Genomic arrays identify high-risk chronic lymphocytic leukemia with genomic complexity: a multi-center study.

Complex karyotype (CK) identified by chromosome-banding analysis (CBA) has shown prognostic value in chronic lymphocytic leukemia (CLL). Genomic arrays offer high-resolution genome-wide detection of copy-number alterations (CNAs) and could therefore be well equipped to detect the presence of a CK. Current knowledge on genomic arrays in CLL is based on outcomes of single center studies, in which different cutoffs for CNA calling were used. To further determine the clinical utility of genomic arrays for CNA assessment in CLL diagnostics, we retrospectively analyzed 2293 arrays from 13 diagnostic laboratories according to established standards. CNAs were found outside regions captured by CLL FISH probes in 34% of patients, and several of them including gains of 8q, deletions of 9p and 18p (p<0.01) were linked to poor outcome after correction for multiple testing. Patients (n=972) could be divided in three distinct prognostic subgroups based on the number of CNAs. Only high genomic complexity (high-GC), defined as ≥5 CNAs emerged as an independent adverse prognosticator on multivariable analysis for time to first treatment (Hazard ratio: 2.15, 95% CI: 1.36-3.41; p=0.001) and overall survival (Hazard ratio: 2.54, 95% CI: 1.54-4.17; p<0.001; n=528). Lowering the size cutoff to 1 Mb in 647 patients did not significantly improve risk assessment. Genomic arrays detected more chromosomal abnormalities and performed at least as well in terms of risk stratification compared to simultaneous chromosome banding analysis as determined in 122 patients. Our findings highlight genomic array as an accurate tool for CLL risk stratification.

Detection of Fusion Genes to Determine Minimal Residual Disease in Leukemia Using Next-Generation Sequencing.

BACKGROUND: Measuring minimal residual disease (MRD), the persistence of leukemic cells after treatment, is important for monitoring leukemia recurrence. The current methods for monitoring MRD are flow cytometry, to assess aberrant immune phenotypes, and digital droplet PCR (ddPCR), to target genetic aberrations such as single-nucleotide variants and gene fusions. We present the performance of an RNA-based next-generation sequencing (NGS) method for MRD gene fusion detection compared with ddPCR. This method may have advantages, including the capacity to analyze different genetic aberrations and patients in 1 experiment. In particular, detection at the RNA level may be highly sensitive if the genetic aberration is highly expressed. METHODS: We designed a probe-based NGS panel targeting the breakpoints of 11 fusion genes previously identified in clinical patients and 2 fusion genes present in cell lines. Blocking probes were added to prevent nonspecific enrichment. Each patient RNA sample was diluted in background RNA, depleted for rRNA and globin mRNA, converted to cDNA, and prepared for sequencing. Unique sequence reads, identified by unique molecular identifiers, were aligned directly to reference transcripts. The same patient and cell-line samples were also analyzed with ddPCR for direct comparison. RESULTS: Our NGS method reached a maximum sensitivity of 1 aberrant cell in 10 000 cells and was mostly within a factor of 10 compared with ddPCR. CONCLUSIONS: Our detection limit was below the threshold of 1:1000 recommended by European Leukemia Net. Further optimizations are easy to implement and are expected to boost the sensitivity of our method to diagnostically obtained ddPCR thresholds.

Targeted RNA-Sequencing Enables Detection of Relevant Translocations and Single Nucleotide Variants and Provides a Method for Classification of Hematological Malignancies-RANKING.

BACKGROUND: Patients with hematological malignancies (HMs) carry a wide range of chromosomal and molecular abnormalities that impact their prognosis and treatment. Since no current technique can detect all relevant abnormalities, technique(s) are chosen depending on the reason for referral, and abnormalities can be missed. We tested targeted transcriptome sequencing as a single platform to detect all relevant abnormalities and compared it to current techniques. MATERIAL AND METHODS: We performed RNA-sequencing of 1385 genes (TruSight RNA Pan-Cancer, Illumina) in bone marrow from 136 patients with a primary diagnosis of HM. We then applied machine learning to expression profile data to perform leukemia classification, a method we named RANKING. Gene fusions for all the genes in the panel were detected, and overexpression of the genes EVI1, CCND1, and BCL2 was quantified. Single nucleotide variants/indels were analyzed in acute myeloid leukemia (AML), myelodysplastic syndrome and patients with acute lymphoblastic leukemia (ALL) using a virtual myeloid (54 genes) or lymphoid panel (72 genes). RESULTS: RANKING correctly predicted the leukemia classification of all AML and ALL samples and improved classification in 3 patients. Compared to current methods, only one variant was missed, c.2447A>T in KIT (RT-PCR at 10-4), and BCL2 overexpression was not seen due to a t(14; 18)(q32; q21) in 2% of the cells. Our RNA-sequencing method also identified 6 additional fusion genes and overexpression of CCND1 due to a t(11; 14)(q13; q32) in 2 samples. CONCLUSIONS: Our combination of targeted RNA-sequencing and data analysis workflow can improve the detection of relevant variants, and expression patterns can assist in establishing HM classification.

Early detection and evolution of preleukemic clones in therapy-related myeloid neoplasms following autologous SCT.

Therapy-related myeloid neoplasms (tMNs) are severe adverse events that can occur after treatment with autologous hematopoietic stem cell transplantation (ASCT). This study aimed to investigate the development of tMNs following ASCT at the molecular level by whole-exome sequencing (WES) and targeted deep sequencing (TDS) in sequential (pre-) tMN samples. WES identified a significantly higher number of mutations in tMNs as compared with de novo myelodysplastic syndrome (MDS) (median 27 vs 12 mutations; P = .001). The mutations found in tMNs did not carry a clear aging-signature, unlike the mutations found in de novo MDS, indicating a different mutational mechanism. In some patients, tMN mutations were identified in both myeloid and T cells, suggesting that tMNs may originate from early hematopoietic stem cells (HSCs). However, the mutational spectra of tMNs and the preceding malignancies did not overlap, excluding common ancestry for these malignancies. By use of TDS, tMN mutations were identified at low variant allele frequencies (VAFs) in transplant material in 70% of the patients with tMNs. Reconstruction of clonal patterns based on VAFs revealed that premalignant clones can be present more than 7 years preceding a tMN diagnosis, a finding that was confirmed by immunohistochemistry on bone marrow biopsies. Our results indicate that tMN development after ASCT originates in HSCs bearing (pre-)tMN mutations that are present years before disease onset and that post-ASCT treatment can influence the selection of these clones. Early detection of premalignant clones and monitoring of their evolutionary trajectory may help to predict the development of tMNs and guide early intervention in the future.

Inferior survival for patients with malignant peripheral nerve sheath tumors defined by aberrant TP53.

Malignant peripheral nerve sheath tumor is a rare and aggressive disease with poor treatment response, mainly affecting adolescents and young adults. Few molecular biomarkers are used in the management of this cancer type, and although TP53 is one of few recurrently mutated genes in malignant peripheral nerve sheath tumor, the mutation prevalence and the corresponding clinical value of the TP53 network remains unsettled. We present a multi-level molecular study focused on aberrations in the TP53 network in relation to patient outcome in a series of malignant peripheral nerve sheath tumors from 100 patients and 38 neurofibromas, including TP53 sequencing, high-resolution copy number analyses of TP53 and MDM2, and gene expression profiling. Point mutations in TP53 were accompanied by loss of heterozygosity, resulting in complete loss of protein function in 8.2% of the malignant peripheral nerve sheath tumors. Another 5.5% had MDM2 amplification. TP53 mutation and MDM2 amplification were mutually exclusive and patients with either type of aberration in their tumor had a worse prognosis, compared to those without (hazard ratio for 5-year disease-specific survival 3.5, 95% confidence interval 1.78-6.98). Both aberrations had similar consequences on the gene expression level, as analyzed by a TP53-associated gene signature, a property also shared with the copy number aberrations and/or loss of heterozygosity at the TP53 locus, suggesting a common "TP53-mutated phenotype" in as many as 60% of the tumors. This was a poor prognostic phenotype (hazard ratio = 4.1, confidence interval:1.7-9.8), thus revealing a TP53-non-aberrant patient subgroup with a favorable outcome. The frequency of the "TP53-mutated phenotype" warrants explorative studies of stratified treatment strategies in malignant peripheral nerve sheath tumor.

Genetic Screening Test to Detect Translocations in Acute Leukemias by Use of Targeted Locus Amplification.

BACKGROUND: Over 500 translocations have been identified in acute leukemia. To detect them, most diagnostic laboratories use karyotyping, fluorescent in situ hybridization, and reverse transcription PCR. Targeted locus amplification (TLA), a technique using next-generation sequencing, now allows detection of the translocation partner of a specific gene, regardless of its chromosomal origin. We present a TLA multiplex assay as a potential first-tier screening test for detecting translocations in leukemia diagnostics. METHODS: The panel includes 17 genes involved in many translocations present in acute leukemias. Procedures were optimized by using a training set of cell line dilutions and 17 leukemia patient bone marrow samples and validated by using a test set of cell line dilutions and a further 19 patient bone marrow samples. Per gene, we determined if its region was involved in a translocation and, if so, the translocation partner. To balance sensitivity and specificity, we introduced a gray zone showing indeterminate translocation calls needing confirmation. We benchmarked our method against results from the 3 standard diagnostic tests. RESULTS: In patient samples passing QC, we achieved a concordance with benchmarking tests of 81% in the training set and 100% in the test set, after confirmation of 4 and nullification of 3 gray zone calls (in total). In cell line dilutions, we detected translocations in 10% aberrant cells at several genetic loci. CONCLUSIONS: Multiplex TLA shows promising results as an acute leukemia screening test. It can detect cryptic and other translocations in selected genes. Further optimization may make this assay suitable for diagnostic use.

MYC expression and translocation analyses in low-grade and transformed follicular lymphoma.

AIMS: Low-grade follicular lymphoma (FL) (grade 1/2, FL1/2) has an annual risk of transformation of ≈3%, which is associated with aberrations in CDKN2A/B, TP53, and MYC. As in diffuse large B-cell lymphoma, high MYC expression in transformed FL (tFL) might predict a MYC breakpoint. METHODS AND RESULTS: We quantified MYC expression by immunohistochemistry and digital analysis in 41 paired biopsies from 20 patients with FL1/2 with subsequent transformation and in four isolated biopsies of tFL. As controls, 28 biopsies of FL1/2 without transformation (median follow-up of 105 months) and nine biopsies of FL3A/B were analysed. In the 20 FL1/2-tFL pairs, MYC expression was significantly higher in tFL than in the initial FL1/2 biopsies (median 54% versus 6%; 7% in FL3A, and 35% in FL3B). MYC breaks (MYC-R) were detected in eight of 21 (38%) tFLs analysed by fluorescence in-situ hybridization (FISH), with a median MYC score of 86%. In two of the analysed tFL cases, the translocation was already detected in antecedent FL1/2. MYC partners were immunoglobulin (IG) loci in three of eight cases (one IGL, one IGH, and one IGK) and non-IG in five of eight cases (two PAX5, one BCL6, and two unknown). Of the eight MYC-R+ cases, six were BCL2+/MYC+ double-hit, one was BCL2+/BCL6+/MYC+ triple-hit, and one was MYC+ single-hit. All three IG-MYC+ cases showed a MYC expression level of >85%, whereas the five cases with a non-IG MYC partner had a wider range of expression (median 68%, range 13-86%). Among the 13 MYC-R- tFLs, two groups with almost dichotomous MYC expression could be observed (three cases showed ≥90% MYC expression), suggesting alternative mechanisms of MYC activation. CONCLUSIONS: we show an increase in MYC expression from FL1/2 to tFL. MYC breakpoints were present in ≈40% of the cases, which is markedly higher than in de novo DLBCL. MYC expression was uniformly high in cases with an IG-MYC translocation but much more heterogeneous and in part independent of the presence of a MYC break in non-IG-MYC and MYC-negative cases.

Guidelines for genomic array analysis in acquired haematological neoplastic disorders.

Genetic profiling is important for disease evaluation and prediction of prognosis or responsiveness to therapy in neoplasia. Microarray technologies, including array comparative genomic hybridization and single-nucleotide polymorphism-detecting arrays, have in recent years been introduced into the diagnostic setting for specific types of haematological malignancies and solid tumours. It can be used as a complementary test or depending on the neoplasia investigated, also as a standalone test. However, comprehensive and readable presentation of frequently identified complex genomic profiles remains challenging. To assist diagnostic laboratories, standardization and minimum criteria for clinical interpretation and reporting of acquired genomic abnormalities detected through arrays in neoplastic disorders are presented.

The entire miR-200 seed family is strongly deregulated in clear cell renal cell cancer compared to the proximal tubular epithelial cells of the kidney.

Despite numerous studies reporting deregulated microRNA (miRNA) and gene expression patterns in clear cell renal cell carcinoma (ccRCC), no direct comparisons have been made to its presumed normal counterpart: the renal proximal tubular epithelial cells (PTECs). The aim of this study was to determine the miRNA expression profiles of 10 ccRCC-derived cell lines and short-term cultures of PTEC and to correlate these with their gene expression and copy-number profiles. Using microarray-based methods, a significantly altered expression level in ccRCC cell lines was observed for 23 miRNAs and 1630 genes. The set of miRNAs with significantly decreased expression levels include all members of the miR-200 family known to be involved in the epithelial to mesenchymal transition process. Expression levels of 13 of the 47 validated target genes for the downregulated miRNAs were increased more than twofold. Our data reinforce the importance of the epithelial to mesenchymal transition process in the development of ccRCC.

Cas9-directed long-read sequencing to resolve optical genome mapping findings in leukemia diagnostics.

Leukemias are genetically heterogeneous and diagnostics therefore includes various standard-of-care (SOC) techniques, including karyotyping, SNP-array and FISH. Optical genome mapping (OGM) may replace these as it detects different types of structural aberrations simultaneously and additionally detects much smaller aberrations (500 bp vs 5-10 Mb with karyotyping). However, its resolution may still be too low to define clinical relevance of aberrations when they are located between two OGM labels or when labels are not distinct enough. Here, we test the potential of Cas9-directed long-read sequencing (LRS) as an additional technique to resolve such potentially relevant new findings. From an internal Bionano implementation study we selected ten OGM calls that could not be validated with SOC methods. Per variant we designed crRNAs for Cas9 enrichment, prepared libraries and sequenced them on a MinION/GridION device. We could confirm all aberrations and, importantly, the actual breakpoints of the OGM calls were located between 0.2 and 5.5 kb of the OGM-estimated breakpoints, confirming the high reliability of OGM. Furthermore, we show examples of redefinition of aberrations between labels that enable judgment of clinical relevance. Our results suggest that Cas9-directed LRS can be a relevant and flexible secondary technique in diagnostic workflows including OGM.

A single center analysis of nucleophosmin in acute myeloid leukemia: value of combining immunohistochemistry with molecular mutation analysis.

Mutations of nucleophosmin 1 are frequently found in acute myeloid leukemia and lead to aberrant cytoplasmic accumulation of nucleophosmin protein. Immunohistochemical staining is therefore recommended as the technique of choice in front-line screening. In this study, we assessed the sensitivity and specificity of immunohistochemistry on formalin-fixed bone marrow biopsies compared with gold standard molecular analysis to predict nucleophosmin 1 mutation status in 119 patients with acute myeloid leukemia. Discrepant cases were further characterized by gene expression analyses and fluorescence in situ hybridization. A large overlap between both methods was observed. Nevertheless, nine patients demonstrated discordant results at initial screening. Five cases demonstrated nuclear staining of nucleophosmin 1 by immunohistochemistry, but a nucleophosmin 1 mutation by molecular analysis. In two cases this could be attributed to technical issues and in three cases minor subpopulations of myeloblasts had not been discovered initially. All tested cases exhibited the characteristic nucleophosmin-mutated gene expression pattern. Four cases had cytoplasmic nucleophosmin 1 staining and a nucleophosmin-mutated gene expression pattern without a detectable nucleophosmin 1 mutation. In two of these cases we found the chromosomal translocation t(3;5)(q25;q35) encoding the NPM-MLF1 fusion protein. In the other discrepant cases the aberrant cytoplasmic nucleophosmin staining and gene expression could not be explained. In total six patients (5%) had true discordant results between immunohistochemistry and mutation analysis. We conclude that cytoplasmic nucleophosmin localization is not always caused by a conventional nucleophosmin 1 mutation and that in the screening for nucleophosmin 1 abnormalities, most information will be obtained by combining immunohistochemistry with molecular analysis.

Transcriptomic subtyping of malignant peripheral nerve sheath tumours highlights immune signatures, genomic profiles, patient survival and therapeutic targets.

BACKGROUND: Malignant peripheral nerve sheath tumour (MPNST) is an aggressive orphan disease commonly affecting adolescents or young adults. Current knowledge of molecular tumour biology has been insufficient for development of rational treatment strategies. We aimed to discover molecular subtypes of potential clinical relevance. METHODS: Fresh frozen samples of MPNSTs (n = 94) and benign neurofibromas (n = 28) from 115 patients in a European multicentre study were analysed by DNA copy number and/or transcriptomic profiling. Unsupervised transcriptomic subtyping was performed and the subtypes characterized for genomic aberrations, clinicopathological associations and patient survival. FINDINGS: MPNSTs were classified into two transcriptomic subtypes defined primarily by immune signatures and proliferative processes. "Immune active" MPNSTs (44%) had sustained immune signals relative to neurofibromas, were more frequently low-grade (P = 0.01) and had favourable prognostic associations in a multivariable model of disease-specific survival with clinicopathological factors (hazard ratio 0.25, P = 0.003). "Immune deficient" MPNSTs were more aggressive and characterized by proliferative signatures, high genomic complexity, aberrant TP53 and PRC2 loss, as well as high relative expression of several potential actionable targets (EGFR, ERBB2, EZH2, KIF11, PLK1, RRM2). Integrated gene-wise analyses suggested a DNA copy number-basis for proliferative transcriptomic signatures in particular, and the tumour copy number burden further stratified the transcriptomic subtypes according to patient prognosis (P < 0.01). INTERPRETATION: Approximately half of MPNSTs belong to an "immune deficient" transcriptomic subtype associated with an aggressive disease course, PRC2 loss and expression of several potential therapeutic targets, providing a rationale for molecularly-guided intervention trials. FUNDING: Research grants from non-profit organizations, as stated in the Acknowledgements.

Targeted exome sequencing in clear cell renal cell carcinoma tumors suggests aberrant chromatin regulation as a crucial step in ccRCC development.

Clear cell renal cell carcinomas are characterized by 3p loss, and by inactivation of Von Hippel Lindau (VHL), a tumorsuppressor gene located at 3p25. Recently, SETD2, located at 3p21, was identified as a new candidate ccRCC tumor-suppressor gene. The combined mutational frequency in ccRCC tumors of VHL and SETD2 suggests that there are still undiscovered tumor-suppressor genes on 3p. We screened all genes on 3p for mutations in 10 primary ccRCC tumors using exome-sequencing. We identified inactivating mutations in VHL, PBRM1, and BAP1. Sequencing of PBRM1 in ccRCC-derived cell lines confirmed its frequent inactivation in ccRCC. PBRM1 encodes for BAF180, the chromatin targeting subunit of the SWI/SNF complex. BAP1 encodes for BRCA1 associated protein-1, involved in histone deubiquitination. Taken together, the accumulating data suggest an important role for aberrant chromatin regulation in ccRCC development.

Microarray-based genomic profiling as a diagnostic tool in acute lymphoblastic leukemia.

In acute lymphoblastic leukemia (ALL) specific genomic abnormalities provide important clinical information. In most routine clinical diagnostic laboratories conventional karyotyping, in conjunction with targeted screens using e.g., fluorescence in situ hybridization (FISH), is currently considered as the gold standard to detect such aberrations. Conventional karyotyping, however, is limited in its resolution and yield, thus hampering the genetic diagnosis of ALL. We explored whether microarray-based genomic profiling would be feasible as an alternative strategy in a routine clinical diagnostic setting. To this end, we compared conventional karyotypes with microarray-deduced copy number aberration (CNA) karyotypes in 60 ALL cases. Microarray-based genomic profiling resulted in a CNA detection rate of 90%, whereas for conventional karyotyping this was 61%. In addition, many small (< 5 Mb) genetic lesions were encountered, frequently harboring clinically relevant ALL-related genes such as CDKN2A/B, ETV6, PAX5, and IKZF1. From our data we conclude that microarray-based genomic profiling serves as a robust tool in the genetic diagnosis of ALL, outreaching conventional karyotyping in CNA detection both in terms of sensitivity and specificity. We also propose a practical workflow for a comprehensive and objective interpretation of CNAs obtained through microarray-based genomic profiling, thereby facilitating its application in a routine clinical diagnostic setting.

Comorbidities and malignancies negatively affect survival in myelodysplastic syndromes: a population-based study.

Population-based studies that contain detailed clinical data on patients with myelodysplastic syndrome (MDS) are scarce. This study focused on the real-world overall survival (OS) of MDS patients in association with comorbidities, specifically malignancies. An observational population-based study using the HemoBase registry was performed, including all patients with MDS diagnosed between 2005 and 2017 in Friesland, a Dutch province. Detailed information about diagnosis, patient characteristics, previous treatment of malignancies, and comorbidities according to the Charlson Comorbidity Index (CCI) was collected from electronic health records. Patients were followed up until June 2019. Kaplan-Meier plots and Cox regression analyses were used to study survival differences. In the 291 patients diagnosed with MDS, the median OS was 25.3 months (95% confidence interval [CI], 20.3-30.2). OS was significantly better for patients with CCI score <4, age <65 years, female sex, and low-risk MDS. Fifty-seven patients (20%) had encountered a prior malignancy (excluding nonmelanoma skin cancer), and a majority (38 patients; 67%) were therapy related. Both therapy-related and secondary MDSs were associated with worse OS (hazard ratio, 1.51; 95% CI, 1.02-2.23 and 1.58; 95% CI, 0.95-2.65, respectively), as compared with de novo MDS patients (P = .04). Patients in remission at time of MDS diagnosis had a similar median OS compared with patients with de novo MDS (25.5 vs 28.3 months). This population-based study involving all newly diagnosed MDS patients over a 13-year period in Friesland showed that multiple comorbidities, including previous malignancies, are associated with shorter OS. OS was not related to the use of radiotherapy or chemotherapy.

Mantle cell lymphomas with concomitant MYC and CCND1 breakpoints are recurrently TdT positive and frequently show high-grade pathological and genetic features.

Chromosomal breakpoints involving the MYC gene locus, frequently referred to as MYC rearrangements (MYC - R+), are a diagnostic hallmark of Burkitt lymphoma and recurrent in many other subtypes of B-cell lymphomas including follicular lymphoma, diffuse large B-cell lymphoma and other high-grade B-cell lymphomas and are associated with an aggressive clinical course. In remarkable contrast, in MCL, only few MYC - R+ cases have yet been described. In the current study, we have retrospectively analysed 16 samples (MYC - R+, n = 15, MYC - R-, n = 1) from 13 patients and describe their morphological, immunophenotypic and (molecular) genetic features and clonal evolution patterns. Thirteen out of fifteen MYC - R+ samples showed a non-classical cytology including pleomorphic (centroblastic, immunoblastic), anaplastic or blastoid. MYC translocation partners were IG-loci in 4/11 and non-IG loci in 7/11 analysed cases. The involved IG-loci included IGH in 3 cases and IGL in one case. PAX5 was the non-IG partner in 2/7 patients. The MYC - R+ MCL reported herein frequently displayed characteristics associated with an aggressive clinical course including high genomic-complexity (6/7 samples), frequent deletions involving the CDKN2A locus (7/10 samples), high Ki-67 proliferation index (12/13 samples) and frequent P53 expression (13/13 samples). Of note, in 4/14 samples, SOX11 was not or only focally expressed and 3/13 samples showed focal or diffuse TdT-positivity presenting a diagnostic challenge as these features could point to a differential diagnosis of diffuse large B-cell lymphoma and/or lymphoblastic lymphoma/leukaemia.

Germline and somatic NF1 mutations in sporadic and NF1-associated malignant peripheral nerve sheath tumours.

Malignant peripheral nerve sheath tumours (MPNSTs) are a malignancy occurring with increased frequency in patients with neurofibromatosis type 1 (NF1). In contrast to the well-known spectrum of germline NF1 mutations, the information on somatic mutations in MPNSTs is limited. In this study, we screened NF1, KRAS, and BRAF in 47 MPNSTs from patients with (n = 25) and without (n = 22) NF1. In addition, DNA from peripheral blood and cutaneous neurofibroma biopsies from, respectively, 14/25 and 7/25 of the NF1 patients were analysed. Germline NF1 mutations were detected in ten NF1 patients, including three frameshift, three nonsense, one missense, one splicing alteration, and two large deletions. Somatic NF1 mutations were found in 10/25 (40%) NF1-associated MPNSTs, in 3/7 (43%) neurofibromas, and in 9/22 (41%) sporadic MPNSTs. Large genomic copy number changes accounted for 6/10 and 7/13 somatic mutations in NF1-associated and sporadic MPNSTs, respectively. Two NF1-associated and 13 sporadic MPNSTs did not show any NF1 mutation. A major role of the KRAS and BRAF genes was ruled out. The spectrum of germline NF1 mutations in neurofibromatosis patients with MPNST is different from the spectrum of somatic mutations seen in MPNSTs. However, the somatic events share common characteristics with the NF1-related and the sporadic tumours.

Dexamethasone-based therapy for childhood acute lymphoblastic leukaemia: results of the prospective Dutch Childhood Oncology Group (DCOG) protocol ALL-9 (1997-2004).

BACKGROUND: A population-based cohort of children aged 1-18 years with acute lymphoblastic leukaemia (ALL) was treated with a dexamethasone-based protocol (Dutch Childhood Oncology Group [DCOG] ALL-9). We aimed to confirm the results of the most effective DCOG ALL protocol for non-high-risk (NHR) patients to date (ALL-6), compare results with ALL-7 and ALL-8, and study prognostic factors in a non-randomised setting. METHODS: From Jan 1, 1997, until Nov 1, 2004, patients with ALL were treated according to the ALL-9 protocol in eight Dutch academic centres with their affiliated peripheral hospitals. Patients were stratified into NHR and high risk (HR) groups. HR criteria were white-blood-cell count of 50,000 cells per microL or more, T-cell phenotype, mediastinal mass, CNS or testicular involvement, and Philadelphia chromosome or MLL rearrangement; patients who did not fulfil these criteria were deemed to be NHR. The NHR group was treated with a three-drug induction (dexamethasone, vincristine, and asparaginase) for 6 weeks, medium-dose methotrexate for 3 weeks, then maintenance therapy. HR patients received a four-drug induction (as for the NHR patients plus daunorubicin) for 6 weeks, high-dose methotrexate for 8 weeks, and two intensification courses before receiving maintenance therapy. Triple intrathecal medication was given 13 times in NHR patients, 15 times in HR patients (17 times for patients with initial CNS involvement). No patient received cranial irradiation. Maintenance therapy was given until 109 weeks for all patients and consisted of mercaptopurine and methotrexate for 5 weeks, alternated with dexamethasone and vincristine for 2 weeks. Kaplan-Meier analysis was done on an intention-to-treat basis with event-free survival as the primary endpoint. This trial is registered at trialregister.nl, number NTR460/SNWLK-ALL-9. FINDINGS: 859 patients were recruited to the study. Complete remission was achieved in 592 (98.5%) of the 601 patients in the NHR group and 250 (96.9%) of the 258 in the HR group. Five patients in the NHR group and four in the HR group died during induction. Median follow-up for patients alive was 72.2 (range 4.8-132.7) months as of August, 2008. 5-year event-free survival was 81% (SE 1%) in all patients: 84% (2%) in NHR patients, and 72% (3%) in HR patients. Isolated CNS relapses occurred in 22 (2.6%) of 842 patients. In a multivariate analysis, DNA index was the strongest predictor of outcome (<1.16 vs >or=1.16; relative risk 0.42, 95% CI 0.22-0.78), followed by age (1-9 vs >or=10 years; 2.23, 1.60-3.11) and white-blood-cell count (<50,000 vs >or=50,000 cells per microL; 1.60, 1.13-2.26). INTERPRETATION: The overall results of the dexamethasone-based DCOG ALL-9 protocol are better than those of our previous Berlin-Frankfurt-Münster-based protocols ALL-7 and ALL-8. The results for NHR patients were achieved with high cumulative doses of dexamethasone and vincristine, but without the use of anthracyclines, etoposide, cyclophosphamide, or cranial irradiation, therefore minimising the risk of side-effects. FUNDING: Dutch Health Insurers.

Not type of induction therapy but consolidation with allogeneic hematopoietic cell transplantation determines outcome in older AML patients: A single center experience of 355 consecutive patients.

Therapeutic decision making is often challenging in older AML patients. We collected retrospective data of 355 consecutive AML patients (≥60 years) who were treated with intensive chemotherapy (IC) (n = 155), hypomethylating agents (HMA) (n = 83), or best supportive care (BSC) (n = 117) between 2002 and 2017. Overall survival (OS) and response rates after therapy were analyzed. Multivariate Cox regression was performed to analyze the impact of different treatment strategies on survival. The median OS was not significantly different between patients treated with IC or HMA (14.9 vs 10.9 months; HR = 1.32, p = 0.076)), despite a difference in complete remission rate (59% after IC vs 35% after HMA). Patients who received a allogeneic hematopoietic cell transplantation (allo HCT) after treatment with IC or HMA had a significant survival benefit compared to patient who didn't proceed to allo HCT (median OS 65 vs 8 months, respectively, p < 0.001). The type of induction therapy (i.e. IC or HMA) did not impact on survival after allo HCT (48 vs 65 months, respectively, p = 0.440). In conclusion, consolidation with an allo HCT provides a significant benefit for older AML patients independent of upfront treatment with IC or HMA. Our data suggest that more older patients should be considered for an allo HCT.