t(X;5)(q13;q33) is a very rare but recurrent translocation observed in hematological malignancies. Only two myeloid cases have been reported in the literature to date. In one case, it was demonstrated that the t(X;5) involves the inactive X chromosome and is associated with silencing of autosomal genes located on 5q. Here, we report four additional cases. Overall, t(X;5) is frequently associated with chronic myelomonocytic leukemia (CMML) (4/6 cases, 67%) and may have functional consequences similar to those of del(5q). In contrast to del(5q) in CMML, which is often associated with a complex karyotype, t(X;5) is most often observed as an isolated abnormality and may define a distinct subgroup with favorable genetic features. Additional cases and functional studies are needed to confirm our observations.

Extramedullary acute myeloid leukemia (eAML) is a rare form of myeloid neoplasm characterized by leukemic infiltration outside the bone marrow (BM). Despite its prognostic significance, eAML is often underdiagnosed and poorly characterized at molecular level. We performed a comprehensive genomic and immunogenomic profiling on paired BM and extramedullary specimens from 26 eAML patients, alongside over 400 AML cases without extramedullary involvement and 97 healthy controls. Clonal branching from BM was observed in 38.5% of extramedullary sites, frequently involving actionable mutations in FLT3 , IDH2 and NPM1 genes. Both compartments were enriched in RAS pathway mutations and class II HLA losses, suggesting active immunoediting mechanisms driving eAML development. Strikingly all relapsed cases acquired FLT3 aberrations, highlighting therapeutic opportunities. These findings underpin the need for improved detection and routine genomic profiling, including targeted sequencing of suspected extramedullary lesions. ### Competing Interest Statement The authors have declared no competing interest. Leukemia Research Foundation, https://ror.org/036wcgd38, Specialized Program of Research Excellence in Acute Myeloid Leukemia, ,

t(X;20)(q13;q13) is a very rare but recurrent translocation observed in myeloid neoplasms such as myelodysplastic neoplasm (MDS), myeloproliferative neoplasm (MPN), and acute myeloid leukemia (AML). While only nine cases have been reported previously,1-5 we have collected data (including gene sequencing data) on a large series of 25 cases of myeloid neoplasms with t(X;20). By retrospectively screening databases at 13 French cytogenetic laboratories, we identified 25 cases of t(X;20)(q13;q13) (Figure 1A,B; Table S1). The study was approved by the local institutional review board (AP-HP (Paris, France); reference: BPD2018DIA008) and complied with the tenets of the Declaration of Helsinki. All the patients provided their written, informed consent to participation. Bone marrow smears (available for 16 of the 25 patients) were blind-reviewed by two independent expert cytologists (J-FL and CS). To obtain R- or G-banded chromosomes, cells were prepared from bone marrow using standard techniques. All the karyotypes were reviewed by the members of the Groupe Francophone de Cytogénétique Hématologique and classified according to the International System for Human Cytogenetic Nomenclature (ISCN 2020). A complex karyotype was defined as the presence of three or more clonal chromosomal abnormalities (CAs). Standard fluorescence in situ hybridization was performed on metaphase and interphase nuclei, using the commercial probes XIST (Xq13.2)/CCPX (CytoTest, Rockville, MD, USA), 20q12 (PTPRT)/20q13 (MYBL2) (Cytocell, Cambridge, UK), WCP20 and WCPX (Metasystems, Altlußheim, Germany). Fourteen patient samples were sequenced with a next-generation sequencing myeloid panel (Supplemental Information). For one center (Pitié-Salpêtrière, between 2015 and 2022), we were able to calculate the exact prevalence of t(X;20) in MDS (0.4%; 2 out of 421) and in MDS or unexplained cytopenia (0.1%; 2 out of 1283). All 25 patients were female. The median age was 78 (range: 60–91). Five of the 23 patients with available clinical data had received chemotherapy for follicular lymphoma (case #1), multiple myeloma (case #16), breast cancer (case #19), ovarian cancer (case #14) or AML (case #24) before the t(X;20) was detected. The initial diagnoses were MDS (n = 12, including ring sideroblasts in cases #2, #8, and #18), cytopenia (n = 6), AML (n = 3), and MPN with eosinophilia (n = 1). Three myelograms were hemodiluted so a morphologic diagnosis could not be made. The morphologic review of 16 samples confirmed the diagnoses for 11 cases of MDS and one case of AML and led to the reclassification of two out of four cases of cytopenia as unclassifiable MDS (MDS-U, cases #6 and #21) (Figure S1). The two reviewers observed an abnormal chromatin clumping in 6/16 (37%) cases, but no specific feature of the t(X;20). Finally, the four remaining cases of cytopenia were considered to be clonal cytopenia of undetermined significance (CCUS). In the 14 cases of MDS and 4 cases of CCUS, the karyotype was never complex; the t(X;20) was the only CA in 12 of the 18 cases (67%). In the other 6 cases, the additional CAs were del(5q) (3 out of 18, 17%; none in CCUS), del(20q) (2 out of 18, 11%; none in CCUS), and trisomy (tri) 8 (1 out of 18, 5%). The t(X;20) was subclonal in only one of the 18 cases (5%). In cases presenting with both t(X;20) and del(20q), the two CAs were always in independent clones. All the cases of AML had complex karyotypes with 3 to 7 CAs. The t(X;20) was present in the major clone in all three patients (Tables S1 and S2). In the cohort as a whole, the most common associated CA was del(5q) (6 out of 25, 24%)—which was variously clonal, subclonal, or present in independent clones—and followed by del(20q) (3 out of 25, 12%) or tri8 (2/25, 8%). It is noteworthy that in one patient (case #24), tri8 was present in 24 of the 25 (96%) mitoses observed at the time of AML diagnosis. Four months later (during the first-line treatment), tri8 was still observed in three mitoses independently of t(X;20); this might correspond to residual AML blasts. At relapse 2 years later, tri8 reappeared (additionally with tetrasomy 8), whereas t(X;20) was no longer observed (Figure S2). In all 14 tested cases, the breakpoint on the X chromosome was proximal to XIST (Xq13.2), and the breakpoint on chromosome 20 was distal to MYBL2 on 20q13 (Figure 1C,D). Molecular data revealed mutations in TET2 (n = 7 out of 14, 50%), SF3B1 (n = 3, 21%), DNMT3A (n = 3, 21%), RUNX1 (n = 2, 14%), and U2AF1 (n = 2, 14%) (Table S1). Of note, the 3 patients with SF3B1mut also had a del(5q); and the only CCUS case tested by NGS had mutations in TET2 and DNMT3A. No TP53 mutations were observed. We also detected mutations in SRSF2, BCOR, BCORL1, IDH1, EP300, NRAS, and ETV6 (n = 1 each). Regarding MDS and CCUS, the cytogenetic category for the Revised International Prognostic Scoring System (IPSS-R) score was intermediate in all but the three cases of MDS with t(X;20) and del(5q) (with a favorable score).6 The median MDS IPSS-R score was 2.25 (range, 1–4). Of the 18 scores, 15 (83%) were classified as low risk, one was classified as very low risk, and two were classified as intermediate risk (Table S1 and Figure 1E). With a median follow-up time of 17 months (95% CI: 10–37), the median overall survival was not reached, and 69% of the patients were alive at 5 years. To the best of our knowledge, the present series of t(X;20) cases is the largest yet. As previously described, all patients were female, and the median age was 78. The majority (64%) of t(X;20) cases corresponded to MDS. The morphological evidence for MDS was not always easy to interpret: two of the four morphologically reviewed cases of cytopenia were reclassified as MDS. However, four cases of cytopenia had no morphological evidence of MDS. According to the fourth edition of the World Health Organization's classification of hematolymphoid tumors, t(X;20)(q13;q13) is not one of the CAs that defines MDS-U. In the fifth edition, patients with isolated t(X;20)(q13;q13) are classified as having CCUS. In our series of MDS/CCUS cases, t(X;20) was frequently isolated (in 67% of cases) and was associated with a low-risk MDS IPSS-R in 83% of cases. Overall, t(X;20) was associated with cases of MDS/CCUS (82%) with a favorable prognosis. In a few cases, t(X;20) was observed in AML but only included in a complex karyotype. Our series included a case of MPN with hypereosinophilia. t(X;20) was frequently isolated or present in the major clone, and may be an early event. The most frequent associated CA was del(5q) (24%). Interestingly, when associated with del(20q) (12%), t(X;20) was always present in an independent clone. This is in line with the possible silencing of 20q genes by a position effect of the XIST gene on der(20).2 The spreading of X inactivation to the 20q region provides an alternate mechanism for del(20q).4 Indeed, in all the tested cases, XIST gene was translocated on the der(20), and all 20q genes stayed on the long arm of der(20). In conclusion, t(X;20)(q13;q13) is a rare translocation observed in older women. It may be an early event. t(X;20)(q13;q13) is often isolated and is mostly associated with MDS/CCUS with a favorable prognosis. It is found less frequently in cases of AML and MPN. The most commonly associated CAs are del(5q) and del(20q), and the latter is always in an independent clone. We therefore suggest that isolated t(X;20) should be classified in a good cytogenetic IPSS-R category (score = 1), rather than in an intermediate cytogenetic category (score = 2). F.N-K. and M.M. designed the study. N.A., E.C., M-A.C-R., A.D., V.E., B.G., A.I., C.L., M-J.M., D.P., J.Q., C.T., and L.V. provided samples and clinical data. J-F.L and C.S performed the morphological review. L.S. performed additional FISH analysis. P.H. performed NGS sequencing. E.C. performed statistical analysis. F.N-K., M.M., D.G., and M-B.T. analyzed data. F.N-K. wrote the manuscript. M.M. and M-B.T. reviewed the manuscript. All co-authors approved the manuscript. The authors declare no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Data S1. Supporting information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

BackgroundTatton-Brown-Rahman syndrome (TBRS; OMIM 615879), also known as DNA methyltransferase 3 alpha (DNMT3A)-overgrowth syndrome (DOS), was first described by Tatton-Brown in 2014. This syndrome is characterised by overgrowth, intellectual disability and distinctive facial features and is the consequence of germline loss-of-function variants inDNMT3A, which encodes a DNA methyltransferase involved in epigenetic regulation. Somatic variants ofDNMT3Aare frequently observed in haematological malignancies, including acute myeloid leukaemia (AML). To date, 100 individuals with TBRS with de novo germline variants have been described. We aimed to further characterise this disorder clinically and at the molecular level in a nationwide series of 24 French patients and to investigate the correlation between the severity of intellectual disability and the type of variant.MethodsWe collected genetic and medical information from 24 individuals with TBRS using a questionnaire released through the French National AnDDI-Rares Network.ResultsHere, we describe the first nationwide French cohort of 24 individuals with germline likely pathogenic/pathogenic variants inDNMT3A, including 17 novel variants. We confirmed that the main phenotypic features were intellectual disability (100% of individuals), distinctive facial features (96%) and overgrowth (87%). We highlighted novel clinical features, such as hypertrichosis, and further described the neurological features and EEG results.ConclusionThis study of a nationwide cohort of individuals with TBRS confirms previously published data and provides additional information and clarifies clinical features to facilitate diagnosis and improve care. This study adds value to the growing body of knowledge on TBRS and broadens its clinical and molecular spectrum.

TP53 aberrations are a major predictive factor of resistance to chemoimmunotherapy in chronic lymphocytic leukemia (CLL), and an assessment of them before each line of treatment is required for theranostic stratification. Acquisition of subclonal TP53 abnormalities underlies the evolution of CLL. To better characterize the distribution, combination, and impact of TP53 variants in CLL, 1,056 TP53 variants collected from 683 patients included in a multicenter collaborative study in France were analyzed and compared to UMD_CLL, a dataset built from published articles collectively providing 5,173 TP53 variants detected in 3,808 patients. Our analysis confirmed the presence of several CLL-specific hotspot mutations, including a two-base pair deletion in codon 209 and a missense variant at codon 234, the latter being associated with alkylating treatment. Our analysis also identified a novel CLL-specific variant in the splice acceptor signal of intron 6 leading to the use of a cryptic splice site, similarly utilized by TP53 to generate p53psi, a naturally truncated p53 isoform localized in the mitochondria. Examination of both UMD_CLL and several recently released large-scale genomic analyses of CLL patients confirmed that this splice variant is highly enriched in this disease when compared to other cancer types. Using a TP53-specific single-nucleotide polymorphism, we also confirmed that copy-neutral loss of heterozygosity is frequent in CLL. This event can lead to misinterpretation of TP53 status. Unlike other cancers, CLL displayed a high proportion of patients harboring multiple TP53 variants. Using both in silico analysis and single molecule smart sequencing, we demonstrated the coexistence of distinct subclones harboring mutations on distinct alleles. In summary, our study provides a detailed TP53 mutational architecture in CLL and gives insights into how treatments may shape the genetic landscape of CLL patients.

TP53 aberrations, including somatic mutations of TP53 gene and 17p deletion, are a major predictive factor of resistance to fludarabine-based chemotherapy in chronic lymphocytic leukemia (CLL) and remain an adverse prognostic factor in the chemofree era. The detection of deletion 17p and TP53 gene mutations has become an integral part of routine diagnostic and should be performed before any administration of treatment.1 TP53 mutations typically occur all along the DNA-binding domain of the p53 protein. However, the functional role or the effect of each TP53 variant remains elusive. In this study, we aimed at characterizing the profile of the TP53 variants in CLL, their distributions, and a correlation with clinical data. To this end, we retrospectively analyzed a large collection of 568 CLL-associated TP53 variants in 336 patients compiled from centers affiliated with the French Innovative Leukemia Organization-CLL (FILO). Fluorescence in situ hybridization analysis for del(17p) status was available for 207 patients, of which 108 (52%) harbored a 17p deletion. Based on the IGHV mutation status, most patients belong to a high risk group as 73% (172/236) were IGHV unmutated (Table S1). TP53 mutational status was assessed from blood samples using NGS technologies (either Illumina or Ion Torrent technologies) covering exons 2–11, with a sensitivity allowing the detection of subclones with variant allele frequency (VAF) >1%. Both amplicon- and capture-based approaches available in commercial kits or custom gene panels including the TP53 gene were used. Polymorphisms were carefully excluded using the gnomAD database and the new TP53 SNP data included in the most recent version of the UMD_TP53 database.2 Classification of TP53 variant pathogenicity was performed in accordance with ACMG criteria and based on population data and TP53-specific functional information previously described.3 Among the 568 TP53 somatic mutations, the majority were missense variants (n = 429, 75%) (Table S2). Using ACMG criteria from the UMD_TP53 database, 518 variants were classified as pathogenic, 42 were likely pathogenic, and 8 were variants of unknown significance. As expected, TP53 variants were predominantly located in the DNA-binding domain, with classical CpG-related hot spots at codons 175, 248, and 273 (Figure 1A). Nevertheless, an unusual mutation hotspot occurring at codon 234 was identified. A total of 25 single nucleotide variants at codon 234 was observed in 24 patients with one patient harboring 2 mutations: 22 mutations p.Tyr234Cys (c.701A>G), 2 mutations p.Tyr234Ser (c.701A>C), and 1 mutation p.Tyr234His (c.700T>C). It was the second most common missense variant of our cohort (25/568; 4.4%) and ranked fifth in the entire CLL subset of the UMD_TP53 database (Figure 1A, and Figure S1A). Mutations at codon 234 were found predominantly at a subclonal level (median of variant allele frequency VAF = 11% [1.8%–51%]) but with VAFs distributed in the same range as those observed for other hotspot codons (Figure 1B). Analysis of the UMD_TP53 database showed that the frequency of mutations occurring at hotspot positions 175, 248, and 273 was similar to that observed in other cancers, while the frequency of mutations at codon 234 reached 4% in CLL and was very low (less than 1%) in the other malignancies (Figure 1C and Figure S1B), suggesting that this mutation is highly specific of CLL cases. Both in silico predictive algorithms and structural studies suggested that variant p.Tyr234Cys was deleterious, which was confirmed by our functional analysis.4 Indeed, the overexpression of p.Tyr234Cys by transfection in the p53 null H1299 cell line was unable to induce growth arrest, similar to p.Arg175His, while wild-type p53 or p.Thr312Ser (a variant without loss of function) leads to a profound reduction of colony number (Figure S2A). On the other hand, in a luciferase assay, p.Tyr234Cys was unable to transactivate a reporter gene with the CDKN1 response element (Figure S2B). In line with these results, the large-scale analysis performed by Kotler et al. showing the relative fitness score for each TP53 variant (defining its capacity to induce growth arrest in H1299 cells) indicates that the majority of TP53 variants at position 234 are dysfunctional (Figure S2C).5 Altogether, these results indicate that TP53 variant p.Tyr234Cys is devoid of any transactivating and antiproliferative activities. In the present study, CLL patients frequently harbored multiple subclones with different TP53 mutations. A single TP53 variant was detected in 66% (n = 222) of the patients (SM-patients), whereas two, three, or more TP53 variants were found respectively in 17% (n = 57), 10% (n = 33), and 7% (n = 24) of the patients (polymutated patients) (Table S2 and Figure S3A). Interestingly, while mutations at classical TP53 hot spot positions (codons 175, 248, or 273) were observed both as single and associated mutations, we noticed that variants at codon 234 were found mostly in polymutated patients (82% cases), highlighting an important intratumoral heterogeneity in cases harboring this mutation (Figure 1D). Strikingly, analysis of the variants associated with mutations at codon 234 reveals a strong association with a splicing mutation c.673-2A (p < .0001, Fischer test) (Figure 1E). The frequency of this mutation was 2.6% (15/568) in the entire cohort and rose up to 37.5% (9 cases) in the 24 patients with mutation at codon 234. In contrast, it was associated with mutation at codon 248 or codon 273 in only one case. Consistently, the reanalysis of sequencing data from published cohorts gathering altogether 272 TP53 mutated patients showed a frequency of 3.3% of mutation at codon 234 (nine cases), among them 5 were associated with the mutation at position c.673-2A (62.5%).6-9 Mutation at position c.673–2A at the splice acceptor site in exon-7 generates a p53 truncated protein10 that functionally and molecularly resembles the naturally occurring alternative p53 splice variant, p53-psi. Shirole et al. have shown that "p53-psi like" proteins lack canonical p53 tumor suppressor activities but promote cancer cell proliferation, survival, and metastasis.11 These mutants can localize to the mitochondria where they promote tumor phenotypes by binding and activating the mitochondria inner pore permeability regulator, Cyclophilin D (CypD). However, the functional relationship between mutation at position c.673–2A>G and p.Tyr234Cys or whether one of these mutations promote the acquisition of the other remains elusive. Clinical data were available for 222 patients. TP53 sequencing was performed before treatment for 81 patients (37%). The type of treatment significantly impacted the nature of the TP53 variant and the number of associated mutations in patients. Indeed, variants at codons 175 and 248 were observed in both treated and untreated patients, whereas 100% of patients who harbored a variant at codon 234 had been treated (Figure 1F). Moreover, while most treatment-naïve patients had a single mutation (n = 72/81; 88%), nearly half of previously treated patients (n = 66/141; 47%) had more than one TP53 variant (from 2 to 11 mutations) (p < .0001), showing that the exposition to a therapy promotes the clonal evolution of TP53 (Figure S3B and C). Fifty-nine percent (53 of 89) of patients who received one line of chemoimmunotherapy (fludarabine, cyclophosphamide, and rituximab [FCR] or bendamustine and rituximab [BR]) had a single mutation, while those who received only continuous chlorambucil treatment (CLB) were mostly polymutated (32 of 44; 77%, p = .0012 with Yates correction), suggesting that CLB has a stronger capacity to drive clonal diversification (Figure 1G). Furthermore, variants at codon 234 were found exclusively in treated patient (24 of 141; 17%) and in 43% of patients treated with CLB (19 of 44). In contrast, only 5.7% of patients treated with FCR and/or BR harbored this mutation (5 of 89) (Figure 1H). Eventually, considering the association between mutation at codon 234 and c.673-2A, the nine patients displaying the two mutations were all treated with CLB, while this co-occurrence was not found in patients treated with fludarabine and/or BR. The co-occurrence of the two mutations was significantly associated with CLB treatment (p < .0001) with an odds ratio of 26 (95% CI: 10.7–69.3). Taken together, our data support the role of the alkylating agent CLB in the appearance and/or the selection of mutations at codon 234 and possibly the truncating mutation c.672-2a. No pre-CLB treatment sample was available for patients with Tyr234Cys mutations to test for TP53 mutation status. However, in contrast to the other hot spot mutations such as those at codon 175 or 248, the absence of variants at position 234 in any of the untreated patients in our database strongly supports the possibility that this mutation is associated with the mutagenic effect of CLB. Furthermore, the absence of this variant in a recent cohort of CLL patients in which none received CLB argues against the hypothesis of the specific selection of this mutation in CLL. This finding is reminiscent of the association between exposure to certain carcinogens (aflatoxin B1 in hepatocellular carcinoma and benzo(a)pyrene in lung cancer) and TP53 hot spot variants in codon 249 or 157.12 Whether or not chlorambucil adduct occurs at codon 234 or the splicing site 6.732-2A of the TP53 gene remains to be tested. This study was supported by Cancéropole Ille de France (convention n°2019-1-EMERG-22-INSERM 6-1) to Thierry Soussi and Fanny Baran-Marszak. Bernard Leroy for the management of the UMD_TP53 database. The authors declare no potential conflicts of interest. Conception and design: Florence Cymbalista, Fanny Baran-Marszak, and Thierry Soussi. Acquisition of data (acquired and managed patients, provided facilities, etc.): Grégory Lazarian, Floriane Theves, Myriam Hormi, Rémi Letestu, Virginie Eclache, Audrey Bidet, Pascale Cornillet-Lefebvre, Frédéric Davi, Eric Delabesse, Marie-Hélène Estienne, Pascaline Etancelin, Olivier Kosmider, Sophie Laibe, Laurence Lode, Marc Muller, Nathalie Nadal, Dina Naguib, Cédric Pastoret, Stephanie Poulain, Pierre Sujobert, Lauren Veronese, Samia Imache, Valérie Lefebvre, Florence Cymbalista, Fanny Baran-Marszak, and the members of the French Innovative Leukemia Organization. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Grégory Lazarian, Floriane Theves, Florence Cymbalista, Fanny Baran-Marszak, and Thierry Soussi. Writing, review, and/or revision of the manuscript: Grégory Lazarian Florence Cymbalista, Fanny Baran-Marszak, and Thierry Soussi. Study supervision: Florence Cymbalista, Fanny Baran-Marszak, and Thierry Soussi. Data S1. Supporting information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

BACKGROUND:Patients with paroxysmal nocturnal hemoglobinuria (PNH) have a clonal population of blood cells deficient in glycosylphosphatidylinositol-anchored (GPI-anchored) proteins, most of the time resulting from a mutation in the X-linked gene PIGA. We report a patient with PNH resulting from a rare biallelic PIGT mutation on chromosome 20.CASE SUMMARY:A 47-year-old man was referred to our hospital for febrile pancytopenia. The patient reported a history of recurrent urticaria and arthralgia and he presented during 3 mo recurrent acute dermo-hypodermitis and aseptic meningitidis. Based on clinical cases published with PIGT-PNH, with clinically typical PNH and autoinflammatory symptoms, we treated our patients with repeated infusions of eculizumab to decrease autoinflammatory symptoms and then we performed an allogeneic stem cell transplantation (allo-SCT) with a mismatched unrelated donor. Our patient experienced no acute Graft vs Host disease (GvHD) and a moderate chronic GvHD and is now considered cured at 24 mo after allo-SCT.CONCLUSION:This case report suggests that allo-SCT should be considered to cure PIGT-PNH patients.

Large granular lymphocytic leukemia (LGLL) is a rare clonal lymphoproliferative disorder from T or NK origin. PURPOSE to report on the diagnostic and therapeutic management of LGLL investigated in the university hospital at Nancy, France. METHODS retrospective (7 years) collection of clinical and biological data and patients' cohort analysis. RESULTS Eight out of fifteen patients presented with neutropenia, including five profound neutropenia (neutrophils < 500 × 109/L). Four patients had an infection. Two patients have rheumatoid arthritis and an associated Felty's syndrome, one a Sweet syndrome. Two also suffered from chronic Lymphocytic Leukemia, and one from a diffuse large B-cell lymphoma. Twelve patients had LGLL-T and 3 had a chronic LGLL-NK. Eleven out of twelve patients had a clonal LGLL-T when polymerase chain reaction assessed. No KIR clonality was sought among the 3 LGL-NK patients. Five patients out of fifteen received immunosuppressive treatment. CONCLUSION Although using simple and robust investigations, our series demonstrates a high heterogeneity in LGLL detection and assessment.

The STI571 prospective randomised trial (SPIRIT) French trial is a four-arm study comparing imatinib (IM) 400 mg versus IM 600 mg, IM 400 mg + cytarabine (AraC), and IM 400 mg + pegylated interferon alpha2a (PegIFN-α2a) for the front-line treatment of chronic-phase chronic myeloid leukaemia (CML). Long-term analyses included overall and progression-free survival, molecular responses to treatment, and severe adverse events. Starting in 2003, the trial included 787 evaluable patients. The median overall follow-up of the patients was 13.5 years (range 3 months to 16.7 years). Based on intention-to-treat analyses, at 15 years, overall and progression-free survival were similar across arms: 85%, 83%, 80%, and 82% and 84%, 87%, 79%, and 79% for the IM 400 mg ( N = 223), IM 600 mg ( N = 171), IM 400 mg + AraC ( N = 172), and IM 400 mg + PegIFN-α2a ( N = 221) arms, respectively. The rate of major molecular response at 12 months and deep molecular response (MR4) over time were significantly higher with the combination IM 400 mg + PegIFN-α2a than with IM 400 mg: p = 0.0001 and p = 0.0035, respectively. Progression to advanced phases and secondary malignancies were the most frequent causes of death. Toxicity was the main reason for stopping AraC or PegIFN-α2a treatment.

Obesity has been associated with an increased risk of developing acute myeloblastic leukaemia (AML). The outcome of AML patients could thus be dependent on their nutritional status that can be evaluated by the simple measurement of serum albumin (SA) and body mass index (BMI). These two parameters could have a value as prognostic factors to guide patients' management. We evaluated the association between SA levels, BMI, and survival, evaluated as overall survival (OS) and event‐free survival. Furthermore, we investigated the association between BMI, SA, and other prognostic factors of interest in AML. This retrospective single‐center study included 159 patients diagnosed with AML at Nancy Hospital between 2005 and 2013, treated with aracytine and anthracycline. Forty‐four percent of patients presented with normal weight while 56% were obese/overweight. Serum albumin levels were <30 g/L for 49 patients, and ≥30 g/L for 110. Thirty‐four patients with low SA levels were also obese. Favourable OS was associated with SA levels ≥30 g/L (HR = 0.467; 95% CI 0.230‐0.946; P = .034) but was not impacted by the BMI. Serum albumin levels appear to be an independent prognostic factor in AML and a better parameter than BMI for evaluating the nutritional status of patients at diagnosis.

B-cell prolymphocytic leukemia (B-PLL) is a rare hematological disorder whose underlying oncogenic mechanisms are poorly understood. Our cytogenetic and molecular assessments of 34 patients with B-PLL revealed several disease-specific features and potential therapeutic targets. The karyotype was complex (≥3 abnormalities) in 73% of the patients and highly complex (≥5 abnormalities) in 45%. The most frequent chromosomal aberrations were translocations involving MYC [t(MYC)] (62%), deletion (del)17p (38%), trisomy (tri)18 (30%), del13q (29%), tri3 (24%), tri12 (24%), and del8p (23%). Twenty-six (76%) of the 34 patients exhibited an MYC aberration, resulting from mutually exclusive translocations or gains. Whole-exome sequencing revealed frequent mutations in TP53, MYD88, BCOR, MYC, SF3B1, SETD2, CHD2, CXCR4, and BCLAF1. The majority of B-PLL used the IGHV3 or IGHV4 subgroups (89%) and displayed significantly mutated IGHV genes (79%). We identified 3 distinct cytogenetic risk groups: low risk (no MYC aberration), intermediate risk (MYC aberration but no del17p), and high risk (MYC aberration and del17p) (P = .0006). In vitro drug response profiling revealed that the combination of a B-cell receptor or BCL2 inhibitor with OTX015 (a bromodomain and extra-terminal motif inhibitor targeting MYC) was associated with significantly lower viability of B-PLL cells harboring a t(MYC). We concluded that cytogenetic analysis is a useful diagnostic and prognostic tool in B-PLL. Targeting MYC may be a useful treatment option in this disease.

Waldenström’s macroglobulinemia is a rare B-cell lymphoma. The gold standard treatment for Waldenström’s macroglobulinemia is an anti-CD20 antibody (rituximab) in combination with alkylating agents and dexamethasone. Treatment targeting the B-cell receptor such as ibrutinib (but not idelalisib) is currently approved for treatment of patients with relapsed or refractory Waldenström’s macroglobulinemia.

Bone marrow (BM) failure (BMF) in children and young adults is often suspected to be inherited, but in many cases diagnosis remains uncertain. We studied a cohort of 179 patients (from 173 families) with BMF of suspected inherited origin but unresolved diagnosis after medical evaluation and Fanconi anemia exclusion. All patients had cytopenias, and 12.0% presented ≥5% BM blast cells. Median age at genetic evaluation was 11 years; 20.7% of patients were aged ≤2 years and 36.9% were ≥18 years. We analyzed genomic DNA from skin fibroblasts using whole-exome sequencing, and were able to assign a causal or likely causal germ line mutation in 86 patients (48.0%), involving a total of 28 genes. These included genes in familial hematopoietic disorders (GATA2, RUNX1), telomeropathies (TERC, TERT, RTEL1), ribosome disorders (SBDS, DNAJC21, RPL5), and DNA repair deficiency (LIG4). Many patients had an atypical presentation, and the mutated gene was often not clinically suspected. We also found mutations in genes seldom reported in inherited BMF (IBMF), such as SAMD9 and SAMD9L (N = 16 of the 86 patients, 18.6%), MECOM/EVI1 (N = 6, 7.0%), and ERCC6L2 (N = 7, 8.1%), each of which was associated with a distinct natural history; SAMD9 and SAMD9L patients often experienced transient aplasia and monosomy 7, whereas MECOM patients presented early-onset severe aplastic anemia, and ERCC6L2 patients, mild pancytopenia with myelodysplasia. This study broadens the molecular and clinical portrait of IBMF syndromes and sheds light on newly recognized disease entities. Using a high-throughput sequencing screen to implement precision medicine at diagnosis can improve patient management and family counseling.

MLLT10 (previously AF10) was originally described as a fusion partner of the KMT2A (previously MLL) gene in acute myeloid leukaemia (AML) (Beverloo et al, 1995), and was subsequently found to be recurrently rearranged in both AML and T-acute lymphoblastic leukaemia (T-ALL) (Dreyling et al, 1998). PICALM (previously CALM) is the most frequent MLLT10 translocation partner in T-ALL, where it is found in 6-7% of cases (Ben Abdelali et al, 2013). The list of MLLT10 partners in T-ALL now also comprises NAP1L1 (Zhang et al, 2012), HNRNPH1, DDX3X (Brandimarte et al, 2013) and XPO1 (Bond et al, 2014). While DDX3X-MLLT10 has been estimated to occur in about 3% of adult T-ALL cases (Brandimarte et al, 2014), all other fusion transcripts have to date been described in single cases only, and their precise incidence remains to be defined. The NAP1L1-MLLT10 rearrangement was originally discovered by whole genome sequencing of leukaemic blasts from a child with early thymic precursor (ETP) ALL (Zhang et al, 2012). Diagnostic karyotypes of two T-ALL patients revealed similar t(10;12) translocations (Table 1), leading us to suspect the presence of NAP1L1-MLLT10. Expression of the transcript was confirmed by reverse transcription polymerase chain reaction (RT-PCR) (Fig 1A), and direct sequencing revealed fusion of NAP1L1 exon 14 and MLLT10 exon 4 in both cases (Fig 1B). CD34− CD1a− CD3− cCD3+ CD5+ CD7+ CD4weak CD8− CD10− TCRAB− TCRGD− MPO− CD117− CD13+ CD33+ HLA-DR+ CD34− CD1aweak CD2− CD3+ CD5+ CD7+ CD4+ CD8− CD10+ TCRGD+ CD13− CD33− CD117− Persistent MRD positivity (3%) post-induction and consolidation phases of treatment. Underwent SCT 7 months post-diagnosis. Remained in remission from leukaemia but died 2 years post-SCT due to chronic GVHD. Clinical details of the NAP1L1-MLLT10 patients are shown in Table 1. Strikingly, both were found to have high expression of HOXA9 by quantitative real time RT-PCR (QRT-PCR). Similar to the index case, Patient A had an ETP-ALL-like immunophenotype, with absence of CD1a, and expression of HLA-DR and the CD13 and CD33 myeloid markers (Table 1). Detailed characterization of T-cell receptor gene configuration revealed biallelic rearrangement of TRD and TRG and incomplete rearrangement of the TRB locus, conforming to an immature pre-β-selection immunogenotype (Asnafi et al, 2003). In contrast, Patient B exhibited a mature TCRγδ+ phenotype and absence of expression of myeloid markers. In common with the reported cases of DDX3X-MLLT10 T-ALL (Brandimarte et al, 2014), both cases had NOTCH1-activating mutations. Notably, both patients had poor initial treatment responses, with positive minimal residual disease (MRD). Patient B had persistent MRD positivity throughout induction and consolidation therapy, which necessitated allogeneic stem cell transplantation (allo-SCT) 7 months after diagnosis. Although this resulted in molecular remission, the patient ultimately died of SCT-related complications 2 years later while in remission from leukaemia. The follow-up of Patient A is at an early stage, and latest assessment revealed significant persistent MRD positivity following the consolidation phase. We sought to determine the incidence of the NAP1L1-MLLT10 translocation in T-ALL by screening 565 cases, comprising 141 patients under 16 years of age and 424 adults, using a specific and sensitive (<1%, data not shown) QRT-PCR assay (Fig 1C). As MLLT10 translocations are normally associated with HOXA locus activation (Brandimarte et al, 2013; Bond et al, 2014), we targeted the screening to 173 patients with elevated HOXA9 at diagnosis (‘HOXA High’ in Fig 1C). We detected no further cases of NAP1L1-MLLT10 in patients for whom leukaemic material was available for testing. We therefore estimate the incidence of the translocation in T-ALL to be 0·35% (2/565 patients), and 1·2% of patients with HOXA9/ABL1 > 0·4 (2/173 patients). HOXA overexpression has not previously been described to be associated with NAP1L1-MLLT10. In order to fully characterize HOX transcription in these cases, we performed Taqman Low-Density Array (TLDA) analysis of diagnostic RNA. This revealed specific deregulation of HOXA gene expression in both samples (Fig 1D). These results, along with the predicted retention of the MLLT10 octapeptide-motif leucine zipper (OM-LZ) domain in the fusion protein, suggest that NAP1L1-MLLT10 is likely to recruit the DOT1 Ligand histone methyltransferase and to activate the HOXA locus by a similar mechanism as PICALM-MLLT10 (Okada et al, 2005). It remains unclear how the activities of MLLT10 fusion partners might contribute to T-ALL biology. Prediction of NAP1L1 (Nucleosome assembly protein 1-like1) function is based on its structural resemblance to NAP1, which is thought to be involved primarily in chromatin modulation. NAP1 has been shown to interact directly with histone H2A-H2B dimers in vitro (Okuwaki et al, 2010), suggesting that NAP1L1 might alter the regulation of histone dynamics. It remains to be determined whether the epigenetic effects of NAP1L1 haploinsufficiency and/or activity of a NAP1L1 fusion protein might contribute to a leukaemic phenotype. In summary, NAP1L1-MLLT10 is a rare recurrent translocation in T-ALL and we report an overall incidence of 0·35%. This may be an underestimation, as our QRT-PCR screening method may not have identified patients with novel breakpoints, and lack of diagnostic material precluded screening in 11% of HOXA-overexpressing patients. Reassuringly, all three cases of NAP1L1-MLLT10 described to date exhibited the t(10;12) translocation by conventional karyotyping, and none of our patient cohort had evidence of chromosome 10 or 12 rearrangement. As with other MLLT10 fusions, NAP1L1-MLLT10 is associated with HOXA deregulation and poor early treatment response. Although one of our patients died of transplant-related complications, the prolonged leukaemia-free survival in this case suggests that these patients should be considered for allo-SCT in first remission, particularly in the event of persistent treatment resistance and/or MRD positivity. JB, AT, AC, AT, MM and TF performed and interpreted diagnostic investigations. TM, ME, AC and CS provided clinical care and interpreted clinical data. JB, VA and EAM analysed and collated data and wrote the manuscript. JB is supported by a Kay Kendall Leukaemia Fund Intermediate Research Fellowship. The Macintyre laboratory is supported by the Association Laurette Fugain and the INCa CARAMELE Translational Research and PhD Programmes. The authors report no conflicts of interest.

INTRODUCTION:Automated haematology analysers may inaccurately determine platelet counts in several circumstances. Spuriously elevated automated platelet counts have been reported in some acute leukaemia (AL) cases because of fragmentation of circulating blast cells (pseudoplatelets). Haemorrhagic diathesis is a common manifestation of AL, which is often caused by severe thrombocytopenia. Therefore, overestimation of the actual platelet count in patients with AL can affect its clinical management. We aimed to detect the frequency of pseudoplatelets in patients with AL. METHODS:Complete blood cell counts were performed on 86 AL patients with three automated analysers (ADVIA 2120, Coulter LH 750 and Sysmex XE-2100D). Platelet counts were also performed by quantitative flow cytometry (QFC). The platelet counts of the automatic analysers were compared to the platelet counts by QFC. Blood smears were checked for the presence of pseudoplatelets. RESULTS:The automated analysers overestimated the platelet count due to the presence of pseudoplatelets in patients with AL. Pseudoplatelets were observed in the blood smears of 11 patients (13%). Three of these patients were near the prophylactic platelet transfusion threshold. CONCLUSION:Spurious increases in automated platelet counts by blast cell fragments are little known but frequent artefacts that should be ruled out by careful examination of peripheral blood smears.

![Figure][1] A 61-year-old male presented with 2 months of bone pain. Complete blood count showed Hb 10 g/dL, white blood cell count 4.5 × 109/L, and platelets 130 × 109/L. The peripheral smear contained visible agglutinates and a blue background color (panel A). Aggregates of amorphous

After antigen recognition, CD5 interacts physically with the antigen receptor on lymphocytes, is rapidly phosphorylated, and delivers a negative signal, thereby avoiding unwanted lymphocyte reactivity to the antigen. An intracellular domain of CD5 is critical to downregulate the antigen receptor–mediated early signaling events. In chronic lymphocytic leukemia (CLL) cells, the B-cell antigen receptor (BCR) repertoire is biased toward autoreactivity suggesting that the BCR helps to select or maintain the malignant clone. We generated a monoclonal antibody against the tyrosine-phosphorylated regulatory domain of CD5 (PCD5) to monitor the activation of the BCR. In B cells, BCR engagement transiently phosphorylated CD5 for less than 60 minutes. CD5 activation followed the same kinetics as BCR internalization and was prevented by incubation with inhibitors of tyrosine kinases (PP2 > PP3 > genistein). Engagement of CD40, CXCR4, and TLR9 failed to activate CD5. In contrast in CLL B cells, CD5 phosphorylation is constitutive and sustained for days. It is abolished at 4 °C and restored after more than 1 hour at 37 °C in culture; it is variably inhibited by tyrosine kinase inhibitors and at variance with non-CLL B cells, which is also inhibited by herbimycin. Anti-PCD5 monoclonal antibody stained all fresh malignant cells from indolent or aggressive stages of 18 of 18 patients with CLL, but not blood B cells from healthy controls. It also stained 2 of 4 samples with CD5+ monoclonal B-cell lymphocytosis but none of 5 samples with CD5+ mantle cell lymphoma (4 by flow cytometry and 1 on pathology section) B-cells. PCD5 is a specific marker of CLL and may reflect an in vivo abnormal configuration or permanent aberrant activation of the BCR complex in this disease.

TransfusionVolume 49, Issue 5 p. 827-828 Thermally induced popcorn red blood cells Marc Muller, Marc Muller From the Service d'Hématologie Biologique, Centre Hospitalier Universitaire, Nancy, France.Search for more papers by this authorSylvain Salignac, Sylvain Salignac From the Service d'Hématologie Biologique, Centre Hospitalier Universitaire, Nancy, France.Search for more papers by this authorJean François Lesesve, Jean François Lesesve From the Service d'Hématologie Biologique, Centre Hospitalier Universitaire, Nancy, France.Search for more papers by this author Marc Muller, Marc Muller From the Service d'Hématologie Biologique, Centre Hospitalier Universitaire, Nancy, France.Search for more papers by this authorSylvain Salignac, Sylvain Salignac From the Service d'Hématologie Biologique, Centre Hospitalier Universitaire, Nancy, France.Search for more papers by this authorJean François Lesesve, Jean François Lesesve From the Service d'Hématologie Biologique, Centre Hospitalier Universitaire, Nancy, France.Search for more papers by this author First published: 21 April 2009 https://doi.org/10.1111/j.1537-2995.2009.02139.xCitations: 3 J.F. Lesesve, Service d'Hématologie Biologique, CHU Nancy, 54511 Vandoeuvre, France; e-mail: [email protected]. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume49, Issue5May 2009Pages 827-828 RelatedInformation