Late cardiac effects of chemotherapy in breast cancer survivors treated with adjuvant doxorubicin: 10-year follow-up

Doxorubicin (Dox), a mainstay of adjuvant breast cancer treatment, is associated with cardiac toxicity in the form of left ventricular dysfunction (LVD), LV diastolic dysfunction, or LV systolic dysfunction. Study objectives were to evaluate the prevalence of LVD in long-term breast cancer survivors treated with Dox and determine if brain-type natriuretic peptide (BNP) may help identify patients at risk for LVD. Patients who participated in prospective clinical trials of adjuvant Dox-based chemotherapy for breast cancer with a baseline left ventricular (LV) ejection fraction evaluation from 1999 to 2006 were retrospectively identified from the St Vincent's University Hospital database. Patients were invited to undergo transthoracic echocardiography, BNP analysis, and cardiovascular (CV) risk factor assessment. LVDD was defined as left atrial volume index >34 mL/m(2) and/or lateral wall E prime <10 m/s, and LVSD as LVEF <50 %. Of 212 patients identified, 154 participated, 19 patients had died (no cardiac deaths), and 39 declined. Mean age was 60.7 [55:67] years. A majority of the patients (128, 83 %) had low CV risk (0/1 risk factors), 21 (13.6 %) had 2 RFs, and 5 (3.2 %) ≥3 RFs. BMI was 27.2 ± 4.9 kg/m(2). Median Dox dose was 240 mg/m(2) [225-298]; 92 patients (59.7 %) received ≤240 mg/m(2) and 62 (40.3 %) > 240 mg/m(2). Baseline LVEF was 68.2 ± 8 %. At follow-up of 10.8 ± 2.2 years, LVEF was 64.4 ± 6 %. Three (1.9 %) subjects had LVEF <50 % and one (0.7 %) had LVDD. Dox >240 mg/m2 was associated with any LVEF drop. BNP levels at follow-up were 20.3 pg/ml [9.9-36.5] and 21.1 pg/ml [9.8-37.7] in those without LVD and 61.5 pg/ml [50-68.4] in those with LVD (p = 0.04). Long-term prospective data describing the impact of Dox on cardiotoxicity are sparse. At over 10 years of follow-up, decreases in LVEF are common, and dose related, but LVD as defined is infrequent (2.6 %). Monitoring with BNP for subclinical LVD needs further evaluation.

Conventional versus hypofractionated high-dose intensity-modulated radiotherapy for prostate cancer: 5-year outcomes of the randomised, non-inferiority, phase 3 CHHiP trial

BACKGROUND: Prostate cancer might have high radiation-fraction sensitivity that would give a therapeutic advantage to hypofractionated treatment. We present a pre-planned analysis of the efficacy and side-effects of a randomised trial comparing conventional and hypofractionated radiotherapy after 5 years follow-up.

METHODS: CHHiP is a randomised, phase 3, non-inferiority trial that recruited men with localised prostate cancer (pT1b-T3aN0M0). Patients were randomly assigned (1:1:1) to conventional (74 Gy delivered in 37 fractions over 7·4 weeks) or one of two hypofractionated schedules (60 Gy in 20 fractions over 4 weeks or 57 Gy in 19 fractions over 3·8 weeks) all delivered with intensity-modulated techniques. Most patients were given radiotherapy with 3-6 months of neoadjuvant and concurrent androgen suppression. Randomisation was by computer-generated random permuted blocks, stratified by National Comprehensive Cancer Network (NCCN) risk group and radiotherapy treatment centre, and treatment allocation was not masked. The primary endpoint was time to biochemical or clinical failure; the critical hazard ratio (HR) for non-inferiority was 1·208. Analysis was by intention to treat. Long-term follow-up continues. The CHHiP trial is registered as an International Standard Randomised Controlled Trial, number ISRCTN97182923.

FINDINGS: Between Oct 18, 2002, and June 17, 2011, 3216 men were enrolled from 71 centres and randomly assigned (74 Gy group, 1065 patients; 60 Gy group, 1074 patients; 57 Gy group, 1077 patients). Median follow-up was 62·4 months (IQR 53·9-77·0). The proportion of patients who were biochemical or clinical failure free at 5 years was 88·3% (95% CI 86·0-90·2) in the 74 Gy group, 90·6% (88·5-92·3) in the 60 Gy group, and 85·9% (83·4-88·0) in the 57 Gy group. 60 Gy was non-inferior to 74 Gy (HR 0·84 [90% CI 0·68-1·03], pNI=0·0018) but non-inferiority could not be claimed for 57 Gy compared with 74 Gy (HR 1·20 [0·99-1·46], pNI=0·48). Long-term side-effects were similar in the hypofractionated groups compared with the conventional group. There were no significant differences in either the proportion or cumulative incidence of side-effects 5 years after treatment using three clinician-reported as well as patient-reported outcome measures. The estimated cumulative 5 year incidence of Radiation Therapy Oncology Group (RTOG) grade 2 or worse bowel and bladder adverse events was 13·7% (111 events) and 9·1% (66 events) in the 74 Gy group, 11·9% (105 events) and 11·7% (88 events) in the 60 Gy group, 11·3% (95 events) and 6·6% (57 events) in the 57 Gy group, respectively. No treatment-related deaths were reported.

INTERPRETATION: Hypofractionated radiotherapy using 60 Gy in 20 fractions is non-inferior to conventional fractionation using 74 Gy in 37 fractions and is recommended as a new standard of care for external-beam radiotherapy of localised prostate cancer.

FUNDING: Cancer Research UK, Department of Health, and the National Institute for Health Research Cancer Research Network.

Targeting treatment resistant cancer stem cells with FKBPL and its peptide therapeutics

FKBPL and its peptide derivatives have already demonstrated well-established inhibitory effects on cancer growth and CD44-dependent anti-angiogenic activity. Since cancer stem cells (CSCs) are CD44 positive, we wanted to explore if these therapeutics could specifically target CSCs in breast and ovarian cancer. In a tumoursphere assay, FKBPL stable overexpression or FKBPL-based peptide (AD-01, preclinical peptide or ALM201, clinical peptide candidate) treatment were highly effective at reducing the CSC population measured by inhibiting tumoursphere forming efficiency in breast and ovarian cancer cell lines and primary breast cancer samples from both solid breast tumours and pleural effusions. Flow cytometry, to assess the ESA+/CD44+/CD24- and ALDH+ cell subpopulations representative of CSCs, validated these results. The ability of AD-01 and ALM201 to inhibit the self-renewal capacity of CSCs was confirmed across three generations, eradicating CSC completely by the third generation (p<0.001). Furthermore, clonogenic assay demonstrated that FKBPL-based peptides mediated CSC differentiation, with a significant decrease in the number of CSCs or holoclones and an associated increase in differentiated cancer cells or meroclones/paraclones. In addition, AD-01 treatment in vitro and in vivo led to a significant reduction in the stem cell markers, Nanog, Sox2 and Oct4 protein and mRNA levels; whilst transfection of FKBPL-targeted siRNAs led to an increase in these markers and in tumoursphere forming potential, highlighting the endogenous role of FKBPL in stem cell signalling. The clinical relevance of this was confirmed using a publically available microarray data set (GSE7390), where, high FKBPL and low Nanog expression were independently associated with improved overall survival in breast cancer patients (log rank test p=0.03; hazard ratio=3.01). Additionally, when AD-01 was combined with other agents, we observed additive activity with the Notch inhibitor, DAPT and AD-01 was also able to abrogate a chemo- and radiotherapy induced enrichment in CSCs. Importantly, using gold standard in vivo limiting dilution assays we demonstrated a delay in tumour initiation and reoccurrence in AD-01 treated xenografts. In summary, FKBPL-based peptides appear to have dual anti-angiogenic and anti-CSC activity which will be advantageous as this agent enters clinical trial.

Inter-Laboratory Evaluation of a Next-Generation Sequencing Panel for Acute Myeloid Leukemia

INTRODUCTION: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder often associated with dismal overall survival. The clinical diversity of AML is reflected in the range of recurrent somatic mutations in several genes, many of which have a prognostic and therapeutic value. Targeted next-generation sequencing (NGS) of these genes has the potential for translation into clinical practice. In order to assess this potential, an inter-laboratory evaluation of a commercially available AML gene panel across three diagnostic centres in the UK and Ireland was performed.

METHODS: DNA from six AML patient samples was distributed to each centre and processed using a standardised workflow, including a common sequencing platform, sequencing chips and bioinformatics pipeline. A duplicate sample in each centre was run to assess inter- and intra-laboratory performance.

RESULTS: An average sample read depth of 2725X (range 629-5600) was achieved using six samples per chip, with some variability observed in the depth of coverage generated for individual samples and between centres. A total of 16 somatic mutations were detected in the six AML samples, with a mean of 2.7 mutations per sample (range 1-4) representing nine genes on the panel. 15/16 mutations were identified by all three centres. Allelic frequencies of the mutations ranged from 5.6 to 53.3 % (median 44.4 %), with a high level of concordance of these frequencies between centres, for mutations detected.

CONCLUSION: In this inter-laboratory comparison, a high concordance, reproducibility and robustness was demonstrated using a commercially available NGS AML gene panel and platform.

Synthetic Lethal Targeting of ARID1A-Mutant Ovarian Clear Cell Tumors with Dasatinib

New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G₁ -S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1Amutant OCCC. Mol Cancer Ther; 15(7); 1472-84. Ó2016 AACR.

An Immunogenetic Signature of Ongoing Antigen Interactions in Splenic Marginal Zone Lymphoma Expressing IGHV1-2*04 Receptors.

Purpose: Prompted by the extensive biases in the immunoglobulin (IG) gene repertoire of splenic marginal-zone lymphoma (SMZL), supporting antigen selection in SMZL ontogeny, we sought to investigate whether antigen involvement is also relevant post-transformation.

Experimental Design: We conducted a large-scale subcloning study of the IG rearrangements of 40 SMZL cases aimed at assessing intraclonal diversification (ID) due to ongoing somatic hypermutation (SHM).

Results: ID was identified in 17 of 21 (81%) rearrangements using the immunoglobulin heavy variable (IGHV)1-2*04 gene versus 8 of 19 (40%) rearrangements utilizing other IGHV genes (P= 0.001). ID was also evident in most analyzed IG light chain gene rearrangements, albeit was more limited compared with IG heavy chains. Identical sequence changes were shared by subclones from different patients utilizing the IGHV1-2*04 gene, confirming restricted ongoing SHM profiles. Non-IGHV1-2*04 cases displayed both a lower number of ongoing SHMs and a lack of shared mutations (per group of cases utilizing the same IGHV gene).

Conclusions: These findings support ongoing antigen involvement in a sizable portion of SMZL and further argue that IGHV1-2*04 SMZL may represent a distinct molecular subtype of the disease.

Genetics and Prognostication in Splenic Marginal Zone Lymphoma: Revelations from Deep Sequencing

Purpose: Mounting evidence supports the clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies.

Experimental Design: We undertook a detailed characterization of the genetic background of splenic marginal zone lymphoma (SMZL), using targeted resequencing and explored potential clinical implications in a multinational cohort of 175 patients with SMZL.

Results: We identified recurrent mutations in TP53 (16%), KLF2 (12%), NOTCH2 (10%), TNFAIP3 (7%), MLL2 (11%), MYD88 (7%), and ARID1A (6%), all genes known to be targeted by somatic mutation in SMZL. KLF2 mutations were early, clonal events, enriched in patients with del(7q) and IGHV1-2*04 B-cell receptor immunoglobulins, and were associated with a short median time to first treatment (0.12 vs. 1.11 years; P = 0.01). In multivariate analysis, mutations in NOTCH2 [HR, 2.12; 95% confidence interval (CI), 1.02–4.4; P = 0.044] and 100% germline IGHV gene identity (HR, 2.19; 95% CI, 1.05–4.55; P = 0.036) were independent markers of short time to first treatment, whereas TP53 mutations were an independent marker of short overall survival (HR, 2.36; 95 % CI, 1.08–5.2; P = 0.03).

Conclusions: We identify key associations between gene mutations and clinical outcome, demonstrating for the first time that NOTCH2 and TP53 gene mutations are independent markers of reduced treatment-free and overall survival, respectively.