Smoldering multiple myeloma (SMM) precedes multiple myeloma (MM). The risk of progression of SMM patients is not uniform, thus different progression-risk models have been developed, although they are mainly based on clinical parameters. Recently, genomic predictors of progression have been defined for untreated SMM. However, the usefulness of such markers in the context of clinical trials evaluating upfront treatment in high-risk SMM (HR SMM) has not been explored yet, precluding the identification of baseline genomic alterations leading to drug resistance. For this reason, we carried out next-generation sequencing and fluorescent in-situ hybridization studies on 57 HR and ultra-high risk (UHR) SMM patients treated in the phase II GEM-CESAR clinical trial (NCT02415413). DIS3, FAM46C, and FGFR3 mutations, as well as t(4;14) and 1q alterations, were enriched in HR SMM. TRAF3 mutations were specifically associated with UHR SMM but identified cases with improved outcomes. Importantly, novel potential predictors of treatment resistance were identified: NRAS mutations and the co-occurrence of t(4;14) plus FGFR3 mutations were associated with an increased risk of biological progression. In conclusion, we have carried out for the first time a molecular characterization of HR SMM patients treated with an intensive regimen, identifying genomic predictors of poor outcomes in this setting.

Background: Chromothripsis is a unique catastrophic event found in 1-5% of chronic lymphocytic leukemia (CLL) patients, associated with poor prognostic factors and short survival (Edelmann et al, 2012; Puente et al, 2015; Leeksma et al, 2021). It is characterized by the presence of multiple genomic rearrangements in one chromosome, resulting in oscillating switches between 2-3 copy number states by chromosomal microarrays (CMA) or next-generation sequencing (Stephens et al, 2011). Optical genome mapping (OGM) is a new method based on the imaging of long DNA molecules (>250kb) labelled at specific motifs, generating a unique pattern. This allows mapping of the genomic location of each molecule and detection of structural and numerical chromosomal abnormalities with high resolution and sensitivity. Aims: 1. To analyze the genome complexity associated with chromothripsis and the patterns of rearrangements detected by OGM; 2. To determine the prognostic impact of chromothripsis in a cohort of CLL patients enriched in complex karyotypes (CK). Methods: A total of 33 patients with chromothripsis detected by CMA were included: 11 with chromothripsis-like patterns (≥7 switches), 22 with classical chromothripsis (≥10 switches). Nine of the cases were studied with OGM (Bionano Genomics). Clinico-biological features and time to first treatment (TTFT) were analyzed. Results were compared with a cohort of 129 patients showing CK without chromothripsis (control group) (Ramos-Campoy et al, 2021). Results: All patients with chromothripsis showed a high genomic complexity (30/33 with CK by chromosome banding analysis and 3/33 classified as complex by CMA [range: 10-24 CNV, being at least three of them >5Mb (range: 3-6)]. Forty-six chromothriptic events were identified. These affected 1-4 chr/patient, but a single chromosome was involved in 25/33 patients (76%). Chromothripsis was distributed throughout all chromosomes, most frequently affecting 3 (n=5), 6 (n=5) and 13 (n=5). The events were mainly multiple losses (25/46) or alternated gains and losses (19/46). Size and coordinates of CNVs detected by OGM and CMA were highly concordant. OGM detected multiple rearrangements cryptic by CMA (median: 9/chromothriptic event). Two patterns of chromothripsis-related rearrangements were identified: one clustered in the chromothriptic region (3/9) and the other involving both chromothriptic and non-chromothriptic chromosomes (range: 1-6 chr) (6/9). One patient showed 36 rearrangements throughout the genome, suggesting the coexistence of chromothripsis and chromoplexy, characterized by the presence of multiple chained translocations (Figure). Patients with chromothripsis showed a higher frequency of TP53del/mut (70% vs 39%; p=0.001) and a shorter TTFT (15m vs 2m; p=0.013) than the control group. No differences were observed for IGHV and del(11q) status. When stratifying patients based on TP53 status, the presence of chromothripsis was not associated with shorter TTFT. In the multivariate analysis including TP53, genomic complexity by CMA and chromothripsis, only TP53 was statistically significant (HR=1.6; p=0.029). Image:Summary/Conclusion: 1. Chromothriptic events involve multiple loci in CLL patients; 2. OGM allows not only the detection of CNV but also the identification of two rearrangement patterns associated with chromothripsis (clustered or involving non-chromothriptic chromosomes) whose clinical impact should be further explored. 3. In CLL patients with CK, the adverse prognosis of chromothripsis is associated with a higher frequency of TP53del/mut.

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:The diagnosis of myeloid malignances includes multiple testing strategies for genetic abnormalities assessment, including gene mutations and copy number variations (CNVs), which is critical for therapeutic decision making. Although next‐generation sequencing (NGS) has been implemented in clinical diagnostic laboratories, it has focused on the detection of single nucleotide variants and small indels, while CNV detection still relies on karyotyping and fluorescence in situ hybridization (FISH).Aims:In the present study, a new strategy was developed and validated to simultaneously detect CNVs and somatic mutations in a series of patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) by using a capture‐based NGS custom panel.Methods:We designed a NGS target‐DNA panel associated to a custom bioinformatic pipeline (Sophia Genetics) for one‐step identification of somatic and germline mutations in 48 genes, as well as CNV. The CNV algorithm was based on the coverage levels across samples within the same batch. We studied the complete CDS or targeted exons of 16 genes sited in chromosome regions 5q14‐q35 (5 genes), 7q22‐q36 (3 genes), 8q12‐q24 (4 genes), 20q11‐q13 (3 genes), 17p13 (1 gene, TP53), as well as other genes in 5p, 7p, 20p and 17q as control regions. We analyzed 45 diagnostic samples of myeloid malignances (39 AML and 6 MDS), that had previously been characterized by FISH and/or karyotyping, as well as by conventional molecular techniques (FLT3‐ITD, NPM1 and CEBPA mutations). Paired‐end sequencing runs were performed on a MiSeq (Illumina) genome sequencer.Results:We detected 133 pathogenic mutations in 42 patients, including all 8 FLT3‐ITD (size 18–204 bp) and 4 mutations in CEBPA. We were also able to detect exon‐level duplications, such as KMT2A (MLL) partial tandem duplication, in 3 cases. Four different CNV types were distinguished by the algorithm: del(5q), del(7q), del(20q) and trisomy 8, showing a full concordance with those alterations identified by FISH probes. All 8 patients with 5q33 deletions (8/8, 100%) were detected by NGS, as well as 5 cases with 7q35 deletion (5/5, 100%), 3 patients with trisomy 8 (3/3, 100%) and 3 with 20q12 deletion (3/3, 100%). None of the patients presented 17p abnormalities by any technique. Moreover, we detected alterations in the copy number of KMT2A gene in 4 of 5 patients with 11q23 alterations (translocation or amplification) identified by FISH and cytogenetics. One case of t(11;19)(q23;p13) was not detected by NGS, which is explained because the breakpoint region of this translocation was not covered by our design. Next panel will overcome this caveat by including the complete CDS of the KMT2A gene. Finally, NGS estimated abnormal copy numbers in three cases of 7q deletions and one trisomy 8, where FISH and cytogenetics had failed to detect any change.Summary/Conclusion:Our study demonstrates that both somatic mutations and targeted CNVs can be accurately detected by NGS covering adequate exons on selected genes. Therefore, this strategy complements conventional methods to identify biomarkers for the diagnosis, prognosis and therapy of myeloid malignances.

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