations governed by the rules of evolution, leading to the emergence and spread of cell lineages with pathogenic mutations. Moreover, somatic evolution can explain the acquisition of mutations conferring drug resistance in the ever-lasting battle for reaching even fitter cell states. Here, we review the current state of the art of somatic cancer evolution and mechanisms of immune control and escape. We also revisit the principles of immunotherapy from the perspective of somatic evolution and discuss the basic rules of resistance to immunotherapies as dictated by evolution.

Leukemic transformation of human cells is a complex process. Here we show that forced expression of MN1 in primitive human cord blood cells maintained on stromal cells in vitro induces a transient, but not serially transplantable, myeloproliferation in engrafted mice. However, cotransduction of an activated HOX gene (NUP98HOXD13) with MN1 induces a serially transplantable acute myeloid leukemia (AML). Further characterization of the leukemic cells generated from the dually transduced cells showed the activation of stem cell gene expression signatures also found in primary human AML. These findings show a new forward genetic model of human leukemogenesis and further highlight the relevance of homeobox transcription factors in the transformation process.

Acute myeloid leukemias (AMLs) are heterogenous diseases often resulting from the acquisition of multiple genetic alterations that deregulate hematopoietic precursor proliferation and block normal differentiation. Chronic myeloid leukemia offers a unique opportunity to identify molecular mechanisms that interfere with normal differentiation in the context of a highly proliferative hematopoietic stem cell clone that produces massive number of functional differentiated myeloid cells due to the presence of a BCR-ABL1 fusion gene. Since the NUP98-HOXA9 (NA9) fusion gene has been identified in some CML blast crisis patients or de novo AML, we asked whether a humanized model of CML progression to AML would result from its lentiviral-mediated introduction into primary CML CD34+ cells from 3 chronic phase CML patients in which >97%, >96%, and 44% of the CD34+ longterm culture-initiating cells were Ph+/BCR-ABL1+. In vitro experiments showed the NA9-transduced cells produced a hugely increased number of granulo-monocytic progenitors in long-term cultures (up to 1000-fold, p=0.03; t-ratio test) and enhanced the serial replating activity of directly clonogenic cells as compared to matched samples of cells transduced with a control vector. In vivo experiments showed that 90% of sublethally irradiated NOD-SCID IL2Rgc-null mice expressing human IL3, GM-CSF and SCF constitutively that were transplanted with these same NA9-transduced cells developed evidence of a progressed myeloproliferative neoplasia. This included tissue infiltrates of eosinophils, basophils and mastocytes, exclusive myeloid differentiation and signs of an imminently fatal leukemia between 8 and 27 weeks post-transplant in 50% of cases, although an excess blast population was not seen in these. RNA-sequencing of cells analyzed just 48 hours after transduction revealed a signature of 53 genes that were more highly expressed in CD34+ CML cells carrying the NA9 fusion gene in comparison to control CML cells. This signature included many genes expressed by hematopoietic stem cells (HSCs), such as 5' HOXA genes, 3' HOXB genes, PBX3, MEIS1,ARID5B, AHR, REL, BMP6, GDF10, SFRP5, PPBP,PLA2G4A suggesting that NA9 induces the expression of a partial HSC program in later CML progenitor types that make up the bulk of the CD34+ CML population. Consistent with this hypothesis, the NA9 signature separated the HSCs, CMPs and GMPs of chronic CML patients as well as those of normal subjects (GSE47927; p=1x10-6, p=2x10-4 respectively). Most genes in the NA9 signature were also found to be over-expressed in CD34+ cells of patients in the accelerated phase and in the blast phase of CML, including ARID5B, AHR, STARD9, TOX, or FOXP1 (p=9.10-8; ANOVA) (GSE4170). The NA9 signature was also significantly enriched in transcripts of genes that are over-expressed in the blasts of AML patients carrying NPM1 mutations or MLL fusions and was predictive of overall survival in the AML cohort of the Cancer Genome Atlas (p=0.02; log-rank test, n=200). This stem cell signature was also associated with an increase in the number of H3K27ac marks (on average 7805 ± 960 peaks for NA9 vs 5888 ± 2739 peaks for control) at 48 hours post-transduction. De novo H3K27ac peaks in NA9+ CD34+ cells were located in proximal and distal enhancers of 993 genes (GREAT parameters TSS ± 2kb from TSS and 100kb max extension). These were significantly enriched genes in the GM-CSF signaling pathway (MySigDB, binomial p-value=2.19x10-8) and that are upregulated in granulocytes and monocytes following LPS exposure. H3K27ac peaks also matched with GATA1, RELA, MEF2A and IKZF1 transcription factor binding sites previously mapped by ChIPSeq experiments (adjusted p-value Overexpression of the NA9 fusion gene in chronic phase CD34+ CML cells thus appears to reprogram the expression of HSC genes as well as those associated with GM-CSF pathway activation and inflammatory responses via H3K27 acetylation of associated loci leading to a picture of advanced accelerated phase/disease progression but not the complete differentiation arrest seen in terminal blast crisis or frank AML. These findings highlight the multiplicity of biologically important molecular alterations that can result from a single epigenetic perturbation but, nevertheless, are insufficient to create an overt AML phenotype. Disclosures Turhan: novartis: Honoraria, Research Funding; Incyte: Consultancy, Honoraria.

Background: Current evidence suggests that genetic and epigenetic abnormalities drive the development of human Acute Myeloid Leukemias (AMLs). However, whether these are sufficient to establish a permanent, self-sustaining AML population, and the potential role of shared perturbed downstream pathways is unknown. We hypothesized that a modest upregulated expression of MYC might play such a role given its commonly increased expression in many AML patients' cells. To test this hypothesis, we assessed the dynamics and types of cells produced in sublethally irradiated NOD-Rag1-/--IL2Rγc-/-(NRG) mice transgenically producing human IL3, GM-CSF and SCF (NRG-3GS mice) following their transplantation with freshly isolated subsets of normal CD34+ cord blood (CB) cells that were first lentivirally transduced with a human MYC cDNA. Results: FACS and Western blot analyses indicated this produced a 2 to 5-fold increase in MYC mRNA and protein levels in MYC-transduced CD34+ CB cells, and 21/22 NRG-3GS mice injected with ≥6,500 of these cells developed a fatal human AML population within 7 weeks. Histological analysis of their bone marrow and spleen cells showed both contained a prominent human CD123+CD33+CD15±CD34-CD14-CD19-CD3- blast population. Additional limiting dilution transplants showed that both the CD34+CD38- cells (enriched for hematopoietic stem cells) and the more differentiated CD34+ GMPs were similarly highly susceptible (at frequencies of 1/14 and 1/46, respectively) and, in both cases, generated progeny that could initiate serially transplantable leukemias with the same phenotypic and transcriptomic features. Comparison to normal CB cells indicated these most closely resembled GMPs, and comparison to pediatric AML patient samples indicated a similarity to myelomonocytic leukemias with enhanced MYC expression. Interestingly, 14 sublethally irradiated NRG mice (the parental strain not producing human 3GS) transplanted with matched aliquots of CD34+ MYC-transduced cells regenerated a normal spectrum of CD19+ lymphoid cells, CD14+ and CD15+ GM cells and readily detectable CD34+ cells for up to 32 weeks of follow-up with no evidence of leukemogenesis. However, transfer of these regenerated human cells into secondary NRG-3GS mice, even after this extended period, enabled their rapid production of a lethal human AML in all 5 mice tested. In contrast, matched aliquots transplanted into 5 NRG recipients produced declining grafts of normal cells. This finding was then exploited to determine which growth factors were responsible for activating the AML program by transplanting NRG mice with CD34+ CB cells transduced with MYC and just a single growth factor, or all 3 as a positive control. In this set of experiments, a lethal human AML was obtained when MYC was paired with human IL3 or GM-CSF (or all 3 together), but not with SCF (or no growth factors). Conclusion: We report here a new in vivo model of MYC-induced human myeloid leukemogenesis that produces a serially transplantable AML closely resembling human pediatric myelomonocytic leukemias with elevated MYC expression. The rapidity, consistency, and high frequency of this transformation process obtained by transducing late granulopoietic as well as early types of normal human CD34+ progenitor cells makes this system highly attractive for future mechanistic and therapeutic testing experiments. The discovery that MYC deregulation alone generates a stable "latent program" that can be rapidly activated by exposure to exogenous growth factors typical of inflammatory states also raises intriguing questions about the potential role of such events in the genesis of AML populations that arise in patients. Disclosures Beer: Karus therapeutics Ltd.: Employment.

Introduction: Despite therapeutic advances, there remains a considerable need for novel therapies for B-cell lymphomas. Although a high proportion of patients (pts) show response to initial therapy, many fail to achieve durable remissions and experience recurrent disease. Agents that target molecular pathways associated with the development and progression of lymphoma are likely to be highly effective and are desirable. The p110δ isoform of the PI3K enzyme is mainly expressed in lymphocytes and has been an attractive therapeutic target, with several PI3Kδ inhibitors demonstrating meaningful efficacy in B-cell lymphomas. Targeting the p110β isoform may further overcome tumor growth and escape mechanisms by mitigating the upregulation of the PI3K/AKT pathway, particularly in PTEN-deficient lymphomas. KA2237 is an oral, potent and selective inhibitor of the PI3K β and δ isoforms. The aim of this first in human, phase I, open-label, single arm study (NCT02679196) was to investigate the safety, tolerability, pharmacokinetic properties and pharmacodynamic effects of KA2237, in order to determine the maximum tolerated dose based on dose limiting toxicity and assess preliminary anti-tumor activity in pts with R/R B-cell lymphoma. Methods: Pts ≥ 18 years (yrs) of age, ECOG ≤ 2, with B-cell lymphoma R/R or intolerant of established therapies (including rituximab) were enrolled using a 3+3 dose escalation (50-400mg) design. KA2237 was given orally on a once daily continuous schedule until progression or unacceptable toxicity. Anti-tumor activity was evaluated by computed tomography and, when available, integrating 18F-FDG positron emission tomography response assessment, at 8, 16 and 24 weeks. Response was assessed according to Lugano 2014 criteria. Pts received PJP prophylaxis. Results: 21 pts with B-cell lymphoma were enrolled (8 DLBCL [diffuse large B-cell], 5 FL [follicular], 3 MCL [mantle cell], 3 CLL/SLL [chronic lymphocytic leukemia/small lymphocytic lymphoma], 1 MZL [marginal zone], 1 WM [Waldenstrom]). Pts received KA2237 at 4 dose levels: 50mg (n=6), 100mg (n=3), 200mg (n=7) and 400mg (n=5) daily; 21 pts were evaluable for safety assessment. Pharmacokinetic profiles were favorable with mean plasma half-life of 17-33 hours, compatible with once daily oral dosing. Median age was 69 yrs (range 48-84) with 76% males; median number of prior therapies was 3 (range 1-6). Median follow up duration was 8.5 months (range 6.9-24.6). Median duration of drug exposure was 82 days (range 10-714 days). 86% of pts experienced treatment-related adverse events (TRAE). 43% of pts experienced a grade ≥ 3 TRAE, with rash (n=3), transaminitis (n=2) and pneumonitis (n=2) occurring in more than 1 pt. 29% discontinued treatment due to a TRAE with pneumonitis (n=2) occurring in more than 1 pt. One grade 5 TEAE (multifocal pneumonia) was observed. 19/21 pts were evaluable for response, ORR was 37% (4 CR, 3 PR). Responses were observed across lymphoma subtypes including DLBCL, FL, CLL and MCL. Responses were often durable (see Figure) and in 2 pts with DLBCL who achieved CR permitted consolidation by autologous stem cell transplantation. Conclusions: KA2237 is an oral, once a day, selective dual inhibitor of PI3K β/δ with a manageable toxicity profile and promising single-agent clinical activity in heavily pretreated R/R B-cell lymphoma. The recommended phase II dose is 200mg daily. The findings of this study support the further evaluation of KA2237. Figure. Disclosures Nastoupil: Novartis: Honoraria; Spectrum: Honoraria; Janssen: Honoraria, Research Funding; Gilead: Honoraria; Genentech, Inc.: Honoraria, Research Funding; Bayer: Honoraria; Celgene: Honoraria, Research Funding; TG Therapeutics: Honoraria, Research Funding. Neelapu:Acerta: Research Funding; Merck: Consultancy, Research Funding; Poseida: Research Funding; Unum Therapeutics: Consultancy, Research Funding; Karus: Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Incyte: Consultancy; Precision Biosciences: Consultancy; BMS: Research Funding; Allogene: Consultancy; Pfizer: Consultancy; Cellectis: Research Funding; Novartis: Consultancy; Celgene: Consultancy, Research Funding; Cell Medica: Consultancy. Fowler:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; ABBVIE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Westin:Janssen: Other: Advisory Board, Research Funding; Genentech: Other: Advisory Board, Research Funding; Juno: Other: Advisory Board; Unum: Research Funding; Kite: Other: Advisory Board, Research Funding; Novartis: Other: Advisory Board, Research Funding; Curis: Other: Advisory Board, Research Funding; Celgene: Other: Advisory Board, Research Funding; 47 Inc: Research Funding; MorphoSys: Other: Advisory Board. Wang:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Acerta Pharma: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding; MoreHealth: Consultancy, Equity Ownership; BioInvent: Consultancy, Research Funding; Aviara: Research Funding; BeiGene: Research Funding; Loxo Oncology: Research Funding; VelosBio: Research Funding; Pulse Biosciences: Consultancy; Juno Therapeutics: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Dava Oncology: Honoraria; Kite Pharma: Consultancy, Research Funding. Beer:Karus therapeutics Ltd.: Employment. Cecil:Karus Therapeutics: Employment. Dow:Karus Therapeutics: Employment. McHale:Karus Therapeutics: Employment. Silva:Karus Therapeutics: Employment. Ward:Karus Therapeutics: Employment. Yavari:Karus Therapeutics: Employment. Shuttleworth:Karus Therapeutics: Employment.

Acute myeloid leukemias (AML) comprise a genetically diverse group of human hematologic malignancies with a generally poor prognosis and modes of pathogenesis that have been difficult to investigate. Historic evidence and more recent findings have identified hematopoietic stem cell programs as major targets of mutations that predispose to and/or initiate a multistep process of transformation. However, details of the changes involved and how they interact in emerging human leukemic cells have been particularly challenging to characterize. This is due to an emerging appreciation of the heterogeneity in events that impact the differentiation processes of normal human hematopoietic cells, as well as difficulties in recreating the full leukemogenic alterations in the actual human cells affected, to avoid discrepancies inherent in studying model organisms. Here we describe several models of de novo human leukemogenesis that illustrate their power and the novel results they can generate. These include our recent discovery of a novel “latent” leukemic state that can be obtained in MYC-transduced human cord blood cells regenerating a normal spectrum of lymphoid and myeloid progeny in transplanted immunodeficient mice, but that can then be rapidly activated to an aggressive form of AML by in vivo exposure to a single human growth factor. These findings portend the future utility of de novo human leukemogenesis models as new platforms for elucidating shared molecular mechanisms responsible for different stages of human leukemogenesis that may be initiated by different mutations or exposure to different microenvironmental conditions. The flexibility and consistency of these models also make them attractive for identifying and testing new treatment strategies targeting mechanisms required for disease manifestation.

Human cord blood (CB) offers an attractive source of cells for clinical transplants because of its rich content of cells with sustained repopulating ability in spite of an apparent deficiency of cells with rapid reconstituting ability. Nevertheless, the clonal dynamics of nonlimiting CB transplants remain poorly understood. To begin to address this question, we exposed CD34+ CB cells to a library of barcoded lentiviruses and used massively parallel sequencing to quantify the clonal distributions of lymphoid and myeloid cells subsequently detected in sequential marrow aspirates obtained from 2 primary NOD/SCID-IL2Rγ(-/-) mice, each transplanted with ∼10(5) of these cells, and for another 6 months in 2 secondary recipients. Of the 196 clones identified, 68 were detected at 4 weeks posttransplant and were often lympho-myeloid. The rest were detected later, after variable periods up to 13 months posttransplant, but with generally increasing stability throughout time, and they included clones in which different lineages were detected. However, definitive evidence of individual cells capable of generating T-, B-, and myeloid cells, for over a year, and self-renewal of this potential was also obtained. These findings highlight the caveats and utility of this model to analyze human hematopoietic stem cell control in vivo.

The guideline group was selected to be representative of UK-based medical experts with an interest in myeloproliferative neoplasms and eosinophilia. PubMed and EMBASE were searched systematically for publications in English until August 2015 using the key words eosinophilia, hypereosinophilia, eosinophilic leukaemia and HES. The writing group produced the draft guideline which was subsequently revised by consensus by members of the General Haematology and Haemato-oncology Task Forces of the British Committee for Standards in Haematology. The guideline was then reviewed by a sounding board of UK haematologists and representatives from the Nordic MPN Study Group, the British Committee for Standards in Haematology (BCSH) and the British Society for Haematology (BSH) Committee and comments were incorporated where appropriate. The ‘GRADE’ system was used to quote levels and grades of evidence, details of which can be found in Table I. The objective of this guideline is to provide healthcare professionals with clear guidance on the investigation and management of eosinophilia.

The increasing number of drugs targeting specific proteins implicated in tumourigenesis and the commercial promotion of relatively affordable genome-wide analyses has led to an increasing expectation among patients with cancer that they can now receive effective personalised treatment based on the often complex genomic signature of their tumour. For such approaches to work in routine practice, the development of correspondingly complex biomarker assays through an appropriate and rigorous regulatory framework will be required. It is becoming increasingly evident that a re-engineering of clinical research is necessary so that regulatory considerations and procedures facilitate the efficient translation of these required biomarker assays from the discovery setting through to clinical application. This article discusses the practical requirements and challenges of developing such new precision medicine strategies, based on leveraging complex genomic profiles, as discussed at the Innovation and Biomarkers in Cancer Drug Development meeting (8th-9th September 2016, Brussels, Belgium).

Better methods to characterize normal human hematopoietic cells with short-term repopulating activity cells (STRCs) are needed to facilitate improving recovery rates in transplanted patients.We now show that 5-fold more human myeloid cells are produced in sublethally irradiated NOD/SCID-IL-2Receptor-γchain-null (NSG) mice engineered to constitutively produce human interleukin-3, granulocyte-macrophage colony-stimulating factor and Steel factor (NSG-3GS mice) than in regular NSG mice 3 weeks after an intravenous injection of CD34 human cord blood cells. Importantly, the NSG-3GS mice also show a concomitant and matched increase in circulating mature human neutrophils. Imaging NSG-3GS recipients of lenti-luciferase-transduced cells showed that human cells being produced 3 weeks posttransplant were heterogeneously distributed, validating the blood as a more representative measure of transplanted STRC activity. Limiting dilution transplants further demonstrated that the early increase in human granulopoiesis in NSG-3GS mice reflects an expanded output of differentiated cells per STRC rather than an increase in STRC detection.NSG-3GS mice support enhanced clonal outputs from human short-term repopulating cells (STRCs) without affecting their engrafting efficiency. Increased human STRC clone sizes enable their more precise and efficient measurement by peripheral blood monitoring.

Several growth factors (GFs) that together promote quiescent human hematopoietic stem cell (HSC) expansion ex vivo have been identified; however, the molecular mechanisms by which these GFs regulate the survival, proliferation. and differentiation of human HSCs remain poorly understood. We now describe experiments in which we used mass cytometry to simultaneously measure multiple surface markers, transcription factors, active signaling intermediates, viability, and cell-cycle indicators in single CD34+ cord blood cells before and up to 2 hours after their stimulation with stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor (5 GFs) either alone or combined. Cells with a CD34+CD38-CD45RA-CD90+CD49f+ (CD49f+) phenotype (∼10% HSCs with >6-month repopulating activity in immunodeficient mice) displayed rapid increases in activated STAT1/3/5, extracellular signal-regulated kinase 1/2, AKT, CREB, and S6 by 1 or more of these GFs, and β-catenin only when the 5 GFs were combined. Certain minority subsets within the CD49f+ compartment were poorly GF-responsive and, among the more GF-responsive subsets of CD49f+ cells, different signaling intermediates correlated with the levels of the myeloid- and lymphoid-associated transcription factors measured. Phenotypically similar, but CD90-CD49f- cells (MPPs) contained lower baseline levels of multiple signaling intermediates than the CD90+CD49f+ cells, but showed similar response amplitudes to the same GFs. Importantly, we found activation or inhibition of AKT and β-catenin directly altered immediate CD49f+ cell survival and proliferation. These findings identify rapid signaling events that 5 GFs elicit directly in the most primitive human hematopoietic cell types to promote their survival and proliferation.