Antitumor Activity of Larotrectinib in Esophageal Carcinoma with NTRK Gene Amplification.

BACKGROUND: Increasing knowledge about the genomic changes underpinning cancer development and growth has led to a rapidly expanding number of individualized therapies that specifically target these changes in a patient's tumor. Here we present a case report of a patient with metastatic esophageal carcinoma whose tumor harbored NTRK1 gene amplification and who received targeted systemic therapy with larotrectinib. At initial diagnosis, the patient presented with tumor obstruction of the middle esophagus, simultaneous liver and lung metastases, UICC IV and WHO performance status 3. MATERIALS AND METHODS: The solid tumor genomic profiling test FoundationOne CDx (F1CDx) was used to detect clinically relevant genomic alterations that, in turn, might identify a targeted therapeutic approach if suggested by the findings. The patient was then treated with larotrectinib and had subsequent follow-up biopsies. RESULTS: Simultaneous biopsies of the primary tumor and liver lesions identified a metastatic squamous cell esophageal carcinoma. Comprehensive genomic profiling obtained from liver metastases identified numerous genomic alterations including amplification of NTRK1. Owing to the reduced performance status of the patient, chemotherapy could not be applied and was denied. Although larotrectinib is only approved for the treatment of cancers with NTRK gene fusions, treatment was started and led to a shrinkage of the primary tumor as well as the liver and lung metastases within 6 weeks according to RECIST criteria accompanied by tumor marker decrease. The NTRK1 gene amplification was below the limit of detection in a subsequent liver biopsy. CONCLUSION: The use of comprehensive genomic profiling, specifically F1CDx, enabled the selection of a targeted therapy that led to a rapid reduction of the tumor and its metastases according to RECIST criteria. This case suggests that larotrectinib is not only effective in NTRK fusions but may be efficacious in cases with gene amplification. KEY POINTS: Advances in precision medicine have revolutionized the treatment of cancer and have allowed oncologists to perform more individualized therapy. This case shows that larotrectinib could also be effective in cases of NTRK amplification of cancer. Today, there is only limited knowledge about NTRK alterations in squamous epithelial carcinoma of the esophagus. Longitudinal tumor sequencing during the course of the disease may allow for the detection of a molecular genetic cause once the tumor progresses. Additional actionable gene alterations may then be identified, which may provide the rationale for a therapy switch.

A meta-analysis of 120 246 individuals identifies 18 new loci for fibrinogen concentration.

Genome-wide association studies have previously identified 23 genetic loci associated with circulating fibrinogen concentration. These studies used HapMap imputation and did not examine the X-chromosome. 1000 Genomes imputation provides better coverage of uncommon variants, and includes indels. We conducted a genome-wide association analysis of 34 studies imputed to the 1000 Genomes Project reference panel and including ∼120 000 participants of European ancestry (95 806 participants with data on the X-chromosome). Approximately 10.7 million single-nucleotide polymorphisms and 1.2 million indels were examined. We identified 41 genome-wide significant fibrinogen loci; of which, 18 were newly identified. There were no genome-wide significant signals on the X-chromosome. The lead variants of five significant loci were indels. We further identified six additional independent signals, including three rare variants, at two previously characterized loci: FGB and IRF1. Together the 41 loci explain 3% of the variance in plasma fibrinogen concentration.

Sex-specific differences in genetic and nongenetic determinants of mean platelet volume: results from the Gutenberg Health Study.

Mean platelet volume (MPV), a measure of platelet size, is a potential biological marker of platelet function. To date, a comprehensive analysis including known genetic and nongenetic factors that determine MPV is still lacking. MPV has been evaluated in 15 010 individuals from the population-based Gutenberg Health Study. Genetic information was available for 4175 individuals. Our results showed that age (ß, 0.0346; 95% confidence interval [CI], 0.0255 to 0.0436), cardiovascular risk factors (CVRFs) such as smoking (ß, 0.178; 95% CI, 0.128 to 0.229), hypertension (ß, 0.05; 95% CI, 0.00289 to .0981), and high glucose level (ß, 0.00179; 95% CI, 0.0006 to 0.00299) were linked with higher MPV in males only. Intake of oral contraceptives (ß, 0.150; 95% CI, 0.0649 to 0.236) and menstruation (ß, 0.123; 95% CI, 0.0231 to 0.224) were strongly associated with higher MPV in females. Seven single nucleotide polymorphisms (SNPs) for females and 4 SNPs for males were associated with higher MPV. The full model, including age, CVRFs, laboratory parameters, medications, and genetic variation, explained 20.4% of the MPV variance in females and 18.6% in males. The curves of cumulative mortality, stratified for sex, showed worse survival for males only with MPV > 9.96 fL vs MPV ≤ 9.96 fL (P < .0001). This study provides evidence for heterogeneity in the profile of determinants for MPV between sexes. The observed interactions between genetic variability, CVRFs, and MPV and its association with the development of cardiovascular disease or thrombotic risk need to be further investigated.

Comprehensive mutational profiling in advanced systemic mastocytosis.

To explore mechanisms contributing to the clinical heterogeneity of systemic mastocytosis (SM) and to suboptimal responses to diverse therapies, we analyzed 39 KIT D816V mutated patients with indolent SM (n = 10), smoldering SM (n = 2), SM with associated clonal hematologic nonmast cell lineage disorder (SM-AHNMD, n = 5), and aggressive SM (n = 15) or mast cell leukemia (n = 7) with (n = 18) or without (n = 4) AHNMD for additional molecular aberrations. We applied next-generation sequencing to investigate ASXL1, CBL, IDH1/2, JAK2, KRAS, MLL-PTD, NPM1, NRAS, TP53, SRSF2, SF3B1, SETBP1, U2AF1 at mutational hotspot regions, and analyzed complete coding regions of EZH2, ETV6, RUNX1, and TET2. We identified additional molecular aberrations in 24/27 (89%) patients with advanced SM (SM-AHNMD, 5/5; aggressive SM/mast cell leukemia, 19/22) whereas only 3/12 (25%) indolent SM/smoldering SM patients carried one additional mutation each (U2AF1, SETBP1, CBL) (P < .001). Most frequently affected genes were TET2, SRSF2, ASXL1, CBL, and RUNX1. In advanced SM, 21/27 patients (78%) carried ≥3 mutations, and 11/27 patients (41%) exhibited ≥5 mutations. Overall survival was significantly shorter in patients with additional aberrations as compared to those with KIT D816V only (P = .019). We conclude that biology and prognosis in SM are related to the pattern of mutated genes that are acquired during disease evolution.

EZH2 mutations are frequent and represent an early event in follicular lymphoma.

Gain of function mutations in the H3K27 methyltransferase EZH2 represent a promising therapeutic target in germinal center lymphomas. In this study, we assessed the frequency and distribution of EZH2 mutations in a large cohort of patients with follicular lymphoma (FL) (n = 366) and performed a longitudinal analysis of mutation during the disease progression from FL to transformed FL (tFL) (n = 33). Mutations were detected at 3 recurrent mutation hot spots (Y646, A682, and A692) in 27% of FL cases with variant allele frequencies (VAF) ranging from 2% to 61%. By comparing VAF of EZH2 with other mutation targets (CREBBP, MLL2, TNFRSF14, and MEF2B), we were able to distinguish patients harboring clonal EZH2 mutation from rarer cases with subclonal mutations. Overall, the high incidence of EZH2 mutations in FL and their stability during disease progression makes FL an appropriate disease to evaluate EZH2 targeted therapy.

BRCC3 mutations in myeloid neoplasms.

Next generation sequencing technologies have provided insights into the molecular heterogeneity of various myeloid neoplasms, revealing previously unknown somatic genetic events. In our cohort of 1444 cases analyzed by next generation sequencing, somatic mutations in the gene BRCA1-BRCA2-containing complex 3 (BRCC3) were identified in 28 cases (1.9%). BRCC3 is a member of the JAMM/MPN+ family of zinc metalloproteases capable of cleaving Lys-63 linked polyubiquitin chains, and is implicated in DNA repair. The mutations were located throughout its coding region. The average variant allelic frequency of BRCC3 mutations was 30.1%, and by a serial sample analysis at two different time points a BRCC3 mutation was already identified in the initial stage of a myelodysplastic syndrome. BRCC3 mutations commonly occurred in nonsense (n=12), frameshift (n=4), and splice site (n=5) configurations. Due to the marginal male dominance (odds ratio; 2.00, 0.84-4.73) of BRCC3 mutations, the majority of mutations (n=23; 82%) were hemizygous. Phenotypically, BRCC3 mutations were frequently observed in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms and associated with -Y abnormality (odds ratio; 3.70, 1.25-11.0). Clinically, BRCC3 mutations were also related to higher age (P=0.01), although prognosis was not affected. Knockdown of Brcc3 gene expression in murine bone marrow lineage negative, Sca1 positive, c-kit positive cells resulted in 2-fold more colony formation and modest differentiation defect. Thus, BRCC3 likely plays a role as tumor-associated gene in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.

Investigation of 305 patients with myelodysplastic syndromes and 20q deletion for associated cytogenetic and molecular genetic lesions and their prognostic impact.

In patients with myelodysplastic syndromes (MDS), sole 20q deletion [del(20q)] is a recurrent favourable abnormality. We studied additional molecular and cytogenetic lesions and their prognostic impact in 305 MDS patients with del(20q) (229 males/76 females; 29-90 years). All patients were investigated by cytomorphology and chromosome banding analysis (CBA), subsets by fluorescence in situ hybridization, molecular mutation screening, and array comparative genomic hybridization (aCGH). By aCGH (n = 30), the minimal common deleted region (CDR) was flanked by PTPRT (20q13·11) and EYA2 (20q13·12). 210 (68·9%) patients had 'early MDS' without blast increase, 95 (31·1%) 'advanced' MDS with blast increase (5-19%). Additional chromosomal abnormalities (ACAs) were detected in 88/305 (28·9%) patients. Patients with advanced MDS more frequently had ACAs (P = 0·003) and had a higher mean number of ACAs (P = 0·020) and of molecular mutations (P = 0·060). Spliceosome mutations were frequent (U2AF1: n = 31/155; 20·0%; SRSF2: n = 31/159; 19·5%; SF3B1mut: n = 8/159; 5·0%). ASXL1mut (25/153; 16·3%) were associated with advanced MDS (P = 0·001). Presence of ≥3 ACAs (P = 0·003) and ASXL1mut (P = 0·002) were associated with worse 2-year survival. In conclusion, the cytogenetic subgroup of MDS with del(20q) has a good prognosis but may be further subclassified by additional cytogenetic and molecular lesions. U2AF1mut is overrepresented in MDS with del(20q), and ASXL1mut is prognostically adverse.

Development and validation of a real-time quantification assay to detect and monitor BRAFV600E mutations in hairy cell leukemia.

The BRAFV600E mutation was recently detected in hairy cell leukemia (HCL) by whole exome sequencing. To make use of this new marker for diagnosis and follow-up of HCL, we developed a BRAFV600Emut-specific quantitative real-time PCR assay and validated it in 117 HCL patients and 102 non-HCL/BRAFwt patients. The cut-off level to discriminate BRAFV600E-positive/-negative cases was set at 0.023% BRAFV600E/BRAFwt. A total of 115 of 117 HCL (98.3%) demonstrated percentage BRAFV600E/BRAFwt above the cut-off (mean, 29.6 ± 41.1). The remaining 2 of 117 HCL with lower percentage BRAFV600E/BRAFwt ratios were also BRAFwt by deep-sequencing technology. Sixteen HCL-variant patients showed percentage BRAFV600E/BRAFwt values corresponding to "non-HCL." Follow-up studies in 19 HCL cases demonstrated a decrease of percentage BRAFV600E/BRAFwt during therapy. The log-reductions as determined by RT-PCR and immunophenotyping correlated significantly (P < .001). In conclusion, we confirmed BRAFmut as a useful marker in HCL, its absence in HCL variant, and developed an RT-PCR-based assay to monitor minimal residual disease in HCL.

Multilineage dysplasia does not influence prognosis in CEBPA-mutated AML, supporting the WHO proposal to classify these patients as a unique entity.

In 2008, the World Health Organization introduced CEBPA (encoding the CCAAT/enhancer binding protein)-mutated acute myeloid leukemia (AML) as a provisional entity. However, the classification of CEBPA-mutated AML with multilineage dysplasia (MLD; ≥ 50% dysplastic cells in 2-3 lineages) remains to be clarified. In the present study, we investigated 108 CEBPA-mutated AML patients for the impact of MLD, karyotype, and additional mutations. MLD(+) patients differed from MLD(-) patients only by lower mean WBC counts, not by biologic characteristics, cytogenetic risk profiles, or additional mutations. Survival was better for female patients, patients < 60 years of age, for intermediate versus adverse karyotypes, and, in the case of FLT3-ITD negativity, biallelic versus monoallelic/homozygous CEBPA mutations. In contrast, 2-year overall survival and event-free survival did not differ significantly between MLD(+) and MLD(-) patients. By univariable Cox regression analysis, sex, age, WBC count, and cytogenetic risk category were related to overall survival, but MLD was not. Therefore, because dysplasia is not relevant for this subtype, CEBPA-mutated AML patients should be characterized only according to mutation status, cytogenetic risk group, or additional mutations.

SRSF2 mutations in 275 cases with chronic myelomonocytic leukemia (CMML).

We analyzed the mutational hotspot region of SRSF2 (Pro95) in 275 cases with chronic myelomonocytic leukemia (CMML). In addition, ASXL1, CBL, EZH2, JAK2V617F, KRAS, NRAS, RUNX1, and TET2 mutations were investigated in subcohorts. Mutations in SRSF2 (SRSF2mut) were detected in 47% (129 of 275) of all cases. In detail, 120 cases had a missense mutation at Pro95, leading to a change to Pro95His, Pro95Leu, Pro95Arg, Pro95Ala, or Pro95Thr. In 9 cases, 3 new in/del mutations were observed: 7 cases with a 24-bp deletion, 1 case with a 3-bp duplication, and 1 case with a 24-bp duplication. In silico analyses predicted a damaging character for the protein structure of SRSF2 for all mutations. SRSF2mut was correlated with higher age, less pronounced anemia, and normal karyotype. SRSF2mut and EZH2mut were mutually exclusive, but SRSF2mut was associated with TET2mut. In the total cohort, no effect of SRSF2mut on survival was observed. However, in the RUNX1mut subcohort, SRSF2 Pro95His had a favorable effect on overall survival. This comprehensive mutation analysis found that 93% of all patients with CMML carried at least 1 somatic mutation in 9 recurrently mutated genes. In conclusion, these data show the importance of SRSF2mut as new diagnostic marker in CMML.

A novel hierarchical prognostic model of AML solely based on molecular mutations.

The karyotype is so far the most important prognostic parameter in acute myeloid leukemia (AML). Molecular mutations have been analyzed to subdivide AML with normal karyotype into prognostic subsets. The aim of this study was to develop a prognostic model for the entire AML cohort solely based on molecular markers. One thousand patients with cytogenetic data were investigated for the following molecular alterations: PML-RARA, RUNX1-RUNX1T1, CBFB-MYH11, FLT3-ITD, and MLL-PTD, as well as mutations in NPM1, CEPBA, RUNX1, ASXL1, and TP53. Clinical data were available in 841 patients. Based on Cox regression and Kaplan-Meier analyses, 5 distinct prognostic subgroups were identified: (1) very favorable: PML-RARA rearrangement (n = 29) or CEPBA double mutations (n = 42; overall survival [OS] at 3 years: 82.9%); (2) favorable: RUNX1-RUNX1T1 (n = 35), CBFB-MYH11 (n = 31), or NPM1 mutation without FLT3-ITD (n = 186; OS at 3 years: 62.6%); (3) intermediate: none of the mutations leading to assignment into groups 1, 2, 4, or 5 (n = 235; OS at 3 years: 44.2%); (4) unfavorable: MLL-PTD and/or RUNX1 mutation and/or ASXL1 mutation (n = 203; OS at 3 years: 21.9%); and (5) very unfavorable: TP53 mutation (n = 80; OS at 3 years: 0%; P < .001). This comprehensive molecular characterization provides a more powerful model for prognostication than cytogenetics.

The molecular profile of adult T-cell acute lymphoblastic leukemia: mutations in RUNX1 and DNMT3A are associated with poor prognosis in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and heterogeneous disease. The diagnosis is predominantly based on immunophenotyping. In addition to known cytogenetic abnormalities molecular mutations were recently identified. Here, 90 adult T-ALL cases were investigated for mutations in NOTCH1, FBXW7, PHF6, CDKN2A, DNMT3A, FLT3, PTEN, and RUNX1 using 454 next-generation amplicon sequencing and melting curve analyses. These data were further complemented by FISH, chromosome banding, array CGH, and CDKN2B promoter methylation analyses. NOTCH1 was the most frequently mutated gene with a 71.1% frequency followed by FBXW7 (18.9%), PHF6 (39.5%), DNMT3A (17.8%), RUNX1 (15.5%), PTEN (10.0%), CDKN2A (4.4%), FLT3-ITD (2.2%), and FLT3-TKD (1.1%). In total, 84/90 (93.3%) cases harbored at least one mutation. Combining these data with CDKN2A/B deletions and CDKN2B methylation status, we detected minimum one aberration in 89/90 (98.9%) patients. Survival analyses revealed the subtype as defined by the immunophenotype as the strongest independent prognostic factor. When restricting the survival analysis to the early T-ALL subtype, a strong association of RUNX1 (P = 0.027) and DNMT3A (P = 0.005) mutations with shorter overall survival was observed. In conclusion, RUNX1 and DNMT3A are frequently mutated in T-ALL and are associated with poor prognosis in early T-ALL.

Next-generation sequencing - feasibility and practicality in haematology.

Next-generation sequencing platforms have evolved to provide an accurate and comprehensive means for the detection of molecular mutations in heterogeneous tumour specimens. Here, we review the feasibility and practicality of this novel laboratory technology. In particular, we focus on the utility of next-generation sequencing technology in characterizing haematological neoplasms and the landmark findings in key haematological malignancies. We also discuss deep-sequencing strategies to analyse the constantly increasing number of molecular markers applied for disease classification, patient stratification and individualized monitoring of minimal residual disease. Although many facets of this assay need to be taken into account, amplicon deep-sequencing has already demonstrated a promising technical performance and is being continuously developed towards routine application in diagnostic laboratories so that an impact on clinical practice can be achieved.

CEBPA double-mutated acute myeloid leukaemia harbours concomitant molecular mutations in 76·8% of cases with TET2 and GATA2 alterations impacting prognosis.

Acute myeloid leukaemia (AML) with CEBPA mutations is listed as a provisional entity in the current World Health Organization classification. A difference in clinical outcome between single- (sm) and double-mutated (dm) cases has been reported, whereupon CEBPAdm cases were shown to be associated with better overall survival (OS). The occurrence and prognostic impact of concomitant molecular mutations in addition to CEBPAdm has not been assessed until now with exception of GATA2 mutations. Here, we investigated a cohort of 95 AML CEBPAdm cases for concomitant mutations. TET2 was found to be most frequently mutated (34·0%) gene, followed by GATA2 (21·0%), WT1 (13·7%), DNMT3A (9·6%), ASXL1 (9·5%), NRAS (8·4%), KRAS (3·2%), IDH1/2 (6·3%), FLT3-internal tandem duplication (6·3%), FLT3-tyrosine kinase domain (2·1%), NPM1 (2·1%), and RUNX1 (1/94). Patients harbouring additional mutations in the TET2 gene showed significantly worse OS than TET2 wild-type cases (P = 0·035), whereas GATA2-mutated patients showed improved OS (P = 0·032). Serial analyses were performed for 39 CEBPAdm cases with concomitant mutations. Here, we observed that CEBPA mutations present the primary pathogenetic event in the majority of cases (76·9%). Further, a distinct gene expression profile (GEP) was confirmed for CEBPAdm versus CEBPAsm or CEBPA wild-type cases while no significant changes in GEP were observed related to additional mutations within the CEBPAdm AML.

Whole-exome sequencing identifies somatic mutations of BCOR in acute myeloid leukemia with normal karyotype.

Among acute myeloid leukemia (AML) patients with a normal karyotype (CN-AML), NPM1 and CEBPA mutations define World Health Organization 2008 provisional entities accounting for approximately 60% of patients, but the remaining 40% are molecularly poorly characterized. Using whole-exome sequencing of one CN-AML patient lacking mutations in NPM1, CEBPA, FLT3-ITD, IDH1, and MLL-PTD, we newly identified a clonal somatic mutation in BCOR (BCL6 corepressor), a gene located on chromosome Xp11.4. Further analyses of 553 AML patients showed that BCOR mutations occurred in 3.8% of unselected CN-AML patients and represented a substantial fraction (17.1%) of CN-AML patients showing the same genotype as the AML index patient subjected to whole-exome sequencing. BCOR somatic mutations were: (1) disruptive events similar to the germline BCOR mutations causing the oculo-facio-cardio-dental genetic syndrome; (2) associated with decreased BCOR mRNA levels, absence of full-length BCOR, and absent or low expression of a truncated BCOR protein; (3) virtually mutually exclusive with NPM1 mutations; and (4) frequently associated with DNMT3A mutations, suggesting cooperativity among these genetic alterations. Finally, BCOR mutations tended to be associated with an inferior outcome in a cohort of 422 CN-AML patients (25.6% vs 56.7% overall survival at 2 years; P = .032). Our results for the first time implicate BCOR in CN-AML pathogenesis.

Ruxolitinib as potential targeted therapy for patients with JAK2 rearrangements.

JAK2 fusion genes are rare but recurrent abnormalities associated with diverse, clinically heterogeneous hematologic malignancies. Here we assess the JAK1/2 inhibitor ruxolitinib as therapy for patients with JAK2-rearrangement associated myeloproliferative neoplasms (MPN). Ruxolitinib-treated Ba/F3 cells transformed to IL3 independence by ETV6-JAK2 showed reduced proliferation and survival (IC(50) = 370 nM) compared with KG1A or Ba/F3 cells transformed by BCR-ABL1, SPBN1-FLT3 and ZMYM2-FGFR1 (IC(50) > 10 µM for all). Inhibition was associated with reduced phosphorylation of ETV6-JAK2, ERK, STAT5 and AKT. Primary cell growth from 2 patients with JAK2 rearrangement and one patient with JAK2 amplification was assessed in methylcellulose assays. Reduced colony growth was seen for all patients in ruxolitinib-treated cultures compared with healthy controls (n=7). Fluorescence in situ hybridization showed reduced growth of JAK2-rearrangement positive colonies compared to JAK2-rearrangement negative colonies. Our data, therefore, provide evidence that ruxolitinib is a promising therapy for treatment of patients with JAK2 fusion genes.

Three novel cytogenetically cryptic EVI1 rearrangements associated with increased EVI1 expression and poor prognosis identified in 27 acute myeloid leukemia cases.

In acute myeloid leukemia (AML), increased ecotropic virus integration site 1 protein homolog (EVI1) gene expression is prognostically unfavorable. Subsets of cases show 3q26 rearrangements, such as inv(3)(q21q26)/t(3;3)(q21;q26), frequently accompanied by chromosome 7 abnormalities. We investigated whether cytogenetically cryptic EVI1 rearrangements may cause EVI1 overexpression in myeloid malignancies without 3q26 abnormalities and investigated 983 patients with AML (n = 606) or myelodysplastic syndromes (MDS; n = 377) with normal karyotype (CN-AML/CN-MDS, n = 594) or chromosome 7 abnormalities (n = 389) for EVI1 rearrangements using interphase FISH. We identified cytogenetically cryptic EVI1 rearrangements in 27 patients (19 AML, 8 MDS): inv(3)(p24q26) [n = 10]; t(3;21)(q26;q11) [n = 9]; and der(7)t(3;7)(q26;q21) [n = 8]. Elevated EVI1 expression was detected in nearly all cases with cryptic EVI1 rearrangements: Median %EVI1/ABL1 was 92.8 (range: 29.8-146.1) in inv(3)(p24q26), 104.9 (41.4-176.3) in t(3;21)(q26;q11), and 101.8 (4.4-210.4) in der(7)t(3;7)(q26;q21). This was similar to median %EVI1/ABL1 of 73.9 (range: 7.3-585.6) in an independent cohort of inv(3)(q21q26)/t(3;3)(q21;q26) and 67.1 (2.3-410.7) in other 3q26/EVI1 rearrangements. Healthy controls showed median EVI1 expression of 0.5 (range: 0.0-5.8). Using SNP microarray and sequencing analyses, the breakpoints of der(7)t(3;7)(q26;q21) were assigned to CDK6 and centromeric of EVI1, and of t(3;21)(q26;q11) to be within EVI1 and NRIP1. Median overall survival in patients with cryptic EVI1 rearrangements was short, comparable to patients with inv(3)(q21q26)/t(3;3)(q21;q26) or other EVI1 rearrangements. Cryptic EVI1 rearrangements contribute to explain the clinical heterogeneity of CN-AML and are associated with elevated EVI1 expression and an unfavorable prognosis. Screening for cryptic EVI1 rearrangements by FISH may be particularly appropriate in CN-AML with elevated EVI1 expression or in AML/MDS patients with chromosome 7 abnormalities.

R453Plus1Toolbox: an R/Bioconductor package for analyzing Roche 454 Sequencing data.

UNLABELLED: The R453Plus1Toolbox is an R/Bioconductor package for the analysis of 454 Sequencing data. Projects generated with Roche's data analysis software can be imported into R allowing advanced and customized analyses within the R/Bioconductor environment for sequencing data. Several methods were implemented extending the current functionality of Roche's software. These extensions include methods for quality assurance and annotation of detected variants. Further, a pipeline for the detection of structural variants, e.g. balanced chromosomal translocations, is provided. AVAILABILITY: The R453Plus1Toolbox is implemented in R and available at http://www.bioconductor.org/. A vignette outlining typical workflows is included in the package. CONTACT: h.klein@uni-muenster.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Molecular analyses of 15,542 patients with suspected BCR-ABL1-negative myeloproliferative disorders allow to develop a stepwise diagnostic workflow.

We investigated 15,542 patients with suspected BCR-ABL1- negative myeloproliferative or myelodysplastic/myeloproliferative neoplasm (including 359 chronic myelomonocytic leukemia) by a molecular marker set. JAK2V617F was detected in the suspected categories as follows: polycythemia vera 88.3%, primary myelofibrosis 53.8%, essential thrombocythemia 50.2%, and not further classifiable myeloproliferative neoplasms 38.0%. JAK2 exon 12 mutations were detected in 40.0% JAK2V617F-negative suspected polycythemia vera, MPLW515 mutations in 13.2%JAK2V617F-negative primary myelofibrosis and 7.1% JAK2V617F-negative essential thrombocythemia. TET2 mutations were distributed across all entities but were most frequent in suspected chronic myelomonocytic leukemia (77.8%). CBL mutations were identified in suspected chronic myelomonocytic leukemia (13.9%), primary myelofibrosis (8.0%), and not further classifiable myeloproliferative neoplasm (7.0%). This leads to a stepwise workflow for suspected myeloproliferative neoplasms starting with JAK2V617F and investigating JAK2V617F-negative patients for JAK2 exon 12 or MPL mutations, respectively. In cases in which a myeloproliferative neoplasm cannot be established, analysis for TET2, CBL and EZH2 mutations may be indicated.

Use of CBL exon 8 and 9 mutations in diagnosis of myeloproliferative neoplasms and myelodysplastic/myeloproliferative disorders: an analysis of 636 cases.

We analyzed 636 patients with diverse myeloproliferative neoplasms or myelodysplastic/myeloproliferative neoplasms for mutations of the Casitas B-cell lymphoma gene (CBL(mut)) in exons 8 and 9 and performed correlations to other genetic alterations. CBL(mut) were detected in 63 of 636 (9.9%) of these selected patients. CBL(mut) were more frequent in myelodysplastic/myeloproliferative neoplasms than myeloproliferative neoplasms (51 of 328, 15.5% vs. 12 of 291, 4.1%; P<0.001). Frequency was 48 of 278 (17.3%) in chronic myelomonocytic leukemia and 3 of 33 (9.1%) in unclassifiable myelodysplastic/myeloproliferative neoplasms. CBL(mut) was not detected in polycythemia vera, primary myelofibrosis, essential thrombocythemia, or refractory anemia with ring sideroblasts and marked thrombocytosis. CBL(mut) were underrepresented in JAK2(V617F) mutated as compared to JAK2V617(wt) cases (P<0.001), and mutually exclusive of JAK2exon12(mut) and MPLW515(mut). CBL(mut) were associated with monosomy 7 (P=0.008) and TET2(mut) (P=0.003). In chronic myelomonocytic leukemia, CBL(mut) had no significant impact on survival outcomes. Therefore, CBL(mut) are frequent in chronic myelomonocytic leukemia, absent in classical myeloproliferative neoplasms, and are only exceptionally found in coincidence with JAK-STAT pathway activating mutations.