Patterns of genomic aberrations suggest that Burkitt lymphomas with complex karyotype are distinct from other aggressive B-cell lymphomas with MYC rearrangement.

We previously showed that complex karyotypes (CK) and chromosome 13q abnormalities have an adverse prognostic impact in childhood Burkitt lymphomas/leukemias (BL) and diffuse large B-cell lymphomas (DLBCL). The aim of our study was to identify recurrent alterations associated with MYC rearrangements in aggressive B-cell lymphomas with CK. Multicolor fluorescence in situ hybridization (M-FISH) was performed in 84 patient samples (59 adults and 25 children), including 37 BL (13 lymphomas and 24 acute leukemias), 12 DLBCL, 28 B-cell lymphomas with intermediate features (DLBCL/BL), 4 B-cell precursor acute lymphoblastic leukemias (BCP-ALL), and 3 unclassifiable B-cell lymphomas. New (cytogenetically undetected) abnormalities were identified in 80% of patients. We also refined one-third of the chromosomal aberrations detected by karyotyping. M-FISH proved to be more useful in identifying chromosomal partners involved in unbalanced translocations and in revealing greater complexity of 13q rearrangements. Most of the newly identified or refined recurrent alterations involved 1q, 13q and 3q (gains/losses), 7q and 18q (gains), or 6q (losses), suggesting that these secondary aberrations may play a role in lymphomagenesis. Several patterns of genomic aberrations were identified: 1q gains in BL, trisomies 7 in DLBCL, and 18q-translocations in adult non-BL. BCP-ALL usually displayed an 18q21 rearrangement. BL karyotypes were less complex and aneuploid than those of other MYC-rearranged lymphomas. BCP-ALL and DLBCL/BL were associated with a higher rate of early death than BL and DLBCL. These findings support the categorization of DLBCL/BL as a distinct entity and suggest that BL with CK are indeed different from other aggressive MYC-rearranged lymphomas, which usually show greater genetic complexity. © 2012 Wiley Periodicals, Inc.

Genomic characterization of Imatinib resistance in CD34+ cell populations from chronic myeloid leukaemia patients.

To ascertain genomic alterations associated with Imatinib resistance in chronic myeloid leukaemia, we performed high resolution genomic analysis of CD34(+) cells from 25 Imatinib (IM) resistant and 11 responders CML patients. Using patients' T-cells as reference, we found significant association between number of acquired cryptic copy number alterations (CNA) and disease phase (p=0.036) or loss of IM response for patients diagnosed in chronic phase (CP) (p=0.04). Recurrent cryptic losses were identified on chromosomes 7, 12 and 13. On chromosome 7, recurrent deletions of the IKZF1 locus were detected, for the first time, in 4 patients in CP.

Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(2;11)(p21;q23) translocation.

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.

Cryptic and partial deletions of PRDM16 and RUNX1 without t(1;21)(p36;q22) and/or RUNX1-PRDM16 fusion in a case of progressive chronic myeloid leukemia: a complex chromosomal rearrangement of underestimated frequency in disease progression?

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the presence in leukemic stem cells of the Philadelphia chromosome (Ph) and the formation of the BCR-ABL1 fusion. Untreated, the disease progresses to accelerate phase and blast crisis in which hematopoietic differentiation has become arrested. CML progression is frequently associated with cytogenetic evidence of clonal evolution, defined as additional chromosomal aberrations. We here report a CML resistant to tyrosine kinase inhibitors that rapidly progressed to blastic phase. At this time, array CGH performed on CD34(+) cells revealed cryptic partial deletions of both PRDM16 and RUNX1 and duplication of the der(21) chromosome. These genomic rearrangements were confirmed by FISH with probes targeting the deletion on chromosome 21 (24 kb), and with BAC probes flanking the deletion on 1p36 (220 kb). However, no cryptic t(1;21)(p36;q22) and/or RUNX1-PRDM16 were detected, suggesting that these deletions are the residual hallmarks of a more complex mechanism of chromosomal rearrangement, as indicated by the additional inversion of the region bounded by 1p36.32 and 1p36.12 breaks. At the molecular level, these abnormalities lead to the overexpression of the PR-domain negative oncogenic isoform of PRDM16, associated with two deleted copies within the runt domain of C-teminal aberrant RUNX1. These events are not detectable by conventional cytogenetic and molecular strategies, and may be of underestimated frequency in disease progression.

Mechanisms of genesis of variant translocation in chronic myeloid leukemia are not correlated with ABL1 or BCR deletion status or response to imatinib therapy.

Many published studies have indicated that various mechanisms could be involved in the genesis of variant chronic myelogeneous leukemia (CML) translocations. These are mainly one-step or two-step mechanisms, associated or not with deletions adjacent to the translocation junction on der(9) or der(22) chromosomes (or both). Based on the mechanism of genesis, it has been suggested that the complexity may affect the occurrence of ABL1 and BCR deletions (either or both), or may be associated with the CML disease course, and thus could determine the response to imatinib therapy. Through a retrospective molecular cytogenetic study of 41 CML patients with variant Philadelphia chromosome (Ph), we explored the genesis of these variant rearrangements and analyzed the correlation with deletion status and imatinib efficiency. Our results confirmed that the one-step mechanism is the most frequent, evidenced in 30 of 41 patients (73%); 3 patients demonstrated other more complex multistep events and 8 patients (19.5%) harbored ABL1 or BCR deletions that are not significantly associated with the complexity of translocation genesis. We also found no association between one-step, two-step, or multistep mechanisms and the response to imatinib therapy.

RUNX1 DNA-binding mutations and RUNX1-PRDM16 cryptic fusions in BCR-ABL+ leukemias are frequently associated with secondary trisomy 21 and may contribute to clonal evolution and imatinib resistance.

Acquired molecular abnormalities (mutations or chromosomal translocations) of the RUNX1 transcription factor gene are frequent in acute myeloblastic leukemias (AMLs) and in therapy-related myelodysplastic syndromes, but rarely in acute lymphoblastic leukemias (ALLs) and chronic myelogenous leukemias (CMLs). Among 18 BCR-ABL+ leukemias presenting acquired trisomy of chromosome 21, we report a high frequency (33%) of recurrent point mutations (4 in myeloid blast crisis [BC] CML and one in chronic phase CML) within the DNA-binding region of RUNX1. We did not found any mutation in de novo BCR-ABL+ ALLs or lymphoid BC CML. Emergence of the RUNX1 mutations was detected at diagnosis or before the acquisition of trisomy 21 during disease progression. In addition, we also report a high frequency of cryptic chromosomal RUNX1 translocation to a novel recently described gene partner, PRDM16 on chromosome 1p36, for 3 (21.4%) of 14 investigated patients: 2 myeloid BC CMLs and, for the first time, 1 therapy-related BCR-ABL+ ALL. Two patients presented both RUNX1 mutations and RUNX1-PRDM16 fusion. These events are associated with a short survival and support the concept of a cooperative effect of BCR-ABL with molecular RUNX1 abnormalities on the differentiation arrest phenotype observed during progression of CML and in BCR-ABL+ ALL.

Hyperdiploidy is a common finding in monoclonal gammopathy of undetermined significance and monosomy 13 is restricted to these hyperdiploid patients.

PURPOSE: Two pathways, hyperdiploid and nonhyperdiploid, are proposed for progression to plasma cell neoplasia. Implication of monosomy 13 (Delta13) is unclear in monoclonal gammopathy of undetermined significance (MGUS), and data on DNA content of plasma cells [DNA index (DI)] are rare. EXPERIMENTAL DESIGN: We ascertained DI in 169 multiple myeloma (MM) and 96 MGUS patients. Interphase fluorescence in situ hybridization (FISH) coupled to cytoplasmic staining of specific Ig (cIg-FISH) was done to look for trisomies and to ascertain Delta13. RESULTS: Hyperdiploidy and hypodiploidy were found in 54% and 11.5% of MGUS patients and in 59.5% and 25% of MM patients, respectively. In MGUS patients tested using probes for odd chromosomes, cIg-FISH showed association between trisomies for chromosomes 3, 7, 9, 11, or 15 and hyperdiploidy. Delta13 was found in 45.3% and 24.6% of MM and MGUS patients, respectively. Most Delta13 cases observed in MGUS were found within hyperdiploid clones, 38% versus 11% in hypodiploid cases, in sharp contrast with the occurrence of Delta13 in MM patients, 31.9% and 76.3%, respectively. That peculiar distribution of Delta13 according to DI persisted with other thresholds used to ascertain hyperdiploidy, such as DI >or= 1.05. A strong relationship between IgA peak and hypodiploidy (P = 0.007) was only observed in MM, whereas lambda light chain was significantly associated with hypodiploidy in MGUS (P = 0.001) and MM (P = 0.05). Hyperdiploidy shows similar pattern in MGUS and MM. CONCLUSION: This fits well a hyperdiploid pathway leading to MM after a preceding MGUS stage. Yet-to-be-determined secondary event(s) needs to occur for the transition to MM, unrelated to changes in chromosome number or to loss of chromosome 13. In contrast, the "nonhyperdiploid" pathway needs to be clarified further because hypodiploidy is less common in MGUS than in MM and Delta13 is rare in hypodiploid MGUS patients compared with hypodiploid MM patients.

Bone morphogenetic protein antagonist gene NOG is involved in myeloproliferative disease associated with myelofibrosis.

In a case with secondary myelofibrosis occurring after essential thrombocythemia, cytogenetic analysis revealed an isolated translocation t(X;17)(q27;q22) in all cells. We found that a bacterial artificial chromosome (BAC) encompassing the breakpoint on chromosome 17 long arm contained only one gene, NOG. We therefore investigated the occurrence of this rare breakpoint in myeloproliferative disorders (MPDs). We identified three more patients with a 17q abnormality in MPDs: myelofibrosis with myeloid metaplasia (MMM); chronic myeloid leukemia positive for t(9;22)(q34;q11) with additional t(4;17)(p15;q22) at diagnosis; and myelofibrosis complicating polycythemia vera. All three cases exhibited a split of BACs containing NOG. The protein encoded by NOG, noggin, acts as an antagonist to bone morphogenetic secreted protein 2 and 4 (BMP2 and BMP4). A comparative analysis of gene expression on Agilent 22K oligonucleotide microarrays in purified CD34+ cells from the blood of MMM patients showed significant downregulation of BMPR2, BMPR1B, BMP2, and BMP8; upregulation of BMP3 and BMP10; and a trend to lower expression of NOG. Thus, given that expression and release of BMPs are important in the induction of osteosclerosis and angiogenic activity, the observed BMP deregulations could be triggered by potential NOG genetic alterations in the four cases here described, and may contribute to the myelofibrotic process characterized by bone marrow stromal reaction including collagen fibrosis, osteosclerosis, and angiogenesis.

Plasma cell growth fraction using Ki-67 antigen expression identifies a subgroup of multiple myeloma patients displaying short survival within the ISS stage I.

The current most powerful prognostic model in Multiple Myeloma (MM) combines beta-2 microglobulin (b2m) with albumin, corresponding to the International Staging System (ISS). However, the prognosis of patients within the ISS stage I (high albumin and low b2m) may vary. Ki-67 is a nuclear protein associated with cell proliferation. We retrospectively evaluated the percentage of bone marrow plasma cells expressing Ki-67 antigen (Ki-67 index) in a series of 174 untreated MM patients at diagnosis. Median survival was 51, 41 and 20 months respectively, and median Ki-67 index was 3.0%, 6.1% and 6.5% in ISS stages I, II, and III respectively. Independently of ISS, Ki-67 index > or =4% was highly predictive of adverse prognosis. Ki-67 index correlated with markers of intrinsic malignancy and with markers of tumour burden. Within ISS stage I, median survival was of 31 months (RR of death 2.65) in patients with Ki-67 index > or =4%. Eventually, the combination of Ki-67 with b2m produced an efficient prognostic model, which appeared most effective in our series when compared with b2m and KI-67 with chromosome 13 deletion models. In this series, we demonstrated that a proliferation marker provides clear-cut additional survival prognostic information to b2m into the ISS model.

High WT1 expression after induction therapy predicts high risk of relapse and death in pediatric acute myeloid leukemia.

PURPOSE: To determine whether minimal residual disease (MRD) measured by Wilms' tumor gene 1 (WT1) expression is a prognostic marker in pediatric acute myeloid leukemia (AML), we quantified WT1 transcript by real-time quantitative-polymerase chain reaction in 92 AML at diagnosis and during follow-up. PATIENTS AND METHODS: Patients (median age, 6 years; cytogenetics, favorable 27%, intermediate 59%, poor 13%) were treated between 1995 and 2002 and enrolled in Leucémie aiguë Myéloblastique Enfant (LAME) 89/91, LAME 99 pilot study and Acute Promyelocytic Leukemia French collaborative protocols. With a median follow-up of 26 months, event-free survival was 56% with a standard deviation (SD) of 5% and overall survival of 62.5% with an SD of 6%. WT1 copy number was normalized by TATA box binding protein gene transcripts and expressed as WT1/TBP x 1,000 ratio. Median WT1 ratio in normal patient controls was 12 (range, 0 to 57). A level over two SD than normal bone marrow controls (ie, WT1 ratio > 50), was considered as significant overexpression. RESULTS: At diagnosis, WT1 overexpression was detected in 78% of patients (72 of 92 patients; median copy ratio, 2231). The WT1 values were significantly higher (P = .01) in favorable cytogenetics and lower (P < .0001) in M5-FAB subtype, 11q23 rearrangements (P < .001), and infants (P = .003) and demonstrate a strong correlation with fusion transcript AML1-ETO, PML-RARalpha expression. After induction treatment, WT1 ratio was analyzed in 46 of 72 patients and found above 50 in nine of 36 patients and five of 25 patients at D35-50 and 3 to 5 months, respectively. WT1 ratio > 50 after induction is an independent prognostic risk factor of relapse (P = .002) and death (P = .02). CONCLUSION: WT1 quantification is an informative molecular marker for MRD in pediatric AML and is now performed as prospective analysis in ELAM02 protocol.

Cryptic 6p21.3 duplications and triplication involving HMGA1 partially masked by add 6p in four cases of myelodysplasia.

Rearrangements of 6p are frequent in both myeloid and lymphoid malignant hematological disorders. High-mobility group AT-hook 2 (HMGA2) rearrangements have been described in myelofibrosis with myeloid metaplasia (MMM) and also in myelodysplasia. High-mobility group A proteins are nonhistone nuclear proteins that bind DNA and regulate the transcriptional activity of many genes. We used FISH, with bacterial artificial chromosome RP11-513I15 probe, to study 16 cases of myeloid malignancies with chromosome 6 short arm rearrangements, most of them following myeloproliferative disorders. Among these we found two 6p21.3 duplications and one 6p21.3 triplication involving HMGA1 in four cases of myelodysplasia with and without myelofibrosis. In these four cases, duplications and triplication were partially masked at the cytogenetic level by a derivative chromosome 6 resulting from translocation with another chromosome. HMGA1 proteins have been recently found overexpressed in human leukemias, but to our knowledge this is the first reported duplication of HMGA1.

Familial occurrence of thymoma and autoimmune diseases with the constitutional translocation t(14;20)(q24.1;p12.3).

Thymomas are low-grade epithelial cancers of the thymus whose prevalence varies between 0.1/100,000 and 0.4/100,000. Familial occurrence of thymoma is very rare. We studied a family bearing the constitutional chromosome translocation t(14;20)(q24;p12), 3 of whose members had a thymoma. In this family, among 27 patients, 11 had the translocation: 3 had thymoma and 4 others had 5 different autoimmune diseases: type 1 diabetes mellitus, Graves' disease, pernicious anemia, primitive Sjögren disease, and autoimmune pancytopenia. FISH studies allowed us to be more specific about the translocation breakpoints. The 14q24 breakpoint was in intron 5 of RAD51L1, and the 20p12 breakpoint was 100 kb telomeric to BMP2. RAD51L1 is a tumor-suppressor gene belonging to the RAD51 family, already implicated in many tumors (uterine leiomyomas, pseudo-Meigs syndromes, pulmonary chondroid hamartomas) and involved in recombinational repair of DNA double-strand breaks. BMP2 belongs to the TGFbeta superfamily, and the BMP2-BMP4 genes are involved in thymocyte differentiation by blocking progression from CD4-CD8- to CD4+CD8+ while maintaining a sufficient pool of immature precursors. Dysregulation of RAD51L1 and/or BMP2 may explain this familial occurrence of thymomas and autoimmune diseases. Using QRT-PCR, we studied the expression of BMP2 in 20 sporadic thymomas and found various levels of expression that may be associated with autoimmune diseases.

Irregular nuclear shape of bone marrow plasma cells defines a multiple myeloma subgroup related to hypodiploidy and to short survival.

Morphological changes of plasma cells (PC) are common in multiple myeloma (MM). Loss of round or oval nuclear shape has been related to cell malignancy in human, and we looked for the occurrence of such morphological change on PC from bone marrow (BM) smears in a retrospective series of 169 MM patients at diagnosis. Nuclear shape changes of PC differed according to the patients (notch, dumb-bell, folded or monocytoid appearance), even in the same patient; all subtypes were pooled and defined as PC with irregular nuclear shape (PCIN). A significant number of PCIN (>/=5% of all BMPC) was found at diagnosis in 20.7%. Median survival was of 22 months for patients with >/=5% PCIN, and 41 months for others (p=0.0001). Significant relationship was observed with prognostic parameters related intrinsic malignancy of the tumour process but not with beta-2-microglobulin (b2m). A clear-cut relationship was found also between PCIN and hypodiploidy (p=0.0001), but not with deletion of chromosome 13. This study emphasises the relationship between PCIN, an easy-to-ascertain marker of intrinsic malignancy of the tumour process, and adverse prognosis.

Role of multiplex FISH in identifying chromosome involvement in myelodysplastic syndromes and acute myeloid leukemias with complex karyotypes: a report on 28 cases.

Chromosomal abnormalities are found by conventional cytogenetic (CC) analysis in about 50% of myelodysplastic syndromes (MDS) and 70% of acute myeloid leukemias (AML). When cytogenetic abnormalities are complex, multiplex fluorescence in situ hybridization (M-FISH) can help clarify complex chromosomal abnormalities and identify rearrangements with prognostic value or cryptic translocations, which could be preliminary steps in identifying new genes. We studied by M-FISH 28 cases of MDS and AML with complex chromosomal abnormalities, 10 of them were therapy-related. M-FISH allowed the characterization of unidentified chromosomal material in 26 cases (93%). One or several unbalanced rearrangements were observed in 27 cases (96%), generally interpreted as deletions or additional material by CC. Among those translocations, 4 involved 3 chromosomes. Eighteen cryptic translocations undetected by CC were found in 13 cases. By FISH analysis using locus specific probes, TP53 deletion, additional copies of MLL, and additional copies or deletions of RUNX1/AML1 were observed in 16, 4, and 3 cases, respectively. Thus, M-FISH is an important tool to characterize complex chromosomal abnormalities which identified unbalanced and cryptic translocations in 96% and 46% of the cases studied, respectively. Complementary FISH helped us identify involvement of TP53, MLL, and RUNX1/AML1 genes in 82% of cases, confirming their probable role in leukemogenesis.

Factors predicting molecular and cytogenetic response in chronic myeloid leukemia patients treated with imatinib.

We studied 94 clinically heterogeneous chronic myeloid leukemia (CML) patients and found that the duration of treatment with interferon-alpha (IFN-alpha) prior to imatinib therapy may not improve response to imatinib for patients in chronic phase but a shorter period between CML diagnosis and the initiation of imatinib is predictive for a better molecular response to therapy (p<0.05).

Abnormal cytogenetics and significant bone marrow plasmacytosis are predictive of early progression and short survival in patients with low tumor mass asymptomatic multiple myeloma.

In order to evaluate the prognostic factors for progression and survival in patients with a low tumor mass asymptomatic multiple myeloma (MM), we studied 59 patients who had a long term follow-up. Cytogenetic abnormalities (using conventional cytogenetics) were observed in 14 out of 45 analyzable patients (31%). An abnormal karyotype and a bone marrow (BM) plasmacytosis > 15% were found to be adverse prognostic factors for progression in univariate and multivariate analysis.