Clonal chromosome abnormalities in Philadelphia-negative cells could concern chronic myeloid leukemia patients treated by tyrosine kinase inhibitors. The European LeukemiaNet distinguishes -7/del(7q) ...abnormalities as a "warning". However, the impact of clonal chromosome abnormalities, and specifically those of -7/del(7q), in Philadelphia-negative cells on clinical outcomes is unclear and based on case-reports showing morphological dysplasia and increased risk of acute myeloid leukemia, suggesting the coexistence of chronic myeloid leukemia and high-risk myelodysplastic syndrome. The aim of this study was to determine whether the impact of -7/del(7q) clonal chromosome abnormalities in Philadelphia-negative cells on the clinical outcome is different from that of other types of abnormalities, and we argue for an underlying associated high-risk myelodysplastic syndrome. Among 102 chronic myeloid leukemia patients with clonal chromosome abnormalities in Philadelphia-negative cells with more than a median of 6 years of follow up, patients with -7/del(7q) more frequently had signs of dysplasia, a lower cumulative incidence of deep molecular response and often needed further treatment lines, with the consequent impact on event-free and progression-free survival. Morphological features of dysplasia are associated with myelodysplastic syndrome/acute myeloid leukemia mutations and compromise the optimal response to tyrosine kinase inhibitors, irrespectively of the type of clonal chromosome abnormalities in Philadelphia-negative cells. However, mutation patterns determined by next-generation sequencing could not clearly explain the underlying high-risk disease. We hereby confirm the pejorative prognostic value of -7/del(7q) clonal chromosome abnormalities in Philadelphia-negative cells and suggest that myelodysplastic features constitute a warning signal that response to tyrosine kinase inhibitors may be less than optimal.
Posttranscriptional modifications of histones play important roles in the control of chromatin structure and transcription. H3K4 (histone H3 lysine 4) methylation by the SET domain of the ...trithorax-group protein MLL (mixed-lineage leukemia) is important for the control of homeobox (HOX) gene expression during development. MLL is tethered to the HOXA locus through interaction of its amino-terminus with menin. MLL fusion proteins associated with human leukemia contain the menin interaction peptide and frequently recruit H3K79 (histone H3 lysine 79) methylation activity. This allows sustained expression of HOXA genes important for cellular transformation. We have characterized a novel recurrent chromosomal aberration, inv(11)(p15q23), as an isolated chromosomal abnormality in 2 patients with acute myeloid leukemia. This aberration is predicted to result in the expression of an NUP98 (nucleoporin 98 kDa)–MLL fusion protein that is unable to interact with menin. As expected, low levels of HOXA gene expression were observed in the patients' samples. This fusion protein is predicted to participate in cellular transformation by activating MLL targets other than HOXA genes.
The ETV6/GOT1 fusion, resulting from t(10;12) (q24;p13), has been recently described in a myelodysplastic syndrome. We reported a second case of t(10;12)-positive myelodysplastic syndrome in whom ...fluorescent in situ hybridization confirmed the non-random translocation but molecular biology analyses revealed a ETV6/GOT1 chimera varying from the first case described.
AML relapse is often associated with a clonal evolution at the cytogenetic and molecular level and therefore represents a challenge for the treatment. Targeted sequencing is now usually done at ...diagnosis in AML, as only a small core group of genes is frequently mutated in AML and MDS. This approach, contrary to Whole Genome Sequencing, is cheaper and allows the detection of variant allele fractions as low as 2% with a rapid turnover and high sequencing coverage and depths.
In this study we analyzed the karyotype and the mutations occuring in a panel of genes in 31 AML patients at diagnosis and at the relapse.
The Illumina TruSight Myeloid Sequencing Panel® was used that covers all exons of 15 genes and hotspots of 39 additionnal genes involved in myeloid malignancies. Three different pipelines were used for data analysis including GATK, Picard, Samtools, VarScan reference tools. We tried to assess the clonality and the clonal evolution patterns at relapse. Five different clonal evolution patterns including cytogenetic and molecular analyses were observed: (1) Stability, defined by absence of clonal change, (2) Gain, strictly defined by acquisition of additional variations (mutations or cytogenetic alterations), (3) Loss, strictly defined by loss of variants or regression, (4) Gain and Loss, indicating the combination of both “Gain” and “Loss” patterns, (5) Emergence, defined by the emergence of alterations that is unrelated to those found at diagnosis.
The cohort included 20 men and 11 women with a median age of 59 years old for which frozen DNA was available both at diagnosis and relapse that were referred in a single center. The elapsed time between the first remission and relapse (TTR) was 1.6 to 113.8 months. Almost all patients relapsed prematurely (less than 12 months). The overall survival and the survival after relapse were respectively 23.7 months and 9.1 months.
The cytogenetic evolution patterns observed at relapse were the following: Stability (40%), Gain (27%), Loss (31%), Gain and Loss (4%), Emergence (<1%). The mutation evolution patterns observed at relapse were the following: Stability (26%), Gain (23%), Loss (32%), Gain and Loss (16%), Emergence (3%) . No correlation between cytogenetic and mutation pattern was observed.
Patients without change at relapse (“Stability” pattern) had less mutated genes at diagnosis than in the other patterns (p=0.009). Whereas patients with the “Gain and Loss” pattern had more mutated genes at diagnosis than the other patients (≥4 mutated genes, p=0.001). Thirty five percent of the alterations acquired at relapse (“Gain” and “Gain and Loss” patterns) were found in tumor suppressor genes: TP53 was mutated in 60% of these patients, but mutations were also observed in signaling genes (25% of the patients). Thirty eight percent of the alterations lost at relapse (“Loss” and “Gain and Loss” patterns) are found in signaling genes (i.e FLT3, KIT, KRAS, NRAS ) and 23% in myeloid transcription factors (i.e CEBPA, RUNX1, GATA2 ). It was also been observed that 47% of mutations thatwere observed in each group at diagnosis and relapse were localized in DNA methylation and chromatin modification genes (i.e DNMT3A, IDH1/2, TET2, ASXL1, EZH2 ).
Interestingly, at diagnosis, mutations of DNA methylation and spliceosome complex genes were preferentially observed in the “Gain” pattern group (p=0.040). In contrast, mutations of signaling genes were preferentially mutated at diagnosis in the “Loss” group (p=0.020).
Even if not statistically significant, we observed that evolution and prognosis seemed to be different among the different patterns. The “Gain” pattern tends to have shorter overall survival (median 14.7 months vs 22.9 months in Stability group) whereas in the “Loss” group, overall survival appeared longer (median 34.1 months). Moreover, patients in the “Gain” group were significantly older (p=0.017), and were classified as AML with myelodysplasia-related changes in most of the cases.
To our knowledge, our work is the first reporting multiplex targeted NGS sequencing in AML at diagnosis and relapse. The comparison of clonal landscape between diagnosis and relapse allowed us to define 5 evolution patterns that differed in terms of mutation landscape and evolution.
Even if these data must be confirmed on a larger cohort, our study shows the feasibility of clonal analysis using targeted NGS at diagnosis and relapse in AML and its potential prognosis interest in clinical routine.
No relevant conflicts of interest to declare.
Most chromosomal translocations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) involve oncogenes that are either up-regulated or form part of new chimeric genes. The ...t(2;11)(p21;q23) translocation has been cloned in 19 cases of MDS and AML. In addition to this, we have shown that this translocation is associated with a strong up-regulation of miR-125b (from 6- to 90-fold). In vitro experiments revealed that miR-125b was able to interfere with primary human CD34+ cell differentiation, and also inhibited terminal (monocytic and granulocytic) differentiation in HL60 and NB4 leukemic cell lines. Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation, and myeloid neoplasms carrying the t(2;11) translocation define a new clinicopathological entity.
The aim of our study was to analyze the factors contributing to heterogeneity of prognosis in patients with hyperdiploidy>50 chromosomes (HD>50), a group of B-cell precursor acute lymphoblastic ...leukemia with favorable outcome. The 541 HD>50 patients registered prospectively in the 58951 European Organisation for Research and Treatment of Cancer (EORTC) Children's Leukemia Group (CLG) trial, identified by karyotype (446 patients) and by DNA index (DI) (490 patients), had a 6-year event-free survival (EFS) of 89.0% (standard error SE = 1.5%) and a 6-year overall survival (OS) of 95.9% (SE = 0.9%). The strongest prognostic factor was the modal number of chromosomes (MNC): the 6-year EFS of 51-53, 54-57, and 58-66 MNC groups were 80%, 89%, and 99%, respectively (P < .0001). Ploidy assessed by DI was also a favorable factor: the higher the DI, the better the outcome. The 6-year EFS of the 3 subgroups of DI < 1.16/≥1.16-<1.24/≥1.24 were 83%, 90%, and 95%, respectively (P = .009). All usual combinations of trisomies (chromosomes 4, 10, 17, 18) were significant favorable factors but had lower EFS when MNC was lower than 58. In multivariate analysis, MNC remained the strongest factor. Consequently, the best indicator for excellent outcome was ploidy assessed by karyotype because patients with 58-66 chromosomes stood every chance of being cured (OS of 100% at 6-year follow-up) with less-intensive therapy. This trial was registered at www.clinicaltrials.gov as #NCT00003728. Registered: http://www.eortc.org/, http://clinicaltrials.gov/show/NCT00003728.
•Patients with 58-66 chromosomes have 99% event-free survival and 100% overall survival in the 58951 EORTC-CLG study.•The higher the ploidy, the better the prognosis in the 58951 EORTC-CLG study.
The term « double » or « triple hit lymphoma » is commonly used to describe mature B cell neoplasms with either BCL2 and/or MYC and/or BCL6 gene rearrangements. These rare and aggressive lymphomas ...with high Ki67 expression are categorized as « B-cell lymphomas unclassified, with features intermediate between diffuse B-cell lymphoma and Burkitt lymphoma » category in the WHO 2008 classification. To our knowledge, only 2 cases of lymphoma have been described with four specific genetic events (quadruple hit) involving MYC, BCL2, BCL6 and CCND1 genes (Bacher U. et al., Genes Chromosomes Cancer 2011). We describe here the third observation.
A patient, a 79 years old man, suffering from paraesthesias for 4 months, was admitted for polyneuritis in a context of poor general condition. Clinical examination showed the presence of numerous axillary, supraclavicular, mediastinal and inguinal lymphadenopathy, neuro-meningeal invasion and skin infiltration. The biopsy of a skin nodule of left arm revealed an infiltration consisting in large proliferating cells (Ki67 80%) stained by anti-CD20, BCL2 and BCL6 antibodies, but not with CD10 nor CD23, consistent with a diffuse large B-cell lymphoma (DLBCL), NOS diagnosis. Blood cell count showed 8.1 G/L leukocytes, 13.2 g/dL hemoglobin, 166 G/L platelets. LDH and β-2 microglobulin were elevated (989 U/I, and 9.14 mg/L respectively). Blood cell film examination showed the presence of 28% of pathological basophil lymphocytes (medium sized, with slightly clumped chromatin and frequent prominent nucleoli). Flow cytometry revealed that these cells expressed a monotypic lambda immunoglobulin light chain and were CD19, CD20, FMC7, CD22 positive, with a dim CD5 positivity and no CD10 staining. The negativity of CD23 associated to strong CD148 staining (Miguet et al, J Proteome Res2009) were in favour of the diagnosis of variant pleomorphic mantle cell lymphoma (MCL).
Cytogenetic study performed in the WBC found a complex hyperdiploid karyotype (47 chromosomes) with a t(3;22) translocation involving BCL6 and IGL genes, structural abnormality of a chromosome 8 resulting in juxtaposition of 5’MYC and BCL2 in FISH (with break of the MYC probe in FISH), a derivative chromosome 18 from a t(14;18) translocation with fusion of 5’IGH and BCL2, and a t(11;14) complex translocation involving IGH and CCND1 (54% of the nuclei). Overexpression of cyclin D1 was detected in the WBC by RQ-PCR, as well as in the skin lesion using immunochemistry. Others numeral (trisomy 12) and structural abnormalities (involving the 1, 7, 14 and 21 chromosomes) were also detected.
The patient was treated with a chemotherapy combining rituximab, ifosfamide, cytosine arabinoside and intrathecal methotrexate. He is still alive 5 months after the diagnosis.
This third case of quadruple hit lymphoma confirms the complexity of the classification of such aggressive malignancies. Initial rearrangement of the CCND1 gene characterizes MCL that may harbour in very rare cases additional rearrangements of MYC or BCL6. Conversely, cyclin D1 overexpression is considered a rare feature in DLBCL. Recently, Ok CY et al. (Cancer 2014) proposed to reclassify DLBCL with expression of cyclin D1 and CCND1 chromosomal rearrangement and CD5 positivity as aggressive pleomorphic MCL variant. But no case with rearrangement of 2 (or more) genes (BCL2 and/or MYC and/or BCL6) was described in this study.
Juskevicius D et al. (Am J Surg Pathol 2014) suggest the existence of a “gray zone” in which morphologic, clinical and genetic features are insufficient to segregate CD5- and SOX11-negative lymphomas with overexpression of cyclin D1/ translocation involving CCND1between blastoid MCL from cyclin D1-positive DLBCL.
In our case, immunophenotyping of circulating cells (CD5+, CD148++) as well as genetic and molecular features (translocation involving CCND1 and IGH, and overexpression of cyclin D1) allow us to diagnose a probable genetically unstable aggressive pleomorphic MCL variant with rearrangement of several extra genetic hits.
No relevant conflicts of interest to declare.
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