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There is a growing body of evidence that therapy-related myeloid neoplasms (t-MNs) with driver gene mutations arise in the background of clonal hematopoiesis (CH) under the positive selective pressure of chemo- and radiation therapies. Uncovering the exposure relationships that provide selective advantage to specific CH mutations is critical to understanding the pathogenesis and etiology of t-MNs. In a systematic analysis of 416 patients with t-MN and detailed prior exposure history, we found that TP53 mutations were significantly associated with prior treatment with thalidomide analogs, specifically lenalidomide. We demonstrated experimentally that lenalidomide treatment provides a selective advantage to Trp53-mutant hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, the effect of which was specific to Trp53-mutant HSPCs and was not observed in HSPCs with other CH mutations. Because of the differences in CK1α degradation, pomalidomide treatment did not provide an equivalent level of selective advantage to Trp53-mutant HSPCs, providing a biological rationale for its use in patients at high risk for t-MN. These findings highlight the role of lenalidomide treatment in promoting TP53-mutated t-MNs and offer a potential alternative strategy to mitigate the risk of t-MN development.

Introduction While clonal hematopoiesis (CH) can precede the development of AML, preleukemic CH can persist after attaining remission. In rare cases, non-leukemic CH can also emerge after remission. Long-term clonal dynamics of persistent CH or emerging CH (together, post-remission CH) in AML patients during consolidation therapies or maintenance therapies is not well understood. Furthermore, the clinical implications of post-remission CH have not been systematically studied. Since AML patients receive various types of post-remission consolidation therapies, including allogeneic stem cell transplant (allo-SCT), analysis of clonal dynamics of post-remission CH offers unique opportunity to study the response of CH to therapies. Such knowledge may be translated into the development of therapeutic strategy targeting CH. Here, we analyzed multiple longitudinal samples from AML patients after remission and analyzed clonal behavior of post-remission CH during consolidation and maintenance therapies. Methods We studied 164 AML patients who attained complete remission (CR) after induction therapies and analyzed baseline and CR marrow by targeted deep sequencing of 295 genes (median 403x depth). Among the patients who were identified to have post-remission CH, we analyzed multiple longitudinal marrows and tracked clonal dynacmis of post-remission CH. We defined “persistent CH” when patients had mutations that were originally detected in AML and persisted after CR with variant allele frequency (VAF) > 2.5%. On the other hand, we defined “ emerging CH” when the patients had new mutations arising after attaining CR. Results Among the 164 AML patients, 79 (48%) patients were found to have post-remission CH at first CR. Of those, we were able to analyze multiple post-remission marrows in 54 patients (median 6 samples/patient, IQR 4-7.75),of which 44 (66%) had persistent CH and 1 (1.9%) had emerging CH, and 9 (17%) had both. The median age of these 54 patients cohort was 59 (IQR: 51-68). 34 (63%) patients attained CR after intensive chemotherapies (IC, idarubicin with high-dose cytarabine based), whereas 20 (37%) patients received low-intensity chemotherapies as induction (hypomethylating agent [HMA]-based N=3, low dose cytarabine-based N=17). All 34 patients treated with IC induction subsequently received consolidation chemotherapies with high-dose cytarabine (HDAC) regimens for median 2 cycles (IQR 2-3.25). Among the 20 patients treated with low-intensity chemotherapies, all patients received consolidation therapies with HMA-based regimen. A subset of patients received maintenance therapies under clinical trials: lenalidomide (N = 1), venetoclax (N = 2), and nivolumab (N = 1). 29 patients underwent allo-SCT (23 with myeloablative conditioning [MAC], and 6 with reduced-intensity conditioning [RIC]). The most frequently mutated genes for persistent CH were DNMT3A (N = 23), followed by TET2 (N = 17), IDH2 (N = 10), and SRSF2 (N = 10). For emerging CH, we observed emergence of JAK2 (N = 2), RUNX1 (N = 2), TET2 (N = 2), BRAF (N = 1), BCOR (N = 1), TP53 (N = 1), KDM6A (N = 1), and NRAS (N = 1). Longitudinal clonal analysis revealed that post-remission CH persisted in 52 of 54 (96%) patients during consolidation and maintenance therapies. Neither cytarabine based chemotherapies, HMA, venetoclax, lenalidomide, and nivolumab, reduced the VAF of post-remission CH, except in 2 (4%) patients, in which post-remission CH (IDH2 and TET2 mutations) were cleared with high dose cytarabine regimen. Although consolidation or maintenance therapies did not reduce post-remission CH, 17 out of 19 (89.4%) patients who underwent for allo-SCT had clearance of post-remission CH with both MAC and RIC. Overall, post-remission CH did not impact the risk of relapse or overall survival. Furthermore, post-remission CH did not affect long-term blood counts (neutrophil counts, hemoglobin, and platelet counts). Conclusion Post-remission CH persisted long-term in AML patients during consolidation and maintenance therapies without having significant impact on blood counts, relapse, and survival. Therapies with cytarabine-based chemotherapies and HMA did not affect the clonal size of post-remission CH, suggesting that these therapies may not be useful to treat CH in pre-leukemic context either. The novel therapeutic approach is warranted to target CH. Disclosures DiNardo: celgene: Consultancy, Honoraria; abbvie: Consultancy, Honoraria; syros: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; daiichi sankyo: Honoraria; agios: Consultancy, Honoraria; medimmune: Honoraria; jazz: Honoraria. Kadia: Bioline RX: Research Funding; Celgene: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; AbbVie: Consultancy, Research Funding. Ravandi: Selvita: Research Funding; Macrogenix: Consultancy, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Xencor: Consultancy, Research Funding; Menarini Ricerche: Research Funding; Cyclacel LTD: Research Funding. Konopleva: Astra Zeneca: Research Funding; Agios: Research Funding; Ablynx: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Eli Lilly: Research Funding; Forty-Seven: Consultancy, Honoraria; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Calithera: Research Funding. Kantarjian: Takeda: Honoraria; AbbVie: Honoraria, Research Funding; BMS: Research Funding; Jazz Pharma: Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Amgen: Honoraria, Research Funding; Cyclacel: Research Funding; Daiichi-Sankyo: Research Funding; Immunogen: Research Funding; Astex: Research Funding; Agios: Honoraria, Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Champlin: Johnson and Johnson: Consultancy; Sanofi-Genzyme: Research Funding; Actinium: Consultancy. Garcia-Manero: Onconova: Research Funding; H3 Biomedicine: Research Funding; Astex: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Amphivena: Consultancy, Research Funding; Merck: Research Funding. Takahashi: Symbio Pharmaceuticals: Consultancy.

Allosteric inhibitors of mutant IDH1 or IDH2 induce terminal differentiation of the mutant leukemic blasts and provide durable clinical responses in approximately 40% of acute myeloid leukemia (AML) patients with the mutations. However, primary resistance and acquired resistance to the drugs are major clinical issues. To understand the molecular underpinnings of clinical resistance to IDH inhibitors (IDHi), we perform multipronged genomic analyses (DNA sequencing, RNA sequencing and cytosine methylation profiling) in longitudinally collected specimens from 60 IDH1- or IDH2-mutant AML patients treated with the inhibitors. The analysis reveals that leukemia stemness is a major driver of primary resistance to IDHi, whereas selection of mutations in RUNX1/CEBPA or RAS-RTK pathway genes is the main driver of acquired resistance to IDHi, along with BCOR, homologous IDH gene, and TET2. These data suggest that targeting stemness and certain high-risk co-occurring mutations may overcome resistance to IDHi in AML. The regulation of resistance to IDH inhibitors in acute myeloid leukaemia is not completely understood. Here the authors reveal with integrative multi-omics analyses that stemness features are major drivers of primary resistance, while high-risk mutations drive acquired resistance.

Clonal haematopoiesis (CH) in patients with acute myeloid leukaemia (AML) may persist beyond attaining complete remission. From a consecutive cohort of 67 patients with nucleophosmin 1-mutated (NPM1mut ) AML, we identified 50 who achieved NPM1mut clearance and had parallel multicolour flow cytometry (MFC) and next generation sequencing (NGS). In total, 13 (26%) cleared all mutations, 37 (74%) had persistent CH frequently involving DNA methyltransferase 3α (DNMT3A,70%), tet methylcytosine dioxygenase 2 (TET2, 27%), isocitrate dehydrogenase 2 (IDH2, 19%) and IDH1 (11%). A small number (

Clonal diversity is a consequence of cancer cell evolution driven by Darwinian selection. Precise characterization of clonal architecture is essential to understand the evolutionary history of tumor development and its association with treatment resistance. Here, using a single-cell DNA sequencing, we report the clonal architecture and mutational histories of 123 acute myeloid leukemia (AML) patients. The single-cell data reveals cell-level mutation co-occurrence and enables reconstruction of mutational histories characterized by linear and branching patterns of clonal evolution, with the latter including convergent evolution. Through xenotransplantion, we show leukemia initiating capabilities of individual subclones evolving in parallel. Also, by simultaneous single-cell DNA and cell surface protein analysis, we illustrate both genetic and phenotypic evolution in AML. Lastly, single-cell analysis of longitudinal samples reveals underlying evolutionary process of therapeutic resistance. Together, these data unravel clonal diversity and evolution patterns of AML, and highlight their clinical relevance in the era of precision medicine., Understanding the evolutionary trajectory of cancer samples may enable understanding resistance to treatment. Here, the authors used single cell sequencing of a cohort of acute myeloid leukemia tumours and identify features of linear and branching evolution in tumours.

PurposeThis study aimed to evaluate the utility of inflammation-based prognostic scores (IBPS) and systemic immune-inflammation index (SII) in the treatment of oral cancer patients.MethodsFor the 183 patients enrolled in this study, IBPS and SII were calculated from peripheral blood samples obtained before and after treatment and at the time of relapse. We examined overall survival (OS) and disease-free survival (DFS) using previously reported cut-off values for IBPS. Cut-off values of neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), platelet-to-lymphocyte ratio (PLR), and prognostic nutritional index (PNI) were analyzed as NLR 1.79, PLR 114.97, LMR 5, and PNI 52.44. The cut-off value for SII was set at 569. OS and DFS were analyzed by Kaplan–Meier methods using the cutoff of each IBPS and SII. Univariate analysis and multivariate analysis using Cox proportional hazards were performed for OS and DFS.ResultsKaplan–Meier methods showed the high-PNI group showed good prognosis including OS and DFS, while the high-SII group displayed poor DFS. Univariate analysis showed that pre-treatment high PNI and low SII were significantly associated with better prognosis. Multivariate analysis identified pre-treatment PNI as independently associated with OS. For DFS, univariate analysis using Cox proportional hazards modeling showed that pre-treatment high NLR and high SII were significantly associated with worse prognosis, while high PNI was significantly associated with better prognosis. Multivariate analysis identified pre-treatment PNI and SII as independently associated with DFS. Parameters of PNI and SII components were compared between pre-treatment, post-treatment and at relapse in the high- and low-PNI groups. PNI was predominantly decreased in both high- and low-PNI groups at post-treatment and at relapse compared to pre-treatment. This trend was also observed for albumin.ConclusionsHigher pre-treatment PNI was associated with better OS, while lower pre-treatment PNI and higher treatment SII were associated with poorer DFS in oral cancer patients. Our data indicated that PNI and SII might offer useful biomarkers for gauging prognosis and the efficacy of conventional therapies.

Background: NPM1 mutations (NPM1 +) occur in ~30% of acute myeloid leukemia (AML), often with co-occurring mutations including isocitrate dehydrogenase mutations (IDH +). Previous studies suggest early acquisition and expansion of IDH1/2 mutations (IDH1 +/IDH2 +) prior to NPM1 + development increase relapse risk. Yet the clonal hierarchy of NPM1 +/IDH + AML, dynamics of IDH + during therapy, and predictive value of IDH1 +/2 + in NPM1 + AML in response to standard intensive (IC) or venetoclax-based (VEN) treatment is less studied. Methods: We identified 71 patients with newly diagnosed NPM1+ AML (index cohort). Longitudinal molecular assessment of bone marrow samples at diagnosis, remission, and relapse was performed using next-generation sequencing (NGS) of genes frequently mutated in AML. Clonality within each sample was inferred using variant allele frequency (VAF) of individual mutations compared with NPM1 +(co-dominant: VAF difference of < 10%; sub-clonal: VAF ≥10% lower). Measurable residual disease (MRD) was assessed using flow cytometry (FC) (MRD FC; lower limit of sensitivity 10 -3-10 -4) and NGS. To validate findings in the index cohort, clinical and genomic data were collected from a publicly available dataset of 39 patients with NPM1 + AML who underwent single cell DNA sequencing (scDNAseq) at our institution. Categorical and continuous variables were assessed using the fisher exact test and the Wilcoxon rank sum test, respectively. Adjustment for multiple comparisons utilized the Benjamini-Horchberg procedure. Time-to-event outcomes were assessed by the log-rank method. Results: Median patient age was 64 years (range 19-84). Thirty-eight patients (54%) received IC (IC: n=30, IC+VEN: n= 8); 33 (46%) received lower-intensity therapy (lower-intensity: n= 6, lower-intensity+VEN: n=27). Common co-occurring mutations at diagnosis included DNMT3A (62%), FLT3 (52%), TET2 (27%), IDH2 (23%), IDH1 (18%), PTPN11 (18%), and NRAS (17%). SRSF2, IDH2, TET2, ASXL1, and DNMT3A mutations preceded or were co-dominant with NPM1 +, while NRAS, PTPN11, and FLT3 mutations were sub-clonal. IDH1 +was sub-clonal in 40% of cases (Fig. 1A). In the scDNAseq cohort IDH1 + was sub-clonal in 40% (n=3/8); IDH2 + preceded NPM1 +in 75% (n=9). Compared with IDH1 +, IDH2 + was more likely to precede or be co-dominant with NPM1 + (95% vs. 60%, p-value: 0.017). DTA (DNMT3A, TET2, ASXL1) mutations were detected more often in remission versus IDH + (88% vs. 55%, p-value: 0.01). Persistent IDH1 + and IDH2 + were identified in 36% (n=4) and 73% (n=11) of patients. Concurrent MRD FC confirmed clearance of the previous AML clone in 100% of patients, with a pre-leukemic/CH immunophenotype identified in 50% and 80% of cases with persistent IDH1 +or IDH2 +, respectively Patients with IDH + often developed new or persistent mutations in remission compared with patients with wild-type IDH(54% vs. 12%, p-value Patients with NPM1 +/IDH + compared with NPM1 +/IDH - AML in complete remission (N=91) had improved event-free (EFS; median 65 vs. 28 months, p-value: 0.002) and overall survival (OS; median 82 months vs. NR, p-value: 0.02) (Fig. 1B and C), largely driven by NPM1 +/IDH2 + cases, and patients receiving IC. No survival difference was observed based on clonal hierarchy of IDH + (clonal vs sub-clonal) compared with NPM1 + or persistent IDH + in remission with concurrent clearance of NPM1 + and MRD FC. FLT3 mutations were identified at similar frequencies in the NPM1 +/IDH +and NPM1 +/IDH - groups (50% vs. 53%, p-value: 0.84; ITD: 81% vs. 69%, p-value: 0.49), and a similar number of patients receiving VEN (48% vs.43%, p-value: 0.38). Conclusion: IDH1 + and IDH2 + develop at differing timepoints during leukemogenesis in NPM1 + AML. IDH2 + frequently precedes NPM1 and persists in remission, while IDH1 + is more often sub-clonal and cleared in remission. Persistent IDH1 + or IDH2 + frequently correlate with detection of a CH phenotype by MFC in remission, and in the absence of NPM1 + and MRD FC do not influence relapse. Improved EFS and OS was observed with NPM1 +/IDH + AML, identifying a molecular subgroup associated with favorable outcomes to both VEN and IC treatment regimens Figure 1 Figure 1. Disclosures DiNardo: Foghorn: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; ImmuneOnc: Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Agios/Servier: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Forma: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Wang: Stemline Therapeutics: Honoraria. Kadia: Ascentage: Other; AstraZeneca: Other; Liberum: Consultancy; Genfleet: Other; Pfizer: Consultancy, Other; Astellas: Other; Novartis: Consultancy; BMS: Other: Grant/research support; Jazz: Consultancy; Dalichi Sankyo: Consultancy; Genentech: Consultancy, Other: Grant/research support; Cure: Speakers Bureau; Amgen: Other: Grant/research support; Aglos: Consultancy; AbbVie: Consultancy, Other: Grant/research support; Cellonkos: Other; Sanofi-Aventis: Consultancy; Pulmotech: Other. Daver: Gilead Sciences, Inc.: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Hanmi: Research Funding; Sevier: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Trovagene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; Novimmune: Research Funding; Glycomimetics: Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Short: NGMBio: Consultancy; Takeda Oncology: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; AstraZeneca: Consultancy; Astellas: Research Funding; Novartis: Honoraria; Amgen: Consultancy, Honoraria. Khoury: Angle: Research Funding; Kiromic: Research Funding; Stemline Therapeutics: Research Funding. Konopleva: F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Agios: Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; Stemline Therapeutics: Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Rafael Pharmaceuticals: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; KisoJi: Research Funding; Sanofi: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Cellectis: Other: grant support; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Ablynx: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding. Ravandi: AbbVie: Honoraria, Research Funding; Xencor: Honoraria, Research Funding; Taiho: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria; Amgen: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Prelude: Research Funding; Syros Pharmaceuticals: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria. Takahashi: Celgene/BMS: Consultancy; Symbio Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Novartis: Consultancy. Loghavi: Abbvie: Current equity holder in publicly-traded company; Curio Sciences: Honoraria; Gerson Lehrman Group: Consultancy; Guidepoint: Consultancy; Peerview: Honoraria; Qualworld: Consultancy.

Background. Distant metastasis is considerably more frequent in superselective intra-arterial chemoradiotherapy than other radical treatments for advanced oral cancers. However, there is no evidence supporting such claim. The purpose of this study was to report our experience in superselective intra-arterial chemoradiotherapy and conventional surgical management with particular focus on distant metastasis. Methods. One hundred seventy-two patients with oral squamous cell carcinoma in stages III and IV were included in this study. Retrospective analysis for DM rates and background between surgical management and superselective intra-arterial chemoradiotherapy was performed. Results. Distant metastasis developed clinically was detected in 24 out of 141 patients (17.0%) treated surgically and in 6 out of 31 patients (19.4%) treated with superselective intra-arterial chemoradiotherapy. There was no significant difference in the rate of distant metastasis between the 2 groups. Comparison of patients in both groups with and without distant metastasis revealed no differences in age, T classification, N classification, and treatment effect. Neck recurrence was the only significant risk factor for distant metastasis. Conclusion. No significant difference was found in the rate of distant metastasis between patients treated with surgical treatment and superselective intra-arterial chemoradiotherapy, and additional effort is needed to reduce the risk of distant metastasis.

Upon viral infection, the cytoplasmic viral sensor retinoic acid-inducible gene-I (RIG-I) recognizes viral RNA to activate antiviral signaling to induce type I interferon (IFN). RIG-I-like receptors (RLRs) activate antiviral signaling in a tissue-specific manner. The molecular mechanism underlying antiviral signaling in the respiratory system remains unclear. We studied antiviral signaling in the lower respiratory tract (LRT), which is the site of many harmful viral infections. Epithelial cells of the LRT can be roughly divided into two groups: bronchial epithelial cells (BECs) and pulmonary alveolar epithelial cells (AECs). These two cell types exhibit different phenotypes; therefore, we hypothesized that these cells may play different roles in antiviral innate immunity. We found that BECs exhibited higher antiviral activity than AECs. TNF receptor-associated factor 3 (TRAF3) has been shown to be a crucial molecule in RLR signaling. The expression levels of TRAF3 and TRAF5, which have conserved domains that are nearly identical, in the LRT were examined. We found that the bronchus exhibited the highest expression levels of TRAF3 and TRAF5 in the LRT. These findings suggest the importance of the bronchus in antiviral innate immunity in the LRT and indicate that TRAF3 and TRAF5 may contribute to RLR signaling.