Combination of flow cytometry and functional imaging for monitoring of residual disease in myeloma.

The iliac crest is the sampling site for minimal residual disease (MRD) monitoring in multiple myeloma (MM). However, the disease distribution is often heterogeneous, and imaging can be used to complement MRD detection at a single site. We have investigated patients in complete remission (CR) during first-line or salvage therapy for whom MRD flow cytometry and the two imaging modalities positron emission tomography (PET) and diffusion-weighted magnetic resonance imaging (DW-MRI) were performed at the onset of CR. Residual focal lesions (FLs), detectable in 24% of first-line patients, were associated with short progression-free survival (PFS), with DW-MRI detecting disease in more patients. In some patients, FLs were only PET positive, indicating that the two approaches are complementary. Combining MRD and imaging improved prediction of outcome, with double-negative and double-positive features defining groups with excellent and dismal PFS, respectively. FLs were a rare event (12%) in first-line MRD-negative CR patients. In contrast, patients achieving an MRD-negative CR during salvage therapy frequently had FLs (50%). Multi-region sequencing and imaging in an MRD-negative patient showed persistence of spatially separated clones. In conclusion, we show that DW-MRI is a promising tool for monitoring residual disease that complements PET and should be combined with MRD.

Investigating the feasibility of tumour molecular profiling in gastrointestinal malignancies in routine clinical practice.

Background: Targeted capture sequencing can potentially facilitate precision medicine, but the feasibility of this approach in gastrointestinal (GI) malignancies is unknown. Patients and methods: The FOrMAT (Feasibility of a Molecular Characterisation Approach to Treatment) study was a feasibility study enrolling patients with advanced GI malignancies from February 2014 to November 2015. Targeted capture sequencing (mainly using archival formalin-fixed paraffin-embedded diagnostic/resection samples) was carried out to detect mutations, copy number variations and translocations in up to 46 genes which had prognostic/predictive significance or were targets in current/upcoming clinical trials. Results: Of the 222 patients recruited, 215 patients (96.8%) had available tissue samples, 125 patients (56.3%) had ≥16 genes successfully sequenced and 136 patients (61.2%) had ≥1 genes successfully sequenced. Sample characteristics influenced the proportion of successfully sequenced samples, e.g. tumour type (colorectal 70.9%, biliary 52.6%, oesophagogastric 50.7%, pancreas 27.3%, P = 0.002), tumour cellularity (high versus low: 78.3% versus 13.3%, P ≤ 0.001), tumour content (high versus low: 78.6% versus 27.3%, P = 0.001) and type of sample (resection versus biopsy: 82.4% versus 47.6%, P ≤ 0.001). Currently, actionable alterations were detected in 90 (40.5%) of the 222 patients recruited (66% of the 136 patients sequenced) and 2 patients subsequently received a targeted therapy. The most frequently detected currently actionable alterations were mutations in KRAS, BRAF, TP53 and PIK3CA. For the 205 patients with archival samples, the median time to obtain sequencing results was 18.9 weeks, including a median of 4.9 weeks for sample retrieval and 5.1 weeks for sequencing. Conclusions: Targeted sequencing detected actionable alterations in formalin-fixed paraffin-embedded samples, but tissue characteristics are of critical importance in determining sequencing success. Routine molecular profiling of GI tumours outside of clinical trials is not an effective use of healthcare resources unless more targeted drugs become available. ClinicalTrials.gov identifier: NCT02112357.

Prediction of outcome in newly diagnosed myeloma: a meta-analysis of the molecular profiles of 1905 trial patients.

Robust establishment of survival in multiple myeloma (MM) and its relationship to recurrent genetic aberrations is required as outcomes are variable despite apparent similar staging. We assayed copy number alterations (CNA) and translocations in 1036 patients from the NCRI Myeloma XI trial and linked these to overall survival (OS) and progression-free survival. Through a meta-anlysis of these data with data from MRC Myeloma IX trial, totalling 1905 newly diagnosed MM patients (NDMM), we confirm the association of t(4;14), t(14;16), t(14;20), del(17p) and gain(1q21) with poor prognosis with hazard ratios (HRs) for OS of 1.60 (P=4.77 × 10-7), 1.74 (P=0.0005), 1.90 (P=0.0089), 2.10 (P=8.86 × 10-14) and 1.68 (P=2.18 × 10-14), respectively. Patients with 'double-hit' defined by co-occurrence of at least two adverse lesions have an especially poor prognosis with HRs for OS of 2.67 (P=8.13 × 10-27) for all patients and 3.19 (P=1.23 × 10-18) for intensively treated patients. Using comprehensive CNA and translocation profiling in Myeloma XI we also demonstrate a strong association between t(4;14) and BIRC2/BIRC3 deletion (P=8.7 × 10-15), including homozygous deletion. Finally, we define distinct sub-groups of hyperdiploid MM, with either gain(1q21) and CCND2 overexpression (P<0.0001) or gain(11q25) and CCND1 overexpression (P<0.0001). Profiling multiple genetic lesions can identify MM patients likely to relapse early allowing stratification of treatment.

Spatial genomic heterogeneity in multiple myeloma revealed by multi-region sequencing.

In multiple myeloma malignant plasma cells expand within the bone marrow. Since this site is well-perfused, a rapid dissemination of "fitter" clones may be anticipated. However, an imbalanced distribution of multiple myeloma is frequently observed in medical imaging. Here, we perform multi-region sequencing, including iliac crest and radiology-guided focal lesion specimens from 51 patients to gain insight into the spatial clonal architecture. We demonstrate spatial genomic heterogeneity in more than 75% of patients, including inactivation of CDKN2C and TP53, and mutations affecting mitogen-activated protein kinase genes. We show that the extent of spatial heterogeneity is positively associated with the size of biopsied focal lesions consistent with regional outgrowth of advanced clones. The results support a model for multiple myeloma progression with clonal sweeps in the early phase and regional evolution in advanced disease. We suggest that multi-region investigations are critical to understanding intra-patient heterogeneity and the evolutionary processes in multiple myeloma.In multiple myeloma, malignant cells expand within bone marrow. Here, the authors use multi-region sequencing in patient samples to analyse spatial clonal architecture and heterogeneity, providing novel insight into multiple myeloma progression and evolution.

Overexpression of EZH2 in multiple myeloma is associated with poor prognosis and dysregulation of cell cycle control.

Myeloma is heterogeneous at the molecular level with subgroups of patients characterised by features of epigenetic dysregulation. Outcomes for myeloma patients have improved over the past few decades except for molecularly defined high-risk patients who continue to do badly. Novel therapeutic approaches are, therefore, required. A growing number of epigenetic inhibitors are now available including EZH2 inhibitors that are in early-stage clinical trials for treatment of haematological and other cancers with EZH2 mutations or in which overexpression has been correlated with poor outcomes. For the first time, we have identified and validated a robust and independent deleterious effect of high EZH2 expression on outcomes in myeloma patients. Using two chemically distinct small-molecule inhibitors, we demonstrate a reduction in myeloma cell proliferation with EZH2 inhibition, which leads to cell cycle arrest followed by apoptosis. This is mediated via upregulation of cyclin-dependent kinase inhibitors associated with removal of the inhibitory H3K27me3 mark at their gene loci. Our results suggest that EZH2 inhibition may be a potential therapeutic strategy for the treatment of myeloma and should be investigated in clinical studies.

Bi-allelic inactivation is more prevalent at relapse in multiple myeloma, identifying RB1 as an independent prognostic marker.

The purpose of this study is to identify prognostic markers and treatment targets using a clinically certified sequencing panel in multiple myeloma. We performed targeted sequencing of 578 individuals with plasma cell neoplasms using the FoundationOne Heme panel and identified clinically relevant abnormalities and novel prognostic markers. Mutational burden was associated with maf and proliferation gene expression groups, and a high-mutational burden was associated with a poor prognosis. We identified homozygous deletions that were present in multiple myeloma within key genes, including CDKN2C, RB1, TRAF3, BIRC3 and TP53, and that bi-allelic inactivation was significantly enriched at relapse. Alterations in CDKN2C, TP53, RB1 and the t(4;14) were associated with poor prognosis. Alterations in RB1 were predominantly homozygous deletions and were associated with relapse and a poor prognosis which was independent of other genetic markers, including t(4;14), after multivariate analysis. Bi-allelic inactivation of key tumor suppressor genes in myeloma was enriched at relapse, especially in RB1, CDKN2C and TP53 where they have prognostic significance.

Translocations at 8q24 juxtapose MYC with genes that harbor superenhancers resulting in overexpression and poor prognosis in myeloma patients.

Secondary MYC translocations in myeloma have been shown to be important in the pathogenesis and progression of disease. Here, we have used a DNA capture and massively parallel sequencing approach to identify the partner chromosomes in 104 presentation myeloma samples. 8q24 breakpoints were identified in 21 (20%) samples with partner loci including IGH, IGK and IGL, which juxtapose the immunoglobulin (Ig) enhancers next to MYC in 8/23 samples. The remaining samples had partner loci including XBP1, FAM46C, CCND1 and KRAS, which are important in B-cell maturation or myeloma pathogenesis. Analysis of the region surrounding the breakpoints indicated the presence of superenhancers on the partner chromosomes and gene expression analysis showed increased expression of MYC in these samples. Patients with MYC translocations had a decreased progression-free and overall survival. We postulate that translocation breakpoints near MYC result in colocalization of the gene with superenhancers from loci, which are important in the development of the cell type in which they occur. In the case of myeloma these are the Ig loci and those important for plasma cell development and myeloma pathogenesis, resulting in increased expression of MYC and an aggressive disease phenotype.

Single-cell genetic analysis reveals the composition of initiating clones and phylogenetic patterns of branching and parallel evolution in myeloma.

Although intratumor heterogeneity has been inferred in multiple myeloma (MM), little is known about its subclonal phylogeny. To describe such phylogenetic trees in a series of patients with MM, we perform whole-exome sequencing and single-cell genetic analysis. Our results demonstrate that at presentation myeloma is composed of two to six different major clones, which are related by linear and branching phylogenies. Remarkably, the earliest myeloma-initiating clones, some of which only had the initiating t(11;14), were still present at low frequencies at the time of diagnosis. For the first time in myeloma, we demonstrate parallel evolution whereby two independent clones activate the RAS/MAPK pathway through RAS mutations and give rise subsequently to distinct subclonal lineages. We also report the co-occurrence of RAS and interferon regulatory factor 4 (IRF4) p.K123R mutations in 4% of myeloma patients. Lastly, we describe the fluctuations of myeloma subclonal architecture in a patient analyzed at presentation and relapse and in NOD/SCID-IL2Rγ(null) xenografts, revealing clonal extinction and the emergence of new clones that acquire additional mutations. This study confirms that myeloma subclones exhibit different survival properties during treatment or mouse engraftment. We conclude that clonal diversity combined with varying selective pressures is the essential foundation for tumor progression and treatment resistance in myeloma.

Reprogramming axonal behavior by axon-specific viral transduction.

The treatment of axonal disorders, such as diseases associated with axonal injury and degeneration, is limited by the inability to directly target therapeutic protein expression to injured axons. Current gene therapy approaches rely on infection and transcription of viral genes in the cell body. Here, we describe an approach to target gene expression selectively to axons. Using a genetically engineered mouse containing epitope-labeled ribosomes, we find that neurons in adult animals contain ribosomes in distal axons. To use axonal ribosomes to alter local protein expression, we utilized a Sindbis virus containing an RNA genome that has been modified so that it can be directly used as a template for translation. Selective application of this virus to axons leads to local translation of heterologous proteins. Furthermore, we demonstrate that selective axonal protein expression can be used to modify axonal signaling in cultured neurons, enabling axons to grow over inhibitory substrates typically encountered following axonal injury. We also show that this viral approach also can be used to achieve heterologous expression in axons of living animals, indicating that this approach can be used to alter the axonal proteome in vivo. Together, these data identify a novel strategy to manipulate protein expression in axons, and provides a novel approach for using gene therapies for disorders of axonal function.

A novel prognostic model in myeloma based on co-segregating adverse FISH lesions and the ISS: analysis of patients treated in the MRC Myeloma IX trial.

The association of genetic lesions detected by fluorescence in situ hybridization (FISH) with survival was analyzed in 1069 patients with newly presenting myeloma treated in the Medical Research Council Myeloma IX trial, with the aim of identifying patients associated with the worst prognosis. A comprehensive FISH panel was performed, and the lesions associated with short progression-free survival and overall survival (OS) in multivariate analysis were +1q21, del(17p13) and an adverse immunoglobulin heavy chain gene (IGH) translocation group incorporating t(4;14), t(14;16) and t(14;20). These lesions frequently co-segregated, and there was an association between the accumulation of these adverse FISH lesions and a progressive impairment of survival. This observation was used to define a series of risk groups based on number of adverse lesions. Taking this approach, we defined a favorable risk group by the absence of adverse genetic lesions, an intermediate group with one adverse lesion and a high-risk group defined by the co-segregation of >1 adverse lesion. This genetic grouping was independent of the International Staging System (ISS) and so was integrated with the ISS to identify an ultra-high-risk group defined by ISS II or III and >1 adverse lesion. This group constituted 13.8% of patients and was associated with a median OS of 19.4 months.

Microduplication of Xp11.23p11.3 with effects on cognition, behavior, and craniofacial development.

We report an ~1.3 Mb tandem duplication at Xp11.23p11.3 in an 11-year-old boy with pleasant personality, hyperactivity, learning and visual-spatial difficulties, relative microcephaly, long face, stellate iris pattern, and periorbital fullness. This clinical presentation is milder and distinct from that of patients with partially overlapping Xp11.22p11.23 duplications which have been described in males and females with intellectual disability, language delay, autistic behaviors, and seizures. The duplicated region harbors three known X-linked mental retardation genes: FTSJ1, ZNF81, and SYN1. Quantitative polymerase chain reaction from whole blood total RNA showed increased expression of three genes located in the duplicated region: EBP, WDR13, and ZNF81. Thus, over-expression of genes in the interval may contribute to the observed phenotype. Many of the features seen in this patient are present in individuals with Williams-Beuren syndrome (WBS). Interestingly, the SYN1 gene within the duplicated interval, as well as the STX1A gene, within the WBS critical region, co-localize to presynaptic active zones, and play important roles in neurotransmitter release.

Effect of hot water treatments on quality of highbush blueberries.

Highbush blueberries, cv 'Burlington', were treated with 22, 45, 50, or 60 degrees C water for 15 or 30 s along with an untreated control. Fruit were then stored for 0, 1, 2, or 4 wk at 0 degrees C and 2 or 9 d at 20 degrees C prior to evaluation of microbial population and fruit quality. After 4 wk of storage, the hot water treatment at 60 degrees C resulted in 92% marketable berries, followed by 90% at 50 degrees C, 88% at 45 degrees C, and 83% at 22 degrees C compared with 76% in untreated controls. Decay incidence was reduced to 0.6%, 1.2%, 1.4%, or 2.8% with 60, 50, 45, or 22 degrees C water treatments, respectively, compared with 5.1% in controls following 4 wk at 0 degrees C and 2 d at 20 degrees C. After an additional 7 d at 20 degrees C, decay in fruit treated at 60 degrees C for 15 or 30 s remained at 1.8% and 0.4%, respectively, compared to 37.4% in controls. Weight loss of berries treated with hot water was 0.4% against 3.8% in controls, and shriveled and split berries were also reduced compared to controls (P<0.001). Aerobic plate count and yeast and mold count were reduced by 0.45 to 0.7 log at 60 degrees C for 30 s. Botrytis cinerea and Colletotrichum sp. were the dominant fungal pathogens causing decay of Burlington blueberries during storage. Hot water treatments also immediately induced an increase in ethanol and reduced fruit titratable acidity and soluble solids content, but had no significant effect on fruit firmness, pH, or most flavor volatile concentrations.

Gaseous ozone treatment inactivates Listeria innocua in vitro.

AIMS: To investigate the effects of ozone on inactivation of Listeria innocua on solid media. METHODS AND RESULTS: Suspensions of L. innocua ranging from 4.5 x 10(4 )- 6.4 x 10(4) CFU ml(-1) were inoculated onto potato dextrose agar (PDA, pH 5.6 and 6.8) and nutrient agar (NA, pH 6.0 and 6.8), then exposed to gaseous ozone. Variable factors included postinoculation standing time at 20 degrees C before exposure to ozone, ozone concentration, treatment duration and treatment temperature (5 or 20 degrees C). The interaction among ozone concentration, treatment duration, media and temperature in effecting changes in colony-forming units (CFU) was significant. The 100 nl l(-1) ozone treatment for 2 h reduced the microbial populations by 2-3 log CFU ml(-1). Cell viability decreased more rapidly on PDA than on NA. The average time to obtain a 2 log CFU ml(-1) reduction was 1.3 h at 20 degrees C and 2.5 h at 5 degrees C (P < 0.001). CONCLUSIONS: Gaseous ozone effectively inactivates L. innocua at concentrations of 50 and 100 nl l(-1) during short exposure times at both 5 and 20 degrees C. The Gompretz model can be utilized for determining the response of L. innocua to ozone over time. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information on ozone inactivating Listeria spp., which may be imposed on ensuring quality and safety of horticultural produce and food products.

Nonsteroidal anti-inflammatory drug-induced colonic stricture: case report.

Nonsteroidal anti-inflammatory drug-induced colonic strictures are uncommon and usually occur in the proximal ascending colon. We describe the progressive findings of nonsteroidal anti-inflammatory drug-induced strictures in the ascending colon and at the ileocecal valve with subsequent bowel obstruction secondary to intussusception.

An unusual example of Aspergillus species lung disease.

A 37-year-old man developed persistent hemoptysis after sustaining a gunshot wound to the right shoulder and lung. A right upper lobectomy was performed, in which Aspergillus species microorganisms were identified within retained bullet fragments. The role of infected bullet fragments in the pathogenesis of hemoptysis in this patient is discussed.