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In some settings, cancer cells responding to treatment undergo an immunogenic form of cell death that is associated with the abundant emission of danger signals in the form of damage-associated molecular patterns. Accumulating preclinical and clinical evidence indicates that danger signals play a crucial role in the (re-)activation of antitumor immune responses in vivo, thus having a major impact on patient prognosis. We have previously demonstrated that the presence of calreticulin on the surface of malignant blasts is a positive prognostic biomarker for patients with acute myeloid leukemia (AML). Calreticulin exposure not only correlated with enhanced T-cell-dependent antitumor immunity in this setting but also affected the number of circulating natural killer (NK) cells upon restoration of normal hematopoiesis. Here, we report that calreticulin exposure on malignant blasts is associated with enhanced NK cell cytotoxic and secretory functions, both in AML patients and in vivo in mice. The ability of calreticulin to stimulate NK-cells relies on CD11c+CD14high cells that, upon exposure to CRT, express higher levels of IL-15Rα, maturation markers (CD86 and HLA-DR) and CCR7. CRT exposure on malignant blasts also correlates with the upregulation of genes coding for type I interferon. This suggests that CD11c+CD14high cells have increased capacity to migrate to secondary lymphoid organs, where can efficiently deliver stimulatory signals (IL-15Rα/IL-15) to NK cells. These findings delineate a multipronged, clinically relevant mechanism whereby surface-exposed calreticulin favors NK-cell activation in AML patients.

Accumulating evidence indicates that immune checkpoint inhibitors (ICIs) can restore CD8+ cytotoxic T lymphocyte (CTL) functions in preclinical models of acute myeloid leukemia (AML). However, ICIs targeting programmed cell death 1 (PDCD1, best known as PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) have limited clinical efficacy in patients with AML. Natural killer (NK) cells are central players in AML-targeting immune responses. However, little is known on the relationship between co-inhibitory receptors expressed by NK cells and the ability of the latter to control AML. Here, we show that hepatitis A virus cellular receptor 2 (HAVCR2, best known as TIM-3) is highly expressed by NK cells from AML patients, correlating with improved functional licensing and superior effector functions. Altogether, our data indicate that NK cell frequency as well as TIM-3 expression levels constitute prognostically relevant biomarkers of active immunity against AML.

We analyzed bone marrow and peripheral blood samples of adult B-other ALL patients treated consecutively at three major Czech teaching hospitals. Samples were analyzed by cytogenetic methods, gene expression profiling, multiplex ligation-dependent probe amplification (MLPA), and next-generation sequencing (NGS).Fifty-eight B-other ALL patients (notThis study widens existing knowledge of the

In some settings, cancer cells responding to treatment undergo an immunogenic form of cell death that is associated with the abundant emission of danger signals in the form of damage-associated molecular patterns. Accumulating preclinical and clinical evidence indicates that danger signals play a crucial role in the (re-)activation of antitumor immune responses in vivo, thus having a major impact on patient prognosis. We have previously demonstrated that the presence of calreticulin on the surface of malignant blasts is a positive prognostic biomarker for patients with acute myeloid leukemia (AML). Calreticulin exposure not only correlated with enhanced T-cell-dependent antitumor immunity in this setting but also affected the number of circulating natural killer (NK) cells upon restoration of normal hematopoiesis. Here, we report that calreticulin exposure on malignant blasts is associated with enhanced NK cell cytotoxic and secretory functions, both in AML patients and in vivo in mice. The ability of calreticulin to stimulate NK-cells relies on CD11c+CD14high cells that, upon exposure to CRT, express higher levels of IL-15Rα, maturation markers (CD86 and HLA- DR) and CCR7. CRT exposure on malignant blasts also correlates with the upregulation of genes coding for type I interferon. This suggests that CD11c+CD14high cells have increased capacity to migrate to secondary lymphoid organs, where can efficiently deliver stimulatory signals (IL-15Rα/IL- 15) to NK cells. These findings delineate a multipronged, clinically relevant mechanism whereby surface-exposed calreticulin favors NK-cell activation in AML patients. Disclosure Information I. Truxova: None. L. Kasikova: None. C. Salek: None. M. Hensler: None. D. Lysak: None. P. Holicek: None. P. Bilkova: None. M. Holubova: None. X. Chen: None. R. Mikyskova: None. M. Reinis: None. M. Kovar: None. B. Tomalova: None. J.P. Kline: None. L. Galluzzi: None. R. Spisek: None. J. Fucikova: None.

Natural killer (NK) cells constitute the predominant innate lymphocyte subset that mediates the anti-viral and anti-tumor immune responses. NK cells use an array of innate receptors to sense their environment and to respond to infections, cellular stress and transformation. The resulting NK cell activation, including cytotoxicity and cytokine production, is a fundamental component of the early immune response. The most recent discoveries in NK cell biology have stimulated the translational research that has led to remarkable results for the treatment of human malignancies. Therefore, the rapid isolation of NK cells from the peripheral blood or tumor microenvironment and the subsequent assessment of cytolytic function are crucial to the study of their potency and NK cell-mediated immunosurveillance. Here, we provide protocols for NK cell isolation and the assessment of NK cell cytotoxicity using flow cytometry.

In some settings, cancer cells responding to treatment undergo an immunogenic form of cell death that is associated with the abundant emission of danger signals in the form of damage-associated molecular patterns (DAMPs). Accumulating preclinical and clinical evidence indicates that danger signals play a crucial role in the (re-)activation of antitumor immune responses in vivo, thus having a major impact on patient prognosis. We have previously demonstrated that the presence of calreticulin (CRT) on the surface of malignant blasts is a positive prognostic biomarker for patients with acute myeloid leukemia (AML). CRT exposure not only correlated with enhanced T cell-dependent antitumor immunity in this setting but also affected the number of circulating NK cells upon restoration of normal hematopoiesis. Here, we report that CRT exposure on malignant blasts is associated with enhanced NK cell cytotoxic and secretory functions, both in AML patients and in vivo in mice. The ability of CRT to stimulate NK cells relies on CD11c+CD14high cells that, upon exposure to CRT, display higher levels of IL-15Rα, maturation markers (CD86 and HLA-DR), and CCR7. CRT exposure on malignant blasts also correlates with the upregulation of genes coding for type I interferons (IFNs). This suggests that CD11c+CD14high cells have increased capacity to migrate to secondary lymphoid organs, where can efficiently deliver stimulatory signals (IL-15Rα/IL-15) to NK cells. These findings delineate a multipronged, clinically relevant mechanism whereby surface-exposed CRT favors NK cell activation in AML patients. Citation Format: Iva Truxova, Lenka Kasikova, Cyril Salek, Michal Hensler, Daniel Lysak, Peter Holicek, Pavla Bilkova, Monika Holubova, Xiufen Chen, Romana Mikyskova, Milan Reinis, Marek Kovar, Barbora Tomalova, Justin Kline, Lorenzo Galluzzi, Radek Spisek, Jitka Fucikova. Calreticulin exposure on malignant blasts correlates with improved NK cell-mediated cytotoxicity in AML patients [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B96.

Cancer cell death can be perceived as immunogenic by the host only when malignant cells emit immunostimulatory signals (so-called "damage-associated molecular patterns," DAMPs), as they die in the context of failing adaptive responses to stress. Accumulating preclinical and clinical evidence indicates that the capacity of immunogenic cell death to (re-)activate an anticancer immune response is key to the success of various chemo- and radiotherapeutic regimens. Malignant blasts from patients with acute myeloid leukemia (AML) exposed multiple DAMPs, including calreticulin (CRT), heat-shock protein 70 (HSP70), and HSP90 on their plasma membrane irrespective of treatment. In these patients, high levels of surface-exposed CRT correlated with an increased proportion of natural killer cells and effector memory CD4+ and CD8+ T cells in the periphery. Moreover, CRT exposure on the plasma membrane of malignant blasts positively correlated with the frequency of circulating T cells specific for leukemia-associated antigens, indicating that ecto-CRT favors the initiation of anticancer immunity in patients with AML. Finally, although the levels of ecto-HSP70, ecto-HSP90, and ecto-CRT were all associated with improved relapse-free survival, only CRT exposure significantly correlated with superior overall survival. Thus, CRT exposure represents a novel powerful prognostic biomarker for patients with AML, reflecting the activation of a clinically relevant AML-specific immune response.

International audience; It is now clear that human neoplasms form, progress, and respond to therapy in the context of an intimate crosstalk with the host immune system. In particular, accumulating evidence demonstrates that the efficacy of most, if not all, chemo- and radiotherapeutic agents commonly employed in the clinic critically depends on the (re)activation of tumor-targeting immune responses. One of the mechanisms whereby conventional chemotherapeutics, targeted anticancer agents, and radiotherapy can provoke a therapeutically relevant, adaptive immune response against malignant cells is commonly known as “immunogenic cell death.” Importantly, dying cancer cells are perceived as immunogenic only when they emit a set of immunostimulatory signals upon the activation of intracellular stress response pathways. The emission of these signals, which are generally referred to as “damage-associated molecular patterns” (DAMPs), may therefore predict whether patients will respond to chemotherapy or not, at least in some settings. Here, we review clinical data indicating that DAMPs and DAMP-associated stress responses might have prognostic or predictive value for cancer patients.

In spite of continuous research efforts directed at early detection and treatment of pancreatic cancer, the outlook for patients affected by the disease remains dismal. With most cases still being diagnosed at advanced stages, no improvement in survival prognosis is achieved with current diagnostic imaging approaches. In the absence of a dominant precancerous condition, several risk factors have been identified including family history, chronic pancreatitis, smoking, diabetes mellitus, as well as certain genetic disorders such as hereditary pancreatitis, cystic fibrosis, familial atypical multiple mole melanoma, and Peutz–Jeghers and Lynch syndromes. Most pancreatic carcinomas, however, remain sporadic. Current progress in experimental molecular techniques has enabled detailed understanding of the molecular processes of pancreatic cancer development. According to the latest information, malignant pancreatic transformation involves multiple oncogenes and tumor-suppressor genes that are involved in a variety of signaling pathways. The most characteristic aberrations (somatic point mutations and allelic losses) affect oncogenes and tumor-suppressor genes within RAS, AKT and Wnt signaling, and have a key role in transcription and proliferation, as well as systems that regulate the cell cycle (SMAD/DPC, CDKN2A/p16) and apoptosis (TP53). Understanding of the underlying molecular mechanisms should promote development of new methodology for early diagnosis and facilitate improvement in current approaches for pancreatic cancer treatment.

K-ras mutations and allelic losses of tumor suppressors p16 and DPC4 are perceived as potential markers for screening of pancreatic malignancy. In this study, molecular data is compared with survival statistics of the patients and whether they correlate with patients' prognosis is questioned.Fifty three consecutive patients with advanced pancreatic cancer (stage III and IV) who underwent EUS-guided fine needle aspiration (FNA) were enrolled into the study (28 males, 25 females, 63+/-10.5 years). Samples were evaluated on-site for presence of malignant cells. DNA was extracted from Giemsa stained smears using laser microdissection, and mutation status of K-ras and p53 was tested by cycling-gradient capillary electrophoresis (CGCE). In addition, allelic losses of tumor suppressor genes p16 (INK4, CDKN2A) and DPC4 (MADH4, SMAD4) were detected by monitoring the loss of heterozygosity (LOH) at 9p and 18q loci. Molecular data were compared with survival statistics using Kaplan-Meier method.The median survival in K-ras positive group was 7.0+/-2.4 months (95% CI 2.3-11.7) and in K-ras negative group was 10.0+/-0.6 months (95% CI 8.7-11.3). The median survival in p53 positive group was 10.0+/-2.2 months (95% CI 5.6-14.4) and in p53 negative group was 6.0+/-2.5 months (95% CI 1.1-10.9). The median survival in LOH 9p positive group was 9.0+/-5.1 months (95% CI 0-18.9), in LOH 9p negatives was 10.0+/-5.0 months (95% CI 0.2-19.8). The median survival in LOH 18q positive group was 10.0+/-4.2 months (95% CI 1.8-18.2) and in LOH 18q negative group was 3.0+/-1.3 months (95% CI 0.5-5.5). After the adjustment for age using Cox proportional hazards model, none of the evaluated molecular markers was shown to be an independent prognostic marker for survival of patients with pancreatic cancer.None of the studied molecular markers was identified as an independent factor determining survival prognosis.

It has been shown that changes in DNA methylation pattern in patients with acute myeloid leukaemia (AML) may both reflect specific molecular abnormalities or characterize a group of patients without a known molecular aberration. Studying genes that are epigenetically deregulated in different groups of patients may contribute to a more detailed understanding of pathways involved in leukemic transformation. For 15 samples (14 AMLs of diverse clinical and genetic background and pool of CD34+ control samples) SureSelectXT Human Methyl-Seq system (Agilent) was used to interrogate DNA methylation of selected regions (84 megabases in total) of their genomes. Targeted bisulfite libraries were run on a HiSeqTM2000 sequencer (Illumina) using 105 bp paired-end sequencing reads. Methylation status of candidate regions was verified using 454 bisulfite pyrosequencing (Roche). The expression levels of selected genes were assessed with TaqMan Gene Expression Assays (Applied Biosystems). Targeted methylation data was correlated with previously obtained whole-genome bisulfite (WGBS) data for the cross-experiment (WGBS/target enrichment) samples (n=3), taking into account positions with minimal coverage of 10. There was a strong positive correlation (R≥0.97) for all three samples. Pairwise Pearson correlations were computed for all (targeted and WGBS) samples and formed the basis of a hierarchical clustering analysis. The cross-experiment replicates clustered together. From the clinical and molecular characteristics, only CBFβ/MYH11+ patients (n=2) clustered together in all clustering analysis performed (using CpG in/out CGIs or all CpG regardless of location). None of the other molecular abnormalities (i.e. DNMT3A mutations, MLL translocations, NPM1 mutations, FTL3/ITDs and CEPBα mutations) formed clusters. We did not observe any effect of clinical status of AML (de novo, secondary AML with dysplastic changes or relapsed AML). Based on this observation, we extracted genomic regions being uniquely differentially methylated for CBFβ/MYH11+ patients. There was a clear tendency towards hypomethylation in 125 regions out of 182 (69%) displaying lower DNA methylation levels compared to CD34+ pool of controls. All CBFβ/MYH11 differentially methylated regions (DMRs) were uploaded to GREAT (http://bejerano.stanford.edu/great/public/html) and enrichment for genes described as upregulated in inv(16) AML (CBFβ/MYH11+) was seen (reported in Valk et al., NEJM 2004, ID: VALK_AML_CLUSTER_9). This enrichment set comprised 10 genomic regions assigned for 6 genes – MN1, SPARC, ST18, DHRS3, FAM171A and BAHCC1.MN1, SPARC, ST18 and DHRS3 – were chosen for hypomethylation verification by 454 bisulfite pyrosequencing. Altogether 11 CBFβ/MYH11+ (inv(16)), 11 non-inv(16) AML M4, 6 other AML subtypes and 5 healthy control samples were examined by 454 pyrosequencing. Average levels of DNA methylation for selected regions were significantly lower for inv(16) versus non-inv(16) AML M4, other AML subtypes and healthy controls (P We found a new hypomethylation signature in CBFβ/MYH11+ patients. The described hypomethylated regions were assigned to genes known as being overexpressed in inv(16) patients. As for MN1 gene, we can speculate that different mechanism responsible for its overexpression in inv(16) versus non-inv(16) patients may explain discrepancy between its negative prognostic value in cytogenetically normal AML patients and generally good outcome of inv(16) patients. This study was supported by the project (Ministry of Health, Czech Republic) for conceptual development of research organization (00023736, IHBT) and was a part of the COST Action BM0801 (EuGESMA). Disclosures: No relevant conflicts of interest to declare.

AIM: To establish an optimum combination of molecular markers resulting in best overall diagnostic sensitivity and specificity for evaluation of suspicious pancreatic mass. METHODS: Endoscopic ultrasound (EUS)-guided fine needle aspiration cytology (FNA) was performed on 101 consecutive patients (63 males, 38 females, 60 ± 12 years; 81 with subsequently diagnosed pancreatic cancer, 20 with chronic pancreatitis) with focal pancreatic mass. Samples were evaluated on-site by an experienced cytopathologist. DNA was extracted from Giemsa stained cells selected by laser microdissection and the presence of K-ras, p53 and p16 somatic mutations was tested by cycling-gradient capillary electrophoresis (CGCE) and single-strand conformation polymorphism (SSCP) techniques. In addition, allelic losses of tumor suppressor genes p16 (INK4, CDKN2A) and DPC4 (MADH4, SMAD4) were detected by monitoring the loss of heterozygosity (LOH) at 9p and 18q, respectively. RESULTS: Sensitivity and specificity of EUS-guided FNA were 75% and 85%, positive and negative predictive value reached 100%. The remaining 26% samples were assigned as inconclusive. Testing of molecular markers revealed sensitivity and specificity of 70% and 100% for K-ras mutations (P < 0.001), 24% and 90% for p53 mutations (NS), 13% and 100% for p16 mutations (NS), 85% and 64% for allelic losses at 9p (P < 0.001) and 78% and 57% for allelic losses at 18q (P < 0.05). When tests for different molecular markers were combined, the best results were obtained with K-ras + LOH at 9p (92% and 64%, P < 0.001), K-ras + LOH at 18q (92% and 57%, P < 0.001), and K-ras + LOH 9q + LOH 18q (96% and 43%, P < 0.001). When the molecular markers were used as complements to FNA cytology to evaluate inconclusive samples only, the overall sensitivity of cancer detection was 100% in all patients enrolled in the study. CONCLUSION: EUS-guided FNA cytology combined with screening of K-ras mutations and allelic losses of tumor suppressors p16 and DPC4 represents a very sensitive approach in screening for pancreatic malignancy. Molecular markers may find its use particularly in cases where FNA cytology has been inconclusive.

Abstract 1396 Introduction. Mutations in the kinase domain (KD) of BCR-ABL are a known mechanism of resistance to the tyrosine kinase inhibitor (TKI) treatment of patients with chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). The gold standard for the routine examination of mutations in patients with therapy failure is the Sanger sequencing of amplified cDNA region of BCR-ABL KD with the sensitivity of ∼15–20%. More than 100 BCR-ABL KD mutations were described in association with development of resistance to imatinib. It was suggested that early ultra-sensitive detection of KD BCR-ABL mutations, at the time of diagnosis or shortly after imatinib start and before switch to 2nd generation TKI, may predict their subsequent expansion and failure of the treatment if the patient is treated with TKI against which is the type of mutation resistant. Ultradeep amplicon pyrosequencing represents a promising tool to study BCR-ABL mutation development and their expansion under TKI treatment. In our study, the detection of mutations reached the sensitivity of 0.03-0.01%. At this level we may expect detection of artifacts introduced during amplicon library preparation and pyrosequencing. Aims. The aim is to define the threshold for relevant detection of published BCR-ABL mutations (n= 124). Methods. We evaluated 155 CML or Ph+ALL patient samples. As the non-mutated control, the cDNA of KD c-ABL of healthy individuals (n= 9) and cDNA of wild type BCR-ABL from Ph+ cell lines (n=3) were used. To cover cDNA region of kinase domain (950 bp), multiple amplicons with the length up to 400 bp should be prepared to apply 454 technology. The experiments consist of: 1) comparison of two amplicon designs with 3 and 4 fragments covering KD to follow the amplicon-related artifacts; 2) the reproducibility test of 5 samples using 3 and 4 PCR fragments for amplicon library preparation; 3) tests with 2 different reverse transcriptases and Taq polymerases to detect artifactual nucleotide substitutions. Results. Using 454 technology we detected high number of ultralow-level single nucleotide substitutions (∼ 200) with the frequency < 1 in 100 in all samples analyzed including non-mutated controls using 3 and also 4 amplicon design. Surprisingly, we found that almost all low-level variants were T#x2610;#x0025;C or A#x2610;#x0025;G substitutions (Fig. 1), including those that may result in clinically relevant mutations (V288A, M244V, V289A, F317L, F359L, M351T, E355G, E453G). Evaluation of pyrograms suggested that they were unlikely to be errors of pyrosequencing, because they were not necessarily associated with hompolymer regions. The frequency of T#x2610;#x0025;C or A#x2610;#x0025;G low-level variants was lower when using proof-reading Taq polymerase. The pattern of these nucleotide substitutions is similar to that reported in IGH hypermutation analysis in CLL (Campbell et al. PNAS 2008) showing that T#x2610;#x0025;C or A#x2610;#x0025;G substitutions are errors caused by Taq polymerases. This is supported by the studies on PCR fidelity suggesting that Taq polymerase caused this pattern of base substitution even when using a proof-reading enzyme. Thus, the T#x2610;#x0025;C or A#x2610;#x0025;G substitution in BCR-ABL KD should not be evaluated below the level of 1%. The other but less frequent transitions caused by Taq polymerase are G#x2610;#x0025;A or C#x2610;#x0025;T substitutions that were detected in our study with the frequency < 1 in 1000, thus, the variants detected below 0.1% should not be examined as the true-positive result. Conclusion. Mutation data from ultradeep amplicon sequencing of BCR-ABL KD below 1% must be evaluated cautiously to avoid misleading interpretations. Attention must be paid to the fact that T#x2610;#x0025;C or A#x2610;#x0025;G substitution may arise spontaneously in vivo, but there is no way to differentiate in vivo spontaneous mutations from Taq polymerase errors in vitro. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy. Machova Polakova:Novartis: Honoraria, Research Funding; BMS: Honoraria.