The use of targeted sequencing and flow cytometry to identify patients with a clinically significant monocytosis

The diagnosis of chronic myelomonocytic leukaemia (CMML) remains centred on morphology, meaning the distinction from a reactive monocytosis is challenging. Mutational analysis and immunophenotyping have been proposed as potential tools for diagnosis however have not been formally assessed in combination. We aimed to investigate the clinical utility of these technologies by performing targeted sequencing, in parallel to current gold standard techniques, on consecutive samples referred for investigation of monocytosis over a 2-year period (n=283). Results were correlated with the morphological diagnosis and objective outcome measures including overall survival (OS) and longitudinal blood counts. Somatic mutations were detected in 79% of patients, being invariably identified in those with a confirmed diagnosis (99%) though also in 57% of patients with non-diagnostic BM features. The OS in non-diagnostic mutated patients was indistinguishable from those with CMML (p=0.118) and significantly worse than unmutated patients (p=0.0002). On multivariate analysis age, ASXL1, CBL, DNMT3A, NRAS & RUNX1 mutations retained significance. Furthermore, the presence of a mutation was associated with a progressive fall in haemoglobin/platelet levels and increasing monocyte counts compared with mutation negative patients. Of note, the immunophenotypic features of non-diagnostic mutated patients were comparable to CMML patients and the presence of aberrant CD56 was highly specific for detecting a mutation. Overall, somatic mutations are detected at high frequency in patients referred with a monocytosis irrespective of diagnosis. In those without a WHO defined diagnosis, the mutation spectrum, immunophenotypic features and OS are indistinguishable from CMML patients and these patients should be managed as such

Targeted sequencing in DLBCL, molecular subtypes, and outcomes: a Haematological Malignancy Research Network report.

Based on the profile of genetic alterations occurring in tumor samples from selected diffuse large B-cell lymphoma (DLBCL) patients, 2 recent whole-exome sequencing studies proposed partially overlapping classification systems. Using clustering techniques applied to targeted sequencing data derived from a large unselected population-based patient cohort with full clinical follow-up (n = 928), we investigated whether molecular subtypes can be robustly identified using methods potentially applicable in routine clinical practice. DNA extracted from DLBCL tumors diagnosed in patients residing in a catchment population of ∼4 million (14 centers) were sequenced with a targeted 293-gene hematological-malignancy panel. Bernoulli mixture-model clustering was applied and the resulting subtypes analyzed in relation to their clinical characteristics and outcomes. Five molecular subtypes were resolved, termed MYD88, BCL2, SOCS1/SGK1, TET2/SGK1, and NOTCH2, along with an unclassified group. The subtypes characterized by genetic alterations of BCL2, NOTCH2, and MYD88 recapitulated recent studies showing good, intermediate, and poor prognosis, respectively. The SOCS1/SGK1 subtype showed biological overlap with primary mediastinal B-cell lymphoma and conferred excellent prognosis. Although not identified as a distinct cluster, NOTCH1 mutation was associated with poor prognosis. The impact of TP53 mutation varied with genomic subtypes, conferring no effect in the NOTCH2 subtype and poor prognosis in the MYD88 subtype. Our findings confirm the existence of molecular subtypes of DLBCL, providing evidence that genomic tests have prognostic significance in non-selected DLBCL patients. The identification of both good and poor risk subtypes in patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) clearly show the clinical value of the approach, confirming the need for a consensus classification.Bloodwise (grant number 15037) funded the majority of this study. Genetic sequencing was funded by 14M Genomics, a start-up company that ceased trading February 2016. DJH was funded by a clinician scientist fellowship from the MRC and receives core funding from Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute. Some of the analysis in this study was performed on the “Viking” high performance computing cluster at the University of York.1

Molecular subclusters of follicular lymphoma: a report from the UK's Haematological Malignancy Research Network

Follicular lymphoma (FL) is morphologically and clinically diverse, with mutations in epigenetic regulators alongside t(14;18) identified as disease-initiating events. Identification of additional mutational entities confirms this cancer’s heterogeneity, but whether mutational data can be resolved into mechanistically distinct subsets remains an open question. Targeted sequencing was applied to an unselected population-based FL cohort (n = 548) with full clinical follow-up (n = 538), which included 96 diffuse large B-cell lymphoma (DLBCL) transformations. We investigated whether molecular subclusters of FL can be identified and whether mutational data provide predictive information relating to transformation. DNA extracted from FL samples was sequenced with a 293-gene panel representing genes frequently mutated in DLBCL and FL. Three clusters were resolved using mutational data alone, independent of translocation status: FL_aSHM, with high burden of aberrant somatic hypermutation (aSHM) targets; FL_STAT6, with high STAT6 & CREBBP mutation and low aSHM; and FL_Com, with the absence of features of other subtypes and enriched KMT2D mutation. Analysis of mutation signatures demonstrated differential enrichment of predicted mutation signatures between subgroups and a dominant preference in the FL_aSHM subgroup for G(C>T)T and G(C>T)C transitions consistent with previously defined aSHM-like patterns. Of transformed cases with paired samples, 17 of 26 had evidence of branching evolution. Poorer overall survival (OS) in the aSHM group (P = .04) was associated with older age; however, overall tumor genetics provided limited information to predict individual patient risk. Our approach identifies 3 molecular subclusters of FL linked to differences in underlying mechanistic pathways. These clusters, which may be further resolved by the inclusion of translocation status and wider mutation profiles, have implications for understanding pathogenesis as well as improving treatment strategies in the future

Longitudinal expression profiling identifies a poor risk subset of patients with ABC-type Diffuse Large B Cell Lymphoma

Despite the effectiveness of immuno-chemotherapy, 40\cell lymphoma (DLBCL) experience relapse or refractory disease. Longitudinal studies have previously focused on the mutational landscape of relapse but fell short of providing a consistent relapse-specific genetic signature. In our study, we have focussed attention on the changes in gene expression profile accompanying DLBCL relapse using archival paired diagnostic/relapse specimens from 38 de novo DLBCL patients. Cell of origin remained stable from diagnosis to relapse in 80\ with only a single patient showing COO switching from ABC to GCB. Analysis of the transcriptomic changes that occur following relapse suggest ABC and GCB relapses are mediated via different mechanisms. We developed a 30-gene discriminator for ABC-DLBCLs derived from relapse-associated genes, that defined clinically distinct high and low risk subgroups in ABC-DLBCLs at diagnosis in datasets comprising both population-based and clinical trial cohorts. This signature also identified a population of \lt;60-year-old patients with superior PFS and OS treated with Ibrutinib-R-CHOP as part of the PHOENIX trial. Altogether this new signature adds to the existing toolkit of putative genetic predictors now available in DLBCL that can be readily assessed as part of prospective clinical trials

Additional file 1 of Zinc white marker paint in Mondrian’s neoplastic paintings

Additional file 1. Supplementary material. 1. Locations of the samples taken from the nine paintings. 2.Reexamination of the cross sections from the paintings dated 1921