Additional Genetic Abnormalities Research Articles

Comprehensive Molecular Profiling of NPM1-Mutated Acute Myeloid Leukemia Using RNAseq Approach.

Acute myeloid leukemia (AML) is a complex hematologic malignancy with high morbidity and mortality. Nucleophosmin 1 (NPM1) mutations occur in approximately 30% of AML cases, and NPM1-mutated AML is classified as a distinct entity. NPM1-mutated AML patients without additional genetic abnormalities have a favorable prognosis. Despite this, 30-50% of them experience relapse. This study aimed to investigate the potential of total RNAseq in improving the characterization of NPM1-mutated AML patients. We explored genetic variations independently of myeloid stratification, revealing a complex molecular scenario. We showed that total RNAseq enables the uncovering of different genetic alterations and clonal subtypes, allowing for a comprehensive evaluation of the real expression of exome transcripts in leukemic clones and the identification of aberrant fusion transcripts. This characterization may enhance understanding and guide improved treatment strategies for NPM1mut AML patients, contributing to better outcomes. Our findings underscore the complexity of NPM1-mutated AML, supporting the incorporation of advanced technologies for precise risk stratification and personalized therapeutic strategies. The study provides a foundation for future investigations into the clinical implications of identified genetic variations and highlights the importance of evolving diagnostic approaches in leukemia management.

Navigating the spectrum of double-outlet right ventricle presentations: Outcomes from a contemporary cohort based on subtypes.

Our aim in this study was to investigate the prenatal and postnatal prognosis of double outlet right ventricle (DORV) cases diagnosed prenatally by analyzing the outcomes based on the subtype. This study is a retrospective chart review. Cases diagnosed with fetal DORV by prenatal ultrasound in the maternal-fetal medicine department of our hospital between 2014 and 2022 were included. Data on maternal characteristics, fetal echocardiographic features (type of DORV), pregnancy and neonatal outcomes (termination of pregnancy [TOP], intrauterine fetal death [IUD], neonatal death [NND], death in infancy (IND), survival) were collected and analyzed. Ninety-nine cases of prenatally diagnosed cases of DORV were included. The prenatal diagnosis was right in 97% of the liveborn fetuses. The cases were classified into subtypes, including transposition of great arteries (TGA), Fallot, ventricular septal defect (VSD), remote, and heterotaxy types. The cohort consisted of 32.3% TGA type, 19.1% fallot type, 11.1% VSD type, 2% remotetype, and 35.3% heterotaxy type of DORV. An additional cardiac anomaly was observed in 87% and an extra-cardiac anomaly was observed in 54% of the cases. When we excluded the cases with heterotaxy type but without any chromosomal abnormality, additional genetic abnormalities were detected in 42% of the remaining cases. Outcome of pregnancy was livebirth in 68/99 (68.7%), IUFD in 5/99 (5.1%), and TOP in 26/99 (26.3%). Postnatal cardiac surgical repair was performed in 48 cases. Survival among livebirths was 39/68 (57.3%). Twenty-nine neonates or infants who had additional cardiac anomalies and/or genetic abnormalities died before any surgical intervention. The postoperative survival rate was 39/48 (81.2%). The prognosis in DORV depends on the anatomical subtype, the presence, and severity of associated anomalies. Survival increases in isolated cases without any additional structural or genetic anomalies.

KIT exon 17 mutations are predictive of inferior outcome in pediatric acute myeloid leukemia with RUNX1::RUNX1T1.

Pediatric core binding factor acute myeloid leukemia (CBF-AML), although considered a favorable risk subtype, exhibits variable outcomes primarily driven by additional genetic abnormalities, such as KIT mutations. In this study, we examined the prognostic impact of KIT mutations in 130 pediatric patients with CBF-AML, treated uniformly at a single center over 4years (2017-2021). KIT mutations were detected via next-generation sequencing using a myeloid panel comprising 52 genes for most patients. Our findings revealed that KIT mutations were present in 31% of CBF-AML cases. Exon 17 KIT mutation was most commonly (72%) seen with notable occurrences at the D816 and N822 residue in 48% and 39% of cases, respectively. The 3-year cumulative incidence of relapse (CIR) and overall survival (OS) for patients with exon 17 KIT mutation were 36% and 40%, respectively, and was significantly worse in comparison to other site KIT mutations (3-year CIR: 11%; OS: 64%) and without KIT mutation (3-year CIR: 13%; OS:71%). Notably, the prognostic impact of KIT mutations was prominent in patients with RUNX1::RUNX1T1, but not in those with CBFB::MYH11 fusion. Additionally, a high KIT variant-allele frequency (VAF) (>33%) predicted for a higher disease relapse; 3-year CIR of 40% for VAF greater than 33% versus 7% for VAF less than 33%. When adjusted for site of KIT mutation and end-of-induction measurable residual disease, VAF greater than 33% correlated with poor OS (hazard ratio [HR]: 4.4 [95% CI: 1.2-17.2], p=.034). Exon 17 KIT mutations serve as an important predictor of relapse in RUNX1::RUNX1T1 pediatric AML. In addition, a high KIT VAF may predict poor outcomes in these patients. These results emphasize the need to incorporate KIT mutational analysis into risk stratification for pediatric CBF-AML.

Whole Genome and Transcriptome Sequencing of 21 Paired Chronic and Blast Phase CML Cases: Acquisition of Genomic Alterations, Changes in the Transcriptomic Profiles and Occurrence of B-Cell Receptor Rearrangements

Background: Since the introduction of tyrosine kinase inhibitor therapy survival has substantially improved in CML. However, a subset of patients become resistant and progress to blast phase. Still the underlying genomic changes are not completely understood. Cohort & Methods: We studied 21 CML patients with paired sample material available from diagnosis in chronic phase (CP) and from blast phase (BP). All patients received a tyrosine kinase inhibitor starting at diagnosis of CML CP. Samples were analyzed by cytomorphology, chromosome banding analysis, WGS (100x, 2x151bp) and WTS (50 Mio reads, 2x101bp). B-cell receptor (BCR) rearrangements were analyzed with MiXCR software. The transcriptomic phenotype (TP) of each CP and BP sample was estimated by mapping their profiles to representative cases diagnosed with B-ALL, T-ALL, MDS, CMML, AML and CML CP. Results: The median time between diagnosis of CP and BP was 14.7 months (range: 4.7 - 137 months). While 18/21 CML in CP showed a TP corresponding to CML CP, one case each showed a TP corresponding to CMML, AML and B-ALL, respectively, although bone marrow blast counts were 6.5%, 5.5% and 5.5%, respectively. Of note, the case with the CMML-TP harbored a p190 BCR:: ABL1 isoform and also resembled CMML in bone marrow morphology. These three cases progressed to BP after 5.8, 5.3, and 8.9 months. The TP in BP corresponded to B-ALL in 13 cases, while 4, 3, and 1 case showed an AML-, CML CP- and MDS-TP, respectively (figure). In CP recurrent genomic alterations in addition to the BCR:: ABL1 fusion were mutations in ASXL1 (n=5), DNMT3A, TET2, RUNX1, and MGA (2 cases each). In BP the genomic landscape was more complex. Additional genetic alterations compared to CP were detected in all cases. Recurrently gained mutations in BP were mutations in WT1, BCOR, and SETD2 (2 cases each). The gain of copy number alterations (CNA) was more frequent. Recurrent CNA were: loss 9p (n=6), gain Ph-chromosome (n=4), loss 7q (n=3), gain 21q (n=3), gain 1q, loss 3q, gain 8q, loss 12p, and loss 13q in 2 patients each. Only three balanced structural variants were gained: a MECOM-, a NUP98:: HHEX- and a KMT2A:: MLLT3-rearrangement. Further, in 11/21 BP resistance mutations within the BCR:: ABL1 kinase domain were detected. The variant allele frequency (VAF) of RUNX1 mutations increased in BP in both cases with RUNX1 mutation present in CP (5% to 42%, time to BP: 4.7 months; 8% to 59%, time to BP: 11.7 months). DNMT3A and MGA mutations were about stable (VAF CP/BP: DNMT3A: 48%/37%, 33%/46%; MGA: 52%/36%, 40%/52%), while TET2 mutations were stable in one case (46%/49%) and decreased in the second (57%/27%). ASXL1 mutations were stable in 2 cases with an AML-TP in BP and lost in 3/5 cases with a B-ALL-TP in BP. BP with a lymphoid TP differed substantially with respect to the genomic profile from BP with myeloid TP. WTS data revealed a clonal BCR rearrangement in 8/13 BP with B-ALL-TP, of which 4 rearrangements are predicted to be productive. Further, CDKN2A/B deletions, IKZF1 deletions, and SETD2 mutations were only found in BP with B-ALL-TP. Thus, CML in BP with a B-ALL-TP showed a genomic landscape similar to B-ALL with BCR:: ABL1-rearrangement. In contrast CML BP cases with an AML-TP (n=4) had gained either rearrangements typically found in AML such as a KMT2A:: MLLT3 rearrangement and a MECOM rearrangement or acquired mutations in RUNX1, BCOR and WT1, or a CN-LOH 11p in combination with a WT1 VAF increase from 51% to 94%. Cases showing still a CML CP-TP (n=3) in BP acquired rather few additional genetic abnormalities (mutations in BCOR, WT1, CTCF; NUP98:: HHEX-rearrangement, gain of Ph-chromosome, loss of 11p, 17q), which were more myeloid in appearance. Conclusions: 1) Extensive genetic profiling indicated a substantial clonal evolution in the progression from CP to BP CML including loss of ASXL1 mutations, expansion of RUNX1 mutated clones, multiple CNA, and the frequent acquisition of a BCR rearrangement in BP with a transcriptomic phenotype resembling B-ALL. 2) A subset of CML cases in CP already showed a transcriptomic phenotype resembling acute leukemia indicating a rapid progression to BP. 3) The presence of a RUNX1 mutated subclone or a clonal BCR rearrangement seem to represent a warning signal in CML CP. 4) The prognostic validity of an extended genetic profiling including ASXL1, RUNX1, BCR rearrangement, and transcriptional signature in CML CP needs to be assessed in forthcoming prospective studies.

Integration of RNA Sequencing, Whole Exome Sequencing, and Flow Cytometry Into Routine Diagnostic Workup of Pediatric Lymphomas

The study was conducted to assess the feasibility of integrating state-of-the-art sequencing techniques and flow cytometry into diagnostic workup of pediatric lymphoma. RNA sequencing (RNAseq), whole exome sequencing, and flow cytometry were implemented into routine diagnostic workup of pediatric biopsies with lymphoma in the differential diagnosis. Within 1 year, biopsies from 110 children (122 specimens) were analyzed because of suspected malignant lymphoma. The experience with a standardized workflow combining histology and immunohistochemistry, flow cytometry, and next-generation sequencing technologies is reported. Flow cytometry was performed with fresh tissue in 83% (102/122) of specimens and allowed rapid diagnosis of T-cell and B-cell non-Hodgkin lymphomas. RNAseq was performed in all non-Hodgkin lymphoma biopsies and 42% (19/45) of Hodgkin lymphoma samples. RNAseq detected all but one of the translocations found by fluorescence in situ hybridization and PCR. RNAseq and whole exome sequencing identified additional genetic abnormalities not detected by conventional approaches. Finally, 3 cases are highlighted to exemplify how synergy between different diagnostic techniques and specialists can be achieved. This study demonstrates the feasibility and discusses the added value of integrating modern sequencing techniques and flow cytometry into a workflow for routine diagnostic workup of lymphoma. The inclusion of RNA and DNA sequencing not only supports diagnostics but also will lay the ground for the development of novel research-based treatment strategies for pediatric lymphoma patients.

The Investigation of Cerebroplacental Ratio and Some Cranial Biometric Measurements in Fetuses with Isolated Congenital Heart Disease

Objective: In this study, we aimed to compare the measurements of some fetal cranial structures and Doppler parameters between the groups with congenital heart disease and healthy pregnant women. Materials and Methods: The study included 30 patients with intrauterine congenital heart disease and 30 healthy pregnant women. Fetuses with additional structural and genetic abnormalities were excluded from the study. In both groups, biparietal diameter (BPD), head circumference (HC), transcerebellar diameter (TCD), middle cerebral artery pulsatility index (MCA-PI), umbilical artery pulsatility index (UA-PI), umbilical artery resistance index (UA-RI), and umbilical artery systole/diastole ratio (UA S/D), as well as superior-inferior diameter of the cavum septum pellucidum (CSP) were measured by ultrasound. The cerebroplacental ratio (CPR), which is an indicator of the protective effect on the brain, was calculated. Data were statistically compared between the two groups, and P < 0.05 was considered statistically significant. Results: A statistically significant difference was found between the groups in UA-RI and UA S/D values. The UA-RI and UA S/D values were significantly higher in the patient group than in the control group (P=0.048; P < 0.001). HC values were lower in the congenital heart disease group, and this result was statistically significant (P=0.047). There was no statistically significant difference between groups in BPD, HC, and TCD Z-scores. There was no statistically significant difference between groups in MCA-PI < 5th percentile, UA-PI > 95th percentile, and CPR < 1 scores. Conclusion: Congenital heart disease may cause chronic fetal hypoxia during the intrauterine process and lead to changes in the fetomaternal circulation. This study suggests that there may be relationships between fetal cranial biometric retardation and cerebral perfusion changes.

Impact of additional genetic abnormalities at diagnosis of chronic myeloid leukemia for first-line imatinib-treated patients receiving proactive treatment intervention.

The BCR::ABL1 gene fusion initiates chronic myeloid leukemia (CML); however, evidence has accumulated from studies of highly selected cohorts that variants in other cancer-related genes are associated with treatment failure. Nevertheless, the true incidence and impact of additional genetic abnormalities (AGA) at diagnosis of chronic phase (CP)-CML is unknown. We sought to determine whether AGA at diagnosis in a consecutive imatinib-treated cohort of 210 patients enrolled in the TIDEL-II trial influenced outcome despite a highly proactive treatment intervention strategy. Survival outcomes including overall survival, progression-free survival, failure-free survival, and BCR::ABL1 kinase domain mutation acquisition were evaluated. Molecular outcomes were measured at a central laboratory and included major molecular response (MMR, BCR::ABL1 ≤0.1%IS), MR4 (BCR::ABL1 ≤0.01%IS), and MR4.5 (BCR::ABL1 ≤0.0032%IS). AGA included variants in known cancer genes and novel rearrangements involving the formation of the Philadelphia chromosome. Clinical outcomes and molecular response were assessed based on the patient's genetic profile and other baseline factors. AGA were identified in 31% of patients. Potentially pathogenic variants in cancer-related genes were detected in 16% of patients at diagnosis (including gene fusions and deletions) and structural rearrangements involving the Philadelphia chromosome (Ph-associated rearrangements) were detected in 18%. Multivariable analysis demonstrated that the combined genetic abnormalities plus the EUTOS long-term survival clinical risk score were independent predictors of lower molecular response rates and higher treatment failure. Despite a highly proactive treatment intervention strategy, first-line imatinib-treated patients with AGA had poorer response rates. These data provide evidence for the incorporation of genomically-based risk assessment for CML.

Use of trio whole exome sequence in an adolescent with unexplained splenomegaly: a case report of CBL syndrome

CBL syndrome is a diagnosis typically made in childhood, which features neuroectodermal, cardiac and haematological abnormalities due to heterozygous germline CBL mutation. When complicated by additional genetic abnormalities, it confers a risk for JMML (due to CBL uniparental disomy), and in adulthood for MDS and AML (due to acquired copy-neutral loss of heterozygosity of the CBL allele, or additional single nucleotide polymorphisms) 1 and therefore will be encountered by both adult and paediatric haematologists.

Clinical significance of clonal evolution in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a hematological malignancy characterized by the Philadelphia (Ph) chromosome, which is formed by a t (9;22)(q34;q11) translocation. The aberrant activation of the ABL1 tyrosine kinase is caused by the BCR::ABL1 fusion gene on the Ph chromosome, leading to significant leukemic cell proliferation. CML is typically diagnosed in the chronic phase with few clinical symptoms and progresses to a blast crisis within years. CML acquires additional genetic abnormalities on top of BCR::ABL1 fusion during clonal evolution. ASXL1 mutations are found in the chronic phase, with a frequency of approximately 20%, whereas other mutations are rare. Most blast crisis cases have additional genetic abnormalities, including frequent ASXL1 and RUNX1 mutations. Recent studies have revealed that a subset of these genetic mutations affects the sensitivity of tyrosine kinase inhibitors to leukemic cells as well as patient prognosis, indicating applications for patient stratification and individualized treatment.

Termination of pregnancy after a prenatal diagnosis of congenital diaphragmatic hernia: Factors influencing the parental decision process.

To evaluate the incidence of termination of pregnancies (TOP) and factors associated with the decision for TOP in prenatally detected congenital diaphragmatic hernia (CDH). Single-centre retrospective cohort includes all prenatally detected CDH cases born between January 2009 and December 2021. Parental factors, such as parity, and fetal characteristics, such as disease severity, were collected. Descriptive statistics were used to present the data. Differences between terminated and continued pregnancies were analysed. The study population consisted of 278 prenatally detected CDH cases of which 80% detected <24weeks of gestation. The TOP rate was 28% in cases that were detected <24weeks of gestation. Twenty continued pregnancies resulted in either intrauterine fetal demise (n=6), preterm birth <24weeks (n=2), or comfort care after birth (n=12). The survival rate was 70% in the remaining 195 live born cases. Factors associated with the decision for TOP were additional fetal genetic or anatomical abnormalities (p<0.0001) and expected severity of pulmonary hypoplasia in left-sided CDH (p=0.0456). The decision to terminate a pregnancy complicated by fetal CDH depends on the severity of pulmonary hypoplasia and the presence of additional abnormalities. This emphasises the importance of early referral to expertise centres for detailed evaluation and multidisciplinary counselling.

Identification of Phenotypic Subgroups of Acute Myeloid Leukemia, Defined By Differentiation According to Who 2022 Classification

Background: The recently published WHO classification 2022 has extended genetic findings as the diagnostic basis for acute myeloid leukemia with defining genetic abnormalities (AML-DGA) resulting in 13 such entities leaving only a minority of cases not genetically defined, i.e. AML, defined by differentiation (AML-DBD). Clustering of flow cytometrically (FC) analyzed immunophenotypes may help to identify subgroups within AML-DBD allowing to search for additional genetic abnormalities which in future WHO classifications may qualify for AML-DGA. Aim: To identify subgroups within AML-DBD based on immunophenotypes analyzed by FC and to elucidate their genetic background applying panel sequencing. Methods: In a multicenter study comprising four institutions in North America, Europe and Asia cases with AML-DBD according to WHO 2022 classification were selected which had been diagnosed applying broad antigen panels by FC and broad gene panels by NGS. Expression data by FC on 30 surface and cytoplasmic antigens were collected and rated as negative, dim, partial and bright. Cluster analysis was performed to identify subgrops of cases with similar phenotpyes. Patterns of gene mutations within these subgroups were assessed. Results: A total of 234 AML-DBD were identified and included in the study. Nine antigens were found to be differentially expressed and to contribute to defining subgroups: MPO, CD34, CD11b, CD14, CD36, CD45, CD15, CD64 and CD65. Clustering of cases appyling expression categories of these nine antigens resulted in the identification of seven subgroups with clear differences in the antigen expression patterns (figure, panel A). Groups 1 and 3, different from all other groups, both showed bright expression of MPO and CD34 but only group 3 in addition showed bright CD36 expression (figure, panel B). Groups 2 and 4 also brightly expressed CD34 but showed only dim and no expression, respectively, of MPO. Conversely, groups 5 and 6 showed bright MPO expression with heterogeneous and largely no expression, respectively, of CD34. Group 5 exclusively showed bright expression of CD45 and CD64 in line with a monocytic phenotype. Group 7 was characterized by merely no or dim only expression of the nine antigens found differentially expressed between these seven groups. Interestingly, there has been no clear correlation between these seven groups and cytomorphologic findings besides AML with maturation occuring frequently in groups 1 (51% of cases) and 4 (67%) and AML without maturation in group 7 (57%). NGS revealed mutations in seven genes, which are not used by the WHO 2022 classification to define AML-DGA, to occur at different frequencies among the seven subgroups: DDX41(non germline), DNMT3A, FLT3-ITD, IDH1, IDH2, KMT2A-PTD and NRAS. In groups 1 to 4 20% to 39% of cases showed DDX41 mutations but no cases did so in groups 5 to 7. DNMT3A mutation frequencies varied among all groups between 17% and 44%. FLT3-ITD was found in 50% of group 5 but in only 7% to 28% of other groups. Mutations in IDH1 and IDH2 were found in all groups at frequencies ranging from 17% to 44%. While in the majority of groups frequencies of IDH2 mutations exceeded those of IDH1 mutations this was not the case in groups 3 and 5. KMT2A-PTD were found at a particularly high frequency of 44% in group 7 with other groups showing frequencies between 5% and 33%. NRAS mutations were found overall most infrequently with frequencies in groups ranging from 4% to 17%. Conclusions: Clustering of flow cytometric data revelad seven subgroups of AML-DBD with different antigen expression patterns in nine differentially expressed genes. Results of panel sequencing revealed differences in the genetic background of the seven groups but at the same time showed overlaps in mutation patterns between groups. Following the insights in the genetic basis of hematopoietic neoplasms gained in recent years and its application as principles in the WHO classification 2022 it is anticipated that the seven subgroups of AML-DBD identified in the present study feature additional genetic abnormalities besides the mutations described here. Whole genome and transcriptome sequencing will help identifying these additional abnormalities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

RUNX1 Mutated AML and MDS: Similarities, Differences and Molecular Factors Leading to Disease Progression

Background: Mutations in the myeloid transcription factor RUNX1 are frequently detected in AML and MDS. Differences have been described regarding additional cytogenetic and molecular genetic abnormalities, however the molecular factors responsible for progression of RUNX1 mutated MDS to AML are yet not fully elucidated. Aim: Analysis of genetic (1) similarities and (2) differences of RUNX1 mutated AML and MDS cases, (3) identification of factors potentially causing disease progression from RUNX1 mutated MDS to AML. Methods: Whole-genome sequencing (WGS) was performed for 773 AML and 747 MDS patients (median coverage 100x). 151bp paired-end reads were generated on NovaSeq 6000 and HiSeqX machines (Illumina, San Diego, CA). As no sample specific normal tissue was available, a so-called Tumor/Unmatched normal (TUN) workflow was used to reduce technical artefacts and germline calls. All reported p-values are two-sided and were considered significant at p<0.05. Results: Mutations in RUNX1 (RUNX1mut) were detected at comparable frequencies in AML and MDS (AML: 97/773 cases, 13%; MDS: 71/747, 10%). In MDS, an association of the RUNX1mut frequency with higher blast counts was observed (0-5% blasts: 15/419 with RUNX1mut, 4%; ≥5,<10%: 16/175, 9%; ≥10,<20%: 40/153, 26%). However, blast count did not influence overall survival (OS) in RUNX1mut subgroups (0-5% blasts: 18 months (m); ≥5,<10%: 25 m; ≥10,<20%: 18 m; AML: 21 m). The variant allele frequency (VAF) of RUNX1mut differed significantly dependent on the blast count (median VAF in cases <5% blasts: 0.22; ≥5%: 0.42; p<0.001). Regarding cytogenetic abnormalities, RUNX1mut MDS showed a higher frequency of normal karyotype (NK) compared to AML (66% vs. 38%, p<0.001) but similar frequencies of trisomies (18% vs. 22%) and complex karyotype (3% vs. 5%), although differences regarding the trisomy type were detected (MDS: only trisomy 8; AML: trisomies 8, 11 and 13). In 10% of AML cases with RUNX1mut a rearrangement involving MECOM was detected (5% GATA::MECOM, 5% other MECOM rearrangements), which was absent in MDS with RUNX1mut. Similarly, KMT2A-PTD and FLT3-ITD alterations were detected in AML but not in MDS with RUNX1mut (KMT2A-PTD: 7% in AML vs. 0% in MDS, p=0.021; FLT3-ITD: 13% vs. 0%, p=0.001). By contrast, TP53mut were found at comparably low frequencies (MDS: 4/71, 6%; AML: 3/97, 3%). For both AML and MDS, a strong association of RUNX1mut with mutations in spliceosome genes (SF3B1, SRSF2, U2AF1, ZRSR2) was detected (AML: 58/97 = 60% in RUNX1mut vs. 139/676 = 21% in RUNX1wild type (wt); p<0.001; MDS: 52/71 = 73% vs. 346/676 = 51%, p<0.001). However, while SRSF2mut in MDS were associated with increasing blast counts similar to RUNX1mut (0-5% blasts: 25/419 with SRSF2mut, 6%; ≥5,<10%: 130/175, 17%; ≥10,<20%: 50/153, 32%), SF3B1mut were negatively correlated to blast counts and thus to RUNX1mut (0-5% blasts: 179/419, 43%; ≥5,<10%: 28/175, 16%; ≥10,<20%: 11/153, 7%). In the total AML cohort, in 24/773 of cases a CN-LOH involving RUNX1 was detected, 21 of these also showed a RUNX1mut (22% (21/97) of RUNX1mut cases). By contrast, in the total MDS cohort in only 2/747 cases a CN-LOH covering RUNX1 was found, none of them harboring a RUNX1mut. The VAF of cases with RUNX1mut + CN-LOH (RUNX1mutLOH) was >0.5 for all cases and was significantly higher than the VAF of RUNX1mut cases without CN-LOH (0.89 (range 0.54-0.97) vs. 0.42 (range 0.05-0.85), p<0.001). Moreover, cases with RUNX1mutLOH were found to be significantly associated with FLT3-ITD (RUNX1mutLOH, 6/21 = 29%; RUNX1mut without CN-LOH, 7/76 = 9%; p=0.032). Conclusions: (1) RUNX1mut occur at similar frequencies in AML and MDS and genetically show an MDS signature, reflected e.g. by association with mutations in spliceosome genes. OS is similar in all subgroups independent of blast counts. (2) In MDS, RUNX1mut are associated with higher blast counts, thus potentially functioning as driver towards progression to AML. (3) Additional cytogenetic/molecular genetic alterations that might function as progression factors from RUNX1mut MDS to AML include: (i) karyotype, especially MECOM rearrangements, trisomy 11 and trisomy 13 (ii) KMT2A-PTD, (iv) FLT3-ITD, (v) and CN-LOH covering RUNX1 (Figure). (4) By contrast, TP53mut do not seem to play a role in progression of RUNX1 mutated MDS to AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Retrospective Study of B Lymphoblastic Leukemia to Assess the Prevalence of TEL/AML1 in South India: A Study of 214 Cases and Review of Literature

Abstract Introduction Translocation t(12;21)(p13;q22), a recurrent and an invisible chromosomal abnormality, resulting in TEL/AML1 gene fusion, associated with good prognosis, has been described to be a common abnormality, in children with B-acute lymphoblastic leukemia (B-ALL). Objectives The initial observation of very few TEL/AML1 positive patients at this center on testing by fluorescence in situ hybridization (FISH) led to study the prevalence of the abnormality, compare with the global distribution, and evaluate clinical, pathological, molecular, and cytogenetic features in TEL/AML1 positive patients. Materials and Methods A retrospective study of all B-ALL patients tested for TEL/AML1 gene fusion during the period January 2009 to November 2020 was undertaken. Clinicopathological, molecular, cytogenetic, treatment, and follow-up details were collected. All publications dealing with TEL/AML1 gene rearrangement were reviewed post Google and PubMed search. Results TEL/AML1gene rearrangement was assessed by FISH in 178 patients and by reverse transcription polymerase chain reaction in 36 patients and detected as the sole abnormality in 8.4% patients with additional genetic abnormalities noted on FISH evaluation. Normal karyotype was noted in 14/18 (77.7%) of these patients and 2 had complex karyotype. Complete blood count revealed hemoglobin to range from 35 to 116 g/L (median: 74 g/L), white blood count: 1.01–110×109/L (median: 7.8×109/L), platelet counts: 10–115×109/L (median: 42×109/L), blast count in peripheral smear: 0–98% (median: 41%). Immunophenotyping demonstrated 94.4% were CD34 positive, common acute lymphoblastic leukemia associated antigen (CALLA) positive with aberrant expression of CD13, CD33, CD56, singly or in combination in 58.8%. Conclusion TEL/AML1 fusion is rare in Indian patients with B-ALL and appears to be much rarer in our region. The detection of relevant specific abnormalities is of fundamental importance in B-ALL patients and these geographic variations can be used in defining management policies.

Clinicopathologic and survival correlates of embryonal rhabdomyosarcoma driven by RAS/RAF mutations.

Embryonal rhabdomyosarcoma (ERMS) is the most common subtype of rhabdomyosarcoma (RMS). Among RMS subtypes, ERMS is associated with a favorable outcome with an overall survival of 70% at 5 years for localized disease. The molecular profile of ERMS is heterogeneous, including mostly point mutations in various genes. Therapeutic strategies have remained relatively consistent irrespective of the molecular abnormalities. In this study, we focus on a homogeneous RAS/RAF mutated ERMS subset and correlate with clinicopathologic findings. Twenty-six cases (16 males and 10 females) were identified from screening 98 ERMS, either by targeted DNA sequencing (MSK-IMPACT) or by Sanger sequencing. Fourteen (54%) cases had NRAS mutations, 6 (23%) had KRAS mutations, 5 (19%) had HRAS mutations, and 1 case (4%) had BRAF mutation. Median age at diagnosis was 8 years (range 1-70) with two-thirds occurring in the children. Tumor sites varied with H&N and GU sites accounting for 62% of cases. RAS isoform hot spot mutations predominated: NRAS p.Q61K (57%), KRAS p.G12D (67%), and HRAS (codons 12, 14, and 61). Additional genetic abnormalities were identified in 85% of the RAS-mutated cases. At last follow-up, 29% of patients died of disease and 23% were alive with disease. The 3-year and 5-year survival rates were 75% and 61% respectively. In conclusion, RAS mutations occur in 27% of ERMS, with NRAS mutations encompassing half of the cases. Overall RAS-mutant RMS does not correlate with age or site, but most tumors show an undifferentiated and spindle cell morphology.

Entospletinib (ENTO) and Decitabine (DEC) Combination Therapy in Older Newly Diagnosed (ND) Acute Myeloid Leukemia (AML) Patients with Mutant TP53 or Complex Karyotype Is Associated with Poor Response and Survival: A Phase 2 Sub-Study of the Beat AML Master Trial

Entospletinib (ENTO) and Decitabine (DEC) Combination Therapy in Older Newly Diagnosed (ND) Acute Myeloid Leukemia (AML) Patients with Mutant TP53 or Complex Karyotype Is Associated with Poor Response and Survival: A Phase 2 Sub-Study of the Beat AML Master Trial

Immune Senescence-Related Gene Expression Profile in CD4+ T-Lymphocytes of HTLV-1 Asymptomatic Carriers and Patients with Adult T-Cell Leukemia/Lymphoma (ATLL): A Brazilian Preliminary Study

Introduction: Adult T-cell leukemia/lymphoma (ATLL) is a rare and aggressive neoplasm caused by the human T-lymphotropic virus type 1 (HTLV-1). It is estimated, worldwide, that at least 5-10 million people carry HTLV-1 and 2-5% out of them will develop ATLL. Previously, our group demonstrated an increase of cells in G0/G1 phase of cell cycle and aneuploidy in CD4+ T-cells in HTLV-1 asymptomatic carriers [1]. These findings may reflect an adverse intracellular environment, caused by genetic stress due to viral particles inserted into host DNA. Intracellular mechanisms aiming to control and prevent replication of cells carrying genetic aberrations in genes involved in cell cycle regulation, DNA repair and apoptosis could be activated to hold cell division while DNA damage is repaired. Based on this hypothesis, delay of cell cycle in G0/G1 may be a step in the process of oncogenesis [1]. Herein, we did a pilot study in order to analyze the pattern of expression of a set of genes involved in cell cycle control and senescence in CD4+ T-lymphocytes of HTLV-1 infected individuals searching for additional genetic abnormalities in this setting.Methods: Peripheral blood samples were tested obtained from five HTLV-1 asymptomatic carriers and four ATLL patients for this pilot study. T-CD4+ cells were isolated in magnetic column followed by RNA extraction. Subsequently, it was converted into cDNA for the assays of the real-time quantitative polymerase chain reaction (qRT-PCR) in microplates of 96 wells previously customized with 44 senescence related genes pursued from ThermoFischer Scientific. A pool of five samples from healthy individuals (control group) was used as a calibrator. RNA transcription was measured using 7500 Fast real time PCR system (Applied Biosystems) and data were collected by the 7500 software v2.0.5 (Applied Biosystems). The expression levels of the target genes were calculated using the Livak and Schmittgen (2001) method [2]. The mRNA expression was normalized using the β-glucuronidase (GUSB) gene (Applied Biosystems, cod. Hs99999908_m1) and those genes with expression ≥ 2x or ≤ 2x in comparison to control group were considered as differentially expressed and were chosen to be validated in a secondary cohort of thirty HTLV-1 infected individuals.Results: In HTLV-1 asymptomatic carriers the median age was 55 years (37-62 years) and 20% (1/5) of them were males, in ATLL patients: 45 years (38-66 years) and 100% (4/4) were females, while the control group had median age of 43 years (22-62 years) and 60% (3/5) were males. Among the ATLL group, 50% (2/4) were classified as smoldering, 25% (1/4) were acute and 25% (1/4) were lymphomatous, according to Shimoyama classification [3]. COL3A1, SPARC and TWIST1 genes were overexpressed and CDKN1A, ID1, IFNG and TERT were suppressed in HTLV-1 asymptomatic carriers when compared to healthy controls. HTLV-1 infected and ATLL patients showed TWIST1 and COL3A1 genes overexpressed and IFNG, CDKN1A and TERT repressed (Figure 1 and 2). The ALDH1, FN1, NOX4 and COL1A1 genes did not amplified.Conclusion: These preliminaries results showed for the first time that HTLV-1 asymptomatic carriers present a set of genes differentially expressed in T-CD4+ cells, especially SPARC, TWIST1, COL3A1, CDNK1A and IFNG. These results will be confirmed in a validation cohort. We expected that this data shed some light on the comprehension of the cell microenvironment of the T-CD4+ lymphocyte in HTLV-1 asymptomatic carriers. Furthermore, we will be able to understand potential mechanisms associated with leukemogenesis in chronic infection by this virus, explaining eventual progression to ATLL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impact of Concomitant Genetic Abnormalities on Sorafenib Therapy in FLT3-ITD Positive Acute Myeloid Leukemia Undergoing Allo-HSCT

AimIn this study, we investigated the co-occurring mutation landscape in acute myeloid leukemia (AML) patients with FLT3 internal tandem duplication (FLT3-ITD), and explored whether post-allogenic hematopoietic stem cell transplantation (allo-HSCT) sorafenib maintenance therapy could improve the outcomes of FLT3-ITD AML patients combined with other co-occurring genetic abnormalities.MethodsA total of 456 FLT3-ITD AML patients receiving first allo-HSCT were included in this study. Gene mutations were detected using direct sequencing or next generation sequencing. Fusion genes were detected using a 53-gene polymerase chain reaction panel. The frequency of each genetic abnormality was investigated, and the mutation landscape was investigated. The primary outcome of this study was 3-year cumulative incidence of relapse. The secondary outcomes were 3-year overall survival (OS), disease-free survival (DFS) and non-relapse mortality (NRM). The outcomes were compared between patients who received post-HSCT sorafenib maintenance and those who did not in the whole population and in subgroups referring to co-occurring mutations.ResultsA total of 190 patients received post transplantation sorafenib maintenance therapy (sorafenib group) and 266 patients did not (control group). Of the patients receiving sorafenib pre-transplantation, the median duration of sorafenib therapy was 106 days (IQR 68 - 132) in the sorafenib group, and 99 days (IQR 71 - 142) in the control group (p = 0.883). Of the patients receiving post-transplantation sorafenib maintenance therapy, sorafenib was initiated at a median of 30 days (IQR, 30 - 52 days) after allo-HSCT, and continued for a median of 148 days (IQR, 112 - 150).Median follow-up time was 38.7 months (IQR, 28.1 - 47.9). Thirty-four patients in the sorafenib group and 93 patients in the control group relapsed. The 3-year cumulative incidence of relapse was 18.0% (95% CI 13.1% - 24.3%) in the sorafenib group and 36.1% (95% CI 30.5% - 42.3%) in the control group (HR 0.43, 95% CI 0.29 - 0.64; p < 0.001). A total of 126 patients died, including 39 in the sorafenib group and 87 in the control group. Three-year DFS was 75.8% in the sorafenib group and 57.5% in the control group (HR 0.47, 95% CI 0.34 - 0.66; p < 0.001). Three-year OS was 79.5% for patients in the sorafenib group and 68.4% for patients in the control group (HR 0.57, 95% CI 0.39 - 0.83, p = 0.004).Gene mutations were detected using a 12-mutation panel direct sequencing including FLT3-ITD, FLT3-TKD, NPM1, KIT, DNMT3A, CEBPA, ASXL1, TP53, TET2, IDH1, IDH2 and RUNX1 in all patients, and a 127-gene panel new generation sequencing in 188 patients.Except FLT3, a total of 920 co-occurring gene mutations and 147 cytogenetic abnormalties were detected. The co-occurring mutations that presented in at least 10% the cases were NPM1 (31.6%), DNMT3A (15.4%), TET2 (11.4%), and CEBPA (10.5%). There were 10.5% patients presented with both NPM1 and DNMT3A mutations (Triple-mutated AML patients), and 13.8% patients combined with at least one additional genetic abnormality classified as adverse according to the 2017 ELN risk stratification other than FLT3-ITD. Cytogenetic abnormalities presented in more than 1% the patients were RUNX1-RUNX1T1, which occurred in 23 patients (5.0%), followed by +8 (2.9%), complex karyotype (2.6%), CBFB-MYH11 (2.4%), DEK-NUP214 (1.3%) and -y (1.3%).Patients combined with NPM1(p = 0.009 and 0.009), DNMT3A (p = 0.036 and 0.086), triple-mutated AML patients (p = 0.030 and 0.027), and patients with at least one additional adverse abnormality (p = 0.014 and 0.005) benefit significantly in DFS and OS from post-transplantation sorafenib maintenance, but not those with CEBPA (p = 0.669 and 0.576) or TET2 (p = 0.375 and 0.178) mutations.ConclusionPost transplantation sorafenib maintenance therapy can improve the prognosis of FLT3-ITD AML patients combined with DNMT3A or NPM1, patients with triple-mutated AML, and patients combined with at least one additional adverse abnormalities, but not of those combined with CEBPA or TET2 mutations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clonally-Related Composite Classic Hodgkin Lymphoma and Follicular Lymphoma

Abstract Introduction/Objective Composite classic Hodgkin lymphoma and follicular lymphoma (CHLFL), defined as CHL and FL occurring simultaneously at the same site, is rare and poorly understood. While both Hodgkin/Reed-Sternberg (HRS) cells and FL are thought to be derived from germinal center B-cells, the relationship between CHL and FL when coexistent is unclear. Here, we present two cases of CHLFL and show that the CHL and FL components have a clonal relationship by FISH. Methods/Case Report Case #1 is a 50-year-old man with abdominal and mediastinal lymphadenopathy. An excised mesenteric lymph node showed two distinct components diagnostic for FL, grade 1-2 and CHL. Case #2 is a 63-year- old woman with a history of FL with transformation to diffuse large B-cell lymphoma. Cytogenetic studies showed a complex karyotype with an add(9p), del(10q), and trisomy 16. Post-treatment imaging revealed left axillary adenopathy. An excised axillary lymph node showed CHL with peripheral areas of FL, grade 3A. Both cases had areas of typical FL with BCL2-positive phenotype and no significant CD30/CD15 expression. HRS cells were CD45/CD20-negative, expressed CD30 (strong), CD15, and PAX5, and were present in a mixed inflammatory background. No EBV RNA was present by in situ hybridization. Interestingly, HRS cells in case #1 expressed both BCL6 and BCL2. FISH was performed in both cases. Case #1 had a BCL2 rearrangement in 48% of FL nuclei and in 100% of HRS cells. In case #2, targeted probes were used based on prior cytogenetic results. Here, 47% of FL nuclei and 44% of HRS cells had a 16p duplication; additionally, 32% of HRS cells had an unbalanced IGH rearrangement with loss of the IGH variable region, suggesting possible clonal evolution. No rearrangement of BCL2 or BCL6 was present. An additional 27 CHLFL cases from the literature were reviewed. CHLFL was mostly nodal and occurred in late adulthood in patients with or without a history of FL. It presented at advanced clinical stage, with a 5-year overall survival of 22%. BCL2 expression in HRS cells was common. Bone marrow involvement was 45% (5/11) and consisted of FL exclusively. Five of six tested cases demonstrated BCL2/IGH rearrangement in both FL and HRS cells. Results (if a Case Study enter NA) NA Conclusion Composite CHL and FL are often clonally related and may share a common progenitor B-cell origin – likely a germinal center B-cell – from which additional genetic abnormalities are acquired to develop two distinct lymphomas.

IMMUNE SENESCENCE-RELATED GENE EXPRESSION PROFILE IN CD4+ T-LYMPHOCYTES OF HTLV-1 ASYMPTOMATIC CARRIERS AND PATIENTS WITH ADULT T-CELL LEUKEMIA/LYMPHOMA (ATLL): A BRAZILIAN PRELIMINARY STUDY

ATLL is a rare and aggressive neoplasm caused by the human T-lymphotropic virus type 1 (HTLV-1). It is estimated that at least 5-10 million people carry HTLV-1 and 2-5% out of them will develop ATLL. Our group demonstrated an increase of cells in G0/G1 phase of cell cycle and aneuploidy in CD4+ T-cells in HTLV-1 asymptomatic carriers. These findings may reflect an adverse intracellular environment, caused by genetic stress due to viral particles inserted into host DNA. Intracellular mechanisms aiming to control and prevent replication of cells carrying genetic aberrations in genes involved in cell cycle regulation, DNA repair and apoptosis could be activated to hold cell division while DNA damage is repaired. Based on this hypothesis, delay of cell cycle in G0/G1 may be a step in the process of oncogenesis. Herein, we did a pilot study in order to analyze the pattern of expression of a set of genes involved in cell cycle control and senescence in CD4+ T-lymphocytes of HTLV-1 infected individuals searching for additional genetic abnormalities in this setting. Peripheral blood samples were tested obtained from five HTLV-1 asymptomatic carriers and four ATLL patients. T-CD4+ cells were isolated in magnetic column followed by RNA extraction. Subsequently, it was converted into cDNA for the assays of the qRT-PCR in microplates of 96 wells customized with 44 senescence related genes. A pool of five samples from healthy individuals (control group) was used as a calibrator. RNA transcription was measured using 7500 Fast real time PCR system ( Applied Biosystems ) and data were collected by the 7500 software v2.0.5 ( Applied Biosystems ). The expression levels of the target genes were calculated using the Livak and Schmittgen (2001) method. The mRNA expression was normalized using the β-glucuronidase ( GUSB ) gene ( Applied Biosystems , cod. Hs99999908_m1) and those genes with expression ≥ 2x or ≤ 2x in comparison to control group were considered as differentially expressed and were chosen to be validated in a secondary cohort of thirty HTLV-1 infected individuals. In HTLV-1 asymptomatic carriers the median age was 55 years (37-62 years) and 20% (1/5) of them were males, in ATLL patients: 45 years (38-66 years) and 100% (4/4) were females, while the control group had median age of 43 years (22-62 years) and 60% (3/5) were males. Among the ATLL group, 50% (2/4) were classified as smoldering, 25% (1/4) were acute and 25% (1/4) were lymphomatous, according to Shimoyama classification. COL3A1, SPARC and TWIST1 genes were overexpressed and CDKN1A, ID1, IFNG and TERT were suppressed in HTLV-1 asymptomatic carriers when compared to healthy controls. HTLV-1 infected and ATLL patients showed TWIST1 and COL3A1 genes overexpressed and IFNG, CDKN1A and TERT repressed. The ALDH1, FN1, NOX4 and COL1A1 genes did not amplified. These preliminaries results showed for the first time that HTLV-1 asymptomatic carriers present a set of genes differentially expressed in T-CD4+ cells, especially SPARC, TWIST1, COL3A1, CDNK1A and IFNG . These results will be confirmed in a validation cohort. We expected that this data shed some light on the comprehension of the cell microenvironment of the T-CD4+ lymphocyte in HTLV-1 asymptomatic carriers. Furthermore, we will be able to understand potential mechanisms associated with leukemogenesis in chronic infection by this virus, explaining eventual progression to ATLL.

New Generation Sequencing of Targeted Genes in Hairy Cell Leukemia Highlighted Mutations Associated to BRAFV600E anda New Recurrent Altered Gene, KDM6A, in Hairy Cell Leukemia Variant Patients

Introduction: BRAFV600E detected in more than 80% of Hairy cell leukemia (HCL) cases, was described as a driver mutation but additional genetic abnormalities seem necessary for the progression of the disease. Within HCL harboring BRAFWT, the differential diagnosis with the variant form of HCL (HCL-v) is complex.Sequencing of targeted genes by next generation sequencing (NGS) has proved its interest for classification and prognosis of lymphoid neoplasms and opens the lead to new perspectives for personalized medicine.Methods : We selected a panel of 21 relevant genes (defined as Trichopanel) based on literature review of whole exome sequencing studies. At diagnosis, we analyzed 20 HCL and 4 HCL-v samples and at relapse 2 HCL and 3 HCL-v. HCL diagnosis was based on hairy cell morphology with the co-expression of CD103, CD123, CD25 and CD11c, and HCL-v on hairy cell morphology with a prominent nucleolus and the lack of CD25 and CD123 expression. DNA was extracted from peripheral blood mononuclear cells isolated from blood (17 samples), bone marrow aspirates (10 samples) or biopsies (spleen: 1 and cutaneous nodule: 1 case). NGS was performed on the Ion Torrent PGMTM device according to the manufacturer' recommendations. The Trichopanel covered 71,020 bases using 712 amplicons. Analyzed genes belong to different gene ontology families: MAPK pathway (BRAF, MAP2K1, DUSP2, MAPK15), epigenetic regulation (ARID1A, ARID1B, EZH2, KDM6A, CREBBP), cell cycle/apoptosis (TP53, CDKN1B, XPO1), homing (KLF2, CXCR4), NOTCH pathway (NOTH1, NOTCH2), NF-kB pathway (MYD88), inflammation (ANXA1), splicing (U2AF1), differentiation (BCOR) and extracellular transport (ABCA8). Data analysis was performed with the Torrent suiteTM software, then variant analysis was performed using an in-house bioinformatics pipeline detailed as previously described1. The ratio of variant allele frequency (rVAF) was pondered according to the % of tumor cells estimated by Flow Cytometry.Results : The Trichopanel was informative for 96% (23/24) of patients and revealed single nucleotide variants (SNVs) in BRAF (18 pts), KLF2 (4 pts), MAP2K1 (3 pts), KDM6A (2 pts), CDKN1B (2 pts), ARID1A (2 pts), CREBBP (2 pts) NOTCH1 (1 pts) and ARID1B (1 pts). BRAFV600E was found in 90% (18/20) of HCL patients and was not detected in HCL-v patients. In one BRAFWT HCL patient, we found a mutation on MAP2K1. In the last case, we found no SNV in the genes sequenced by the Trichopanel. In HCL BRAFV600E patients, other mutations were found in 39% (7/18) of cases: KLF2 (3 pts), CDKN1B (2 pts), NOTCH1 (1 pt), ARID1B (1 pt) and CREBBP (1 pt). All 4 HCL-v patients had SNVs in epigenetic regulation genes e.g.: KDM6A (2 pts), CREBBP (1 pt) or ARID1A (1 pt). KDM6A SNVs were either frameshift deletions or splicing variations with a loss of exon (confirmed by RNA sequencing). We also analyzed at diagnosis and relapse serial samples from 5 patients (2 HCL and 3 HCL-v). In one HCL, patient we observed at diagnosis and at first relapse the same mutational pattern and in the second case, we observed the presence of 2 news sub-clonal mutations (BCORE1430X and XPO1E571K). In the 3 HCL-v patients (2 treated and 1 untreated), no new mutation was observed but variations of the rVAF were identified in 2 cases.Discussion : In addition to BRAFV600E mutations, one third of HCL patients had mutations with a rVAF close to BRAFV600E rVAF. KLF2, which is mutated in 3 patients, is known to play a key role for B-cell homing in lymph nodes and inhibition of NF-kB pathway. KLF2275N, KLF2275T, KFL2T271I variants found in those patients have been previously described in splenic zone marginal lymphoma (SMZL) as inhibitory mutations, possibly suggesting a genetic similarity between both diseases2. We also describe new mutations targeting KDM6A, a lysine demethylase protein, in HCL-v disease. Stop-gain KDM6A mutations have been already described in 2 HCL patients with atypical clinical features3,4. Those mutations and the 2 mutations found in our series leads to the loss of the highly-conserved C-terminal region (included Jumomji and Zinc binding domains) which is essential for its demethylase activity. Loss of KDM6A activity may sensibilise tumor cells to demethylating agents such as 5-azacytidine or EZH2 inhibitors5.Conclusion : the Trichopanel is a useful tool for the diagnosis, relapse and prognosis of HCL and HCL-v. [Display omitted] DisclosuresTroussard:ABBVIE: Honoraria; JANSSEN: Honoraria; GILEAD: Honoraria; ROCHE: Honoraria. Jardin:Roche: Honoraria; Janssen: Honoraria; celgen: Honoraria.