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Background: Septic shock (SS) and cardiogenic shock (CS) are two types of circulatory shock with a different etiology. Several studies have described the molecular alterations in SS patients, whereas the molecular factors involved in CS have been poorly investigated. We aimed to assess in the whole blood of CS and SS patients, using septic patients without shock (SC) as controls, transcriptomic modifications that occur over 1 week after ICU admission and are common to the two types of shock. Methods: We performed whole blood RNA sequencing in 21 SS, 11 CS, and 5 SC. In shock patients, blood samples were collected within 16 h from ICU admission (T1), 48 h after ICU admission (T2), and at day 7 or before discharge (T3). In controls, blood samples were available at T1 and T2. Gene expression changes over time have been studied in CS, SS, and SC separately with a paired analysis. Genes with p value < 0.01 (Benjamini-Hochberg multiple test correction) were defined differentially expressed (DEGs). We used gene set enrichment analysis (GSEA) to identify the biological processes and transcriptional regulators significantly enriched in both types of shock. Results: In both CS and SS patients, GO terms of inflammatory response and pattern recognition receptors (PRRs) were downregulated following ICU admission, whereas gene sets of DNA replication were upregulated. At the gene level, we observed that alarmins, interleukin receptors, PRRs, inflammasome, and DNA replication genes significantly changed their expression in CS and SS, but not in SC. Analysis of transcription factor targets showed in both CS and SS patients, an enrichment of CCAAT-enhancer-binding protein beta (CEBPB) targets in genes downregulated over time and an enrichment of E2F targets in genes with an increasing expression trend. Conclusions: This pilot study supports, within the limits of a small sample size, the role of alarmins, PRRs, DNA replication, and immunoglobulins in the pathophysiology of circulatory shock, either in the presence of infection or not. We hypothesize that these genes could be potential targets of therapeutic interventions in CS and SS., SCOPUS: ar.j, info:eu-repo/semantics/published

Currently, there is no therapy targeting septic cardiomyopathy (SC), a key contributor to organ dysfunction in sepsis. In this study, we used a machine learning (ML) pipeline to explore transcriptomic, proteomic, and metabolomic data from patients with septic shock, and prospectively collected measurements of high-sensitive cardiac troponin and echocardiography. The purposes of the study were to suggest an exploratory methodology to identify and characterise the multiOMICs profile of (i) myocardial injury in patients with septic shock, and of (ii) cardiac dysfunction in patients with myocardial injury. The study included 27 adult patients admitted for septic shock. Peripheral blood samples for OMICS analysis and measurements of high-sensitive cardiac troponin T (hscTnT) were collected at two time points during the ICU stay. A ML-based study was designed and implemented to untangle the relations among the OMICS domains and the aforesaid biomarkers. The resulting ML pipeline consisted of two main experimental phases: recursive feature selection (FS) assessing the stability of biomarkers, and classification to characterise the multiOMICS profile of the target biomarkers. The application of a ML pipeline to circulate OMICS data in patients with septic shock has the potential to predict the risk of myocardial injury and the risk of cardiac dysfunction., SCOPUS: ar.j, info:eu-repo/semantics/published

Cystic fibrosis (CF) is a hereditary disease, with 70% of patients developing a proteinopathy related to the deletion of phenylalanine 508. CF is associated with multiple organ dysfunction, chronic inflammation, and recurrent lung infections. CF is characterized by defective autophagy, lipid metabolism, and immune response. Intracellular lipid accumulation favors microbial infection, and autophagy deficiency impairs internalized pathogen clearance. Myriocin, an inhibitor of sphingolipid synthesis, significantly reduces inflammation, promotes microbial clearance in the lungs, and induces autophagy and lipid oxidation. RNA-seq was performed in Aspergillusfumigatus-infected and myriocin-treated CF patients&rsquo, derived monocytes and in a CF bronchial epithelial cell line. Fungal clearance was also evaluated in CF monocytes. Myriocin enhanced CF patients&rsquo, monocytes killing of A. fumigatus. CF patients&rsquo, monocytes and cell line responded to infection with a profound transcriptional change, myriocin regulates genes that are involved in inflammation, autophagy, lipid storage, and metabolism, including histones and heat shock proteins whose activity is related to the response to infection. We conclude that the regulation of sphingolipid synthesis induces a metabolism drift by promoting autophagy and lipid consumption. This process is driven by a transcriptional program that corrects part of the differences between CF and control samples, therefore ameliorating the infection response and pathogen clearance in the CF cell line and in CF peripheral blood monocytes.

Although chemotherapeutic agents have been used for decades, the mechanisms of action, mechanisms of resistance, and the best treatment schedule remain elusive. Mitomycin C (MMC) is the gold standard treatment for non-muscle-invasive bladder cancer (NMIBC). However, it is effective only in a subset of patients, suggesting that, aside from cytotoxicity, other mechanisms could be involved in mediating the success of the treatment. Here, we showed that MMC promotes immunogenic cell death (ICD) and in vivo tumor protection. MMC-induced ICD relied on metabolic reprogramming of tumor cells toward increased oxidative phosphorylation. This favored increased mitochondrial permeability leading to the cytoplasmic release of mitochondrial DNA, which activated the inflammasome for efficient IL-1β (interleukin-1β) secretion that promoted dendritic cell maturation. Resistance to ICD was associated with mitochondrial dysfunction related to low abundance of complex I of the respiratory chain. Analysis of complex I in patient tumors indicated that low abundance of this mitochondrial complex was associated with recurrence incidence after chemotherapy in patients with NMIBC. The identification of mitochondria-mediated ICD as a mechanism of action of MMC offers opportunities to optimize bladder cancer management and provides potential markers of treatment efficacy that could be used for patient stratification.

Chronic Myeloid Leukemia (CML) is a stem cell cancer that arises when t(9;22) translocation occurs in a hematopoietic stem cells. This event results in the expression of the BCR-ABL1 fusion gene, which codes for a constitutively active tyrosine kinase that is responsible for the transformation of a HSC into a CML stem cell, which then gives rise to a clonal myeloproliferative disease. The introduction of Tyrosine Kinase Inhibitors (TKIs) has revolutionized the management of the disease. However, these drugs do not seem to be able to eradicate the malignancy. Indeed, discontinuation trials (STIM; TWISER; DADI) for those patients who achieved a profound molecular response showed 50% relapsing within 12 months. We performed a comparative analysis on 15 CML patients and one B-ALL patient, between the standard quantitative reverse-transcriptase PCR (qRT–PCR) and our genomic DNA patient-specific quantitative PCR assay (gDNA qPCR). Here we demonstrate that gDNA qPCR is better than standard qRT–PCR in disease monitoring after an average follow-up period of 200 days. Specifically, we statistically demonstrated that DNA negativity is more reliable than RNA negativity in indicating when TKIs therapy can be safely stopped.

Background: We developed a macrophage-based treatment relying on ex vivo transduction of autologous hematopoietic stem and progenitor cells (HSPC) to express immune-payloads within the TME. Our ATMP (Temferon) targets IFN-a, an immune-modulatory molecule counteracting also neo-angiogenesis and tumor growth, to a subset of Tie2-expressing, tumor-infiltrating macrophages known as TEMs. Materials and Methods: TEM-GBM is an open-label, Phase I/IIa dose-escalation study evaluating safety and efficacy of Temferon in up to 21 newly diagnosed glioblastoma patients with unmethylated MGMT promoter. Key eligibility criteria include age 18-70 years, ECOG 0-1 and KPS >70%, and adequate cardiac, renal, hepatic and pulmonary function. Important exclusion criteria include the presence of active autoimmune disease or receipt of any oral or parenteral chemotherapy or immunotherapy within 2 years of screening. Autologous CD34+ HSPC are mobilized with lenograstim and plerixafor, collected by apheresis, purified and transduced ex vivo with a 3 rd generation lentiviral vector encoding for IFN-a2. Transgene expression is confined to TEMs by the Tie2 promoter and post-transcriptional regulation by microRNA-126 thus achieving tumor specificity. The study evaluates safety and biological activity of Temferon in 7 cohorts of three patients each, where escalating doses of Temferon are co-administered with a fixed CD34+ cell dose of non-manipulated supporter cells following a sub-myeloablative conditioning regimen (Thiotepa + BCNU or + Busulfan). The primary endpoints for this study are: Engraftment of Temferon over the first 90 DaysThe proportion of patients achieving hematologic recovery by Day +30 from ASCTShort-term tolerability of Temferon; stable blood counts and absence of cytopenias, absence of significant organ toxicities (> grade 2); absence of Replication Competent Lentivirus The figure below reports the TEM-GBM study design. Results: As of 28th June 2021, 18 patients have been enrolled; 15 received Temferon (D+0) with follow-up of 30 - 697 days. There was rapid engraftment and hematological recovery after the conditioning regimen. Median neutrophil and platelet engraftment occurred at D+13 and D+12 for patients in cohort 1-3 and D+16 and D+15 for patients assigned to cohort 4 and 5, respectively. Temferon-derived differentiated cells, as determined by the presence of vector genomes in the DNA of peripheral blood and bone marrow cells, were found within 14 days post treatment and persisted subsequently, albeit at lower levels (up to 18 months). Very low concentrations of IFNa were detected in the plasma (average 7.8 pg/ml at D+30; baseline < LLOQ) and in the cerebrospinal fluid (average 1.6 pg/ml at D+30; baseline < LLOQ), suggesting tight regulation of transgene expression. Seven deaths occurred: six at D+241, +322, +340, +402, +478, +646 after Temferon administration due to disease progression, and one at D+60 due to complications following the conditioning regimen. Nine patients had progressive disease (PD; range D-12 to +239). SAEs include infections, venous thromboembolism, brain abscess, hemiparesis, GGT elevation and poor performance status compatible with autologous stem cell transplantation, concomitant medications and PD. Four patients underwent second surgery. These recurrent tumors had gene-marked cells present and increased expression of IFN-responsive gene signatures compared to diagnosis, indicative of local IFNa release by TEMs. In one patient, a stable lesion (as defined by MRI) had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased IFN-response signature than in a progressing lesion. The T-cell immune repertoire changed with evidence for expansion of tumor-associated clones. Tumor microenvironment characterization by scRNA and TCR sequencing is ongoing. Conclusion: These interim results show that Temferon is generally well tolerated by patients, with no dose limiting toxicities identified to date. The results provide initial evidence of Temferon's potential to activate the immune system and reprogram the tumor microenvironment (TME), as predicted by preclinical studies. Figure 1 Figure 1. Disclosures Naldini: Genenta Science: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Temferon is an ex vivo gene therapy consisting of autologous HSPCs genetically modified to deliver IFN-α2 within the tumor microenvironment (TME) by Tie-2 expressing macrophages. TEM-GBM is an open-label, Phase I/IIa dose-escalation study evaluating safety and efficacy of Temferon in up to 21 newly diagnosed GBM patients with unmethylated MGMT. Autologous HSPCs are transduced with a LVV encoding for IFN-a2 gene. As of 30th April 2021, 18 patients have been enrolled; 13 received Temferon (D+0) with follow-up of 8 – 662 days. After conditioning and Temferon infusion, a rapid engraftment and hematological recovery occurred, with median neutrophil and platelet engraftment at D+13 and D+12, respectively. No dose limiting toxicities were reported. Temferon-derived cells were found within 14 days post treatment and persisted albeit at lower levels in the long-term. Five deaths occurred: one at +478, three at +322, +340 and +402 days due to PD, and the fourth at +60 due to complications following the conditioning regimen. Eight patients had PD (-12 to +239). SAEs include respiratory tract infections, pulmonary embolism, CMV and C.Diff infections, febrile neutropenia, hemiparesis, seizure, brain abscess, worsening of performance status and respiratory failure compatible with ASCT, concomitant medications and PD. Four patients underwent second surgery. Recurrent tumors had gene-marked cells present and increased expression of ISGs compared to diagnosis, indicative of local IFNa release by TEMs. In one patient, a stable lesion had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased IFN-response signature than in a progressing lesion. Characterization of T-cell immune repertoire suggests the expansion of tumor-associated clones. TME characterization by scRNA and TCR sequencing is ongoing. Interim results show that Temferon is well tolerated, with no dose limiting toxicities identified to date and provide initial evidence of potential immune system activation within the TME.

Temferon is a macrophage-based treatment relying on ex-vivo transduction of autologous HSPCs to express immune-payloads within the TME. Temferon targets the immune-modulatory molecule IFN-a, to a subset of tumor infiltrating macrophages known as Tie-2 expressing macrophages (TEMs) due to the Tie2 promoter and a post-transcriptional regulation layer represented by miRNA-126 target sequences. As of 31st May 2021, 15-patients received Temferon (D+0) with follow-up of 3 – 693 days. After conditioning neutrophil and platelet engraftment occurred at D+13 and D+13.5, respectively. Temferon-derived differentiated cells, as determined be the number of vector copy per genome, were found within 14 days post treatment and persisted albeit at lower levels up to 18-months. Very low concentrations of IFN-a in the plasma (8.7 pg/ml-D+30) and in the CSF (1.6 pg/ml-D+30) were detected, suggesting tight regulation of transgene expression. Five-deaths occurred at D+322, +340, +402, +478 and +646 due to PD, and one at D+60 due to complications following the conditioning regimen. Eight-patients had progressive disease (range: D-11 to +239) as expected for this tumor type. SAEs include GGT elevation (possibly related to Temferon) and infections, venous thromboembolism, brain abscess, hemiparesis, seizures, anemia and general physical condition deterioration, compatible with ASCT, concomitant medications and PD. Four-patients underwent 2ndsurgery. Recurrent tumors had gene-marked cells and increased expression of ISGs compared to first surgery, indicative of local IFNa release by TEMs. In one patient, a stable lesion had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased ISGs than in the progressing lesion, detected in the same patient. Tumor-associated clones expanded in the periphery. TME characterization by scRNA and TCR-sequencing is ongoing. To date, Temferon is well tolerated, with no DLTs identified. The results provide initial evidence of Temferon potential to activate the immune system of GBM patients, as predicted by preclinical studies.

BACKGROUND The authors describe a family with a high penetrance of plasma cell dyscrasias, suggesting inheritance of an autosomal dominant risk allele. METHODS The authors performed whole-exome sequencing and reported on a combined approach aimed at the identification of causative variants and risk loci, using the wealth of data provided by this approach. RESULTS The authors identified gene mutations and single-nucleotide polymorphisms of potential significance, and pinpointed a known risk locus for myeloma as a potential area of transmissible risk in the family. CONCLUSIONS To the authors' knowledge, the current study is the first to provide a whole-exome sequencing approach to such cases, and a framework analysis that could be applied to further understanding of the inherited risk of developing plasma cell dyscrasias. Cancer 2017. © 2017 American Cancer Society.

Transplantation of hematopoietic cells from a healthy individual (allogeneic hematopoietic cell transplantation (allo-HCT)) demonstrates that adoptive immunotherapy can cure blood cancers: still, post-transplantation relapses remain frequent. To explain their drivers, we analyzed the genomic and gene expression profiles of acute myeloid leukemia (AML) blasts purified from patients at serial time-points during their disease history. We identified a transcriptional signature specific for post-transplantation relapses and highly enriched in immune-related processes, including T cell costimulation and antigen presentation. In two independent patient cohorts we confirmed the deregulation of multiple costimulatory ligands on AML blasts at post-transplantation relapse (PD-L1, B7-H3, CD80, PVRL2), mirrored by concomitant changes in circulating donor T cells. Likewise, we documented the frequent loss of surface expression of HLA-DR, -DQ and -DP on leukemia cells, due to downregulation of the HLA class II regulator CIITA. We show that loss of HLA class II expression and upregulation of inhibitory checkpoint molecules represent alternative modalities to abolish AML recognition from donor-derived T cells, and can be counteracted by interferon-gamma or checkpoint blockade, respectively. Our results demonstrate that the deregulation of pathways involved in T cell-mediated allorecognition is a distinctive feature and driver of AML relapses after allo-HCT, which can be rapidly translated into personalized therapies.

Background Septic shock is the most severe complication of sepsis and this syndrome is associated with high mortality. Treatment of septic shock remains largely supportive of hemodynamics and tissue perfusion. Early changes in organ function assessed by the Sequential Organ Function Assessment (SOFA) score are highly predictive of the outcome. However, the individual patient’s response to supportive therapy is very heterogeneous, and the mechanisms underlying this variable response remain elusive. The aim of the study was to investigate the transcriptome of whole blood in septic shock patients with different responses to early supportive hemodynamic therapy assessed by changes in SOFA scores within the first 48 h from intensive care unit (ICU) admission. Methods We performed whole blood RNA sequencing in 31 patients: 17 classified as responders (R) and 14 as non-responders (NR). Gene expression was investigated at ICU admission (time point 1, or T1), comparing R with NR [padj

Current understanding of acute myeloid leukemia (AML) assumes a developmental hierarchy, in which a minor fraction of primitive and quiescent leukemia stem cells (LSC) sustain clonal propagation of disease. These therapy-resistant LSC may be the basis of relapse, as supported by data correlating the presence of LSC gene expression signatures at diagnosis with poor prognosis (Ng et al, Nature 2016). Novel approaches tracing LSC fates at single cell level before, during and after chemotherapy (CTX) are needed to confirm their biological relevance and derive new, LSC-focused diagnostic and therapeutic strategies. We and others have previously linked key LSC properties, such as quiescence and therapy resistance, to complex transcriptional regulation orchestrated by miR-126 (Lechman et al, Cancer Cell 2016). Furthermore, we provided proof of concept that LSCs could be prospectively isolated as miR-126(high) cells exploiting a lentiviral reporter vector capturing miR-126 bioactivity in live cells with single cell resolution. To extend these studies, we transduced primary blasts from n=3 AML patients (pts) carrying NPM1 and FLT3-ITD mutations with the miR-126 reporter, followed by xenografting (PDX). Blasts showed intra-tumor heterogeneity (ITH) in terms of miR-126 activity, with a minor fraction identified as miR-126(high). Limiting dilution secondary transplantation of FACS sorted miR-126(high) and -(low) blasts proved strong enrichment of repopulating activity within the miR-126(high) compartment in all 3 pts. On the contrary, no LSC enrichment could be verified in the CD34+CD38- fraction in 1 patient, suggesting that high miR-126 activity represents a more robust LSC identifier than commonly used surface markers. Next, we investigated the impact of daunorubicin and cytarabine CTX on miR-126-reporter+ blasts (n=3 AML) in PDX. Surprisingly, overall miR-126 activity diminished in post CTX residual AML compared to controls, compatible with a loss of blast quiescence. CTX accentuated ITH by uncovering a subset of blasts with very high levels of miR-126, distinct from the bulk population, which may correspond to residual quiescent LSC (Fig A). We then performed bulk RNA sequencing on miR-126(high) and miR-126(low) subsets from CTX and control PDX. While miR-126(low) blasts from both groups expressed markers of myeloid differentiation, miR-126(high) blasts were enriched for published hematopoietic stem cell hallmark signatures. Integrating differentially expressed genes between miR-126(high) and-(low) subsets at steady state and post CTX, we extrapolated a novel 8 gene signature associated with miR-126(high) blasts. Of note, patients from the AML TCGA PanCancer Atlas Cohort (n=161) harboring overexpression in one or more of these 8 genes had significantly decreased overall survival (10 vs 19 months, Logrank test p-value = 0.018). To further test whether our 8-gene miR-126(high) signature reveals ITH in patients, we performed single cell RNA sequencing (scRNAseq) of AML patient BM aspirates at diagnosis (n= 6). Blasts were identified based on the detection of mutated NPM1 transcripts in single cells. In 5 out of 7 patients expression of the miR-126 signature mapped to specific clusters of blasts identified by unsupervised shared nearest neighbor algorithm, confirming that it identifies ITH in patient samples. Interestingly, we detected miR-126 signature(high) blasts in pts with poor prognosis (n=4) and not in those with favorable outcome (n=2) (Fig B). To investigate ITH across longitudinal samples, we next performed scRNAseq of residual AML from a representative patient assessed early after CTX. In line with our PDX CTX model displaying increased miR-126 ITH, blasts on day14 of induction CTX segregated into 2 different clusters: cluster 1 containing LSC-like cells with miR-126(high) signature and similar transcriptional profile to the diagnosis counterpart; cluster 2, instead, was composed of actively cycling blasts. Residual blasts at day30, in addition to uniformly expressing the miR-126(high) signature, differed from diagnosis and day14 blasts by displaying cell cycle quiescence and induction of oxidative phosphorylation genes (Fig C). In summary, we have set up and applied PDX LSC modeling to clinically relevant patient samples to address AML intra-tumor heterogeneity and to pinpoint novel relevant transcriptomic features of LSC at diagnosis and after chemotherapy. Figure Disclosures Gentner: Genenta Science: Consultancy, Equity Ownership, Research Funding.

Background In mice MEOX2/TCF15 heterodimers are highly expressed in heart endothelial cells and are involved in the transcriptional regulation of lipid transport. In a general population, we investigated whether genetic variation in these genes predicted coronary heart disease (CHD). Results In 2027 participants randomly recruited from a Flemish population (51.0 % women; mean age 43.6 years), we genotyped six SNPs in MEOX2 and four in TCF15. Over 15.2 years (median), CHD, myocardial infarction, coronary revascularisation and ischaemic cardiomyopathy occurred in 106, 53, 78 and 22 participants. For SNPs, we contrasted CHD risk in minor-allele heterozygotes and homozygotes (variant) vs. major-allele homozygotes (reference) and for haplotypes carriers (variant) vs. non-carriers. In multivariable-adjusted analyses with correction for multiple testing, CHD risk was associated with MEOX2 SNPs (P ≤ 0.049), but not with TCF15 SNPs (P ≥ 0.29). The MEOX2 GTCCGC haplotype (frequency 16.5 %) was associated with the sex- and age-standardised CHD incidence (5.26 vs. 3.03 events per 1000 person-years; P = 0.036); the multivariable-adjusted hazard ratio [HR] of CHD was 1.78 (95 % confidence interval, 1.25–2.56; P = 0.0054). For myocardial infarction, coronary revascularisation, and ischaemic cardiomyopathy, the corresponding HRs were 1.96 (1.16–3.31), 1.87 (1.20–2.91) and 3.16 (1.41–7.09), respectively. The MEOX2 GTCCGC haplotype significantly improved the prediction of CHD over and beyond traditional risk factors and was associated with similar population-attributable risk as smoking (18.7 % vs. 16.2 %). Conclusions Genetic variation in MEOX2, but not TCF15, is a strong predictor of CHD. Further experimental studies should elucidate the underlying molecular mechanisms. Electronic supplementary material The online version of this article (doi:10.1186/s12863-015-0272-2) contains supplementary material, which is available to authorized users.

INTRODUCTION: Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) represents an effective form of adoptive immunotherapy for Acute Myeloid Leukemia (AML), thanks to the antitumor effect mediated by donor immune cells infused as part of the graft. Unfortunately, post-transplantation relapses remain a frequent observation, warranting further investigation on AML immunobiology. Our group demonstrated that relapses after partially HLA-incompatible HSCT are frequently due to the outgrowth of immune-resistant mutant AML clones characterized by genomic loss the mismatched histocompatibility determinants targeted by alloreactive donor T cells, and have thus gained a selective advantage upon pressure from the transplanted immune system, in a process called “leukemia immunoediting” (Vago et al., N Engl J Med, 2009). In the present study, we took advantage of high-throughput technologies to profile post-transplantation AML relapses with no genomic loss of HLA, to identify novel targets of the leukemia immunoediting process. METHODS: Serial AML samples were collected and FACS-purified from 9 patients at diagnosis, relapse after sole chemotherapy and relapse after allo-HSCT, and employed for whole transcriptome profiling using Illumina HumanHT12 microarrays. Deregulated genes were identified by pair-wise LIMMA analysis, and unsupervised enrichment analysis was performed interrogating the Gene Ontology database. High-throughput results were confirmed in a validation cohort of 12 patients by ad hoc designed qPCR assays, immunophenotypic analyses and functional experiments. In particular, co-cultures were performed using as responders peripheral blood lymphocytes harvested from patients after allo-HSCT, and as stimulators and targets their respective AML blasts collected at diagnosis or at relapse: read-outs included antigen-specific T cell activation, cytotoxicity, and cytokine release. RESULTS: Amongst all the genes expressed by purified AML blasts, a 110-gene signature (p CONCLUSIONS: Our results demonstrate that the deregulation of immune-related processes, and in particular of molecules and pathways involved in T cell-mediated allo-recognition, are pervasive and distinctive features of AML relapses after allo-HSCT. Identification of patient-specific mechanisms of immune evasion might be translated into personalized therapeutic approaches, tailored to restore or circumvent inefficient antigen presentation and T cell costimulation. Disclosures Bonini: MolMed S.p.A.: Consultancy.

Introduction Imatinib mesylate (IM) is the first line therapy against Chronic Myeloid Leukemia (CML), effectively prolonging overall survival. Because discontinuation of treatment is associated with molecular relapse, IM is required indefinitely to maintain operational cure. To evaluate the degree of response to therapy and to highlight the persistence of the disease after treatment, patients should be monitored routinely. The gold standard for diagnosing CML is the cytogenetic analysis, a direct not-sensitive method to detect Ph-positive cells. Quantitative real-time RT-PCR (qRT-PCR) provides highly sensitive detection of BCR-ABL1 transcripts, but mRNA levels are not directly related to the number of leukemic cells and cannot detect transcriptionally silent leukemic stem cells. Methods Here we will propose a new sensitive approach to directly detect the number of leukemic cells using a DNA-based biomarker specific for each patient. We applied targeted next-generation sequencing for the identification of genomic BCR-ABL1 fusion junctions, and we developed a sensitive new approach to detect the number of leukemic cells by a DNA Q-PCR assay based on the genomic break-point, with a formula to calculate the number of Ph+ cells. The percentage of the leukemic cells (LC) was calculated using the following formula: %LC= (2/(2Δct+1))*100, where ΔCt is the difference between the amplification cycles of the BCR-ABL1 and BCR reactions. The number of LC was calculated by multiplying the total number of cells analyzed in each sample by the percentage of LC calculated by the ΔCt formula. We then defined a limit of quantization and a limit of sensitivity in the evaluation of minimal residual disease (MRD), as described by guidelines for the detection of MRD by genomic Q-PCR in acute lymphoblastic leukemia (ALL). We defined a “quantitative range” of detection, the portion of the standard curve in which the MRD levels can be quantified reproducibly and accurately, and we defined the “limit of sensitivity”, the lowest MRD level that still can be detected, although not in all replicates. We thus calculated the exact number of leukemic cells only when the MRD fell within the range of quantization. The detection of MRD at the limit of sensitivity was indicated as positive but not quantified. Results We monitored eight CML patients treated with Imatinib for 8 years. We tested the same samples by patient specific Q-PCR, and in parallel by cytogenetic analysis and by standard qRT-PCR. In all samples positive for chimeric transcripts we measured corresponding chimeric genomic DNA (gDNA) by Q-PCR, confirming the sensitivity of the Q-PCR method. According to conventional criteria, undetectable levels of BCR-ABL1 mRNA assessed by qRT-PCR are indicative of complete molecular response (CMR), but in 33.3% (45/135) samples with undetectable levels of mRNA, we detected the persistence of transcriptionally-silent leukemic cells. However, we never found samples negative by gDNA Q-PCR and positive by RNA-based qRT-PCR (Figure 1). Thirty-six of 135 samples were also analyzed cytogenetically until the achievement of CCyR. As expected, Ph+cells were detected only in 25% (9/36) and 22,2% (8/36) of samples by CBA and I-FISH, respectively, whereas BCR-ABL1 mRNA was detected by qRT-PCR in 83.3% (30/36) of samples and Ph+ cells were detected by genomic Q-PCR in 91.7% (33/36) of samples (Figure 1). Finally, the separation of different cell populations from blood and bone marrow revealed the presence of a population of transcriptionally silent cancer stem cells. The gDNA based Q-PCR analysis performed on highly purified (immunomagnetically selected ) CD34+and CD3+ cells confirmed the presence of a population of transcriptionally silent cancer stem cells. Conclusions The demonstration of positive gDNA Q-PCR in 33.3% of samples negative for the RNA qRT-PCR could partially explain why some patients lose MMR and CMR and others do not, when IM is discontinued for brief periods. The gDNA based Q-PCR could be used to supplement conventional techniques, providing clinicians with additional information about disease status and response in determining whether to stop or alter therapy. Acknowledgments to AIRC and AIL. Disclosures: No relevant conflicts of interest to declare.

Introduction Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) is the most effective therapy for many patients with Acute Myeloid Leukemia (AML). Still, post-transplantation relapse remains an unmet clinical need, and our insights into its biological determinants are limited. Based on the “leukemia immunoediting” hypothesis, post-transplantation relapse may result from the outgrowth of immune-resistant leukemic variants upon the selective pressure of the transplanted immune system, verging on the intrinsic ability of tumor cells to alter their genomic and transcriptional profile. Our group provided a compelling proof of this model, demonstrating that upon partially HLA-incompatible allo-HSCT, AML blasts frequently undergo genomic loss of the mismatched HLA, evading donor immune control and resulting in clinical relapse (Vago et al, N Engl J Med, 2009). Besides its biological and medical relevance, this proof-of-principle discovery paved the way to the identification of novel mechanisms of leukemia relapse. Methods Serial AML samples at time of diagnosis, relapse after sole chemotherapy, and relapse after allo-HSCT were collected longitudinally in 9 patients. AML blasts were FACS-purified and gene expression profile was performed using Illumina HumanHT12 v4.0 Expression Bead chips. Deregulated genes and signatures were identified by pairwise LIMMA analysis. Gene Ontology and Gene Set Enrichment Analysis curated databases were interrogated to identify deregulated processes. Validation of high-throughput results was performed by molecular HLA typing, ad hoc designed locus- and allele-specific qPCR, immunophenotypic analysis and functional ex vivo assays. Results Pairwise enrichment analysis of the patient samples demonstrated that 18/29 (62%) biological processes significantly deregulated in AML at post-transplantation relapse were immune-related, whereas after sole chemotherapy no immune-related process resulted deregulated. Pairwise analysis of triplicate samples at diagnosis and post-transplantation relapse allowed the identification of a 149 gene signature (p Conclusions Our data demonstrate that not only genomic HLA haplotype loss, but also transcriptional HLA Class II downregulation can be at the basis of AML relapse after partially mismatched HSCT, and provide further evidence on how tightly post-transplantation relapse and immune evasion may be linked. Moreover, the novel mechanism we described depends on CIITA downregulation, and may in turn be linked to methylation of its promoter, potentially providing a novel biological rationale for the documented efficacy of demethylating agents as salvage therapy for relapses after allo-HSCT. Disclosures: Bonini: MolMed SpA: Consultancy.