Angela Hamblin

Presence of multiple recurrent mutations confers poor trial outcome of relapsed/refractory CLL.

Although TP53, NOTCH1, and SF3B1 mutations may impair prognosis of patients with chronic lymphocytic leukemia (CLL) receiving frontline therapy, the impact of these mutations or any other, alone or in combination, remains unclear at relapse. The genome of 114 relapsed/refractory patients included in prospective trials was screened using targeted next-generation sequencing of the TP53, SF3B1, ATM, NOTCH1, XPO1, SAMHD1, MED12, BIRC3, and MYD88 genes. We performed clustering according to both number and combinations of recurrent gene mutations. The number of genes affected by mutation was ≥ 2, 1, and 0 in 43 (38%), 49 (43%), and 22 (19%) respectively. Recurrent combinations of ≥ 2 mutations of TP53, SF3B1, and ATM were found in 22 (19%) patients. This multiple-hit profile was associated with a median progression-free survival of 12 months compared with 22.5 months in the remaining patients (P = .003). Concurrent gene mutations are frequent in patients with relapsed/refractory CLL and are associated with worse outcome.

Ruxolitinib vs best available therapy for ET intolerant or resistant to hydroxycarbamide

Treatments for high-risk essential thrombocythemia (ET) address thrombocytosis, disease-related symptoms, as well as risks of thrombosis, hemorrhage, transformation to myelofibrosis and leukemia. Patients resistant/intolerant to hydroxycarbamide (HC) have a poor outlook. MAJIC (ISRCTN61925716) is a randomized phase II trial of ruxolitinib (JAK1/2 inhibitor) vs Best Available Therapy (BAT) in ET and polycythemia vera (PV) patients resistant or intolerant to HC. Here findings of MAJIC-ET are reported, where the modified intention-to-treat population included 58 & 52 patients randomized to receive ruxolitinib or BAT respectively. There was no evidence of improvement in complete response within 1 year reported in 27 (46.6%) patients treated with ruxolitinib vs 23 (44.2%) with BAT (P=.40). At 2 years rates of thrombosis, hemorrhage and transformation were not significantly different, however some disease-related symptoms improved in patients receiving ruxolitinib relative to BAT. Molecular responses were uncommon; there were two complete molecular responses (CMR) and one partial molecular response (PMR) in CALR positive ruxolitinib-treated patients. Transformation to myelofibrosis occurred in one CMR patient, presumably due to the emergence of a different clone raising questions about the relevance of CMR in ET patients. Grade 3&4 anemia occurred in 19% & 0% of ruxolitinib vs 0% (both grades) BAT arm, grade 3&4 thrombocytopenia in 5.2% & 1.7% of ruxolitinib vs 0% (both grades) of BAT treated patients. Rates of discontinuation or treatment switching did not differ between the two trial arms. The MAJIC-ET trial suggests that ruxolitinib is not superior to current second-line treatments for ET.Treatments for high-risk essential thrombocythemia (ET) address thrombocytosis, disease-related symptoms, as well as risks of thrombosis, hemorrhage, transformation to myelofibrosis, and leukemia. Patients resistant/intolerant to hydroxycarbamide (HC) have a poor outlook. MAJIC (ISRCTN61925716) is a randomized phase 2 trial of ruxolitinib (JAK1/2 inhibitor) vs best available therapy (BAT) in ET and polycythemia vera patients resistant or intolerant to HC. Here, findings of MAJIC-ET are reported, where the modified intention-to-treat population included 58 and 52 patients randomized to receive ruxolitinib or BAT, respectively. There was no evidence of improvement in complete response within 1 year reported in 27 (46.6%) patients treated with ruxolitinib vs 23 (44.2%) with BAT (P = .40). At 2 years, rates of thrombosis, hemorrhage, and transformation were not significantly different; however, some disease-related symptoms improved in patients receiving ruxolitinib relative to BAT. Molecular responses were uncommon; there were 2 complete molecular responses (CMR) and 1 partial molecular response in CALR-positive ruxolitinib-treated patients. Transformation to myelofibrosis occurred in 1 CMR patient, presumably because of the emergence of a different clone, raising questions about the relevance of CMR in ET patients. Grade 3 and 4 anemia occurred in 19% and 0% of ruxolitinib vs 0% (both grades) in the BAT arm, and grade 3 and 4 thrombocytopenia in 5.2% and 1.7% of ruxolitinib vs 0% (both grades) of BAT-treated patients. Rates of discontinuation or treatment switching did not differ between the 2 trial arms. The MAJIC-ET trial suggests that ruxolitinib is not superior to current second-line treatments for ET. This trial was registered at www.isrctn.com as #ISRCTN61925716.

Clinical applicability and cost of a 46-gene panel for genomic analysis of solid tumours: Retrospective validation and prospective audit in the UK National Health Service

Background Single gene tests to predict whether cancers respond to specific targeted therapies are performed increasingly often. Advances in sequencing technology, collectively referred to as next generation sequencing (NGS), mean the entire cancer genome or parts of it can now be sequenced at speed with increased depth and sensitivity. However, translation of NGS into routine cancer care has been slow. Healthcare stakeholders are unclear about the clinical utility of NGS and are concerned it could be an expensive addition to cancer diagnostics, rather than an affordable alternative to single gene testing. Methods and findings We validated a 46-gene hotspot cancer panel assay allowing multiple gene testing from small diagnostic biopsies. From 1 January 2013 to 31 December 2013, solid tumour samples (including non-small-cell lung carcinoma [NSCLC], colorectal carcinoma, and melanoma) were sequenced in the context of the UK National Health Service from 351 consecutively submitted prospective cases for which treating clinicians thought the patient had potential to benefit from more extensive genetic analysis. Following histological assessment, tumour-rich regions of formalin-fixed paraffin-embedded (FFPE) sections underwent macrodissection, DNA extraction, NGS, and analysis using a pipeline centred on Torrent Suite software. With a median turnaround time of seven working days, an integrated clinical report was produced indicating the variants detected, including those with potential diagnostic, prognostic, therapeutic, or clinical trial entry implications. Accompanying phenotypic data were collected, and a detailed cost analysis of the panel compared with single gene testing was undertaken to assess affordability for routine patient care. Panel sequencing was successful for 97% (342/351) of tumour samples in the prospective cohort and showed 100% concordance with known mutations (detected using cobas assays). At least one mutation was identified in 87% (296/342) of tumours. A locally actionable mutation (i.e., available targeted treatment or clinical trial) was identified in 122/351 patients (35%). Forty patients received targeted treatment, in 22/40 (55%) cases solely due to use of the panel. Examination of published data on the potential efficacy of targeted therapies showed theoretically actionable mutations (i.e., mutations for which targeted treatment was potentially appropriate) in 66% (71/107) and 39% (41/105) of melanoma and NSCLC patients, respectively. At a cost of £339 (US

Outcome of Azacitidine Therapy in Acute Myeloid Leukemia Is not Improved by Concurrent Vorinostat Therapy but Is Predicted by a Diagnostic Molecular Signature.

449) per patient, the panel was less expensive locally than performing more than two or three single gene tests. Study limitations include the use of FFPE samples, which do not always provide high-quality DNA, and the use of “real world” data: submission of cases for sequencing did not always follow clinical guidelines, meaning that when mutations were detected, patients were not always eligible for targeted treatments on clinical grounds. Conclusions This study demonstrates that more extensive tumour sequencing can identify mutations that could improve clinical decision-making in routine cancer care, potentially improving patient outcomes, at an affordable level for healthcare providers.

The 100 000 Genomes Project: bringing whole genome sequencing to the NHS

Purpose: Azacitidine (AZA) is a novel therapeutic option in older patients with acute myeloid leukemia (AML), but its rational utilization is compromised by the fact that neither the determinants of clinical response nor its mechanism of action are defined. Co-administration of histone deacetylase inhibitors, such as vorinostat (VOR), is reported to improve the clinical activity of AZA, but this has not been prospectively studied in patients with AML. Experimental Design: We compared outcomes in 259 adults with AML (n = 217) and MDS (n = 42) randomized to receive either AZA monotherapy (75 mg/m2 × 7 days every 28 days) or AZA combined with VOR 300 mg twice a day on days 3 to 9 orally. Next-generation sequencing was performed in 250 patients on 41 genes commonly mutated in AML. Serial immunophenotyping of progenitor cells was performed in 47 patients. Results: Co-administration of VOR did not increase the overall response rate (P = 0.84) or overall survival (OS; P = 0.32). Specifically, no benefit was identified in either de novo or relapsed AML. Mutations in the genes CDKN2A (P = 0.0001), IDH1 (P = 0.004), and TP53 (P = 0.003) were associated with reduced OS. Lymphoid multipotential progenitor populations were greatly expanded at diagnosis and although reduced in size in responding patients remained detectable throughout treatment. Conclusions: This study demonstrates no benefit of concurrent administration of VOR with AZA but identifies a mutational signature predictive of outcome after AZA-based therapy. The correlation between heterozygous loss of function CDKN2A mutations and decreased OS implicates induction of cell-cycle arrest as a mechanism by which AZA exerts its clinical activity. Clin Cancer Res; 23(21); 6430–40. ©2017 AACR.

Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations.

In partnership with NHS England, Genomics England’s ambitious plans to embed genomic medicine into routine patient care are well underway. Clare Turnbull and colleagues discuss its progress

State of the Art Review: New Insights in T Cell Prolymphocytic Leukemia

SF3B1, SRSF2, and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34+ cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology.

Clinically actionable mutation profiles in patients with cancer identified by whole-genome sequencing.

T cell ProLymphocytic Leukemia (T-PLL) is a rare lymphoid malignancy and is typified by its aggressive presentation and chemo-resistance. Given its rarity, haemato-oncologists are likely to manage only a handful of cases across their career and therefore making the diagnosis is the initial challenge. This diagnosis is critical, as recent data suggests that choosing specific treatment options, such as alemtuzumab, is important in improving outcomes.

IDENTIFICATION OF ABERRANTLY SPLICED GENES AND DEREGULATED PATHWAYS/GENE ONTOLOGY THEMES IN MYELODYSPLASTIC SYNDROME PATIENTS WITH SPLICING FACTOR GENE MUTATIONS

Next-generation sequencing (NGS) efforts have established catalogs of mutations relevant to cancer development. However, the clinical utility of this information remains largely unexplored. Here, we present the results of the first eight patients recruited into a clinical whole-genome sequencing (WGS) program in the United Kingdom. We performed PCR-free WGS of fresh frozen tumors and germline DNA at 75× and 30×, respectively, using the HiSeq2500 HTv4. Subtracted tumor VCFs and paired germlines were subjected to comprehensive analysis of coding and noncoding regions, integration of germline with somatically acquired variants, and global mutation signatures and pathway analyses. Results were classified into tiers and presented to a multidisciplinary tumor board. WGS results helped to clarify an uncertain histopathological diagnosis in one case, led to informed or supported prognosis in two cases, leading to de-escalation of therapy in one, and indicated potential treatments in all eight. Overall 26 different tier 1 potentially clinically actionable findings were identified using WGS compared with six SNVs/indels using routine targeted NGS. These initial results demonstrate the potential of WGS to inform future diagnosis, prognosis, and treatment choice in cancer and justify the systematic evaluation of the clinical utility of WGS in larger cohorts of patients with cancer.