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Complete set of CGH results of advanced breast cancer in 31 tumor cases

Genomic imbalances in 31 formalin-fixed and paraffin-embedded primary tumors of advanced breast cancer were analyzed by microarray-based comparative genomic hybridization (matrix-CGH). A DNA chip was designed comprising 422 mapped genomic sequences including 47 proto-oncogenes, 15 tumor suppressor genes, as well as frequently imbalanced chromosomal regions. Analysis of the data was challenging due to the impaired quality of DNA prepared from paraffin-embedded samples. Nevertheless, using a method for the statistical evaluation of the balanced state for each individual experiment, we were able to reveal imbalances with high significance, which were in good concordance with previous data collected by chromosomal CGH from the same patients. Owing to the improved resolution of matrix-CGH, genomic imbalances could be narrowed down to the level of individual bacterial artificial chromosome and P1-derived artificial chromosome clones. On average 37 gains and 13 losses per tumor cell genome were scored. Gains in more than 30% of the cases were found on 1p, 1q, 6p, 7p, 8q, 9q, 11q, 12q, 17p, 17q, 20q, and 22q, and losses on 6q, 9p, 11q, and 17p. Of the 51 chromosomal regions found amplified by matrix-CGH, only 12 had been identified by chromosomal CGH. Within these 51 amplicons, genome database information defined 112 candidate genes, 44 of which were validated by either PCR amplification of sequence tag sites or DNA sequence analysis.

PURPOSE DNA-hypomethylating agents are studied in combination with other epigenetic drugs, such as histone deacetylase inhibitors or differentiation inducers (eg, retinoids), in myeloid neoplasias. A randomized, phase II trial with a 2 × 2 factorial design was conducted to investigate the effects of the histone deacetylase inhibitor valproate and all- trans retinoic acid (ATRA) in treatment-naive elderly patients with acute myeloid leukemia (AML). PATIENTS AND METHODS Two hundred patients (median age, 76 years; range, 61-92 years) ineligible for induction chemotherapy received decitabine (20 mg/m2 intravenously, days 1 to 5) alone (n = 47) or in combination with valproate (n = 57), ATRA (n = 46), or valproate + ATRA (n = 50). The primary endpoint was objective response, defined as complete and partial remission, tested at a one-sided significance level of α = .10. Key secondary endpoints were overall survival, event-free survival, and progression-free survival and safety. RESULTS The addition of ATRA resulted in a higher remission rate (21.9% with ATRA v 13.5% without ATRA; odds ratio, 1.80; 95% CI, 0.86 to 3.79; one-sided P = .06). For valproate, no effect was observed (17.8% with valproate v 17.2% without valproate; odds ratio, 1.06; 95% CI, 0.51 to 2.21; one-sided P = .44). Median overall survival was 8.2 months with ATRA v 5.1 months without ATRA (hazard ratio, 0.65; 95% CI, 0.48 to 0.89; two-sided P = .006). Improved survival was observed across risk groups, including patients with adverse cytogenetics, and was associated with longer response duration. With valproate, no survival difference was observed. Toxicities were predominantly hematologic, without relevant differences between the 4 arms. CONCLUSION The addition of ATRA to decitabine resulted in a higher remission rate and a clinically meaningful survival extension in these patients with difficult-to-treat disease, without added toxicity.

Background TP53 mutations are associated with adverse outcome of AML treated with cytarabine-based regimens. Interestingly, DNA-hypomethylating agents (HMAs) induce a higher response rate in TP53-mutated (MUT) compared to TP53 wildtype (WT) AML (Welch et al., N. Engl. J. Med. 2016, Döhner et al., Leukemia 2018). We conducted a randomized phase II trial (NCT00867672, 2x2 factorial design) asking whether the in vitro cooperativity of DAC with VPA or ATRA translates into clinical benefit. While VPA added to DAC affected neither objective response rate (ORR) nor overall survival (OS), ATRA significantly improved ORR and OS, without added toxicity (Lübbert et al., J. Clin. Oncol. 2020). Preclinical data suggest that HMAs and ATRA have cooperative effects also in TP53 MUT AML. We therefore performed a post-hoc analysis to determine the predictive impact of TP53 status. Patients and Methods Key inclusion criteria: newly diagnosed AML pts >60 yr (non-M3) unfit for induction, ECOG performance status (PS) 0-2. Treatment: DAC 20 mg/m 2 day 1-5 (arms A/B/C/D), VPA p.o. from day 6 (arms B/D), ATRA p.o. day 6-28 (arms C/D) of each 28-day course. For TP53 mutation analyses, the Illumina TruSight Myeloid Sequencing Panel was used for library preparation and an Illumina MiSeq device for sequencing. Key endpoints: ORR (CR/CRi/PR, ELN 2010 criteria) and OS. Original sample size calculation of a total of 200 patients (pts) was based on the primary endpoint ORR (Lübbert et al., Haematologica 2012). ORR was analyzed with logistic regression, OS with Cox regression. Odds ratios (OR) for the effect on ORR and hazard ratios (HR) for the effect on death with 95% confidence intervals (CI) are presented in the genetic subgroups TP53 MUT and TP53 WT including tests for interactions (TFI) between treatment and TP53. These are post-hoc exploratory analyses, hence p-values have to be interpreted in a descriptive sense. Results Between 12/2011 and 2/2015, 200 pts were randomized and treated. Information on TP53 status was available for 168 of 200 pts (84%); 155 of them (92%) had died at last follow-up (June 2021). 61% of pts were aged >75 years (range 61-92), ECOG PS 0/1/2: 19/62/19% (a single pt had a PS of 3); 53% had an HCT-CI >3, 19% WBC >30.000/µl, 30% adverse genetics (ELN 2010), 51% an antecedent hematologic disorder. TP53 aberrations were detected in 42 pts (25%), with 1 (n=27) or 2 mutations (n=12, median variant allele frequency 44%, range, 1.3-99%) in 39 pts, and TP53 deletions in 3 additional pts. The 42 pts with TP53 MUT showed a higher ORR (23.8%) than the 126 pts with TP53 WT (ORR 15.1%), with an OR of 2.04 (95% CI 0.83-4.98), p=0.12. OS (irrespective of treatment) in the TP53 MUT v WT pts was not different (HR, adjusted for treatment: 1.14 [95% CI 0.78-1.66], p=0.51; Fig. A). In both genetic groups, the addition of ATRA had a favorable effect on ORR (ATRA v no ATRA in TP53 MUT: 31.3% v 19.2%, OR 1.91 [95% CI 0.45-8.03]; ATRA v no ATRA in TP53 WT: 18.8% v 10.5%, OR 1.98 [95% CI 0.70-5.61]), TFI p=0.97 (Fig. B). A positive effect of ATRA on OS in both genetic groups was reflected by a median OS of 6.0 v 4.5 months (ATRA v no ATRA in TP53 MUT: HR 0.75 [95% CI 0.38-1.48]), and a median OS of 8.9 v 4.7 months (ATRA v no ATRA in TP53 WT: HR 0.58 [95% CI 0.39-0.86], all results adjusted for VPA, ECOG, HCT-CI, sLDH, Hb), TFI p=0.49 (Fig. C). VPA did not affect ORR in either of the 2 genetic groups (VPA v no VPA in TP53 MUT: 21.7% v 26.3%, OR 0.76 [95% CI 0.18-3.21]; VPA v no VPA in TP53 WT: 16.7% v 13.3%, OR 1.34 [95% CI 0.5-3.61]), TFI p=0.53. The impact of VPA on OS differed between TP53 MUT pts (VPA v no VPA: median OS of 4.2 v 5.3 months, HR 1.31 [95% CI 0.69-2.48], and TP53 WT pts (VPA v no VPA: median OS of 8.4 v 4.8 months, HR 0.67 [95% CI 0.46-0.99], all results adjusted for ATRA, ECOG, HCT-CI, sLDH, Hb; TFI p=0.08, Fig. C). Conclusions Our results confirm the reported higher response rate to DAC in pts with TP53 MUT compared to TP53 WT; the addition of ATRA led to a higher ORR. Improved OS with ATRA was observed particularly in TP53 WT pts. In contrast, VPA did not affect the ORR in either genetic group; TP53 WT pts may benefit from VPA regarding OS. Our exploratory post-hoc results need confirmation in other trials, e.g. in the DECIDER-2 study (adding ATRA or placebo to the recently approved dual treatment of a HMA combined with venetoclax). Cooperative effects of HMAs and retinoids deserve a deeper mechanistic understanding, which may have implications not only for AML but also for other malignancies with impaired TP53. Figure 1 Figure 1. Disclosures Becker: BMS: Honoraria; Pierre Fabre Pharma: Honoraria; Servier: Honoraria; MSD: Honoraria; Novartis: Honoraria. Crysandt: Incyte: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees. Thol: Abbvie: Honoraria; Pfizer: Honoraria; Astellas: Honoraria; Novartis: Honoraria; BMS/Celgene: Honoraria, Research Funding; Jazz: Honoraria. Heuser: Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolremo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; BergenBio: Research Funding; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer Pharma AG: Research Funding; Astellas: Research Funding. Goetze: Abbvie: Other: Advisory Board; BMS/Celgene: Other: Advisory Board, Research Funding. Schlenk: Agios: Honoraria; Astellas: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria; Daiichi Sankyo: Honoraria, Research Funding; Abbvie: Honoraria; Hexal: Honoraria; Neovio Biotech: Honoraria; Novartis: Honoraria; Pfizer: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding; AstraZeneca: Research Funding; Boehringer Ingelheim: Research Funding. Salih: Synimmune GmbH: Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Celgene: Honoraria; BMS: Honoraria. Schittenhelm: Takeda: Other: advisory board; Astellas: Other: advisory board; BMS: Other: advisory board; University of Tuebingen: Patents & Royalties: patent for ASPP2k. Müller-Tidow: Pfizer: Research Funding; Janssen: Consultancy, Research Funding; Bioline: Research Funding. Germing: Novartis: Honoraria, Research Funding; Jazz Pharmaceuticals: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Other: advisory activity, Research Funding. Giagounidis: Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Wäsch: Amgen: Consultancy, Honoraria; Pfizer: Consultancy; Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy; BMS/Celgene: Consultancy; Gilead: Consultancy. Döhner: Jazz Roche: Consultancy, Honoraria; Celgene/BMS: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria, Other: Advisory Board; Agios and Astex: Research Funding; Abbvie: Consultancy, Honoraria; Janssen: Honoraria, Other: Advisory Board; Astellas: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Ganser: Novartis: Honoraria; Jazz Pharmaceuticals: Honoraria; Celgene: Honoraria. Döhner: Astellas: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Oxford Biomedicals: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Research Funding; Helsinn: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Berlin-Chemie: Consultancy, Honoraria; Astex: Consultancy, Honoraria; Agios: Consultancy, Honoraria, Research Funding; Ulm University Hospital: Current Employment; Jazz: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria; GEMoaB: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding. Hackanson: Roche: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Boehringer-Ingelheim: Consultancy, Honoraria; MSD: Consultancy, Honoraria. Lübbert: Cheplapharm: Other: study drug (ATRA); TEVA: Other: study drug (valproic acid); Janssen: Consultancy, Other: study drug (decitabine), Research Funding; Syros: Consultancy, Honoraria; Aristopharm: Other: study drug ; Imago BioSciences: Consultancy, Other: study support with study drug; AbbVie: Consultancy, Honoraria; Astex: Consultancy, Honoraria. OffLabel Disclosure: ATRA, in non-M3 AML valproic acid, in non-M3 AML

Background: DNA-hypomethylating agents are providing a very well-accepted backbone for non-intensive combination treatment of AML/MDS patients (pts), and an in vivo synergism has been demonstrated for the azacitidine+venetoclax combination in the VIALE-A trial (DiNardo et al., EHA 2020). The DAC+ATRA combination also resulted in an improved response rate and survival compared to DAC without ATRA (DECIDER trial, Lübbert et al., J. Clin. Oncol. 2020), also in pts with prior hematologic disorder (mostly MDS); no benefit was seen when valproic acid (VPA) was added to DAC (2x2 factorial design). In a previous study, we had investigated the outcome of elderly pts with oligoblastic AML (i.e. with 20-30% bone marrow blasts, defined as MDS RAEBt according to the French-American-British classification) treated with either DAC or best supportive care within the EORTC 06011 phase III trial (Becker et al., Ann. Hematol. 2015), observing a median overall survival (OS) of 8.0 months (mths) in DAC-treated RAEBt pts. We now hypothesized that the outcome of pts with oligoblastic AML may be improved by the addition of ATRA to DAC. Therefore, in the present exploratory subgroup analysis, pts from the DECIDER cohort with 20-30% bone marrow blasts were analyzed for clinical outcome. Patients and Methods: Key inclusion criteria: newly diagnosed pts >60 years (yr), unfit for induction with non-M3 AML (WHO, de novo or after antecedent hematologic disorder [AHD], therapy-associated [t]AML), ECOG performance status (PS) 0-2. Treatment: DAC 20 mg/m2 day 1-5 (treatment arms A/B/C/D), ATRA p.o. day 6-28 (arms C/D), VPA p.o. continuously from day 6 (arms B/D), of each 28-day course (repeated until relapse/progression, prohibitive toxicity, withdrawal or death). Key endpoints: objective response rate (ORR): CR/CRi/PR, overall (OS) and event-free survival (EFS). Sample size calculation was based on the primary endpoint ORR, assuming an ORR of 25% in arm A (Lübbert et al., Haematologica 2012). For a power of 80% (test in this phase II study at 1-sided alpha=0.1) for an increase of ORR to 40% with ATRA or VPA, 176 pts were necessary, planned sample size 200. Between 12/2011 and 2/2015, 200 pts were randomized and treated. Efficacy analyses were performed in the intention-to-treat (ITT) population. ATRA was investigated by comparing arms C+D vs arms A+B, VPA by comparing arms B+D vs arms A+C, ORR was analyzed with logistic regression estimating odds ratios (OR), OS/EFS with Cox regression estimating hazard ratios (HR), each with 95% confidence intervals (CI), and presented with descriptive two-sided p values of the tests of no treatment effect. Central hematopathologic review (blinded as to treatment arms) was conducted by an independent morphologist. Results: In 56/200 pts of the DECIDER cohort, bone marrow blasts were 20-30% (median, 25%). The number of pts in the randomized arms were: 13 in arm A, 21 in arm B, 9 in arm C, 13 in arm D. Baseline pt characteristics were as follows: male 77%, median age: 75 yr (range 61-88), median WBC: 3400/µl (range 500-52,600), adverse genetics (ELN 2010) present in 25%, ECOG 2 in 13%, comorbidities (HCT-CI) ≥ 3 in 48%, AHD in 68%, tAML in 11% (only slight random imbalances across randomized treatment arms). A median of 5 DAC courses were administered (per arm: 2/5/11/4). Six pts attained a CR, 7 pts a CRi, and 1 pt a PR, resulting in an ORR of 25% (arm A: 7.7%, arm B: 28.6%, arm C: 33.3%, arm D: 30.8%, respectively). Effect on ORR of ATRA vs no ATRA (31.8 vs 20.6%): OR 1.85, CI [0.54,6.37], p=0.33; and of VPA vs no VPA (29.4 vs 18.2%): OR 1.93, CI [0.51,7.24], p=0.33. With 40 deaths out of 56 pts, median OS was 9.5 mths (arm A: 7.6 mths, arm B: 8.9 mths, arm C: 37.2 mths, arm D: 11.2 mths, respectively). Effect on OS of ATRA vs no ATRA (12.5 vs 7.6 mths median OS): HR 0.47, CI [0.24,0.94], p=0.032 (after adjustment for PS, HCT-CI, WBC, LDH, genetic risk: HR 0.42, CI [0.19,0.90], p=0.025); and of VPA vs no VPA (10.0 vs 8.4 mths median OS): HR 0.99, CI [0.51,1.92], p=0.98: A comparable benefit on EFS of ATRA vs no ATRA (but not VPA vs no VPA) was observed. Conclusion: In elderly pts with oligoblastic AML ineligible for induction chemotherapy, the addition of ATRA, but not VPA, to DAC resulted in a clinically meaningful survival benefit; OS of pts receiving DAC without ATRA was very similar to that observed in a previous study. It is tempting to speculate that the combination of an HMA with a retinoid such as ATRA may also be active in MDS pts with excess of blasts. Disclosures Jost: JAZZ: Other: travel support; Roche: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Celgene: Other: travel support. Thol:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Heuser:Amgen: Research Funding; Bayer: Consultancy, Research Funding; BerGenBio ASA: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Stemline Therapeutics: Consultancy; Janssen: Consultancy; PriME Oncology: Honoraria; Karyopharm: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy; Astellas: Research Funding; Roche: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Götze:Celgene: Research Funding. Schlenk:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accomodations, Expenses, Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Roche: Research Funding; AstraZeneca: Research Funding; PharmaMar: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Döhner:Sunesis Pharmaceuticals: Research Funding; Abbvie: Consultancy; Agios: Consultancy; Amgen: Consultancy, Research Funding; Astellas Pharma: Consultancy; Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; Arog: Research Funding; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy; Janssen: Consultancy, Honoraria; Daiichi Sankyo: Honoraria; Celgene: Consultancy, Honoraria; Novartis: Honoraria, Research Funding. Salih:Synimmune: Consultancy, Research Funding; Philogen: Consultancy; Medigene: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Schittenhelm:Pfizer: Consultancy; Astellas: Consultancy. Mueller-Tidow:Jose-Carreras-Siftung: Research Funding; Wilhelm-Sander-Stiftung: Research Funding; BMBF: Research Funding; Deutsche Krebshilfe: Research Funding; Deutsche Forschungsgemeinschaft: Research Funding; Janssen-Cilag Gmbh: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Research Funding; Daiichi Sankyo: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer AG: Research Funding. Brugger:MorphoSys: Current Employment. Bug:Jazz: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Hexal: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Eurocept: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Sanofi: Other: Travel; Neovii: Other: Travel. Wäsch:Pfizer: Consultancy; Amgen: Consultancy; Janssen: Consultancy. Ganser:Celgene: Consultancy; Novartis: Consultancy. Döhner:AstraZeneca: Consultancy, Honoraria; Sunesis: Research Funding; Roche: Consultancy, Honoraria; Pfizer: Research Funding; Oxford Biomedicals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Helsinn: Consultancy, Honoraria; Jazz: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Astex: Consultancy, Honoraria; Astellas: Consultancy, Honoraria, Research Funding; AROG: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; GEMoaB: Consultancy, Honoraria. OffLabel Disclosure: ATRA is approved for APL treatment but not for non-APL AML

Background Germinal center-derived B cell lymphomas are tumors of the lymphoid tissues representing one of the most heterogeneous malignancies. Here we characterize the variety of transcriptomic phenotypes of this disease based on 873 biopsy specimens collected in the German Cancer Aid MMML (Molecular Mechanisms in Malignant Lymphoma) consortium. They include diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt’s lymphoma, mixed FL/DLBCL lymphomas, primary mediastinal large B cell lymphoma, multiple myeloma, IRF4-rearranged large cell lymphoma, MYC-negative Burkitt-like lymphoma with chr. 11q aberration and mantle cell lymphoma. Methods We apply self-organizing map (SOM) machine learning to microarray-derived expression data to generate a holistic view on the transcriptome landscape of lymphomas, to describe the multidimensional nature of gene regulation and to pursue a modular view on co-expression. Expression data were complemented by pathological, genetic and clinical characteristics. Results We present a transcriptome map of B cell lymphomas that allows visual comparison between the SOM portraits of different lymphoma strata and individual cases. It decomposes into one dozen modules of co-expressed genes related to different functional categories, to genetic defects and to the pathogenesis of lymphomas. On a molecular level, this disease rather forms a continuum of expression states than clearly separated phenotypes. We introduced the concept of combinatorial pattern types (PATs) that stratifies the lymphomas into nine PAT groups and, on a coarser level, into five prominent cancer hallmark types with proliferation, inflammation and stroma signatures. Inflammation signatures in combination with healthy B cell and tonsil characteristics associate with better overall survival rates, while proliferation in combination with inflammation and plasma cell characteristics worsens it. A phenotypic similarity tree is presented that reveals possible progression paths along the transcriptional dimensions. Our analysis provided a novel look on the transition range between FL and DLBCL, on DLBCL with poor prognosis showing expression patterns resembling that of Burkitt’s lymphoma and particularly on ‘double-hit’ MYC and BCL2 transformed lymphomas. Conclusions The transcriptome map provides a tool that aggregates, refines and visualizes the data collected in the MMML study and interprets them in the light of previous knowledge to provide orientation and support in current and future studies on lymphomas and on other cancer entities. Electronic supplementary material The online version of this article (10.1186/s13073-019-0637-7) contains supplementary material, which is available to authorized users.

Chromosomal translocations affecting the MYC oncogene are the biological hallmark of Burkitt lymphomas but also occur in a subset of other mature B-cell lymphomas. If accompanied by a chromosomal break targeting the BCL2 and/or BCL6 oncogene these MYC translocation-positive (MYC(+)) lymphomas are called double-hit lymphomas, otherwise the term single-hit lymphomas is applied. In order to characterize the biological features of these MYC(+) lymphomas other than Burkitt lymphoma we explored, after exclusion of molecular Burkitt lymphoma as defined by gene expression profiling, the molecular, pathological and clinical aspects of 80 MYC-translocation-positive lymphomas (31 single-hit, 46 double-hit and 3 MYC(+)-lymphomas with unknown BCL6 status). Comparison of single-hit and double-hit lymphomas revealed no difference in MYC partner (IG/non-IG), genomic complexity, MYC expression or gene expression profile. Double-hit lymphomas more frequently showed a germinal center B-cell-like gene expression profile and had higher IGH and MYC mutation frequencies. Gene expression profiling revealed 130 differentially expressed genes between BCL6(+)/MYC(+) and BCL2(+)/MYC(+) double-hit lymphomas. BCL2(+)/MYC(+) double-hit lymphomas more frequently showed a germinal center B-like gene expression profile. Analysis of all lymphomas according to MYC partner (IG/non-IG) revealed no substantial differences. In this series of lymphomas, in which immunochemotherapy was administered in only a minority of cases, single-hit and double-hit lymphomas had a similar poor outcome in contrast to the outcome of molecular Burkitt lymphoma and lymphomas without the MYC break. Our data suggest that, after excluding molecular Burkitt lymphoma and pediatric cases, MYC(+) lymphomas are biologically quite homogeneous with single-hit and double-hit lymphomas as well as IG-MYC and non-IG-MYC(+) lymphomas sharing various molecular characteristics. peerReviewed

Translocations affecting chromosome subband 6p25.3 containing the IRF4 gene have been recently described as characteristic alterations in a molecularly distinct subset of germinal center B-cell-derived lymphomas. Secondary changes have yet only been described in few of these lymphomas. Here, we performed array-comparative genomic hybridization and molecular inversion probe microarray analyses on DNA from 12 formalin-fixed paraffin-embedded and two fresh-frozen IRF4 translocation-positive lymphomas, which together with the previously published data on nine cases allowed the extension of copy number analyses to a total of 23 of these lymphomas. All except one case carried chromosomal imbalances, most frequently gains in Xq28, 11q22.3-qter, and 7q32.1-qter and losses in 6q13-16.1, 15q14-22.31, and 17p. No recurrent copy-neutral losses of heterozygosity were observed. TP53 point mutations were detected in three of six cases with loss of 17p. Overall this study unravels a recurrent pattern of secondary genetic alterations in IRF4 translocation-positive lymphomas.

A single nucleotide polymorphism-chip analysis of 98 cases of aggressive B-cell lymphomas revealed a recurrent deletion at 19p13 in nine of the cases. Six further cases with deletions encompassing this region were found in array-comparative genomic hybridization data of 295 aggressive B-cell lymphomas from a previous study. Three cases even showed a homozygous deletion, suggesting a tumor suppressor gene in the deleted region. Two genes encoding members of the tumor necrosis factor superfamily (TNFSF) were located in the minimally deleted region, that is, TNFSF7 and TNFSF9. As no mutations were found within the coding exons of the remaining alleles in the lymphomas with heterozygous deletions, we speculate that the deletions may mostly function through a haploinsufficiency mechanism. The cases with deletions encompassed both diffuse large B-cell lymphomas and Burkitt lymphomas, and a deletion was also found in a Hodgkin lymphoma cell line. Thus, TNFSF7 and TNFSF9 deletions are recurrent genetic lesions in multiple types of human lymphomas.

We identified regions of DNA copy number changes that are significantly associated with metastasis and clinical outcome in patients with clear cell renal cell carcinoma.We analyzed 53 primary clear cell renal cell carcinomas, including 31 metastasized and 22 nonmetastasized tumors, by array comparative genomic hybridization with a median resolution of 1 to 1.5 Mbp. To validate copy number aberrations with potential prognostic value we performed fluorescence in situ hybridization analysis using commercially available fluorescent probes.We identified 5 recurrent chromosomal aberrations that were significantly associated with metastasis, including gains of 1q21.3, 12q13.12, 12q13.3q14.1 and 20q11.21q13.2, and loss of 9p21.3p24.1. The most prominent of them with the highest OR for metastatic risk were loss of 9p21.3p24.1, and gains of 1q21.3 and 20q11.21q13.32. Eight alterations involving chromosomes 7, 9, 12, 16 and 20 significantly correlated with shortened cancer specific survival. The lowest p values on Kaplan-Meier analysis showed losses of 9p21.3p24.1 and 9q32q33.1, and gains of 7q36.3 and 20q11.21q13.32. Fluorescence in situ hybridization done in the same cohort for the 4 select regions 1q21.3, 7q36.3, 9p21.3p24.1 and 20q11.21q13.32 clearly confirmed the results of array comparative genomic hybridization.Data suggest that specific chromosomal alterations in clear cell renal cell carcinoma can be used to predict metastasis and cancer specific survival in patients with clear cell renal cell carcinoma. It seems possible to design a combined fluorescence in situ hybridization assay based on these genetic targets for outcome prediction, which can be used for routine diagnostics.

Lymphomas are assumed to originate at different stages of lymphocyte development through chromosomal aberrations. Thus, different lymphomas resemble lymphocytes at distinct differentiation stages and show characteristic morphologic, genetic, and transcriptional features. Here, we have performed a microarray-based DNA methylation profiling of 83 mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) characterized for their morphologic, genetic, and transcriptional features, including molecular Burkitt lymphomas and diffuse large B-cell lymphomas. Hierarchic clustering indicated that methylation patterns in maB-NHLs were not strictly associated with morphologic, genetic, or transcriptional features. By supervised analyses, we identified 56 genes de novo methylated in all lymphoma subtypes studied and 22 methylated in a lymphoma subtype–specific manner. Remarkably, the group of genes de novo methylated in all lymphoma subtypes was significantly enriched for polycomb targets in embryonic stem cells. De novo methylated genes in all maB-NHLs studied were expressed at low levels in lymphomas and normal hematopoietic tissues but not in nonhematopoietic tissues. These findings, especially the enrichment for polycomb targets in stem cells, indicate that maB-NHLs with different morphologic, genetic, and transcriptional background share a similar stem cell–like epigenetic pattern. This suggests that maB-NHLs originate from cells with stem cell features or that stemness was acquired during lymphomagenesis by epigenetic remodeling.

The spectrum of entities, the therapeutic strategy, and the outcome of mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) differs between children and adolescents on the one hand and adult patients on the other. Whereas adult maB-NHLs have been studied in detail, data on molecular profiling of pediatric maB-NHLs are hitherto lacking. We analyzed 65 cases of maB-NHL from patients up to 18 years of age by gene expression profiling, matrix comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), and immunohistochemistry. The majority of the analyzed pediatric patients were treated within prospective trials (n = 49). We compared this group to a series of 182 previously published cases of adult maB-NHL. Gene expression profiling reclassified 31% of morphologically defined diffuse large B-cell lymphomas as molecular Burkitt lymphoma (mBL). The subgroups obtained by molecular reclassification did not show any difference in outcome in children treated with the NHL-Berlin-Frankfurt-Muenster (BFM) protocols. No differences were detectable between pediatric and adult mBL with regard to gene expression or chromosomal imbalances. This is the first report on molecular profiling of pediatric B-NHL showing mBL to be much more prominent in children than suggested by morphologic assessment. Based on molecular profiling mBL is a molecularly homogeneous disease across children and adults.

Deregulation of cell signaling pathways controlling cell growth and cell survival is a common feature of all cancers. Although a core repertoire of oncogenic mechanisms is widely conserved between various malignancies, the constellation of pathway activities can vary even in patients with the same malignant disease. Modern molecularly targeted cancer drugs intervene in cell signaling compensating for pathway deregulation. Hence characterizing tumors with respect to pathway activation will become crucial for treatment decisions. Here we have used semi-supervised machine learning methodology to generate signatures of eight oncogene-inducible pathways, which are conserved across epithelial and lymphoid tissues. We combined them to patterns of pathway activity called PAPs for pathway activation patterns and searched for them in 220 morphologically, immunohistochemically and genetically well-characterized mature aggressive B-cell lymphomas including 134 cases with clinical data available. Besides Burkitt lymphoma, which was characterized by a unique pattern, the PAPs identified four distinct groups of mature aggressive B-cell lymphomas across independent gene expression studies with distinct biological characteristics, genetic aberrations and prognosis. We confirmed our findings through cross-platform analysis in an independent data set of 303 mature aggressive B-cell lymphomas.

BACKGROUND: The aim of this study was to determine the impact of a gain of the MALT1 gene on gene expression and clinical parameters in diffuse large B-cell lymphoma. DESIGN AND METHODS: We analyzed 116 cases of diffuse large B-cell lymphoma by fluorescence in situ hybridization, array-based comparative genomic hybridization, and transcriptional profiling. RESULTS: A gain of 18q21 including MALT1 was detected in 44 cases (38%) and was accompanied by a gain of BCL2 in 43 cases. All cases with a 18q21/MALT1 gain showed BCL2 protein whereas 79% in the group without a 18q21/MALT1 gain did so (p

Summary Objectives: Array-comparative genomic hybridization (aCGH) is a high-throughput method to detect and map copy number aberrations in the genome. Multi-step analysis of high-dimensional data requires an integrated suite of bioinformatic tools. In this paperwe detail an analysis pipeline for array CGH data. Methods: We developed an analysis tool for array CGH data which supports single and multi-chip analyses as well as combined analyses with paired mRNA gene expression data. The functions supporting relevant steps of analysis were implemented using the open source software R and combined as package aCGHPipeline. Analysis methods were illustrated using 189 CGH arrays of aggressive B-cell lymphomas. Results: The package covers data input, quality control, normalization, segmentation and classification. For multi-chip analysis aCGHPipeline offers an algorithm for automatic delineation of recurrent regions. This task was performed manuallyup to now. The package also supports combined analysis with mRNA gene expression data. Outputs consist of HTML documents to facilitate communication with clinical partners. Conclusions: The R package aCGHPipeline supports basic tasks of single and multi-chip analysis of array CGH data.

Purpose To identify novel genomic regions of interest in acute myeloid leukemia (AML) with complex karyotypes, we applied comparative genomic hybridization to microarrays (array-CGH), allowing high-resolution genome-wide screening of genomic imbalances. Patients and Methods Sixty AML cases with complex karyotypes were analyzed using array-CGH; parallel analysis of gene expression was performed in a subset of cases. Results Genomic losses were found more frequently than gains. The most frequent losses affected 5q (77%), 17p (55%), and 7q (45%), and the most frequent genomic gains 11q (40%) and 8q (38%). Critical segments could be delineated to genomic fragments of only 0.8 to a few megabase-pairs of DNA. In lost/gained regions, gene expression profiling detected a gene dosage effect with significant lower/higher average gene expression levels across the genes located in the respective regions. Furthermore, high-level DNA amplifications were identified in several regions: 11q23.3-q24.1 (n = 7), 21q22 (n = 6), 11q23.3 (n = 5), 13q12 (n = 3), 8q24 (n = 3), 9p24 (n = 2), 12p13 (n = 2), and 20q11 (n = 2). Parallel analysis of gene expression in critical amplicons displayed overexpressed candidate genes (eg, C8FW and MYC in 8q24). Conclusion In conclusion, a large spectrum of genomic imbalances, including novel recurring changes in AML with complex karyotypes, was identified using array-CGH. In addition, the combined analysis of array-CGH data with gene expression profiles allowed the detection of candidate genes involved in the pathogenesis of AML.

Gliomas are the most frequent primary brain tumors and comprise a group of morphologically, biologically and clinically heterogeneous neoplasms. The different glioma types are associated with distinct genetic aberrations, which may provide useful information for tumor classification as well as prediction of prognosis and response to therapy. To facilitate the molecular classification of gliomas, we established a genomic microarray that consists of bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) clones representing tumor suppressor genes, proto-oncogenes and chromosomal regions frequently gained or lost in gliomas. In addition, reference clones distributed evenly throughout the genome in approximately 15 Mbp intervals were spotted on the microarray. These customized microarrays were used for matrix-based comparative genomic hybridization (matrix CGH) analysis of 70 gliomas. Matrix CGH findings were validated by molecular genetic analyses of candidate genes, loss of heterozygosity studies and chromosomal CGH. Our results indicate that matrix CGH allows for the sensitive and specific detection of gene amplifications as well as low-level copy number gains and losses in clinical glioma samples. Furthermore, molecular classification based on matrix CGH data closely paralleled histological classification and was able to distinguish with few exceptions between diffuse astrocytomas and oligodendrogliomas, anaplastic astrocytomas and anaplastic oligodendrogliomas, anaplastic oligodendrogliomas and glioblastomas, as well as primary and secondary glioblastomas. Thus, matrix CGH is a powerful technique that allows for an automated genomic profiling of gliomas and represents a promising new tool for their molecular classification.

Genomic analyses aimed at the detection of high-level DNA amplifications were performed on 13 widely used pancreatic cancer cell lines and 6 pancreatic tumor specimens. For these analyses, array-based comparative genomic hybridization (Matrix-CGH) onto dedicated microarrays was used. In comparison with chromosomal CGH (eight amplifications), a >3-fold number of DNA amplifications was detected (n = 29). The most frequent amplifications mapped to 7p12.3 (three pancreatic cancer cell lines and three pancreatic tumor specimens), 8q24 (four pancreatic cancer cell lines and one pancreatic tumor specimen), 11q13 (three pancreatic cancer cell lines and three pancreatic tumor specimens), and 20q13 (four pancreatic cancer cell lines and three pancreatic tumor specimens). Genes contained in the consensus regions were MYC (8q24), EGFR (7p12.3), and FGF3 (11q13). In six of seven pancreatic cancer cell lines and pancreatic tumor specimens with 20q13 amplifications, the novel candidate gene NFAT C2, which plays a role in the activation of cytokines, was amplified. Other amplifications also affected genes for which a pathogenetic role in pancreatic carcinoma has not been described, such as BCL10 and BCL6, two members of the BCL family. A subset of amplified genes was checked for overexpression by means of real-time PCR, revealing the highest expression levels for BCL6 and BCL10. Thus, Matrix-CGH allows the detection of a high number of amplifications, resulting in the identification of novel candidate genes in pancreatic cancer.

The genetic hallmark of Burkitt lymphoma (BL) is the t(8;14)(q24;q32) and its variants leading to activation of the MYC oncogene. It is a matter of debate whether true BL without MYC translocation exists. Here, we identified 59 lymphomas concordantly called BL by 2 gene expression classifiers among 753 B-cell lymphomas. Only 2 (3%) of these 59 molecular BL lacked a MYC translocation, which both shared a peculiar pattern of chromosome 11q aberration characterized by interstitial gains including 11q23.2-q23.3 and telomeric losses of 11q24.1-qter. We extended our analysis to 17 MYC-negative high-grade B-cell lymphomas with a similar 11q aberration and showed this aberration to be recurrently associated with morphologic and clinical features of BL. The minimal region of gain was defined by high-level amplifications in 11q23.3 and associated with overexpression of genes including PAFAH1B2 on a transcriptional and protein level. The recurrent region of loss contained a focal homozygous deletion in 11q24.2-q24.3 including the ETS1 gene, which was shown to be mutated in 4 of 16 investigated cases. These findings indicate the existence of a molecularly distinct subset of B-cell lymphomas reminiscent of BL, which is characterized by deregulation of genes in 11q.

The prognosis of germinal center–derived B-cell (GCB) lymphomas, including follicular lymphoma and diffuse large-B-cell lymphoma (DLBCL), strongly depends on age. Children have a more favorable outcome than adults. It is not known whether this is because of differences in host characteristics, treatment protocols, or tumor biology, including the presence of chromosomal alterations. By screening for novel IGH translocation partners in pediatric and adult lymphomas, we identified chromosomal translocations juxtaposing the IRF4 oncogene next to one of the immunoglobulin (IG) loci as a novel recurrent aberration in mature B-cell lymphoma. FISH revealed 20 of 427 lymphomas to carry an IG/IRF4-fusion. Those were predominantly GCB-type DLBCL or follicular lymphoma grade 3, shared strong expression of IRF4/MUM1 and BCL6, and lacked PRDM1/BLIMP1 expression and t(14;18)/BCL2 breaks. BCL6 aberrations were common. The gene expression profile of IG/IRF4-positive lymphomas differed from other subtypes of DLBCL. A classifier for IG/IRF4 positivity containing 27 genes allowed accurate prediction. IG/IRF4 positivity was associated with young age and a favorable outcome. Our results suggest IRF4 translocations to be primary alterations in a molecularly defined subset of GCB-derived lymphomas. The probability for this subtype of lymphoma significantly decreases with age, suggesting that diversity in tumor biology might contribute to the age-dependent differences in prognosis of lymphoma.

Follicular lymphoma (FL) is characterized by a large number of chromosomal aberrations. However, their exact genomic extension and involved target genes remain to be determined. For this purpose, we used array-based intermediate-high resolution genomic profiling in combination with Affymetrix gene expression analysis. Tumor specimens from 128 FL patients were analyzed for the presence of genomic aberrations and the results were correlated to clinical data sets and mRNA expression levels. In 114 (89%) of the 128 analyzed cases, a total of 688 genomic aberrations (384 gains/amplifications and 304 losses) were detected. Frequent genomic aberrations were: -1p36 (18%), +2p15 (24%), -3q (14%), -6q (25%), +7p (19%), +7q (23%), +8q (14%), -9p (16%), -11q (15%), +12q (20%), -13q (11%), -17p (16%), +18p (18%), and +18q (28%). Critical segments of these imbalances were delineated to genomic fragments with a minimum size down to 0.2 Mb. By comparison of these with mRNA gene expression data, putative candidate genes were identified. Moreover, we found that deletions affecting the tumor suppressor gene CDKN2A/B on 9p21 were detected in nontransformed FL grade I-II. For this aberration as well as for -6q25 and -6q26, an association with inferior survival was observed.

Primary mediastinal B-cell lymphoma (PMBL) is an aggressive extranodal B-cell non-Hodgkin's lymphoma with specific clinical, histopathological and genomic features. To characterize further the genotype of PMBL, we analyzed 37 tumor samples and PMBL cell lines Med-B1 and Karpas1106P using array-based comparative genomic hybridization (matrix- or array-CGH) to a 2.8k genomic microarray. Due to a higher genomic resolution, we identified altered chromosomal regions in much higher frequencies compared with standard CGH: for example, +9p24 (68%), +2p15 (51%), +7q22 (32%), +9q34 (32%), +11q23 (18%), +12q (30%) and +18q21 (24%). Moreover, previously unknown small interstitial chromosomal low copy number alterations (for example, -6p21, -11q13.3) and a total of 19 DNA amplifications were identified by array-CGH. For 17 chromosomal localizations (10 gains and 7 losses), which were altered in more than 10% of the analyzed cases, we delineated minimal consensus regions based on genomic base pair positions. These regions and selected immunohistochemistries point to candidate genes that are discussed in the context of NF-kappaB transcription activation, human leukocyte antigen class I/II defects, impaired apoptosis and Janus kinase/signal transducer and activator of transcription (JAK/STAT) activation. Our data confirm the genomic uniqueness of this tumor and provide physically mapped genomic regions of interest for focused candidate gene analysis.

Background The identification of genomic signatures of colorectal cancer for risk stratification requires the study of large series of cancer patients with an extensive clinical follow-up. Multicentric clinical studies represent an ideal source of well documented archived material for this type of analyses. Methods To verify if this material is technically suitable to perform matrix-CGH, we performed a pilot study using macrodissected 29 formalin-fixed, paraffin-embedded tissue samples collected within the framework of the EORTC-GI/PETACC-2 trial for colorectal cancer. The scientific aim was to identify prognostic genomic signatures differentiating locally restricted (UICC stages II-III) from systemically advanced (UICC stage IV) colorectal tumours. Results The majority of archived tissue samples collected in the different centers was suitable to perform matrix-CGH. 5/7 advanced tumours displayed 13q-gain and 18q-loss. In locally restricted tumours, only 6/12 tumours showed a gain on 13q and 7/12 tumours showed a loss on 18q. Interphase-FISH and high-resolution array-mapping of the gain on 13q confirmed the validity of the array-data and narrowed the chromosomal interval containing potential oncogenes. Conclusion Archival, paraffin-embedded tissue samples collected in multicentric clinical trials are suitable for matrix-CGH analyses and allow the identification of prognostic signatures and aberrations harbouring potential new oncogenes.

Background The distinction between Burkitt’s lymphoma and diffuse large-B-cell lymphoma is unclear. We used transcriptional and genomic profiling to define Burkitt’s lymphoma more precisely and to distinguish subgroups in other types of mature aggressive B-cell lymphomas. Methods We performed gene-expression profiling using Affymetrix U133A GeneChips with RNA from 220 mature aggressive B-cell lymphomas, including a core group of 8 Burkitt’s lymphomas that met all World Health Organization (WHO) criteria. A molecular signature for Burkitt’s lymphoma was generated, and chromosomal abnormalities were detected with interphase fluorescence in situ hybridization and array-based comparative genomic hybridization. Results We used the molecular signature for Burkitt’s lymphoma to identify 44 cases: 11 had the morphologic features of diffuse large-B-cell lymphomas, 4 were unclassifiable mature aggressive B-cell lymphomas, and 29 had a classic or atypical Burkitt’s morphologic appearance. Also, five did not have a detectable IG-myc Burkitt’s translocation, whereas the others contained an IG-myc fusion, mostly in simple karyotypes. Of the 176 lymphomas without the molecular signature for Burkitt’s lymphoma, 155 were diffuse large-B-cell lymphomas. Of these 155 cases, 21 percent had a chromosomal breakpoint at the myc locus associated with complex chromosomal changes and an unfavorable clinical course. Conclusions Our molecular definition of Burkitt’s lymphoma clarifies and extends the spectrum of the WHO criteria for Burkitt’s lymphoma. In mature aggressive B-cell lymphomas without a gene signature for Burkitt’s lymphoma, chromosomal breakpoints at the myc locus were associated with an adverse clinical outcome.

A broad range of malignant diseases, such as mantle cell lymphoma (MCL), is associated with complex genomic alterations, demanding multimodal functional testing of candidate genes. To assess such candidate disease genes, we have developed a bidirectional targeted transgenesis tool, which allows well-controlled modulation of individual gene activities within a cellular MCL system. The engineered versatile transgenesis system permits functional analysis of virtually any candidate gene: for tumor suppressor genes by complementation via integration of respective genomic DNA or for oncogenes by inactivation via integrated shRNA coding plasmids. Complementation by genomic DNA ensures wild-type (WT) regulated gene expression, whereas genomic integration of shRNA coding inserts by an advanced RNAi-strategy mediates specific knock-down of gene expression. Site-specific genomic integration of an unmodified BAC, which contains the CDKN2A/B genes absent in the MCL model system, restored CDKN2A/B expression resulting in the inhibition of cell proliferation. CCND1, strongly overexpressed in the model system, was down-regulated via shRNA expression, again inhibiting proliferation. Notably, the presented site-specific shRNA-strategy circumvents interference by IFN-response induced when using other RNAi gene knock-down methods. In conclusion, we here demonstrate that adequate restoration of a range of different gene activities yields in a desired antiproliferative effect in MCL-derived cells. By antagonizing inactivated tumor suppressor genes or activated oncogenes, the presented approach can be readily used for the functional analysis of a broad range of disease-related genetic defects.

Chromosomal aberrations are known to have an impact on the initiation and progression of oral squamous cell carcinoma (OSCC), but individual genes involved in OSCC pathogenesis are poorly described. To elucidate the molecular events underlying oral carcinogenesis, a set of primary OSCC were screened for distinct genetic imbalances by means of array-based comparative genomic hybridisation. For this, a DNA array was used containing 812 genomic targets including oncogenes, tumour-suppressor genes and chromosomal regions frequently altered in human neoplasms. The most frequent aberrations were amplification of MYC, EGFR, CCND1 and PIK3CA, whereas deletions affected TRAILR1 and ATM. Furthermore, a distinct high-level amplification of the fibroblast growth factor receptor 1 (FGFR1) locus was detected in two cases. Detailed FISH analysis on OSCC tissue microarray sections revealed amplification prevalence for FGFR1 of 17.4% (16/92). Furthermore, FGFR1 protein analysis by immunohistochemistry on a TMA containing 178 OSCC found a high FGFR1 expression in tumours of early t-stadium and UICC stage (T1/2 vs. T3/4: p=0.002; SI-II vs. S III-IV: p=0.048). Our results indicate that an increase in FGFR1 expression contributes to oral carcinogenesis at an early stage of development.

Urokinase-type plasminogen activator (UPA) has been implicated in a broad spectrum of pathogenic processes involved in the formation and disruption of atherosclerotic lesions. Up to now, there is no consensus on the contribution of membrane-bound UPA and its receptor CD87 (UPAR) to the development of atherosclerosis. In this study, we determined comparatively the levels of UPAR and UPAR-bound UPA in segments of human coronary and aortic vessels with different degrees of atherosclerotic lesions (macroscopically normal areas, early atherosclerotic lesions, fibrous and calcified plaques). The UPAR content increased progressively with the severity of atherosclerosis. In aortic segments, in which intima and media layers were analyzed separately, the content of UPAR in the intima significantly exceeded the levels measured in the media. Using a detergent-phase separation method with a Triton X-114-containing buffer, we could demonstrate that the levels of membrane (glycosylphosphatidylinositol)-anchored UPAR were significantly higher in the intima of early atherosclerotic lesions as well as in the cap areas of fibrous plaques compared with macroscopically normal areas. However, only 20-25% of the intimal and 30-50% of the medial glycosylphosphatidylinositol-UPAR was occupied by UPA as determined on a molar basis. These data confirm that the overexpression of UPAR in advanced atherosclerotic lesions contributes to lesion development. Whether UPAR's excess over cell surface UPA provides an additional role for this receptor in atherogenesis besides UPA-mediated proteolysis remains to be elucidated.

Tumor samples of 53 patients with t(11;14)-positive mantle cell lymphomas (MCLs) were analyzed by matrix-based comparative genomic hybridization (matrix-CGH) using a dedicated DNA array. In 49 cases, genomic aberrations were identified. In comparison to chromosomal CGH, a 50% higher number of aberrations was found and the high specificity of matrix-CGH was demonstrated by fluorescence in situ hybridization (FISH) analyses. The 11q gains and 13q34 deletions, which have not been described as frequent genomic aberrations in MCL, were identified by matrix-CGH in 15 and 26 cases, respectively. For several genomic aberrations, novel consensus regions were defined: 8p21 (size of the consensus region, 2.4 megabase pairs [Mbp]; candidate genes: TNFRSF10B, TNFRSF10C, TNFRSF10D); 10p13 (2.7 Mbp; BMI1); 11q13 (1.4 Mbp; RELA); 11q13 (5.2 Mbp; CCND1); 13q14 (0.4 Mbp; RFP2, BCMSUN) and 13q34 (6.9 Mbp). In univariate analyses correlating genomic aberrations and clinical course, 8p- and 13q14- deletions were associated with an inferior overall survival. These data provide a basis for further studies focusing on the identification of pathogenetically or clinically relevant genes in MCL.

B cell chronic lymphocytic leukemia (B-CLL) is characterized by a highly variable clinical course. Recurrent chromosomal imbalances provide significant prognostic markers. Risk-adapted therapy based on genomic alterations has become an option that is currently being tested in clinical trials. To supply a robust tool for such large scale studies, we developed a comprehensive DNA microarray dedicated to the automated analysis of recurrent genomic imbalances in B-CLL by array-based comparative genomic hybridization (matrix–CGH). Validation of this chip in a series of 106 B-CLL cases revealed a high specificity and sensitivity that fulfils the criteria for application in clinical oncology. This chip is immediately applicable within clinical B-CLL treatment trials that evaluate whether B-CLL cases with distinct chromosomal abnormalities should be treated with chemotherapy of different intensities and/or stem cell transplantation. Through the control set of DNA fragments equally distributed over the genome, recurrent genomic imbalances were discovered: trisomy of chromosome 19 and gain of the MYCN oncogene correlating with an elevation of MYCN mRNA expression.

DNA amplifications are important mechanisms for proto-oncogene activation. Comparative genomic hybridization (CGH) to metaphase chromosome preparations has revealed amplifications in 10–20% of B-cell lymphomas (B-NHL). We analysed a series of 16 aggressive non-Hodgkin lymphomas by the new approach termed Matrix-CGH (M-CGH) using genomic DNA microarrays as hybridization target. For M-CGH, a dedicated B-cell lymphoma chip was constructed containing 496 genomic targets covering oncogenes, tumor suppressor genes as well as chromosome regions frequently altered in B-NHL. In 10 of 16 samples a total of 15 DNA amplifications were identified. The amplicons included BCL2, REL, CCND1, CCND2, JAK2, FGF4 and MDM2. Four of the 15 amplifications remained undetected by chromosomal CGH. The respective amplicons mapped to bands 2p13, 9p13–p21 and 12q24 and, were confirmed by fluorescence in situ hybridization. Furthermore, for four genomically amplified genes real-time quantitative reverse transcription polymerase chain reaction revealed elevated mRNA expression levels. These data show the superior diagnostic sensitivity of the newly developed diagnostic tool. As only a small portion of the genome (approximately 1.5%) has been analysed by the present DNA array, it is likely that gene amplifications are much more common in aggressive lymphomas than previously assumed.

Recently, DNA microarray technology has opened new avenues for the understanding of lymphomas. By hybridization of cDNA to arrays containing >10,000 different DNA fragments, this approach allows the simultaneous evaluation of the mRNA expression of thousands of genes in a single experiment. Using sophisticated bioinformatic tools, the huge amount of raw data can be clustered resulting in (1) tumor subclassification, (2) identification of pathogenetically relevant genes, or (3) biological predictors for the clinical course. This approach already has provided novel insights into different entities of B-cell non-Hodgkin's lymphomas. Genomic DNA chip hybridization (matrix-CGH) is a complementary approach focussing on genomic aberrations. In this review, we discuss the impact of this new technology both with regard to methodological aspects as well as to novel findings influencing our understanding of lymphomas.

Sixteen dedifferentiated and pleomorphic liposarcomas were analyzed by comparative genomic hybridization (CGH) to genomic microarrays (matrix-CGH), cDNA-derived microarrays for expression profiling, and by quantitative PCR. Matrix-CGH revealed copy number gains of numerous oncogenes, i.e., CCND1, MDM2, GLI, CDK4, MYB, ESR1, and AIB1, several of which correlate with a high level of transcripts from the respective gene. In addition, a number of genes were found differentially expressed in dedifferentiated and pleomorphic liposarcomas. Application of dedicated clustering algorithms revealed that both tumor subtypes are clearly separated by the genomic profiles but only with a lesser power by the expression profiles. Using a support vector machine, a subset of five clones was identified as "class discriminators." Thus, for the distinction of these types of liposarcomas, genomic profiling appears to be more advantageous than RNA expression analysis.

Genome-wide screening for chromosomal imbalances using comparative genomic hybridization (CGH) revealed a wealth of data on previously unrecognized tumor-specific genomic alterations. CGH to microarrays of DNA, an approach termed matrix-CGH, allows detection of genomic imbalances at a much higher resolution. We show that matrix CGH is also feasible from small tissue samples requiring universal amplification of genomic DNA. Because widespread application of matrix-CGH experiments using large numbers of DNA targets demands a high degree of automation, we have developed a protocol for a fully automated procedure. The use of specialized instrumentation for the generation of DNA chips, their hybridization, scanning, and evaluation required numerous alterations and modifications of the initial protocol. We here present the elaboration and testing of automated matrix-CGH. A chip consisting of 188 different genomic DNA fragments, cloned in bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) vectors and immobilized in replicas of 10, was used to assess the performance of the automated protocol in determining the gene dosage variations in tumor cell lines COLO320-HSR, HL60, and NGP. Although ratios of matrix-CGH were highly concordant with results of chromosomal CGH (85%), the dynamic range of the matrix-CGH ratios was highly superior. Investigation of the two amplicons on 8q24 in COLO320-HSR and HL60, containing the MYC gene, revealed a homogeneous amplicon in COLO320-HSR but a heterogeneous amplification pattern in HL60 cells. Although control clones for normalization of the signal ratios can be predicted in cases with defined chromosomal aberrations, in primary tumors such data are often not available, requiring alternative normalization algorithms. Testing such algorithms in a primary high-grade B-cell lymphoma, we show the feasibility of this approach. With the matrix-CGH protocol presented here, robust and reliable detection of genomic gains and losses is accomplished in an automated fashion, which provides the basis for widespread application in tumor and clinical genetics.

Introduction: Clear cell renal cell carcinoma (ccRCC) is the most frequently encountered renal malignancy with a high metastatic potential and the prognosis of patients in this subgroup is very poor. Prognostic parameters which can be used for an individual prognostic evaluation are still not available, So the aim of our study was high resolution screening of genomic imbalances of metastasized and non-metastasized primary ccRCC by array CGH in order to identify regions of DNA copy number changes significantly associated with metastasis and clinical outcome of patients. Methods: We conducted genome-wide copy number profiles in 56 primary ccRCC including 32 metastasized and 24 non-metastasized tumours by array CGH with a median resolution of 2Mbp. The median follow up of the patients was 47 months. Results: Array CGH identified 6 recurrent chromosomal aberrations: gains of 1q21.3, 12q13.11, 12q13.2 and 20q11.21q13.2 and losses of 8p11.23p12 and 9p21.3p24.1, which were significantly associated with metastasis occurrence. In multivariate analysis including these aberrations gains of 1q21.3 and of 20q11.21q13.2 and loss of 9p21.3p24.1 attained as independent predictors for metastasis in our cohort with 81.5% sensitivity and 95.8% specificity of 81.5% and 95.8%, respectively. Kaplan-Meier survival analysis showed that gains on chromosomes 7, 12, 16 and 20q and losses on chromosome 9 have a significant correlation with disease specific survival. Multivariate Cox-regression analysis revealed gains of 7q36.3 and of 20q11.21q13.2 and a loss of 9p21.3p24.1 as independent prognostic factors for patients' outcome of patients. Conclusions: Our data suggest that specific common copy number alterations in ccRCC could serve as independent predictors for metastasis occurrence and cancer specific survival in patients with ccRCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2246.

Background: Chromosomal abnormalities are detectable in virtually all patients with multiple myeloma (MM) by the use of molecular cytogenetic techniques (e.g. fluorescence in-situ hybridization, FISH; comparative genomic hybridization, CGH). Genomic rearrangements play a crucial role in the pathogenesis of clonal plasma cell disorders and can deliver important prognostic information. Microarray-based comparative genomic hybridization (mCGH) is an innovative technique that enables a genome-wide, high-resolution tumor cell screening for chromosomal imbalances in a single experiment. The aim of the present study was the detailed characterization of genomic gains and losses in MM - in particular the delineation of critical regions - by the use of mCGH. Methods: So far, bone marrow specimens from 55 patients with Salmon &Durie stage II or III MM were analyzed. To achieve a plasma cell proportion greater than 50% before hybridization, CD 138-enrichment by immunomagnetic separation was performed whenever necessary. mCGH chips consisting of approximately 6.400 DNA clones, among them ~3.200 clones covering the whole human genome in 1 Mb increments, the remaining forming contigs from specific genomic regions or containing tumor-relevant genes were applied. Results: 52 out of 55 patients (94.5%) were evaluable by mCGH. Gains and losses of chromosomal material were found in 51 out of 52 patients (98%). In one case, mCGH did not exhibit imbalances but +9q34 and +11q25 was diagnosed by FISH for yet unknown reasons. Genomic losses most frequently involved chromosomes 13 (65%), 1p (39%), 16 (31%), 6 (27%), and 12 (21%), while gains commonly affected chromosomes 1q, 9, 11, 15 (46%), and 19 (35%). High-level amplifications were identified on chromosome 16 in two cases (overall three amplicons involving chromosome bands 16p11-p13, 16q12, and 16q21, respectively) as well as on chromosomes 8 (three amplified segments, one of them containing c-myc at 8q24) and 20q (2 amplifications involving bands 20q13.1 and 20q13.3) in one case each. Data on chromosome 13 deletion was consistent with monosomy 13 in 28 out of 34 cases (82.3%). For the remaining 6 cases with a partial 13q loss, no commonly deleted region could be delineated. In contrast, single critical regions were identified on other chromosomes, e.g. chromosome 14 (+14q23.2-q24.3). A small genomic region defined by only four closely adjacent 11q DNA clones was gained in 23 out of 24 (96%) cases with chromosome 11 extra copies. Conclusions: mCGH allows the detection of genomic imbalances including high-level amplifications in almost all patients with MM and enables a precise delineation of critical regions in this disease. Evaluation of mCGH raw data is ongoing and confirmative FISH analyses are currently under way. Supported by grants from the Deutsche José Carreras Leukämie-Stiftung (DJCLS-R04/04) and the Wilhelm Sander-Stiftung (No. 2002.098.1) to P.L.

Follicular Lymphoma (FL), one of the most frequent lymphoma entities in the western world, is characterized by a highly variable clinical course reaching from rapid progression with fatal outcome to cases with long term survival. In a recent study applying chromosomal comparative hybridization (CGH) to FL, in 70% of the cases genomic aberrations were detectable and a loss of genomic material on chromosomal bands 6q25-q27 was the strongest predictor for short overall survival. However, limitations of CGH as a screening method are a restricted genomic resolution to 3–10 Mbp and demanding non-automated evaluation procedures. Thus, high throughput analysis of genomic alterations for risk adapted patient stratification and monitoring within treatment trials should rely on efficient and automated diagnostic techniques. In this study, we used array CGH to a novel generation of DNA Chips containing 2800 genomic DNA probes. Target clones comprised i) contigs mapping to genomic regions of possible pathogenetic relevance in lymphoma (n=610 target clones mapping to e.g. 1p, 2p, 3q, 7q, 9p, 11q, 12q, 13q, 17p, 18q, X); ii) selected oncogenes and tumor suppressor genes (n=686) potentially relevant in hematologic neoplasms; and iii) a large genome-wide cluster of 1502 target DNA clones covering the genome at a distance of app. 2 Mbp (part of the golden path clone set). This chip represents a median genomic resolution of app. 1.5 Mbp. In total, DNAs from 70 FL samples were analyzed and results were compared to data from chromosomal CGH experiments and clinical data sets. The sensitivity of array CGH was considerably higher compared to chromosomal CGH (aberrations in 95% of cases vs 70% of cases). Most frequent aberrations were gain mapping to chromosome arms 2p (21%), 7p (24%), 7q (30%), 12p (17%), 12q (21%), 18p (21%) and 18q (34%) as well as losses mapping to chromosome arms 1p (19%), 6q (23%), 7p (20%), 11q (26%) and 17p (20%). In addition, several genomic aberrations were identified which have not been described before in FL. Currently, these aberrations are characterized in more detail and results will be correlated with the clinical data set. Moreover, three recurrent sites of genomic polymorphisms in human beings affecting chromosomes 5q, 14q and 15q were identified. In conclusion, these data underline the potential of array CGH for the sensitive detection of genomic imbalances in FL.

Chronic lymphocytic leukemia of B-cell type (B-CLL) is characterized by a number of typical genomic aberrations. In comparison to patients with normal karyotypes or 13q deletions patients with high risk imbalances such as deletions of 11q, 17p or unmutated IgVH status have a higher risk for advanced disease and a significantly shorter survival. For a precise mapping of chromosomal imbalances as well as to verify the number of aberrations per case in different genetic subgroups of B-CLL (e.g. del11q, del13q, del17p, +12 or unmutated IgVH status) we performed high resolution genomic profiling using a genomic DNA-chip containing 2.800 probes. Target clones compriseda large genome-wide cluster of clones covering the genome at a distance of approx. 1.5Mb andclones mapping to genomic regions or genes of possible pathogenetic relevance in lymphoma. This chip covers approximately 10% of the human genome. In 93 (70%) of 133 analyzed B-CLL cases 171 genomic imbalances were identified (between 1–7 aberrations/case). Besides the confirmation of known recurrent chromosomal aberrations, previously unknown recurrent imbalances were detectable on 2p (8%), 4p (4%), 7p-q (5%), 10q (5%) and 20p (3%). Most of these imbalances were of larger extension (> 10 Mb) and therefore impeded a further delineation of minimal aberrant regions and the identification of possible candidate genes. The mean number of chromosomal aberrations per case (= genomic complexity) in IgVH unmutated CLLs was approx. 2 times higher than in mutated cases (0,77 vs. 1,58 per case). 84% of samples with > 2 aberrations showed an unmutated IgVH status. Moreover, most of the previously unknown imbalances were identified within this group. A higher genomic complexity was also shown for samples with gain on 2p vs. balanced 2p status (2.2 times higher; 2.5 vs. 1.2) and in samples with del17p vs. balanced 17p status (3.7 times higher; 3.52 vs. 0.95). 11q aberrations had no impact on the number of genomic aberrations per case (1.6 vs. 1.2). Moreover, we found a strong association of 2p gains and an unmutated IgVH status (100%). Array based genomic profiling confirmes the chromosomal aberration structure and underlines the consistency of chromosomal aberration patterns of B-CLL. The biological and prognostic relevance of 2p gains and unmutated IgVH mutational status have to be further investigated.

Genomic imbalances in 31 formalin-fixed and paraffin-embedded primary tumors of advanced breast cancer were analyzed by microarray-based comparative genomic hybridization (matrix-CGH). A DNA chip was designed comprising 422 mapped genomic sequences including 47 proto-oncogenes, 15 tumor suppressor genes, as well as frequently imbalanced chromosomal regions. Analysis of the data was challenging due to the impaired quality of DNA prepared from paraffin-embedded samples. Nevertheless, using a method for the statistical evaluation of the balanced state for each individual experiment, we were able to reveal imbalances with high significance, which were in good concordance with previous data collected by chromosomal CGH from the same patients. Owing to the improved resolution of matrix-CGH, genomic imbalances could be narrowed down to the level of individual bacterial artificial chromosome and P1-derived artificial chromosome clones. On average 37 gains and 13 losses per tumor cell genome were scored. Gains in more than 30% of the cases were found on 1p, 1q, 6p, 7p, 8q, 9q, 11q, 12q, 17p, 17q, 20q, and 22q, and losses on 6q, 9p, 11q, and 17p. Of the 51 chromosomal regions found amplified by matrix-CGH, only 12 had been identified by chromosomal CGH. Within these 51 amplicons, genome database information defined 112 candidate genes, 44 of which were validated by either PCR amplification of sequence tag sites or DNA sequence analysis.