BCR-ABL1 positive AML or CML in blast crisis? A pediatric case report with inv(3) and t(9;22) in the initial clone.

The co-occurrence of an inversion inv(3)(q21q26)/GATA2-MECOM and a Philadelphia translocation t(9;22)(q34;q11)/BCR-ABL1 in the context of chronic myeloid leukemia (CML) in blast crisis or acute myeloid leukemia (AML) has only rarely been described. To our knowledge, this co-occurrence has been reported in six pediatric patients with CML but not in pediatric patients with AML. Here, we report on a 7-year-old girl, who, presented with a t(9;22) and inv(3) in 14 of 15 metaphases and an additional monosomy 7 was detected in 5 of these metaphases (ISCN: 46,​XX,​inv(3)(q21q26),​t(9;22)(q34q11)[9]/45,​idem,​-7[5]/46,​XX[1]). The p190 BCR-ABL1 fusion transcript was detected by multiplex PCR and targeted RNA sequencing. Due to these results, a clear distinction between a CML in blast crisis and a BCR-ABL1 positive AML was not possible. The patient was treated according to the treatment recommendations of the AML-BFM study group and additionally received tyrosine kinase inhibitor therapy (Dasatinib). The treatment with Dasatinib was successful in eliminating the inv(3)/t(9;22) clone, but the ancestral inv(3) clone persisted. Based upon these findings we diagnosed an AML with inv(3) and a secondary acquisition of t(9;22). This treatment as well as an allogenic transplantation has led to a complete remission of the disease up to this date (21 months post diagnosis).

Unbalanced translocation der(5;17) resulting in a TP53 loss as recurrent aberration in myelodysplastic syndrome and acute myeloid leukemia with complex karyotype.

A complex karyotype, detected in myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML), is associated with a reduced median survival. The most frequent chromosomal aberrations in complex karyotypes are deletions of 5q and 17p harboring the tumor suppressor gene TP53. The unbalanced translocation der(5;17) involving chromosome 5q and 17p is a recurrent aberration in MDS/AML, resulting in TP53 loss. We analyzed the karyotypes of 178 patients with an unbalanced translocation der(5;17) using fluorescence R-/G-banding analysis. Whenever possible, fluorescence in situ hybridization (FISH) (n = 138/141), multicolor FISH (n = 8), telomere length measurement (n = 9), targeted DNA sequencing (n = 13), array-CGH (n = 7) and targeted RNA sequencing (n = 2) were conducted. The der(5;17) aberration was accompanied with loss of genetic material in 7q (53%), -7 (27%), gain of 21q (29%), +8 (17%) and - 18 (16%) and all analyzed patients (n = 13) showed a (likely) pathogenic variant inTP53. The der(5;17) cohort showed significantly shortened telomeres in comparison to the healthy age-matched controls (P < .05), but there was no significant telomere shortening in comparison to MDS/AML patients with a complex karyotype without der(5;17). No fusion genes resulted from the unbalanced translocation. This study demonstrates that the unbalanced translocation der(5;17) is associated with a biallelic inactivation of TP53 due to a deletion of TP53 in one allele and a pathogenic variant of the second TP53 allele. Since the breakpoints are located within (near-) heterochromatic regions, alterations of DNA methylation or histone modifications may be involved in the generation of der(5;17).

Frequency and prognostic impact of PAX5 p.P80R in pediatric acute lymphoblastic leukemia patients treated on an AIEOP-BFM acute lymphoblastic leukemia protocol.

PAX5 is a member of the paired box (PAX) family of transcription factors involved in B-cell development. PAX5P80R has recently been described as a distinct genetic B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) subtype with a favorable prognosis in adults. In contrast, an unfavorable outcome has been observed in children. Our aim was to determine the frequency of PAX5P80R in childhood BCP-ALL treated according to the Associazione Italiana Ematologia ed Oncologia Pediatrica-Berlin-Frankfurt-Muenster (AIEOP-BFM) ALL 2000 protocol and to evaluate its clinical significance within this study cohort. The analyses included 1237 patients with ALL treated in the AIEOP-BFM ALL 2000 trial with complete information for copy number variations (CNVs) of IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A, CDKN2B, and ERG. A customized TaqMan genotyping assay was used to screen for PAX5P80R . Sanger sequencing was used to confirm PAX5P80R -positive results as well as to screen for second variants in PAX5. Agilent CGH + SNP arrays (e-Array design 85 320; Agilent Technologies) were performed in PAX5P80R -positive patients to verify additional CNVs. Almost 2% (20/1028) of our BCP-ALL cohort were PAX5P80R -positive. White blood cell counts higher than 50 000/µl as well as male sex were significantly (P < .05) associated with PAX5P80R . Most of the PAX5P80R -positive cases were 10 years of age or older. PAX5P80R -positive samples were enriched for deletions affecting PAX5, IKZF1, CDKN2A, and CDKN2B. Compared to PAX5P80R -wildtype BCP-ALL, PAX5P80R -positive patients showed a significantly reduced 5-year overall survival (P = .042). Further studies should evaluate the interaction of PAX5P80R with other genetic aberrations to further stratify intermediate risk pediatric BCP-ALL.

The identification of pathogenic variants in BRCA1/2 negative, high risk, hereditary breast and/or ovarian cancer patients: High frequency of FANCM pathogenic variants.

NGS-based multiple gene panel resequencing in combination with a high resolution CGH-array was used to identify genetic risk factors for hereditary breast and/or ovarian cancer in 237 high risk patients who were previously tested negative for pathogenic BRCA1/2 variants. All patients were screened for pathogenic variants in 94 different cancer predisposing genes. We identified 32 pathogenic variants in 14 different genes (ATM, BLM, BRCA1, CDH1, CHEK2, FANCG, FANCM, FH, HRAS, PALB2, PMS2, PTEN, RAD51C and NBN) in 30 patients (12.7%). Two pathogenic BRCA1 variants that were previously undetected due to less comprehensive and sensitive methods were found. Five pathogenic variants are novel, three of which occur in genes yet unrelated to hereditary breast and/or ovarian cancer (FANCG, FH and HRAS). In our cohort we discovered a remarkably high frequency of truncating variants in FANCM (2.1%), which has recently been suggested as a susceptibility gene for hereditary breast cancer. Two patients of our cohort carried two different pathogenic variants each and 10 other patients in whom a pathogenic variant was confirmed also harbored a variant of unknown significance in a breast and ovarian cancer susceptibility gene. We were able to identify pathogenic variants predisposing for tumor formation in 12.3% of BRCA1/2 negative breast and/or ovarian cancer patients.

GT198 (PSMC3IP) germline variants in early-onset breast cancer patients from hereditary breast and ovarian cancer families.

GT198, located 470 kb downstream of BRCA1, encodes for the nuclear PSMC3-interacting protein, which functions as co-activator of steroid hormone-mediated gene expression, and is involved in RAD51 and DMC1-mediated homologous recombination during DNA repair of double-strand breaks. Recently, germline variants in GT198 have been identified in hereditary breast and ovarian cancer (HBOC) patients, mainly in cases with early-onset. We screened a cohort of 166 BRCA1/2 mutation-negative HBOC patients, of which 56 developed early-onset breast cancer before the age of 36 years, for GT198 variants. We identified 7 novel or rare GT198 variants in 8 out of 166 index patients: c.-115G>A (rs191843707); c.-70T>A (rs752276800); c.-37A>T (rs199620968); c.-24C>G (rs200359709); c.519G>A p.(Trp173*); c.537+51G>C (rs375509656); c.*24G>A. Three out of 7 identified variants (c.-115G>A, c.519G>A and c.*24G>A) with putative pathogenic impact were found in HBOC patients with breast cancer onset at ≤ 36 years. The nonsense mutation c.519G>A p.(Trp173*) was located within the DNA binding domain of GT198 and is predicted to induce nonsense-mediated mRNA decay. Functional analyses of c.-115G>A, and c.*24A>G indicated an influence of these variants on gene expression. This is the second study that gives evidence for an association between pathogenic GT198 germline variants and early-onset breast cancer in HBOC.

Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase.

Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puroΔtk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.

Array-CGH in childhood MDS.

To study genomic imbalances potentially involved in disease development and/or progression of childhood MDS, array-based comparative genomic hybridization (aCGH) is a helpful tool. Copy number alterations (CNA) of subtle chromosomal regions containing potential candidate genes, e.g., TP53 or RUNX1 can be detected. However, characterizing small and/or heterogeneous tumor subpopulations by high-resolution aCGH within a majority of normal cells is a challenge in MDS and requires validation by independent methods like FISH or quantitative PCR. For the identification of tumor-relevant CNA, the analysis of DNA isolated from purified granulocytes or myeloid populations instead of DNA from whole bone marrow (BM) cells is helpful to overcome some of these limitations.

Induced G1 phase arrest of fast-dividing cells improves the quality of genomic profiles generated by array-CGH.

Genome-wide profiling of copy number alterations by array-based high resolution comparative genomic hybridization (array-CGH) is an important method to ensure the genomic integrity of cells in diverse conditions. We observed that the analysis of genomic profiles, in particular of fast-dividing murine leukemia cell lines, is challenging due to characteristic patterns oscillating around the array-CGH baseline. Here we show array-CGH data can be drastically improved by reducing proliferation rates of cultured cells using deprivation protocols or cell cycle inhibitors. Arresting cell cycle in the G1 phase leads to smoother genomic profiles, and hence to a more reliable detection of copy number alterations.

Constitutional trisomy 8p11.21-q11.21 mosaicism: a germline alteration predisposing to myeloid leukaemia.

Juvenile myelomonocytic leukaemia (JMML) is a unique myeloproliferative disorder of early childhood. Frequently, mutations in NRAS, KRAS, PTPN11, NF1 or CBL are found in these patients. Monosomy 7 is the most common cytogenetic aberration. To identify submicroscopic genomic copy number alterations, 20 JMML samples were analysed by comparative genomic hybridization. Ten out of 20 samples displayed additional submicroscopic alterations. In two patients, an almost identical gain of chromosome 8 was identified. In both patients, fluorescence in situ hybridization confirmed a constitutional partial trisomy 8 mosaic (cT8M). A survey on 27 cT8M patients with neoplasms showed that 21 had myeloid malignancies, and five of these had a JMML. Notably, the region gained in our cases is the smallest gain of chromosome 8 reported in cT8M cases with malignancies so far. Our results dramatically reduce the critical region to 8p11.21q11.21 harbouring 31 protein coding genes and two non-coding RNAs, e.g. MYST3, IKBKB, UBE2V2, GOLGA7, FNTA and MIR486--a finding with potential implications for the role of somatic trisomy 8 in myeloid malignancies. Further investigations are required to more comprehensively determine how constitutional partial trisomy 8 mosaicisms may contribute to leukaemogenesis in different mutational subtypes of JMML and other myeloid malignancies.

Clonal heterogeneity in childhood myelodysplastic syndromes--challenge for the detection of chromosomal imbalances by array-CGH.

To evaluate whether copy number alterations (CNAs) are present that may contribute to disease development and/or progression of childhood myelodysplastic syndromes (MDS), 36 pediatric MDS patients were analyzed using array-based comparative genome hybridization (aCGH). In addition to monosomy 7, the most frequent chromosome aberration in childhood MDS, novel recurrent CNAs were detected. They included a loss of 3p14.3-p12.3, which contains the putative tumor suppressor gene FHIT, a loss of 7p21.3-p15.3, a loss of 9q33.3-q34.3 (D184) and microdeletions in 17p11.2, 6q23 containing MYB, and 17p13 containing TP53. In this small patient cohort, patients without CNA, patients with monosomy 7 only and patients with one CNA in addition to monosomy 7 did not differ in their survival. As expected, all patients with complex karyotypes, including two patients with deletions of TP53, died. A challenge inherent to aCGH analysis of MDS is the low percentage of tumor cells. We evaluated several approaches to overcome this limitation. Genomic profiles from isolated granulocytes were of higher quality than those from bone marrow mononuclear cells. Decreased breakpoint calling stringency increased recognition of CNAs present in small clonal populations. However, further analysis using a custom-designed array showed that these CNAs often did not confirm the findings from 244k arrays. In contrast, constitutional CNVs were reliably detected on both arrays. Moreover, aCGH on amplified DNA from distinct myeloid clusters is a new approach to determine CNAs in small subpopulations. Our results clearly emphasize the need to verify array-CGH results by independent methods like FISH or quantitative PCR.

Analysis of array-CGH data using the R and Bioconductor software suite.

BACKGROUND: Array-based comparative genomic hybridization (array-CGH) is an emerging high-resolution and high-throughput molecular genetic technique that allows genome-wide screening for chromosome alterations. DNA copy number alterations (CNAs) are a hallmark of somatic mutations in tumor genomes and congenital abnormalities that lead to diseases such as mental retardation. However, accurate identification of amplified or deleted regions requires a sequence of different computational analysis steps of the microarray data. RESULTS: We have developed a user-friendly and versatile tool for the normalization, visualization, breakpoint detection, and comparative analysis of array-CGH data which allows the accurate and sensitive detection of CNAs. CONCLUSION: The implemented option for the determination of minimal altered regions (MARs) from a series of tumor samples is a step forward in the identification of new tumor suppressor genes or oncogenes.

Copy number alterations in childhood acute lymphoblastic leukemia and their association with minimal residual disease.

In vivo response to initial therapy, as assessed by determination of minimal residual disease (MRD) after 5 and 12 weeks of treatment, has evolved as a strong prognostic factor in children with acute lymphoblastic leukemia (ALL) treated according to the BFM regime. Individual treatment response may be influenced by copy number alterations (CNA) leading to altered gene expression. We aimed to evaluate CNA using high-resolution array-comparative genomic hybridization (array-CGH) in different treatment-response groups. Leukemic genomic profiles of 25 standard risk (MRD-SR) and 25 high risk (MRD-HR) patients were compared. CNAs were found in 46/50 patients (92%). The most significant difference was a gain of 1q23-qter because of an unbalanced t(1;19), found in 10/25 MRD-SR patients, but in none of the MRD-HR patients (P < 0.001). The most frequent CNAs in the MRD-HR group were deletions of genomic regions harboring the immunoglobulin genes (Ig), e.g., 2p11.2 in 60% of MRD-HR compared to 28% of MRD-SR (P = 0.045). Combining all Ig loci, significantly more MRD-HR than MRD-SR patients displayed deletions (17:8 patients, P = 0.02). Frequency of other CNAs, such as loss of 9p21 or gains of 21q, did not differ strongly between the two patient groups. This is the first study evaluating the clinical significance of CNA as detected by array-CGH in childhood ALL and the first to suggest that such analyses may provide clinically important data. This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Promoter methylation of PARG1, a novel candidate tumor suppressor gene in mantle-cell lymphomas.

BACKGROUND AND OBJECTIVES: Mantle cell lymphoma (MCL), a mature B-cell neoplasm, is genetically characterized by the translocation t(11;14)(q13;q32). However, secondary alterations are required for malignant transformation. The identification of inactivated tumor suppressor genes contributing to the development of MCL may lead to further elucidation of the biology of this disease and help to identify novel targets for therapy. DESIGN AND METHODS: Whole genome microarray-based gene expression profiling on treated versus untreated MCL cell lines was used to identify genes induced by 5-aza-2'-deoxycytidine. The degree of promoter methylation and transcriptional silencing of selected genes was then proven in MCL cell lines and primary cases by methylation-specific polymerase chain reaction (PCR) techniques, real-time PCR and gene expression profiling. RESULTS: After 5-aza-2'-deoxycytidine treatment, we identified more than 1000 upregulated genes, 16 of which were upregulated > or =3-fold. Most of them were not known to be silenced by methylation in MCL. A low expression of ING1, RUNX3 and BNIP3L was observed in three of the five the MCL cell lines. In addition, the expression of PARG1, which is located in the frequently deleted region 1p22.1, was substantially reduced and displayed at least partial promoter methylation in all investigated MCL cell lines as well as in 31 primary MCL cases. INTERPRETATION AND CONCLUSIONS: In summary, we identified interesting novel candidate genes that probably contribute to the progression of MCL and suggest that PARG1 is a strong candidate tumor suppressor gene in MCL.

Assessment of differentiation and progression of hepatic tumors using array-based comparative genomic hybridization.

BACKGROUND & AIMS: To gain more information about the molecular mechanisms leading to dedifferentiation of hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC), high-resolution array-based comparative genomic hybridization (array-CGH) was performed on 24 cases of HCC and 10 cases of HCA. METHODS: DNA chips containing 6251 individual bacterial artificial chromosome/plasmid artificial chromosome clones were used. They allowed for a genome-wide resolution of 1 Mb and an even higher resolution of up to 100 kb for chromosome regions recurrently involved in human tumors and for regions containing known tumor-suppressor genes and oncogenes. RESULTS: Copy number changes on the genomic scale were found by array-based comparative genomic hybridization in all cases. In HCC, gains of chromosomal regions 1q (91.6%), and 8q (58.3%), and losses of 8p (54%) were found most frequently. Hierarchic cluster analysis branched all HCA from HCC. However, in 2 adenomas with a known history of glycogenosis type I and adenomatosis hepatis gains of 1q were found, too. The critically gained region was narrowed down to bands 1q22-23. Although no significant differences in the mean number of chromosomal aberrations were seen between adenomas and well-differentiated carcinomas (2.7 vs 4.6), a significant increase accompanied the dedifferentiation of HCC (14.1 in HCC-G2 and 16.3 in HCC-G2/3; P < .02). Dedifferentiation of HCC also was correlated closely to losses of 4q and 13q (P <.001 and <.005, respectively). CONCLUSIONS: The increased chromosomal instability during dedifferentiation of HCC leads to an accumulation of structural chromosomal aberrations and losses and gains of defined chromosome regions.

Gene-expression profiles and their association with drug resistance in adult acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: From 20-50% of patients with acute myeloid leukemia (AML) are primarily resistant to induction chemotherapy. It has previously been shown that resistance to the first cycle of induction chemotherapy is an independent prognostic factor. We investigated whether resistance to chemotherapy be represented by gene-expression profiles, and which genes are associated with resistance. DESIGN AND METHODS: cDNA microarrays containing approximately 41,000 features were used to compare the gene-expression profile of AML blasts between 33 patients with good or poor response to induction chemotherapy. Data generated by cDNA-arrays were confirmed by quantitative reverse transcription polymerase chain reaction. RESULTS: Using significance analysis of microarrays, we identified a characteristic gene-expression profile which distinguished AML samples from patients with good or poor responses. In hierarchical clustering analysis poor responders clustered together with normal CD34+ cells. Moreover, 13/40 (32.5%) genes highly expressed in poor responders are also overexpressed in hematopoietic stem/progenitor cells. Prediction analysis using 10-fold cross-validation revealed an 80% overall accuracy. Using the treatment-response signature to predict the outcome in an independent test set of 104 AML patients, samples were separated into two subgroups with significantly inferior response rate (43.5% vs. 66.7%, p=0.04), significantly shorter event-free and overall survival (p=0.01 and p=0.03, respectively) in the poor-response compared to in the good-response signature group. In multivariate analysis, the treatment-response signature was an independent prognostic factor (hazard ratio, 2.1, 95% confidence interval 1.2 to 3.6, p=0.006). INTERPRETATION AND CONCLUSIONS: Resistance to chemotherapy in AML can be identified by gene-expression profiling before treatment and seems to be mediated by a transcriptional program active in hematopoietic stem/progenitor cells.