Long-term Relapse-free Survival Research Articles

Long-term relapse-free survival enabled by integrating targeted antibacteria in antitumor treatment

The role of tumor-resident intracellular microbiota (TRIM) in carcinogenesis has sparked enormous interest. Nevertheless, the impact of TRIM-targeted antibacteria on tumor inhibition and immune regulation in the tumor microenvironment (TME) remains unexplored. Herein, we report long-term relapse-free survival by coordinating antibacteria with antitumor treatment, addressing the aggravated immunosuppression and tumor overgrowth induced by TRIM using breast and prostate cancer models. Combining Ag+ release with a Fenton-like reaction and photothermal conversion, simultaneous bacteria killing and multimodal antitumor therapy are enabled by a single agent. Free of immune-stimulating drugs, the agent restores antitumor immune surveillance and activates immunological responses. Secondary inoculation and distal tumor analysis confirm lasting immunological memory and systemic immune responses. A relapse-free survival of >700 days is achieved. This work unravels the crucial role of TRIM-targeted antibacteria in tumor inhibition and unlocks an unconventional route for immune regulation in TME and a complete cure for cancer.

Abstract 45: Driving AMPK signaling in CD4+ CART cells enhances metabolic capacity, memory formation, and anti-leukemia activity

Abstract Background: While Chimeric Antigen Receptor (CAR)T cell therapy has seen great success in pediatric and young adult acute lymphoblastic leukemia, up to 50% of patients will relapse following treatment. Increased memory formation and mitochondrial capacity, particularly in the CD4+ subset, have been linked to improved CART cell function and enhanced in vivo persistence, leading to long-term relapse-free survival. AMP-activated protein kinase (AMPK) is a cellular energy sensor responsible for promoting metabolic efficiency and T cell memory formation. We have previously demonstrated AMPK activity can be increased in human T cells via overexpression of the AMPKγ2 domain. We hypothesized that overexpression of AMPKγ2 in CART cells would enhance their metabolic capacity and improve their anti-leukemia function. Methods: Human CART cells bearing a CD19-CAR utilizing a 41BB costimulatory domain were generated from healthy human donors via lentiviral transduction and co-transduced with AMPKγ2 (AMPK) or an empty vector (EV) control. Flow sorting for the co-transduced populations yielded >95% CD4+ CART cells. CARTs were expanded in IL-2 for approximately 2 weeks, followed by overnight stimulation and subsequent metabolic analysis using the Seahorse Metabolic Analyzer. Memory status was assessed by CD62L and CCR7 co-expression. In vitro CART function was assessed using Zs-green+ NALM6 targets, with loss of green fluorescence over time measured on the Incucyte analyzer. Where indicated, tumor conditioned media was harvested from 72-hour NALM6 cultures. For in vivo experiments, immunodeficient NOD-scid IL2Rγnull (NSG) mice were injected with luciferase+ NALM6 targets, followed one week later by AMPK- or EV-CARTs. Leukemia burden, via IVIS imaging, and survival were followed over time. Results: CD4+ CARTs co-transduced with AMPKγ2 (AMPK-CARTs) increased oxygen consumption, maximal respiration, and spare respiratory capacity following overnight stimulation by ~20% compared to CARTs co-transduced with the EV (EV-CARTs) (p<0.05). Following expansion, CD62L+CCR7+ central memory cells were 4.5-fold more abundant in the AMPK-CART group (2.4-6.2-fold, p<0.01). In vitro, while AMPK-CARTs demonstrated similar cytotoxicity against NALM6 targets as EV-CARTs in standard media, they mediated faster killing of NALM6 targets when challenged in tumor conditioned media (p<0.001). In vivo, recipients of AMPK-CARTs showed a consistent decrease in tumor burden as measured by median radiance values, translating to a significant survival advantage (p<0.05). Conclusions: Increasing AMPK signaling in CD4+ CARTs via overexpression of AMPKγ2 improved metabolic profile, increased memory generation, and enhanced toxicity against leukemia targets both in vitro and in vivo. Together, these features make elevating AMPK signaling an attractive method to increase the efficacy of CART therapy. Citation Format: Erica Braverman, Herbert Schuler, Mengtao Qin, Craig Byersdorfer. Driving AMPK signaling in CD4+ CART cells enhances metabolic capacity, memory formation, and anti-leukemia activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 45.

Long-term prognosis and its associated predictive factors in patients with eosinophilic gastroenteritis.

Eosinophilic gastroenteritis (EGE) is a chronic recurrent disease with abnormal eosinophilic infiltration in the gastrointestinal tract. Glucocorticoids remain the most common treatment method. However, disease relapse and glucocorticoid dependence remain notable problems. To date, few studies have illuminated the prognosis of EGE and risk factors for disease relapse. To describe the clinical characteristics of EGE and possible predictive factors for disease relapse based on long-term follow-up. This was a retrospective cohort study of 55 patients diagnosed with EGE admitted to one medical center between 2013 and 2022. Clinical records were collected and analyzed. Kaplan-Meier curves and log-rank tests were conducted to reveal the risk factors for long-term relapse-free survival (RFS). EGE showed a median onset age of 38 years and a slight female predominance (56.4%). The main clinical symptoms were abdominal pain (89.1%), diarrhea (61.8%), nausea (52.7%), distension (49.1%) and vomiting (47.3%). Forty-three (78.2%) patients received glucocorticoid treatment, and compared with patients without glucocorticoid treatments, they were more likely to have elevated serum immunoglobin E (IgE) (86.8% vs 50.0%, P = 0.022) and descending duodenal involvement (62.8% vs 27.3%, P = 0.046) at diagnosis. With a median follow-up of 67 mo, all patients survived, and 56.4% had at least one relapse. Six variables at baseline might have been associated with the overall RFS rate, including age at diagnosis < 40 years [hazard ratio (HR) 2.0408, 95% confidence interval (CI): 1.0082-4.1312, P = 0.044], body mass index (BMI) > 24 kg/m2 (HR 0.3922, 95%CI: 0.1916-0.8027, P = 0.014), disease duration from symptom onset to diagnosis > 3.5 mo (HR 2.4725, 95%CI: 1.220-5.0110, P = 0.011), vomiting (HR 3.1259, 95%CI: 1.5246-6.4093, P = 0.001), total serum IgE > 300 KU/L at diagnosis (HR 0.2773, 95%CI: 0.1204-0.6384, P = 0.022) and glucocorticoid treatment (HR 6.1434, 95%CI: 2.8446-13.2676, P = 0.003). In patients with EGE, younger onset age, longer disease course, vomiting and glucocorticoid treatment were risk factors for disease relapse, whereas higher BMI and total IgE level at baseline were protective.

Predictors of outcome of trace element levels in acute myeloid leukaemia patients: a prospective study for one year

Abstract Background Trace elements are essential for mammalian metabolism. To date, no data have been presented predicting the relapse-free survival of acute myeloid leukemia (AML) patients based on the level of trace elements in the serum. This prospective cohort study had the following objectives: (i) to quantify essential trace elements in the serum of AML patients.(ii) to examine the predictive values of the trace element levels in newly diagnosed de novo AML patients. Methods 50 patients AML were studied. The control group consisted of 50 patients. Serum levels of the trace elements copper, zinc, copper to zinc ratio and selenium, were measured by spectrophotometry. The associations of trace element with the relapse-free survival of these AML patients were assessed by Kaplan–Meier survival curves. Results AML patients, a control group, and AML subgroups with and without hematological relapse were evaluated for trace element concentrations. AML patients showed higher copper and lower zinc levels than controls. AML patients had increased Cu/Zn ratios. AML patients had reduced selenium levels. Relapsed AML patients had increased copper and Cu to Zn ratios, but decreased zinc and selenium. Copper, zinc, Cu to Zn ratio, and selenium cut-off values for AML relapse and nonrelapse were identified via ROC curve analysis. Kaplan-Meier curves showed that patients with higher copper, lower zinc, higher Cu to Zn ratio, or lower selenium had a shorter estimated mean relapse-free survival time. Conclusions Trace element levels are correlated with long-term relapse-free survival in newly diagnosed AML patients and may serve as predictive biomarkers.

Establishing an AML Ex Vivo Platform to Generate Physiologically Relevant Models

Acute myelogenous leukemia (AML) comprises a highly polyclonal and heterogeneous disease containing leukemic stem cells, progenitors, and differentiated blasts. Despite advances in treatment, the long-term relapse-free survival for AML remains poor. Due to complex mutational profiles and highly divergent subtypes in AML, the use of physiologically relevant, patient-derived models is needed. Traditional cell line models of AML almost exclusively represent differentiated blasts, are homogenous in clonality, and often do not encompass major genetic subtypes. To address these gaps, we have established a culture system that supports the growth of higher-order AML samples ex vivo. By optimizing culture conditions, we successfully cultivated 70% (10/14) of AML disseminated PDX models ex vivo and demonstrated they continue to proliferate after long term cultures. We established a 16-color flow panel to characterize their cell maturity state and show they maintain cell surface heterogeneity. Importantly, these models have not ‘drifted’ transcriptomically after multiple passages. Our platform includes 2 NPM1c mutant and 3 IDH1/2 mutant ex vivo lines, in addition to other subtypes underrepresented in traditional culture methods. Furthermore, we treated AML PDXs in vivo with a clinically-relevant dose of venetoclax + azacytidine (Ven/AZA) for 2 cycles (14 days) and then evaluated post-VEN/AZA cells in our ex vivo assay. Growing these models ex vivo, we show using our flow panel that Ven/AZA treatment shifts the differentiation status from a stem-like state to a more mature-like cell state which is consistent with literature evidence of treatment resistance. Additionally, a second PDX model treated with three cycles of Ven/AZA in vivo and harvested in ex vivo culture conditions maintained both Ven and AZA resistance. Interestingly, compared with the vehicle treatment, Ven/AZA treatment caused resistance to other cell death agents including selective MCL1 and dual Bcl2/xL inhibitors. Lastly, we performed a 7-day cell compound panel screen using common standard of care agents for the treatment of AML. Many of the targeted agent activity correlated with expected genetic subtypes, such as FLT3 inhibitors in FLT3-ITD mutants and venetoclax in WT TP53 models. This established ex vivo platform provides physiologically relevant models to explore novel target biology, better understand compound mechanism of action (MoA), and generate drug resistant models through in vivo driven clinically relevant dose scheduling using standard of care agents.

Unsupervised Clustering Analysis of Regimen and HLA Characteristics Identifies Subgroups with Improved Relapse-Free Survival in Children with ALL and AML Undergoing Umbilical Cord Blood Transplantation

Introduction. Umbilical cord blood transplantation (UCBT) is an efficacious therapeutic intervention for pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). However, contention persists regarding how best to achieve long-term relapse-free survival (RFS). Differences in clinical practice can be attributed to an incompletely understood interplay between graft-versus-leukemia (GvL) effect and graft-versus-host disease (GvHD), as well as an incomplete understanding of how UCBTs differ from those of other stem cell sources. Human leukocyte antigen (HLA) matching is a critical determinant of post-transplant outcome, but factors such as GvHD prophylaxis and total body irradiation (TBI) usage also play a key role. Objective. To use unsupervised clustering algorithms to examine the interplay between HLA matching, immunosuppressive regimens, and conditioning factors with greater power than that conferred by conventional multivariate regression analyses. Methodology. Data pertaining to UCBTs performed for pediatric ALL (n = 908) and AML (n = 417) between 2004 and 2022 was obtained from the Eurocord/EBMT registry. ALL and AML UCBTs were separately subjected to a partitioning around medoids clustering algorithm, which identified k = 15 ALL clusters and k = 11 AML clusters. These clusters were used to form hypotheses for post-hoc testing, which were tested using Kaplan-Meier and competing risks analyses. Results. Clusters of both diseases were frequently divided by usage of ATG, which was administered in 77% of 8/8 ABCDR matched UCBTs. ALL and AML patients who did not receive ATG experienced greater RFS than recipients of ATG, and this appeared to be driven by elimination of relapse mortality in the late (≥5 year) post-transplant setting (Figs 1-2). ALL clusters were frequently divided by use of total body irradiation (TBI), and those featuring TBI regimens conferred RFS comparable or superior to non-TBI regimens; this appeared to be driven by a reduction in relapse mortality within the first three years post-UCBT (P = 0.007). In AML but not ALL, both HLA-C and KIR match levels appeared to be relevant (P = 0.04), despite typing only having been performed for 70% and 24% of AML UCBTs, respectively. Patients receiving a 7/8 ABCDR-matched graft with a single HLA-C mismatch experienced significantly poorer RFS than 8/8 matched UCBTs (P = 0.04), but a concomitant single co-mismatch at HLA-A or HLA-B appeared to restore RFS to levels comparable to 8/8 matched grafts. Conclusions. ATG is over-used in ALL and AML patient-graft pairings where risk of non-relapse mortality (NRM) is low, and this may be creating an avoidable risk of relapse. The conflicting results in published literature may be explained by follow-up time, as the effect does not become apparent until 5 years post-UCBT and many studies do not follow patients beyond this. Incorporation of TBI into ALL myeloablative regimens prevents relapse mortality in the early to mid-post UCBT setting, and this effect was independent of HLA matching. These findings align with the known genetic and biological determinants of these diseases and redouble the importance of typing at HLA-C in AML; administration of TBI in ALL; and a measured approach to the usage of ATG.

How does Nephroblastomatosis impact the prognosis of unilateral Wilms tumor?

We aimed to explore whether Nephroblastomatosis (Nbm) has an effect on the prognosis of Wilms tumor (WT), and compare the relapse-free survival (RFS) and overall survival (OS) after surgery of WT patients with or without Nbm. We retrospectively analyzed the clinical data of children with WT admitted to our department from April 2010 to September 2021. The enrolled patients were divided into two groups according to whether they were combined with Nbm histologically or not, the Nbm group for patients accompany with Nbm and the non-Nbm group for pure WT. All patients underwent a standardized comprehensive treatment according to National Wilms Tumor Study 5. Clinical variables, pathological results, and the prognosis were collected during follow-up. A total of 345 patients were finally enrolled in this study, of which 299 patients in the non-Nbm group and 46 patients in the Nbm group. Univariate Cox analysis revealed that Nbm was not the risk factor of either OS or RFS. The difference of postoperative RFS (P = 0.66) and OS (P = 0.68) between two groups was not significant. Subgroup analysis revealed that the RFS and OS between the non-Nbm group and the Nbm group had no statistical difference under the condition of same stage (low grade and high grade), same histology (favorable histology and unfavorable histology), same surgical approach (nephrectomy and nephron-sparing surgery), with or without lymph node metastasis (P > 0.05). Although Nbm had a strong correlation with the occurrence of WT, a combined Nbm did not increase the risk of metastasis and poor prognosis of WT. After complete surgical resection followed by standardized treatment, the long-term RFS and OS were not different from pure WT. IEC-C-006-A04-V.06 retrospectively registered.

Allogeneic stem cell transplant for myelofibrosis- A retrospective single-center study.

Allogeneic hematopoietic stem cell transplantation (HSCT) is currently the only curative treatment option for myelofibrosis (MF). Despite the benefits of long-term relapse-free survival, HSCT can be associated with substantial treatment-related morbidity and mortality. This is an observational retrospective study of 15 consecutive patients with MF who underwent allogeneic HSCT at a tertiary care center in Northern India between June 2012 and January 2020. The pre-transplant Dynamic International Prognostic Scoring System (DIPSS) and hematopoietic cell transplantation-specific co-morbidity index (HCT-CI) scores were used. The primary endpoints were overall survival (OS) and disease-free survival (DFS), and the secondary endpoints were post-transplant complications (acute and chronic graft-versus-host-disease [GvHD], graft failure [GF], and cytomegalovirus reactivation [CMV]). The OS and DFS in our study were 60% with no relapse at a median follow-up of 364 days (range 7-2,815 days). Twenty-seven percent of patients developed acute GvHD and 27% of patients developed chronic (limited) GvHD. The non-relapse mortality (NRM) was 40%, with the main cause of death being sepsis, followed by acute GvHD. MF remains a challenging condition to treat, with a poor prognosis. Our study showed that reduced toxicity conditioning provided good DFS and OS. Thus, it should be offered to patients with high DIPSS scores. Sepsis was the predominant cause of mortality in this cohort.

Regularized Mixture Cure Models Identify a Gene Signature That Improves Risk Stratification within the Favorable-Risk Group in 2017 European Leukemianet (ELN) Classification of Acute Myeloid Leukemia (Alliance 152010)

Introduction: The 2017 ELN Favorable-risk group comprises specific recurrent cytogenetic and molecular alterations. Assignment to this group has important clinical implications, as allogeneic stem cell transplantation in first complete remission (CR) is not advised due to a relatively good outcome of patients (pts) receiving chemotherapy and transplant-associated mortality. However, not all Favorable-risk pts experience long-term survival and their outcomes are affected by co-occurring genetic features making recognition of pts who would most likely be cured of high importance. Thus, we used our regularized mixture cure model (MCM) that embeds false discovery rate control to identify prognostically relevant transcripts that can distinguish pts cured from pts susceptible to lower- or higher-risk of relapse. Methods: We analyzed 312 pts <60 years with de novo AML classified in the 2017 ELN Favorable-risk group excluding APL who achieved a CR, had RNA-seq data available, and treated on one of 8 CALGB/Alliance frontline protocols with intensive 7+3-based induction chemotherapy. No pt received an allogeneic stem cell transplant in first CR. MCMs assume the population consists of 2 subgroups, cured and susceptible, thus there are 2 regression components, which permit identification of features associated with cure and/or latency of susceptible pts. 'Cured' is synonymous with attaining long-term relapse-free survival and thus pts have a survival probability of 1. We fit univariable MCMs to identify baseline demographic, clinical features, or selected gene mutations related to the probability of being cured and/or to the latency distribution (time to relapse). RNA-seq expression values were candidate covariates in a regularized MCM to identify a parsimonious list of transcripts associated with cure or latency. We then used 2017 ELN Favorable pts in an independent dataset (GSE37642) for validation. Results: The estimated proportion cured (39.5%) was significant (P < 0.001) indicating that a MCM should be fit. Other than % of bone marrow blasts (P = 0.009) and % of blood blasts (P = 0.048), no other pretreatment features were significant in univariable MCMs. CEBPAdouble and TET2 mutations were the only ones significantly associated with latency (P = 0.008 and P = 0.013, respectively), but neither was associated with cure (P = 0.881 and P = 0.507, respectively). Our regularized MCM identified 101 transcripts associated with cure and 71 transcripts associated with latency. Kaplan-Meier curves of cured vs susceptible pts as well as of those susceptible with lower vs higher risk of relapse or death were well separated. We mapped the 168 unique transcripts to GSE37642 Affymetrix HG-U133Plus2 GeneChip for validation and found that 65 cure-related probe sets and 57 latency probe sets mapped to 34 and 32 unique genes, respectively. The validation data yielded AUC=0.788 and C-statistic=0.887, indicating good predictive ability of the selected genes. As desired, the predicted cured group had a survival probability of 1 throughout the observation period (Fig 1), with good separation between predicted susceptibles in the lower- versus higher-risk groups (Fig 2). Cell death and survival was the top molecular/cellular function for both the cure and latency portions of the model with 22 and 20 transcripts involved, respectively, with only BRD2, HLA-A, HLA-B, NAIP, and TXNIP in common. Cell death and survival molecules unique to the latency portion of the model included CD300A, CRNDE, DDX39B, FAS, GPI, HLA-DRA, KLF13, LAT, let-7, LMO2, LSP1, NOTCH1, PLCG2, PSMB8, and U2AF1; those unique to the cure portion included ARID5B, BAG6, BMI1, CCL5, CDK7, DPPA4, ELOVL5, HLA-DRB1, IL18, INTS6, MAP4K4, mir-25, mir-491, NOMO1, NR1H3, RPS16, and TUBB. Conclusion: Our regularized MCM identified important subsets of genes associated with cure and latency in the 2017 ELN Favorable-risk pts. Our results suggest that the Favorable-risk group includes distinct transcriptionally defined subgroups with different biological properties, which may be useful for refining this risk category by identification of pts who might be cured with chemotherapy alone. Given similar treatment with 7+3-based cytotoxic chemotherapy, the identified subgroups (cured, susceptible lower risk, susceptible higher risk) seem to have different sensitivities to 7+3-based therapy. Support: R01LM013879, U10CA180821, U10CA180882, U24CA196171 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Highly Parallel Discovery of Synthetic Knockin Sequences for Enhanced Cancer Immunotherapies

Introduction T cells engineered to express transgenic T cell receptors (TCRs) or chimeric antigen receptors (CARs) have emerged as powerful treatment options for some malignancies. However, while CAR T cells have induced impressive initial response rates in patients with B-precursor acute lymphoblastic leukemia (ALL) and lymphoma, they fail to mediate long-term relapse-free survival in 40-60% of patients and have not been successful in most solid tumors. For both TCR- and CAR-based approaches, T cell persistence and long-term functionality can be diminished by chronic antigen stimulation or tonic signaling. One approach to enhance the efficacy of cell therapies that has been explored by several groups is the overexpression of specific transcription factors (e.g. AP-1/ATF transcription factors such as c-JUN or BATF); however, more systematic gain-of-function screens in human CAR T cells have not yet been done. Methods and Results Here, we introduce CRISPR-based modular pooled knockin (ModPoKI) screens to evaluate hundreds to thousands of different constructs in human T cells and their potential to overcome TCR or CAR T cell dysfunction. We generated two barcoded ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs) to identify those that confer a fitness advantage to TCR or CAR T cells. The libraries were combined with either an NY-ESO-1-specific TCR, an anti-CD19 CAR or a high affinity anti-GD2 CAR that is known to induce tonic signaling-based dysfunction. The libraries and the TCR/CAR were engineered as polycistronic sequences that were non-virally integrated into the TRAC (T cell receptor alpha chain constant) locus of primary human T cells. We performed >20 unique ModPoKI screens including bead-based single stimulation, repetitive stimulation with target cells and tonic stimulation (GD2 CAR model). While constructs containing known AP-1/ATF transcription factors BATF and BATF3 showed increased abundance across multiple screens, transcription factor AP4 (TFAP4) knockin (KI) constructs were more clearly enriched after repetitive stimulation and tonic signaling, suggesting potentially undescribed benefits in exhaustion-prone environments. We next validated single knockins of TFAP4 or a control construct (truncated nerve growth factor receptor, tNGFR) in combination with the GD2 or the CD19 CAR. TFAP4 KI led to increased levels of cytokine release, proliferation and target cell killing compared to conventional CAR T cells. Moreover, TFAP4 KI mediated increased tumor control and survival in an in vivo model. Interestingly, we observed elevated levels of CD25 (IL2RA) expression on TFAP4 KI T cells and enrichment of genes related to IL2/STAT5 pathways which could hint at a potential benefit in situations of IL-2 competition/presence of regulatory T cells. Lastly, we aimed to discover specific combinations of TFs that work in concert to enhance T cell fitness in the setting of tonic CAR signaling. We created a ~10,000-member library (102 x 102 - pairwise combinations of 100 transcription factors plus two controls) cloned into constructs with the GD2 CAR and performed a tonic signaling ModPoKI screen. Analysis of the constructs that increased the most in relative abundance highlighted that several of the top performing constructs included either TFAP4, BATF, BATF3 or a combination of TFAP4 and BATF(3), suggesting that TFAP4 and BATF(3) are key transcription factors that can coordinately drive increased T cell fitness upon repetitive stimulation. Validation analyses confirmed that the combinatorial KI of a BATF-TFAP4 construct confers highest cytotoxic capacity in vitro and in vivo compared to single knockin (BATF-RFP or RFP-TFAP4) or control knockin (RFP-tNGFR) GD2 CAR constructs. Conclusions In conclusion, ModPoKI screens allow for customizable parallel evaluation and functional characterization of hundreds to thousands of different T cell constructs. Using clinically relevant screening modalities, we nominated candidate genes that can synthetically re-write the T cell response to repetitive antigen exposure or tonic signaling and thus have the potential to program more effective TCR or CAR T cell therapies for cancer. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A novel 16-gene alternative mRNA splicing signature predicts tumor relapse and indicates immune activity in stage I–III hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is a lethal disease with high relapse and dismal survival rates. Alternative splicing (AS) plays a crucial role in tumor progression. Herein, we aim to integratedly analyze the relapse-associated AS events and construct a signature predicting tumor relapse in stage I–III HCC. Methods: AS events of stage I–III HCC with tumor relapse or long-term relapse-free survival were profiled to identify the relapse-associated AS events. A splicing network was set up to analyze the correlation between the relapse-associated AS events and splicing factors. Cox regression analysis and receiver operating characteristic curve were performed to develop and validate the relapse-predictive AS signature. Single-sample gene set enrichment analysis (ssGSEA) and the ESTIMATE algorithm were used to assess the immune infiltration status of the HCC microenvironment between different risk subgroups. Unsupervised cluster analysis was conducted to assess the relationship between molecular subtypes and local immune status and clinicopathological features. Results: In total, 2441 ASs derived from 1634 mRNA were identified as relapse-associated AS events. By analyzing the proteins involved in the relapse-associated AS events, 1573 proteins with 11590 interactions were included in the protein–protein interaction (PPI) network. In total, 16 splicing factors and 61 relapse-associated AS events with 85 interactions were involved in the splicing network. The relevant genes involved in the PPI network and splicing network were also analyzed by Gene Ontology enrichment analysis. Finally, we established a robust 16-gene AS signature for predicting tumor relapse in stage I–III HCC with considerable AUC values in all of the training cohort, testing cohort, and entire cohort. The ssGSEA and ESTIMATE analyses showed that the AS signature was significantly associated with the immune status of the HCC microenvironment. Moreover, four molecular subgroups with distinguishing tumor relapse modes and local immune status were also revealed. Conclusion: Our study built a novel 16-gene AS signature that robustly predicts tumor relapse and indicates immune activity in stage I–III HCC, which may facilitate the deep mining of the mechanisms associated with tumor relapse and tumor immunity and the development of novel individualized treatment targets for HCC.

797P Relapse-free survival (RFS) update and first translational analyses of DONIMI, a study testing personalized neoadjuvant domatinostat, nivolumab (NIVO) and ipilimumab (IPI) in stage III melanoma patients (pts) based on the interferon-gamma signature (IFN-γ sign) algorithm

In stage III melanoma, neoadjuvant (neoadj) IPI + NIVO induces high pathologic response rates (pRR 72-78%), which is associated with long-term RFS. Pts with a low baseline IFN-γ sign are less likely to respond (pRR 55%). Domatinostat (DOM), a class I histone deacetylase inhibitor, increased intratumoral T cell infiltration and the IFN-γ sign expression in melanoma. DONIMI tests neoadj combinations of NIVO ± IPI with DOM stratified according to the IFN-γ sign of baseline biopsies in stage IIIB-D nodal melanoma.

Effect of additional cytogenetic abnormalities on survival in arsenic trioxide-treated acute promyelocytic leukemia

AbstractFrontline arsenic trioxide (ATO)–based treatment regimens achieve high rates of long-term relapse-free survival in treating acute promyelocytic leukemia (APL) and form the current standard of care. Refining prognostic estimates for newly diagnosed patients treated with ATO-containing regimens remains important in continuing to improve outcomes and identify patients who achieve suboptimal outcomes. We performed a pooled analysis of exclusively ATO-treated patients at a single academic institution and from the ALLG APML4 and Alliance C9710 studies to determine the prognostic importance of additional cytogenetic abnormalities and/or complex karyotype. We demonstrated inferior event-free survival for patients harboring complex karyotype (hazard ratio [HR], 3.74; 95% confidence interval [CI], 1.63-8.56; P = .002), but not for patients harboring additional cytogenetic abnormalities (HR, 2.13; 95% CI, 0.78-5.82; P = .142). These data support a role for full karyotypic analysis of all patients with APL and indicate a need for novel treatment strategies to overcome the adverse effect of APL harboring complex karyotype.

Modern combined treatment of multiple cerebral and visceral metastases of skin melanoma on the example of clinical observation

Introduction. The article discusses modern methods of combined treatment of patients with cerebral and visceral metastases of melanoma, including drug therapy and radiation therapy, the place of neurosurgery, and also discusses a clinical case of long-term relapse-free survival after effective treatment of multiple intracerebral and extracranial metastases of non-pigmented melanoma without a driver mutations. Purpose. Analysis of the results of the application of modern methods of antitumor treatment of melanoma with metastases to the brain and their effect on survival on the example of a clinical case of a patient with metastatic non-pigmented melanoma without an identified primary focus without driver mutations with multiple metastases to the brain, single metastases to the cervical lymph node and left adrenal gland. Materials and methods. Using a clinical example, a possible sequence of an individual approach to the treatment of a patient with multiple intracerebral metastases of non-pigmented melanoma without a primary identified focus without driver mutations and metastasis to the left adrenal gland is considered, the place of modern methods of treatment and examination. Results. The use of a combination of modern methods of anticancer therapy, including immunotherapy, stereotactic radiosurgery and radiation therapy, has increased the overall and relapse-free survival of patients with metastases of melanoma to the brain and visceral organs, and, moreover, reduces the need in neurosurgical interventions. As confirmation of this, the patient is alive for more than 25 months from the moment of progression with a life expectancy of 3-6 months. Conclusions. Modern methods of anticancer therapy can significantly increase the survival rate of patients with metastases of melanoma to the brain and visceral organs, and the accumulation of clinical experience will contribute to the optimization of approaches in the combined and sequential treatment of metastatic melanoma.

LBA39 Personalized combination of neoadjuvant domatinostat, nivolumab (NIVO) and ipilimumab (IPI) in stage IIIB-D melanoma patients (pts) stratified according to the interferon-gamma signature (IFN-γ sign): The DONIMI study

Neoadjuvant (neoadj) IPI + NIVO induces high pathologic response rates (pRR 72-78%) in stage IIIB-D melanoma which is strongly associated with long-term relapse-free survival (RFS). Pts with a low baseline IFN-γ sign are less likely to respond. The class I histone deacetylase inhibitor domatinostat (DOM) increased intratumoral T cell infiltration and IFN-γ sign expression in melanoma. DONIMI tests neoadj combinations of NIVO ± IPI with DOM stratified according to IFN-γ sign from tumor biopsies.

1075P Regression tree analysis to identify factors associated with relapse-free survival (RFS) in patients with resected stage III BRAF V600E/K–mutant melanoma

The 5-y analysis of the Phase III COMBI-AD trial (NCT01682083) showed long-term RFS benefit with 12 months of adjuvant dabrafenib (dab) plus trametinib (tram) vs placebo (pbo) in patients with resected stage III BRAF V600E/K–mutant melanoma (5-y RFS rate, 52% vs 36%; hazard ratio, 0.51 [95% CI, 0.42-0.61]). A prior regression tree analysis (median follow-up in the dab + tram arm, 44 mo) evaluated 13 patient and clinical characteristics to identify treatment type (dab + tram vs pbo), disease stage, and age as predictive of long-term benefit.

The Role of Comprehensive Mutational Profiling in Predicting Patients Who May Benefit from Autologous Hematopoietic Cell Transplant for Acute Myeloid Leukemia

BACKGROUNDThe use of autologous hematopoietic cell transplantation (autoHCT) as post-remission therapy for acute myeloid leukemia (AML) has become increasingly infrequent, both because of an expanding pool of donors for allogeneic hematopoietic cell transplantation (alloHCT) and because of the unclear benefit of autoHCT over conventional consolidation chemotherapy. Since most trials of autoHCT for AML were performed in an era prior to routine molecular testing at diagnosis, little is known about the impact of recurrent genetic mutations on outcome of autoHCT for AML, and in particular the impact of mutations other than FLT3-ITD, NPM1, and CEBPA. We retrospectively used next-generation sequencing for >400 cancer-related genes to evaluate diagnostic samples of patients (pts) with both intermediate- (int) and favorable- (fav) risk AML who underwent autoHCT, and correlated these findings with clinical outcome. METHODSEligible pts included adults with non-M3 fav- or int-risk AML who underwent autoHCT at the University of California, San Francisco and had available diagnostic bone marrow specimens from which DNA was successfully extracted. Pts were excluded from analysis if they remained relapse-free with <1 year of follow-up available. Mutational profiling via next-generation sequencing was performed via either the Foundation One Heme (n=35) or UCSF 500 (n=28) sequencing platforms. Mutations occurring at a frequency of ≤3% were excluded from analysis. RESULTSOf 61 patients with available diagnostic material and successful DNA extraction, 46 had int-risk and 15 had fav-risk AML. Median age at transplant was 47 years (range 20 - 69) and 38% were male. Median follow-up for this cohort was 3.4 years (range 0.3 - 16.3). For the int-risk cohort, median relapse-free survival (RFS) was not reached (range 0.2 - 16.3), with 25/46 pts (54%) remaining relapse-free. For the fav-risk cohort, median RFS was also not reached (range 0.2 - 9.6), with 10/15 pts (67%) remaining relapse-free. All but 1 pt (98%) had ≥ 1 detectable somatic mutation, with an average of 3.1 somatic mutations per pt in the int-risk cohort and 2.5 somatic mutations per pt in the fav-risk cohort.In the int-risk group, the most commonly mutated genes were NPM1 (21/46; 46% of pts), DNMT3A (16/46; 35%), IDH1/2 (13/46; 28%), NRAS (11/46; 24%) and FLT3-ITD (9/46; 20%) (Figure 1A). Fifty-two percent of pts harbored mutations in either NPM1 and/or FLT3-ITD. Only 4/15 (27%) patients with mutated NPM1 (NPM1mut) and wild-type FLT3-ITD (FLT3-ITDwt) relapsed. Of the 4 NPM1mut/FLT3-ITDwt pts who relapsed, all had co-occurring DNMT3A mutations and 2 had both co-occurring FLT3-TKD and IDH1 mutations. Of pts with FLT3-ITDmut, 6/9 (67%) relapsed. Of the 3 FLT3-ITDmut pts who did not relapse, 2 had co-occurring mutations in IDH1 and the other had a co-occurring FLT3-TKD mutation. The only mutation 100% predictive for non-relapse was PTPN11 (n=6), although it co-occurred with NPM1mut in 5 of 6 cases and never co-occurred with FLT3-ITDmut. In the cohort of pts with wild-type NPM1/FLT3-ITD/CEBPA (n=18), 2/2 (100%) with KMT2A-ELL fusions, 3/5 (60%) with RUNX1mut, 3/4 (75%) with IDH1/2 (2 of which co-occurred with RUNX1 mutations), and 1/4 (25%) with NRASmut relapsed. Overall 7 of the 18 (39%) remain relapse-free.In the fav-risk group, KIT mutations were detected in 5/15 (33%) pts, 3 of whom (60%) remain relapse-free (Figure 1B). The next most frequently mutated genes were ASXL2, EZH2, TET2, and PTEN, each present in 2/15 (13%) pts. Of the 3 pts who relapsed in the absence of a KIT mutation, 1 harbored a DNMT3A mutation, 1 harbored a mutation in TBL1XR1, and 1 had no detectable somatic mutations. Three of the 5 pts who relapsed were ≥ 60 years old. CONCLUSIONSComprehensive genomic profiling may aid risk-adapted decision-making in the management of pts with int- and fav-risk AML considered for autoHCT. In our cohort-similar to conventional consolidation chemotherapy-mutations in FLT3-ITD, NPM1, and CEBPA were the strongest predictors of outcome. The presence of PTPN11 mutations in int-risk AML appears to strongly predict long-term RFS after autoHCT. In a small cohort of fav-risk AML patients, the adverse prognosis of a KIT mutation was overcome by autoHCT in 60% of pts. [Display omitted] DisclosuresMannis:Amgen: Honoraria; Juno: Research Funding; Curis: Honoraria; Agios: Research Funding. Zhong:Foundation Medicine: Employment. He:Foundation Medicine, Inc: Employment, Other: Stock. Nahas:Foundation Medicine inc: Employment, Other: stockholder. Andreadis:Cellerant Therapeutics: Research Funding; Novartis Pharmaceuticals Corporation: Honoraria, Research Funding; Incyte Pharmaceuticals: Research Funding; Genentech Inc.: Employment, Equity Ownership, Honoraria; Astellas: Honoraria; Amgen: Research Funding; Gilead Sciences: Honoraria; Pharmacyclics: Research Funding; Seattle Genetics: Honoraria. Mughal:Foundation Medicine, Inc: Employment, Other: Stock. Logan:Astellas: Research Funding; Shire: Consultancy; Amgen: Consultancy; Novartis: Consultancy, Research Funding; Pharmacyclics: Research Funding; Jazz: Consultancy; Kite: Research Funding; Incyte: Consultancy.

A case of simultaneous abdominal wall metastasis of hepatocellular carcinoma with long-term relapse-free survival after laparoscopic resection

We report our experience of an extremely rare case of a simultaneous extrahepatic metastasis of hepatocellular carcinoma (HCC) with long-term relapse-free survival, treated by laparoscopic resection of an abdominal wall tumor and subsequent radiofrequency ablation (RFA) of an intrahepatic lesion. A 76-year-old man visited a local clinic for right lower abdominal pain. He was treated with antibiotics and the symptom resolved. However, a mass was detected in the same region and he was referred to our hospital for further evaluation. Computed tomography (CT) of the abdomen showed a mass 5 cm in diameter, raising suspicions of an intra-abdominal tumor. Laparoscopic surgery was performed, and the tumor was found in the abdominal wall and completely resected. Histopathological examination yielded a diagnosis of extrahepatic HCC. Post-operative positron emission tomography (PET)-CT showed increased uptake of fluorodeoxyglucose in segment 3 (S3) of the liver. On performing a liver biopsy, HCC was diagnosed. Subsequently, the S3 lesion was treated with radiofrequency ablation. The patient has remained relapse-free for 6 years without further treatments.

Perioperative use of β-blockers in oncological patients

Background. One of the most common responses to surgical stress is the activation of the sympathetic nervous system, leading to a dramatic increase in circulating adrenaline and norepinephrine, which can potentially accelerate the spread of the tumor process in cancer patients. In this regard, it is relevant to review the results of studies devoted to the determination of the antitumor effect of β-blockers and their use in the perioperative treatment of cancer patients.&#x0D; Purpose – to analyze and summarize data on the oncogenic effects of catecholamines and to highlight the experience of using β-blockers in the perioperative treatment of cancer patients. Materials and methods. We used the electronic resources of Vernadsky National Library of Ukraine, Cochrane library and databases (Pubmed, Scopus). We analysed the results of studies conducted before August 2020.&#x0D; Results. A summary of the mechanisms by which excessive release of catecholamines may facilitate metastasis is provided. The analysis of clinical trials devoted to the study of the effects of perioperative use of propranolol and its combination with COX-2 inhibitors in cancer patients is conducted.&#x0D; Conclusions. Short-term perioperative therapy with propranolol and its combination with anti-inflammatory drugs may have antimetastatic effects and neutralize the immunosuppressive effects of surgical stress. Further elucidation of the clinical significance of perioperative propranolol treatment in cancer patients requires a 3–5-year study to assess the long-term relapse-free survival of cancer patients.

Distinct Tumor-Resident Memory HBV-Specific T Cell Responses Correlate with Relapse-Free Survival in Patients with HBV-Associated Hepatocellular Carcinoma

In the context of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), despite a range of possibilities, the antigen specificities of tumor-infiltrating T cells and their relevance to control of is largely unknown. Using highly multiplexed peptide-MHC tetramer staining of unexpanded cells from blood, liver and tumor tissues from 46 HCC patients, we detected 91 different CD8 T cell populations specific for epitopes derived from HBV, tumor-associated and neoantigens (NeoAg), as well as disease-unrelated antigens. Parallel high-dimensional analysis delineated distinct tissue-resident memory T cells (TRM) populations among other highly diverse lymphocytes profiles observed in these compartments. Intratumoral and intrahepatic HBV-specific T cells were particularly enriched for three TRM phenotypes and the majority expressed relatively low levels of PD-1 receptor and TOX gene, inconsistent with reported terminal exhausted T cells (TEX) despite being clonally expanded within tumors. High frequencies of terminal TEX in these tumors was uncommon, whereas patients who had detectable and less exhausted tumor-infiltrating HBV- or NeoAg-specific CD8 TRM had superior long-term relapse-free survival. Thus, non-terminally-exhausted tumor-resident HBV-specific CD8 TRM show hallmarks of active involvement and effective antitumor response, implying that these cells could be harnessed for therapeutic purposes.