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Mantle cell lymphoma (MCL) is a subtype of B-cell lymphoma with a large number of recurrent cytogenetic/molecular aberrations. Approximately 5&ndash, 10% of patients do not respond to frontline immunochemotherapy. Despite many useful prognostic indexes, a reliable marker of chemoresistance is not available. We evaluated the prognostic impact of seven recurrent gene aberrations including tumor suppressor protein P53 (TP53) and cyclin dependent kinase inhibitor 2A (CDKN2A) in the cohort of 126 newly diagnosed consecutive MCL patients with bone marrow involvement &ge, 5% using fluorescent in-situ hybridization (FISH) and next-generation sequencing (NGS). In contrast to TP53, no pathologic mutations of CDKN2A were detected by NGS. CDKN2A deletions were found exclusively in the context of other gene aberrations suggesting it represents a later event (after translocation t(11, 14) and aberrations of TP53, or ataxia telangiectasia mutated (ATM)). Concurrent deletion of CDKN2A and aberration of TP53 (deletion and/or mutation) represented the most significant predictor of short EFS (median 3 months) and OS (median 10 months). Concurrent aberration of TP53 and CDKN2A is a new, simple, and relevant index of chemoresistance in MCL. Patients with concurrent aberration of TP53 and CDKN2A should be offered innovative anti-lymphoma therapy and upfront consolidation with allogeneic stem cell transplantation.

Dicentric chromosomes (DCs) are considered markers of cancer in various malignancies. However, they can be overlooked when conventional analysis or multicolor fluorescence in situ hybridization (mFISH) is used to detect complex karyotypes. We analyzed the karyotypes of 114 patients with acute myeloid leukemia (AML) and complex karyotypes and verified the presence of monosomies by FISH using multi-centromeric probes. Monosomy was detected in 63% of patients by G-banding/mFISH and confirmed in 55% of patients by centromeric FISH. FISH analysis indicated a high frequency of DCs that were previously considered monosomies. In some cases, it was apparent that the derivative monocentric chromosome was a primary DC. DCs were formed mostly by chromosomes 17 and 20. In conclusion, chromosome loss and unbalanced translocation suggest the presence of a hidden DC or its previous existence. DCs undergo several stabilizing changes and can induce other chromosomal aberrations and/or the formation of new DCs. This can result in the clonal evolution of abnormal cells, which is considered an independent prognostic marker of an unfavorable disease course and short survival.

Glioblastomas are deadly neoplasms resistant to current treatment modalities. Fibroblast activation protein (FAP) is a protease which is not expressed in most of the normal adult tissues but is characteristically present in the stroma of extracranial malignancies. FAP is considered a potential therapeutic target and is associated with a worse patient outcome in some cancers. The FAP localization in the glioma microenvironment and its relation to patient survival are unknown. By analyzing 56 gliomas and 15 non-tumorous brain samples, we demonstrate increased FAP expression in a subgroup of high-grade gliomas, in particular on the protein level. FAP expression was most elevated in the mesenchymal subtype of glioblastoma. It was neither associated with glioblastoma patient survival in our patient cohort nor in publicly available datasets. FAP was expressed in both transformed and stromal cells; the latter were frequently localized around dysplastic blood vessels and commonly expressed mesenchymal markers. In a mouse xenotransplantation model, FAP was expressed in glioma cells in a subgroup of tumors that typically did not express the astrocytic marker GFAP. Endogenous FAP was frequently upregulated and part of the FAP+ host cells coexpressed the CXCR4 chemokine receptor. In summary, FAP is expressed by several constituents of the glioblastoma microenvironment, including stromal non-malignant mesenchymal cells recruited to and/or activated in response to glioma growth. The limited expression of FAP in healthy tissues together with its presence in both transformed and stromal cells suggests that FAP may be a candidate target for specific delivery of therapeutic agents in glioblastoma.

Richter syndrome represents the transformation of the chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, most frequently the diffuse large B-cell lymphoma (DLBCL). In this report we describe a patient with CLL, who developed a clonally-related pleomorphic highly-aggressive mantle cell lymphoma (MCL) after five cycles of a fludarabine-based second-line therapy for the first relapse of CLL. Molecular cytogenetic methods together with whole-exome sequencing revealed numerous gene alterations restricted to the MCL clone (apart from the canonical t(11;14)(q13;q32) translocation) including gain of one copy of ATM gene or emergence of TP53, CREBBP, NUP214, FUBP1 and SF3B1 gene mutations. Similarly, gene expression analysis revealed vast differences between the MCL and CLL transcriptome, including overexpression of cyclin D1, downregulation of cyclins D2 and D3, or downregulation of IL4R in the MCL clone. Backtracking analysis using quantitative PCR specifically detecting an MCL-restricted focal deletion of TP53 revealed that the pre-MCL clone appeared in the bone marrow and peripheral blood of the patient approximately 4 years before the clinical manifestation of MCL. Both molecular cytogenetic and sequencing data support the hypothesis of a slow development of the pre-MCL clone in parallel to CLL over several years, and thereby exclude the possibility that the transformation event occurred at the stage of the CLL relapse clone by mere t(11;14)(q13;q32) acquisition.

Dicentric chromosomes (DCs) have been described in many hematological diseases, including acute myeloid leukemia (AML). They are markers of cancer and induce chromosomal instability, leading to the formation of other chromosomal aberrations and the clonal evolution of pathological cells. Our knowledge of the roles and behavior of human DCs is often derived from studies of induced DCs and cell lines. It is difficult to identify all the DCs in the karyotypes of patients because of the limitations of metaphase cytogenetic methods. The aim of this study was to revise the karyotypes of 20 AML patients in whom DCs were found with conventional G-banding or multicolor fluorescence in situ hybridization (mFISH) with (multi)centromeric probes and to characterize the DCs at the molecular cytogenetic level. FISH analyses confirmed 23 of the 29 expected DCs in 18 of 20 patients and identified 13 others that had not been detected cytogenetically. Fourteen DCs were altered by other chromosomal changes. In conclusion, karyotypes with DCs are usually very complex, and we have shown that they often contain more than one DC, which can be missed with conventional or mFISH methods. Our study indicates an association between number of DCs in karyotype and very short survival of patients.

Cytogenetic and molecular genetic data are of paramount importance in the diagnosis, prognosis, and risk stratification of patients with malignant diseases. Sometimes they even directly guide the choice of therapy.1 Disease-specific abnormalities, particularly translocations, can provide essential information to assist the Pathologist and/or Oncologist in assigning a diagnosis. In several diseases, tumour genetics correlate strongly with clinical risk; thus, cytogenetic information may help the Oncologist counsel the patient, choose a specific treatment, and/or modulate treatment intensity. Clinical trials may involve cytogenetic classification of patients to the appropriate treatment regimens. Currently, the provision of specific assays for acquired neoplasiaspecific genomic changes varies among and within countries as a range of laboratories offer diagnostic solid tumour genetics; these may include Cytogenetic, Pathology, Haematology, and Molecular Genetics laboratories. Technical standards and general guidelines for the analysis and the report of results on most solid tumours are lacking. To address these deficits, a tumour best practice meeting with invited tumour experts without conflict of interest was held on 23rd April 2013 in Oxford, United Kingdom. The aim was to produce professional guidelines for tumour genetic laboratories and to incorporate the standards imposed by generic European guidelines,2 regulatory bodies (ISO15189, 2012 Medical laboratories – requirements for quality and competence),3 reporting guidelines,4 ISCN,5 and acquired best practice guidelines, while taking into account the current practice in Europe. The guidelines are aimed principally at giving guidance on the minimum, standard cytogenetic analyses, which are applicable to different types of laboratories operating under different regulatory arrangements and are relevant if more specific recommendations are not available. It was universally acknowledged that information on ancillary techniques in use in most cytogenetic laboratories (eg, RTPCR) or advanced techniques not always extensively performed in all laboratories (eg, next-generation sequencing (NGS)) were considered. The process for developing these evidence-based consensus guidelines included agreement on the need of general uniform rules on solid tumour analysis and reporting, discussion on the architecture of the guidelines, working group formation with different tasks (collection, analysis and comparison of any existing guidelines on this subject, type of tumours to be included according to published data and database consultation, method of analysis to be included, report formulation), circulation of the working group activities, formulation and circulation of the initial recommendations, draft and discussion, final consensus, and approval. It is noted that some elements of the tumour diagnostic service not subject to statute may be varied according to local constraints and agreements. Therefore, these guidelines are minimum requirements and additional professional judgment may be of paramount importance under many circumstances. In addition, as new techniques, particularly NGS, as well as clinical evidence, are becoming available all the time, these should be kept under constant review. Notes: The use of ‘must’ in this document indicates a requirement and the use of ‘should’ indicates a recommendation. Where there appears to be contradiction between available guidelines, the most recently published ones should be taken to apply to all. All diagnostic laboratories should be accredited to national or internationally accepted standards (ISO15189).3,6 Laboratories should participate in an External Quality Assessment Scheme7 in all aspects of their service for which a scheme is available.

Chromosomal translocations are acquired genetic rearrangements in human cancers. Jumping translocations are rare nonreciprocal rearrangements involving the same donor chromosome segment translocated to two or more recipient chromosomes. In this report, we describe a patient with Burkitt lymphoma/leukemia (BL) and a complex karyotype including a t(2;8)(p12;q24), copy-neutral loss of heterozygosity at 17p13.1-p13.3 and 19q13.1-q13.2, trisomy 20, and two uncommon chromosomal aberrations. The first uncommon aberration was a complex rearrangement of chromosome 15 (probably the consequence of chromothripsis) masked by an apparently balanced reciprocal translocation, t(11;15)(p11.2;q21). The second one was a special type of unbalanced "vice versa" jumping translocation, which involved the same acceptor chromosome arm (13q) and various donor chromosome segments. It is unclear whether both atypical rearrangements are the consequence of the TP53 alteration or whether assumed chromothripsis influenced the development of the jumping-like translocation. However, the presence of the t(11;15)(p11.2;q21) in all pathological cells suggests that it occurred in the early stage of the disease, whereas the jumping-like translocation, as an additional change, subsequently accelerated the progression of the disease.

MDS with complex chromosomal aberrations (CCA) are characterized by short survival and a high rate of transformation to AML. A comprehensive genome-wide analysis of bone-marrow cells of 157 adults with newly diagnosed MDS and CCA revealed a large spectrum of nonrandom genomic changes related to the advanced stages of MDS. Chromosome shattering, probably resulting from chromothripsis, was found in 47% of patients. Deleted chromosome 5 was unstable and often involved in different types of cryptic unbalanced rearrangements. No true monosomy 5 was observed. Patients with CCA involving deleted chromosome 5 had an extremely poor prognosis (median overall survival, 2 months).

Chromosome 11 abnormalities are found in many hematological malignancies. In acute myeloid leukemia (AML), a proto-oncogene MLL (11q23.3) is frequently altered. However, rearrangements involving other regions of chromosome 11 have been reported. Therefore, we have characterized the chromosome 11 breakpoints and common deleted and amplified areas in the bone marrow or peripheral blood cells of newly diagnosed patients with AML. Using molecular-cytogenetic methods (multicolor fluorescence in situ hybridization (mFISH), multicolor banding (mBAND), microarrays, and FISH with bacterial artificial chromosome (BAC) probes, chromosome 11 abnormalities were delineated in 54 out of 300 (18%) newly diagnosed AML patients. At least 36 different chromosome 11 breakpoints were identified; two were recurrent (11p15.4 in the NUP98 gene and 11q23.3 in the MLL gene), and three were possibly nonrandom: 11p13 (ch11:29.31-31.80 Mb), 11p12 (ch11:36.75-37.49 Mb) and 11q13.2 (68.31-68.52 Mb). One new MLL gene rearrangement is also described. No commonly deleted region of chromosome 11 was identified. However, some regions were affected more often: 11pter-11p15.5 (n = 4; ch11:0-3.52 Mb), 11p14.1-11p13 (n = 4; ch11:28.00-31.00 Mb) and 11p13 (n = 4; ch11:31.00-31.50 Mb). One commonly duplicated (3 copies) region was identified in chromosomal band 11q23.3-11q24 (n = 9; ch11:118.35-125.00 Mb). In all eight cases of 11q amplification (>3 copies), only the 5' part of the MLL gene was affected. This study highlights several chromosome 11 loci that might be important for the leukemogeneic process in AML.

Myelodysplastic syndrome (MDS), a clonal disorder originating from hematopoietic stem cell, is characterized by a progressive character often leading to transformation to acute myeloid leukemia. We used single nucleotide polymorphism arrays (SNP-A) to identify previously cryptic chromosomal abnormalities such as copy number alterations and uniparental disomies (UPD) in cytogenetically normal MDS. In the aberrant regions, we attempted to localize candidate genes with potential relevance to the disease. Using SNP-A, we analyzed peripheral blood granulocytes from 37 MDS patients. The analysis identified 13 cryptic chromosomal defects in 10 patients (27%). Four UPD (affecting chromosomes 3q, 7q, 17q, and 20p), 5 deletions and 4 duplications were detected. Gene expression data measured on CD34+ cells were available for 4 patients with and 6 patients without SNP-A lesions. We performed an integrative analysis of genotyping and gene expression microarrays and found several genes with an altered expression located in the aberrant regions. The expression microarrays suggested BMP2 and TRIB3 located in 20p UPD as potential candidate genes contributing to MDS. We showed that the genome-wide integrative approach is beneficial to the comprehension of molecular backgrounds of diseases with incompletely understood etiopathology. © 2012 Wiley Periodicals, Inc.

PURPOSE: Treatment of childhood acute lymphoblastic leukemia (ALL) was unified in the year 1986 in the Czech Republic using BFM protocols. PATIENTS AND METHODS: Children were treated in 10 and later on in 8 centers localized at the pediatric departments of the biggest hospitals. More than 1.000 children and adolescents up to the age of 18 years were treated between 1986 and 2002 on three consecutive studies ALL-BFM 83, ALL-BFM 90 and ALL-BFM 95. RESULTS: Event-free survival and overall survival improved gradually from 58/62% on ALL-BFM 83 through 70.5/76.6% on ALL-BFM 90 until 72.1/80.2% on ALL-BFM 95. At the same time the incidence of toxic deaths was decreasing and the success in the achievement of complete remission was increasing. Diagnostics in the Czech Republic improved remarkably in the 1990 with gradual introduction of centralized flow cytometry and molecular genetic analysis in one reference laboratory (CLIP = Childhood Leukemia Investigation Prague), which since then has become an internationally respected research center. CONCLUSIONS: Building up a network of closely collaborating leukemia centers covering the whole country, together with the establishment of reference and research laboratories has paved the way for the implementation of the Czech Pediatric Hematology Working Group (CPH) into the international studies Interfant 99, EsPhALL and for the active role in ALL IC-BFM 2002, the I-BFM-SG international randomized trial for treatment of children and adolescents with non-B ALL starting in 2002.

Posterior polymorphous corneal dystrophy (PPCD) is a rare, bilateral autosomal dominant disorder affecting primarily the corneal endothelium and descemet membrane (DM). The aim of this study was to establish the origin of abnormal endothelium in a patient with PPCD exhibiting cornea graft failure after keratoplasty surgery. A sex-mismatched graft obtained from a patient with PPCD who underwent repeat penetrating keratoplasty and the patient's original cornea were investigated. Combined fluorescent immunohistochemistry for cytokeratin (CK) 19 (a marker of aberrant PPCD endothelium) with fluorescence in situ hybridization (FISH) of the sex chromosomes were used in order to characterize the cells on the posterior graft surface. The pathological endothelium of the failed PPCD cornea revealed strong positivity for CK19 using fluorescent immunohistochemistry. In all the CK19-positive cells, both X and Y chromosomes were simultaneously detected using FISH. The results clearly showed the original cells of the patient (XY), within 3.5 years, almost totally overgrown the posterior corneal surface of the graft (XX). Moreover, an abnormal posterior collagenous layer populated by fibroblast-like cells was observed between DM and the endothelium in the failed graft, but its exact origin could not be established due to the low number of cells. Simultaneous detection of CK19 using fluorescent immunohistochemistry together with the detection of gonosomes using FISH was performed for the first time in the cornea and allowed us to prove that the recurrence of PPCD was caused by pathological abnormal proliferation and migration of recipient cells into donor graft.

Patients with near-tetraploid acute myeloid leukemia (NT-AML) typically have poor survival. We present the case of a 67-year-old Caucasian male with NT-AML M0 who had an unusually long first complete remission of 51 months and an overall survival of 80 months. The only characteristic distinguishing him from other previously described patients with NT-AML was the absence of erythroblastic and/or megakaryocytic dysplasia (EMD) at diagnosis. Molecular-genetic testing for AML fusion transcripts associated with a favorable prognosis (PML/RARα,AML1/ETO, and CBFβ/MYH11) were negative, as were other prognostic markers like MLL-PTD,FLT3-ITD, or mutations of FLT3-D835,NPM1, or CEBPA. Expression studies of ERG,MN1, and EVI1 revealed overexpression of ERG only. The absence of EMD may be a useful prognostic/diagnostic feature of this new rare subtype of NT-AML.

Monosomy 7 and/or deletion of the long arm of chromosome 7 is a common cytogenetic aberration in children with myelodysplastic syndrome (MDS) and is associated with poor outcome. In this report, we present an unusual cytogenetic abnormality leading to loss of both the whole short and whole long arms of chromosome 7, which was found in the bone marrow cells of three pediatric patients with MDS. Using a combination of conventional and molecular cytogenetic methods, a tiny "dot-like" marker chromosome was found and described as der(7)del(7)(p11)del(7)(q11). Together with one previously published case, this chromosomal aberration represents a new rare recurrent karyotypic abnormality involving chromosome 7 in children with MDS.

The ETV6/ABL1 (TEL/ABL) fusion gene is a rare aberration in malignant disorders. Only 19 cases of ETV6/ABL1-positive hematological malignancy have been published, diagnosed with chronic myeloid leukemia, other types of chronic myeloproliferative neoplasm, acute myeloid leukemia or acute lymphoblastic leukemia (ALL). This study reports three new cases (aged 8 months, 5 years, and 33 years) of ALL with the ETV6/ABL1 fusion found by screening 392 newly diagnosed ALL patients (335 children and 57 adults). A thorough review of the literature and an analysis of all published data, including the three new cases, suggest poor prognosis of ETV6/ABL1-positive acute leukemias. The course of the disease in the two pediatric patients is characterized by minimal residual disease monitoring, using quantification of both the ETV6/ABL1 transcript and immunoreceptor gene rearrangements. Eosinophilia could not be confirmed as a hallmark of the ETV6/ABL1-positive disease. Studies of neonatal blood spots demonstrated that, in the child diagnosed at five years, the ETV6/ABL1 fusion initiating the ALL originated prenatally.

Objectives: Patients with near-tetraploid (karyotype: 81 - 103 chromosomes) acute lymphoblastic leukemia (NT-ALL) constitute about 1% of childhood ALL and data reported on them are limited and controversial. The aim of the study was to enlarge the knowledge on these rarely occurring ALL. Methods: The members of the European Group for Immunophenotyping of Leukemias (EGIL) searched retrospectively their databases for NT-ALL patients. Results: We collected data of 36 European children from seven European countries with NT-ALL diagnosed since 1992. All patients reached complete remission (CR) after induction chemotherapy. Their blasts were negative for peroxidase and BCR-ABL1. Ten children were diagnosed as T-cell ALL (T-ALL) EGIL categories (T-I n = 2, T-II n = 2, T-III n = 3, T-IV n = 3) and four displayed various structural chromosomal abnormalities. Eight of 10 T-ALL remained in 1st CR; one died in CR from sepsis and one is alive in 2nd CR. Median survival was 88 (7-213) months. B-cell precursor (BCP) ALL was diagnosed in 26 children. Thirteen were positive for ETV6-RUNX1 and are alive in 1st CR for 32-147 months. Ten children were ETV6-RUNX1 negative and remained in 1st CR for 16-163 months. One girl with hypodiploid and NT metaphases and ETV6-RUNX1-negative BCP-ALL and one of two boys with NT-BCP-ALL not examined for ETV6-RUNX1 died of infection after stem cell transplantation in 2nd/3rd CR. Secondary myelodysplastic syndrome developed in two patients with NT-BCP-ALL. Conclusions: Our data demonstrate immunophenotypic, cytogenetic, and molecular heterogeneity of NT-ALL and favorable prognosis of most NT-ALL across different immunophenotypic and/or genetic ALL subtypes.

Gene amplification is a frequent genetic abnormality in solid tumors, and many oncogenes are activated in this way. In acute myeloid leukemia (AML), a frequent target of gene amplification is chromosome 11, particularly chromosome region 11q23, including the MLL (myeloid/lymphoid leukemia) gene. However, the number of other amplicons from the long arm of chromosome 11 has also been described. Duplication/amplification of chromosome 11 was found by cytogenetic methods in 10 of 119 newly diagnosed patients with AML. The amplification was presented as: amplification including only the 5' segment of the MLL gene (1 patient), trisomy 11 (3 patients), partial trisomy 11q (2 patients), isochromosome 11q (1 patient), and multiple amplification of specific regions (3 patients). In two cases, amplification involved parts of not only long arm but also of short arm of the chromosome 11: 11p15 and 11p11.1 to 11p13.

The prognostic significance of 1q21 gain, del(13)(q14), del(17)(p13), t(4;14)(p16.3;q32), and t(11;14)(q13;q32) detected by interphase fluorescein in situ hybridization (FISH) was studied in a cohort of 91 patients with newly diagnosed multiple myeloma (MM). 1q21 gain was detected in 37 of 91 patients (40.7%). In comparison with patients lacking 1q21 gain, patients with 1q21 gain had significantly shorter progression-free survival (PFS) (14.9 versus 27.4 months; P = .044) and worse 4-year overall survival (OS) (40.1% versus 76.2% of patients; P =

Unusual MLL gene rearrangements were found in bone marrow cells of four patients with acute myeloid leukemia. A combination of conventional and molecular cytogenetic methods were used to describe translocations t(9;12;11)(p22;p13;q23), t(11;19)(q23;p13.3), and t(10;11)(p12;23) and inverted insertion ins(10;11)(p12;q23.3q23.1). Partial nontandem duplication of the MLL gene was identified by reverse transcriptase-polymerase chain reaction in all cases. The duplication, which included MLL exons 2 through 8-9, was interrupted by a cryptic insertion of one or two exons from the respective MLL partner gene: MLLT10, MLLT3, or MLLT1.

Specific gene mutations, loss of heterozygosity, deletions and/or amplifications of entire chromosomal regions and gene silencing have been described in gliomas. 82 samples from 81 patients were investigated to detect the deletion of TP53, RB1, CDKN2A genes, deletion of 1p36 and 19q13.3 region, amplification of EGFR gene, trisomy of chromosome 7 and monosomy of chromosome 10 in glial cells. Dual-colour interphase fluorescence in situ hybridization (I-FISH) with locus-specific and/or chromosome enumeration DNA probes were used for cytogenetic analyses. In the study, molecular cytogenetic analyses were successfully performed in 74 patients (91.3%) and were uninformative in 7 only (8.7%). The cytogenetic analyses were correlated with morphological data and clinical outcome. I-FISH was the essential part of diagnostics. In comparison with the clinical data, the patients' age seems to be a factor more important for the overall survival, rather than cytogenetic findings in glial tumours. The combined deletion of 1p36 and 19q13.3 chromosomal regions predicts longer overall survival for patients with oligodendroglial tumours.

We compared the effect of control genes (CG): total Abelson (total-ABL), beta-2-microglobulin (B2M) and beta-glucuronidase (GUS), recommended in the Europe Against Cancer (EAC) program, on real-time BCR-ABL monitoring in patients with chronic myeloid leukemia (CML). We focused on the stability of CG expressions during therapy and the effect of the CGs on BCR-ABL ability to characterize the disease status and disease prognosis, issues that have not been addressed yet. The results showed B2M as a very convenient CG for BCR-ABL monitoring. On the contrary, the widely used total-ABL was not confirmed as appropriate for normalization of gene expression in CML.

Diffuse astrocytomas and oligodendrogliomas (WHO grade II) are the most common histological subtypes of low-grade gliomas (LGGs). Several molecular and epigenetic markers have been identified that predict tumor progression. Our aim was in detail to investigate the genetic and epigenetic background of LGGs and to identify new markers that might play a role in tumor behavior. Twenty-three patients with oligodendroglioma or oligoastrocytoma (LGO) and 22 patients with diffuse astrocytoma (LGA) were investigated using several molecular-cytogenetic and molecular methods to assess their copy number variations, mutational status and level of promoter methylation. The most frequent findings were a 1p/19q codeletion in 83% of LGO and copy-neutral loss of heterozygosity (CN-LOH) of 17p in 72% of LGA. Somatic mutations in the isocitrate dehydrogenase 1 or 2 (IDH1/IDH2) genes were detected in 96% of LGO and 91% of LGA. The O-6-methylguanine-DNA-methyltransferase (MGMT) promoter was methylated in 83% of LGO and 59% of LGA. MutL homolog 3 (MLH3) promoter methylation was observed in 61% of LGO and 27% of LGA. Methylation of the MGMT promoter, 1p/19q codeletion, mutated IDH1, and CN-LOH of 17p were the most frequent genetic aberrations in LGGs. The findings were more diverse in LGA than in LGO. To the best of our knowledge, this is the first time description of methylation of the MLH3 gene promoter in LGGs. Further studies are required to determine the role of the methylated MLH3 promoter and the other aberrations detected.

During progression of chronic myeloid leukemia (CML) from the chronic to the accelerated phase and/or blast crisis, clonal evolution with nonrandom secondary aberrations such as +8, +Ph, i(17q), +19, -Y, +21, +17, and -7 is frequently observed. Complex chromosomal rearrangements (CCR) are rather rare, and the significance and frequency of different anomalies are poorly understood. The aim of this study was to determine the chromosomes and chromosomal regions which are involved in CCR during progression of the disease and the frequency of nonrandom changes. Conventional cytogenetics, FISH, and multicolor FISH (mFISH) were used to study karyotypes of 18 CML patients with CCR ascertained by G-banding. Most often involved in CCR were chromosomes 2 (x6); 3, 7, and 17 (x5); 1 and 4 (x4); and 5, 6, 11, and 12 (x3); regions 1q, 2q, 5q, 7p, and 17p; and breakpoints 17p11.2 (x3) and 7p15 (x2). There were no recurrent complex translocations. The present findings demonstrate the very high instability of the genome of malignant cells at the chromosomal level. Precise determination of breakpoints involved in CCR can give new dimension to the understanding of genetic mechanisms which play role in progression of malignant disease.

B-chronic lymphocytic leukemia (B-CLL) is the most common adult leukemia. Molecular genetic characterization of B-CLL has made significant progress and typical chromosomal anomalies have been assessed. The most frequent chromosomal abnormalities are deletions at 13q14, 17p13, and 11q22 approximately q23 and trisomy 12. The aim of this study was to establish incidence of chromosomal changes in bone marrow or peripheral blood cells (or both) of B-CLL patients using a molecular cytogenetic method, interphase fluorescence in situ hybridization (I-FISH), and to evaluate the prognostic implications. We performed I-FISH on bone marrow and blood smears from 217 B-CLL patients (124 male, 93 female). Trisomy 12 was found in 35 of the 217 (16%); deletion 13q14 was analyzed in 207 patients and found in 112 (54%). Deletion 17p13 was found in 34 (16%) out of 206 examined. Deletion of 11q23 was analyzed in 56 patients and was present in 7 (12%). Statistical analyses were performed to correlate the molecular-cytogenetic findings with disease status (stable versus progressive), Rai stage, CD38/CD19 antigen coexpression, immunoglobulin variable heavy chain (IgV(H)) mutational pattern, and other clinical and laboratory parameters. No apparent differences in distribution were noted for anomalies +12, del(13)(q14), or del(17)(p13) among patients with stable and progressive disease, and no consistent pattern in the distribution of type of genomic changes were found among various Rai stages and in CD38/CD19-positive or -negative patients. Patients without IgV(H) mutation had a worse prognosis; however, distribution of chromosomal abnormalities identified with FISH was the same for patients with and without IgV(H) mutations.

Telomere length was evaluated by terminal repeat fragment method (TRF) in 50 patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) arising from MDS and in 21 patients with untreated primary AML to ascertain, whether telomere erosion was associated with progression of MDS towards overt leukemia. Heterogeneity of TRF among MDS FAB subgroups ( P =0.004) originated from its shortening in increased number of patients during progression of the disease. Chromosomal aberrations were present in 32% MDS patients with more eroded telomeres ( P =0.022), nevertheless a difference between mean TRF in the subgroups with normal and abnormal karyotype diminished during progression of MDS. A negative correlation between individual TRF and IPSS value ( P =0.039) showed that telomere dynamics might serve as a useful prognostic factor for assessment of an individual MDS patient’s risk and for decision of an optimal treatment strategy.

Ploidy is an important prognostic factor in the risk stratification of multiple myeloma (MM) patients. Patients with MM can be divided into two groups according to the modal number of chromosomes: nonhyperdiploid (NH-MM) and hyperdiploid (H-MM), which has a more favorable outcome. The two ploidy groups represent two different oncogenetic pathways determined at the premalignant stage. The ploidy subtype also persists during the course of the disease, even during progression after the therapy, with only very rare cases of ploidy conversion. The clinical significance of ploidy conversion and its relation to drug resistance have been previously discussed. Here, we describe a female MM patient with a rare change in her ploidy status from H-MM to NH-MM, detected by cytogenetic and molecular cytogenetic examinations of consecutive bone marrow aspirates. We hypothesize that ploidy conversion (from H-MM to NH-MM) is associated with disease progression and acquired resistance to bortezomib/lenalidomide therapy.

The BCR/ABL rearrangement, the molecular hallmark of chronic myelogenous leukaemia (CML), is rare in acute myeloid leukaemia (AML), being detected in approximately 1% of cases. In the vast majority of CML cases the breakpoint on chromosome 22 falls in the so-called major breakpoint cluster region of the BCR gene. Only a few cases of CML with breakpoint in the minor or in the micro bcr region have so far been reported. The micro breakpoint position has been associated mainly with a mild form of CML, defined as Philadelphia chromosome-positive chronic neutrophilic leukaemia (Ph-positive CNL). Using reverse transcription-polymerase chain reaction (RT-PCR) we report a patient with an acute myeloid leukaemia phenotype at diagnosis who showed a BCR/ABL rearrangement with a breakpoint located in the micro bcr region (e19a2 junction). Cytogenetic analysis showed a progression of the malignant clone, finally leading to cells with two Ph chromosomes, trisomy 8, isochromosome 17q and deletion of the long arms of chromosome 7. The findings of chromosomal changes point to a possibility of blast crisis of CML with a clinically silent chronic phase. Immunoprecipitation and auto-phosphorylation assay revealed the expression, by the patient's blast cells, of an abnormal P230 BCR/ABL protein, which showed for the first time that this protein was constitutively activated in primary cells from patients. This finding may contribute to the understanding of the role of the BCR/ABL rearrangements in determining different leukaemia phenotypes ranging from acute lymphoid and myeloid leukaemias to mild chronic neutrophilic leukaemias.

We used microarray profiling to investigate the direct effects of lenalidomide on gene expression in isolated CD14(+) monocytes from 6 patients with del(5q). Our data demonstrate that changes in genes involved the tumor necrosis factor (TNF) signaling pathway and the bone marrow stroma, suggesting that treatment with lenalidomide may help restore the damaged niche and suppress the TNF signaling pathway.Lenalidomide is an effective treatment for patients with del(5q) and myelodysplastic syndrome (MDS) The exact mechanism of lenalidomide function and its impact on the prognosis of patients is not known exactly.We used gene expression profiling to study the effect of lenalidomide therapy in peripheral blood CD14(+) monocytes of 6 patients with del(5q) and MDS.After lenalidomide treatment, genes involved in the tumor necrosis factor (TNF) signaling pathway that were upregulated in the patients before treatment decreased to the healthy control baseline expression level. This change in gene expression, in conjunction with increased expression of repressed genes that affect the stem cell niche (ie, CXCR4 and CRTAP), may exert a positive effect on treated patients. In contrast, we found that increased expression of the ARPC1B gene may have a negative impact on the stability of patient remission.The observed changes in gene expression described here may contribute to the identification of pathways that are affected by lenalidomide, which may help to explain the effects of this drug.

Detection of Flt3-internal tandem duplication (ITD) in acute myelogenous leukemia (AML) blasts at the time of diagnosis is associated with an increased risk of leukemia relapse after chemotherapy. In the present report, we describe two patients with Flt3-abnormalities that were only detectable at the time of relapse. One of the patients had an ITD at the time of diagnosis (duplicated region 41-133 numbered from the start of exon 14 plus CGG insertion), but at the time of relapse a new abnormality (duplicated region 70-230 plus insertion GGGGAGT) was detected as the only Flt3-abnormality. The second patient showed no Flt3-ITD at the time of diagnosis, but ITD was detected at the time of relapse (duplicated region 12-96, no insertion). Our observations of independent Flt3-ITD and the detection of new Flt3-abnormalities at the time of relapse suggest that (i) Flt3-gene abnormalities may be even more important in the pathogenesis of chemotherapy-resistant AML than suggested from previous studies of newly diagnosed AML, and (ii) monitoring of minimal residual disease by using patient-specific real-time polymerase chain reaction assays for Flt3-abnormalities may not be reliable.

Purpose Because cytogenetic data are essential for risk stratification of childhood acute myeloid leukemia (AML), the impact of chromosomal aberrations is crucial. Patients and Methods Data of a large group of patients younger than 18 years treated according to study AML–Berlin-Frankfurt-Münster (BFM) 98 (n = 454), including their cytogenetics, were analyzed. Results The favorable outcome in the subgroups of patients with t(8;21), inv(16), and t(15;17), with an overall survival of 91% (SE, 4%), 92% (SE, 6%), and 87% (SE, 5%), respectively, was confirmed. Within this group, the 5-year probability of event-free survival (pEFS) of all 17 children with t(8;21) and additional aberrations apart from del(9q) or −X/−Y was 100%. As expected, the cytogenetic finding of a complex karyotype (n = 35; pEFS, 33%; SE, 8%) or a monosomy 7 (n = 12; pEFS, 17%; SE, 11%) was associated with a poor outcome. Compared with remaining patients with cytogenetic data (pEFS, 48%; SE, 2%), prognosis in patients with an MLL rearrangement (n = 91) was inferior (pEFS, 34%; SE, 5%; P = .0005). Particularly, children with t(9;11) and additional aberrations (n = 13; pEFS, 31%; SE, 14%) and MLL rearrangements other than t(9;11) and t(11;19) (n = 41; pEFS, 24%; SE, 7%) had an unfavorable outcome. Nine patients with aberrations in 12p showed an adverse prognosis (pEFS, 11%; SE, 10%). The outcome of patients with aberrations of chromosome 5 (n = 13) was better than expected (pEFS, 50%; SE, 13%). Conclusion Because the prognostic value of rare recurrent chromosomal aberrations still has to be elucidated, these data will contribute to future risk stratification for the treatment of pediatric AML.