Background:Myeloid neoplasms (MN) are heterogeneous clonal diseases arising in hematopoietic cells. Although MN are usually sporadic late‐onset cancers, growing evidence suggests that an important number of cases could emerge as a consequence of inherited predisposition. Since many of the genes associated with hereditary myeloid malignancies (HMM) are also affected by somatic mutations in sporadic MN, there is a chance of identifying variants of germline nature when performing Next Generation Sequencing (NGS) tumor testing. Given the clinical relevance of the issue, it is important to correctly classify those suspicious variants as somatic or germline.Aims:We intended to identify hematological patients with potentially inherited variants in a myeloid cohort with tumor‐only NGS data.Methods:We reviewed 332 NGS clinical reports of samples from 299 myeloid patients tested with tumor‐only sequencing during 2018 in our lab. We considered variants detected in BM sample at a 40–60% VAF classified as pathogenic, likely pathogenic or of uncertain significance variants as potentially inherited.On the one hand, for patients with available follow‐up data, we considered as suggestive of inherited origin variants with stable VAF. On the other hand, for patients with available non‐myeloid tissue we sequenced 20 samples including DNA derived from cultured skin fibroblasts (n = 3), cultured bone marrow (BM) fibroblasts (n = 1), hair follicles (n = 5), purified CD3+ T cells (n = 6) and buccal swab (n = 5). Libraries were sequenced using our custom Pan‐Myeloid Panel, which includes 22 genes related to HMM (PMP, 48 genes, capture‐based, SOPHiA GENETICS). All patients signed a written informed consent form for genetic testing, research and tissue banking (Biobank of the University of Navarra). Samples were processed following Standard Operating Procedures approved by the Ethical and Scientific Committee of the University.Results:In total, 130 suspicious variants in HMM‐related genes were identified in tumor sample from 100 patients. Follow‐up data was available for 8 patients harboring a total of 9 variants. Six variants from 5 patients presented stable VAFs and clearance of accompanying variants during follow‐up; this might be due to an inherited event, although germline tissue testing is required for confirmation. Suspicious germline variants were found in ASXL1, DDX41, RUNX1 and IKZF1. Additionally, non‐myeloid tissue was available for 7 patients harboring a total of 8 variants. All 8 (100%) variants were confirmed of inherited origin. Germline variants were detected in ASXL1, DDX41, SH2B3, TET2 and NF1 (Figure 1).Summary/Conclusion:This study shows that NGS panels may incidentally detect inherited variants related to predisposition to MN despite not having been designed for that purpose. Our data supports the importance of considering variants incidentally found upon tumor‐only sequencing as potentially of germline origin. Indeed, one third of our cohort was suspicious of carrying a germline variant. We could confirm as inherited 100% of tested variants from patients with non‐myeloid tissue available. For patients with follow‐up data, VAF may help in discriminating ambiguous variants, but ultimately germline tissue sequencing is needed to determine the nature of the variant.imageThese findings suggest that there is an increasing need for germline tissue testing along with the appropriate genetic counseling associated to it.

Background:The current standard for morphologic complete remission in acute myeloid leukemia (AML) is less than 5% myeloblasts, but mounting data show this criterion is not sufficiently sound. Alternative methods, such as quantitative reverse‐transcription polymerase chain reaction (RT‐qPCR), are widely used to detect molecular responses, but it relies on the initial detection of a fusion transcript, or overexpressed gene. Deeper knowledge of the clonal dynamics of AML could potentially be of clinical utility. A wide scope testing technology is required in order to address the molecular heterogeneity of AML. We reasoned that an appropriate Next Generation Sequencing (NGS) panel could be a useful tool to provide personalized molecular monitoring in patients diagnosed as or progress to AML.Aims:The aim of this study is to evaluate the clinical utility of NGS panel in the prognostic and treatment monitoring in patients diagnosed as or progress to AML.Methods:We studied the genomic alterations of 19 AML cases (13 de novo, and 6 secondary to a preexisting MN) during disease follow‐up; 11 of these patients received hematopoietic stem cell transplantation (HSCT). The 67 samples were tested with our custom Pan‐Myeloid Panel (48 genes, SOPHiA GENETICS). Samples were provided by the Biobank of the University of Navarra and were processed following SOP approved by the Ethical and Scientific Committee of the University. Libraries were pair‐end sequenced on a Miseq sequencer (Illumina). Sequencing data were analyzed by two geneticists with expertise in hematological malignancies.Results:Sequencing data identified genomic clonal markers with clinical utility (i.e. diagnostic, prognostic, and/or predictive value) in 89,5% of cases. In patients not receiving HSCT (n = 8), NGS was useful to classify them in two genetic profiles: those achieving molecular complete remission (mCR) (n = 2) (Figure 1A), and those not responding to treatment and undergoing disease progression (n = 6) (Figure 1B). In the last group, NGS identified pathogenic variants in DDX41, DNMT3A, IDH1, JAK2, NRAS, SRSF2, U2AF1 genes. In patients receiving HSCT (n = 11), NGS also classified patients in two groups: those clearing pathogenic variants upon HSCT (n = 5) (Figure 1C), and those with persisting variants, not achieving mCR (n = 6) (Figure 1D). Again, NGS identified initial clones harboring pathogenic variants, like KRAS, that appeared in the 66% of the patients after HSCT failure. Also mutations in CBL, DNMT3A, FLT3, JAK2, KRAS and SRSF2 genes are present in this cohort of patients.In two cases, NGS either did not detect any clinically relevant variant, or it detected variants only after disease progression; in these two cases an NGS panel was insufficient, and therefore more comprehensive studies are needed (e.g. exomes). Of note, NGS data detected clones harboring pathogenic variants in two patients with negative minimal residual disease (MRD), as measured by flow cytometry (Figure 1D), indicating that NGS could complement current gold standard follow‐up method in some instances.Summary/Conclusion:A 48‐gene panel NGS has been useful for molecular diagnosis, treatment follow‐up, and relapse detection in nearly 90% of the AML cases included in our study (17 of 19). NGS was also useful for following mutational clearance and/or clonal evolution in 12 of 19 patients (63%), including cases undergoing HSCT. According to our data, NGS could be of clinical utility for routine diagnosis and follow‐up in an elevated proportion of AML patients, even complementing immunophenotypic techniques for MRD monitoring in some instances.image

Background: Reversible protein phosphorylation is a dynamic process that regulates virtually all cellular events and is controlled by the opposed activity of protein kinases and protein phosphatases. Although strong emphasis has been placed on the hyperactivation of protein kinases in the onset and progression of human disease, especially cancer, accumulating evidence suggests that abnormal signaling also results from impaired expression or activity of phosphatases. In Large Granular Lymphocyte (LGL) leukemia (LGLL), a rare lymphoproliferative disorder chatracterized by clonal expansion of either cytotoxic T lymphocytes (T-LGLL) or natural killer cells (chronic lymphoproliferative disorder of NK cells, NK-CLPD), constitutive activation of phosphorylation-regulated survival pathways, JAK2/STAT3, PI3K/Akt, and NF-κB pathways among others, has been found to account for the molecular mechanisms sustaining monoclonal cell proliferation and resistance to apoptosis. Interestingly, since several factors participating in such pathways are known to be substrates for protein phosphatases, namely the tyrosine phosphatase SHP-1 and the serine/threonine phosphatase PP2A under normal conditions, it is conceivable that their possible alterations and consequent inability to disrupt hyperactive pathways play a role in LGLL pathogenesis. Aims: In this study, we assessed the ability of small molecules activating SHP-1 and PP2A to abrogate the survival pathways in LGLs by counteracting aberrant signals generated by constitutively activated protein kinases. Methods: Peripheral blood specimens were obtained from untreated patients with LGLL (both T-LGLL and NK-CLPD). Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque (Sigma Aldrich) gradient separation. LGLs were then separated from PBMCs by the immunomagnetic beads (Miltenyi Biotec). LGLs were incubated with SC-43, SHP-1 activator, or alternatively CC11, PP2A activator, at increasing concentrations and different time points. Subsequently, LGLs underwent annexin V–PI flow cytometry to assess the extent of apoptosis or were lysed to monitor the level of SHP-1 and PP2A activity. Moreover, the phosphorylation/activation status and the protein level of factors directly involved in the constitutively activated survival pathways of LGLs were evaluated by Western blot analysis. Results: Both SC-43 and CC11 proved effective at bringing a significant level of caspase-dependent apoptosis in the low micromolar range, also exhibiting a striking synergistic effect when used in combination. Moreover, both compounds selectively activated their target phosphatases, as demonstrated by proper in-vitro phosphatase assays. As to the signaling pathways affected by SC-43 via SHP-1 activation, the phosphorylation status of STAT3 was remarkably decreased, thereby negatively affecting the function of this transcription factor, as shown by the reduced expression of downstream target genes such as Mcl-1, Cyclin D and S1P5. In turn, CC11 caused dephosphorylation of factors taking part in PI3K/Akt and NF-kB pathways, including Akt itself and IKKbeta and IkB, respectively, confirming the role of PP2A as functional antagonist of survival pathways in patients’ LGLs. Summary/Conclusion: Taken together, our results support the newly emerging evidence that the drug-induced activation of SHP-1 and PP2A, already recognized as tumor suppressors in other tumor cells, may serve as a mechanism countering the aberrant pro-survival signals, thus opening up new prospects for treatment of LGLL.

Background:The diagnosis of myeloid neoplasms (MN) has significantly evolved through the last few decades. Nowadays, blood cell morphology, blasts count, cytogenetics and molecular testing are crucial for MN diagnosis, prognosis and therapy. The advent of personalized medicine has drastically influenced the way we currently diagnose and monitor MN, mainly thanks to Next Generation Sequencing (NGS), which is gradually becoming an essential tool to help clinicians with disease management. To this end, most specialized genetic laboratories have implemented NGS panels targeting a number of different genes relevant to MN.Aims:The aim of the present study is to evaluate the performance of four different targeted NGS gene panels based on their technical features and clinical utility.Methods:A total of 32 patient bone marrow samples were accrued: 17 acute myeloid leukemia (AML), 7 myeloproliferative neoplasms (MPN), 6 myelodysplastic syndromes (MDS), 2 chronic myelomonocytic leukemia (CMML). All of them had been tested with conventional molecular testing and harboured a variety of sequence variants, including single nucleotide variants (SNVs), insertions and deletions (indels). These samples were tested with four NGS panels, three were commercially available panels and one custom. The samples were tested as follows: 17 with Trusight Myeloid Panel (TSMP, 54 genes, amplicon based, Illumina), 16 with SureSeq (23 genes, capture‐based, Oxford Gene Technology), 15 with Myeloid Solutions (MYS, 30 genes, capture‐based, SOPHiA GENETICS), and all 32 with our custom Pan‐Myeloid Panel (PMP, 48 genes, capture‐based, SOPHiA GENETICS). Libraries were built following manufacturer's instructions and pair‐end sequenced on a Miseq sequencer (Illumina). Analysis of the sequencing data was carried out with a valid performance at the clinical variant allele frequency (VAF) cut‐off of 5%. Variants were classified by two geneticists with expertise in hematological malignancies; variants classified as “pathogenic” or “likely pathogenic” were kept as clinically relevant.Results:The average sequencing depth was over 5000X in PMP, MYS and TSMP; and over 1600X in SureSeq panel. Nineteen genes are included in all four panels (core myeloid gene set), being ASXL1, CALR, CEBPA, DNMT3A, ETV6, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, RUNX1, TET2, TP53, U2AF1 and WT1. A total of 50 clinically relevant variants were detected by all four panels, and 37 of these fell in one gene of the core myeloid gene set. There are 11 discordant variants between panels, 3 in the core myeloid gene set missed only by SureSeq panel (2 FLT3‐internal tandem duplications (ITD) and one CALR mutation); and 8 mutations only called by the panels that include those genes in their design (Table 1). Of note, 2 additional FLT3‐ITD mutations of 36 bp of length that had been detected by conventional molecular analysis were not called by any of the NGS panels.Summary/Conclusion:After testing the four panels, our data show that there is a high risk of finding different mutations depending on the panel of choice, due both to the panel design and the data analysis method. Based on our data, ITD calling remains a challenge for NGS. This is a major issue, since AML management strongly depends on FLT3‐ITD detection. In addition, NGS testing times are hard to harmonise with turnaround time stablished in current European Leukemia Net guidelines. Therefore, conventional molecular testing might need to be kept in place for the correct diagnosis of MN for now.image