The role of infiltrating lymphocytes in the neo-adjuvant treatment of women with HER2-positive breast cancer.

BACKGROUND: Pre-treatment tumour-associated lymphocytes (TILs) and stromal lymphocytes (SLs) are independent predictive markers of future pathological complete response (pCR) in HER2-positive breast cancer. Whilst studies have correlated baseline lymphocyte levels with subsequent pCR, few have studied the impact of neoadjuvant therapy on the immune environment. METHODS: We performed TIL analysis and T-cell analysis by IHC on the pretreatment and 'On-treatment' samples from patients recruited on the Phase-II TCHL (NCT01485926) clinical trial. Data were analysed using the Wilcoxon signed-rank test and the Spearman rank correlation. RESULTS: In our sample cohort (n = 66), patients who achieved a pCR at surgery, post-chemotherapy, had significantly higher counts of TILs (p = 0.05) but not SLs (p = 0.08) in their pre-treatment tumour samples. Patients who achieved a subsequent pCR after completing neo-adjuvant chemotherapy had significantly higher SLs (p = 9.09 × 10-3) but not TILs (p = 0.1) in their 'On-treatment' tumour biopsies. In a small cohort of samples (n = 16), infiltrating lymphocyte counts increased after 1 cycle of neo-adjuvant chemotherapy only in those tumours of patients who did not achieve a subsequent pCR. Finally, reduced CD3 + (p = 0.04, rho = 0.60) and CD4 + (p = 0.01, rho = 0.72) T-cell counts in 'On-treatment' biopsies were associated with decreased residual tumour content post-1 cycle of treatment; the latter being significantly associated with increased likelihood of subsequent pCR (p < 0.01). CONCLUSIONS: The immune system may be 'primed' prior to neoadjuvant treatment in those patients who subsequently achieve a pCR. In those patients who achieve a pCR, their immune response may return to baseline after only 1 cycle of treatment. However, in those who did not achieve a pCR, neo-adjuvant treatment may stimulate lymphocyte influx into the tumour.

Molecular subtype, biological sex and age shape melanoma tumour evolution.

BACKGROUND: Many cancer types display sex and age disparity in incidence and outcome. The mutational load of tumours, including melanoma, varies according to sex and age. However, there are no tools to explore systematically whether clinical variables such as age and sex determine the genomic landscape of cancer. OBJECTIVES: To establish a mathematical approach using melanoma mutational data to analyse how sex and age shape the tumour genome. METHODS: We model how age-related (clock-like) somatic mutations that arise during cell division, and extrinsic (environmental ultraviolet radiation) mutations accumulate in cancer genomes. RESULTS: Melanoma is driven primarily by cell-intrinsic age-related mutations and extrinsic ultraviolet radiation-induced mutations, and we show that these mutation types differ in magnitude and chronology and by sex in the distinct molecular melanoma subtypes. Our model confirms that age and sex are determinants of cellular mutation rate, shaping the final mutation composition. We show mathematically for the first time how, similarly to noncancer tissues, melanoma genomes reflect a decline in cell division during ageing. We find that clock-like mutations strongly correlate with the acquisition of ultraviolet-induced mutations, but critically, men present a higher number and rate of cell-division-linked mutations. CONCLUSIONS: These data indicate that the contribution of environmental damage to melanoma likely extends beyond genetic damage to affect cell division. Sex and age determine the final mutational composition of melanoma.

Meta-analysis of the molecular associations of mucinous colorectal cancer.

BACKGROUND: Mucinous differentiation occurs in 5-15 per cent of colorectal adenocarcinomas. This subtype of colorectal cancer responds poorly to chemoradiotherapy and has a worse prognosis. The genetic aetiology underpinning this cancer subtype lacks consensus. The aim of this study was to use meta-analytical techniques to clarify the molecular associations of mucinous colorectal cancer. METHODS: This study adhered to MOOSE guidelines. Databases were searched for studies comparing KRAS, BRAF, microsatellite instability (MSI), CpG island methylator phenotype (CIMP), p53 and p27 status between patients with mucinous and non-mucinous colorectal adenocarcinoma. A random-effects model was used for analysis. RESULTS: Data from 46 studies describing 17 746 patients were included. Mucinous colorectal adenocarcinoma was associated positively with KRAS (odds ratio (OR) 1·46, 95 per cent c.i. 1·08 to 2·00, P = 0·014) and BRAF (OR 3·49, 2·50 to 4·87; P < 0·001) mutation, MSI (OR 3·98, 3·30 to 4·79; P < 0·001) and CIMP (OR 3·56, 2·85 to 4·43; P < 0·001), and negatively with altered p53 expression (OR 0·46, 0·31 to 0·67; P < 0·001). CONCLUSION: The genetic origins of mucinous colorectal adenocarcinoma are predominantly associated with BRAF, MSI and CIMP pathways. This pattern of molecular alterations may in part explain the resistance to standard chemotherapy regimens seen in mucinous adenocarcinoma.

Beyond the exome: the role of non-coding somatic mutations in cancer.

The comprehensive identification of mutations contributing to the development of cancer is a priority of large cancer sequencing projects. To date, most studies have scrutinized mutations in coding regions of the genome, but several recent discoveries, including the identification of recurrent somatic mutations in the TERT promoter in multiple cancer types, support the idea that mutations in non-coding regions are also important in tumour development. Furthermore, analysis of whole-genome sequencing data from tumours has elucidated novel mutational patterns and processes etched into cancer genomes. Here, we present an overview of insights gleaned from the analysis of mutations from sequenced cancer genomes. We then review the mechanisms by which non-coding mutations can play a role in cancer. Finally, we discuss recent efforts aimed at identifying non-coding driver mutations, as well as the unique challenges that the analysis of non-coding mutations present in contrast to the identification of driver mutations in coding regions.

A personalised medicine approach for ponatinib-resistant chronic myeloid leukaemia.

BACKGROUND: Chronic myeloid leukaemia (CML) is characterised by the presence of a fusion driver oncogene, BCR-ABL1, which is a constitutive tyrosine kinase. Tyrosine kinase inhibitors (TKIs) are the central treatment strategy for CML patients and have significantly improved survival rates, but the T315I mutation in the kinase domain of BCR-ABL1 confers resistance to all clinically approved TKIs, except ponatinib. However, compound mutations can mediate resistance even to ponatinib and remain a clinical challenge in CML therapy. Here, we investigated a ponatinib-resistant CML patient through whole-genome sequencing (WGS) to identify the cause of resistance and to find alternative therapeutic targets. PATIENTS AND METHODS: We carried out WGS on a ponatinib-resistant CML patient and demonstrated an effective combination therapy against the primary CML cells derived from this patient in vitro. RESULTS: Our findings demonstrate the emergence of compound mutations in the BCR-ABL1 kinase domain following ponatinib treatment, and chromosomal structural variation data predicted amplification of BCL2. The primary CD34(+) CML cells from this patient showed increased sensitivity to the combination of ponatinib and ABT-263, a BCL2 inhibitor with a negligible effect against the normal CD34(+) cells. CONCLUSION: Our results show the potential of personalised medicine approaches in TKI-resistant CML patients and provide a strategy that could improve clinical outcomes for these patients.

Clusterin regulates ß-amyloid toxicity via Dickkopf-1-driven induction of the wnt-PCP-JNK pathway.

Although the mechanism of Aß action in the pathogenesis of Alzheimer's disease (AD) has remained elusive, it is known to increase the expression of the antagonist of canonical wnt signalling, Dickkopf-1 (Dkk1), whereas the silencing of Dkk1 blocks Aß neurotoxicity. We asked if clusterin, known to be regulated by wnt, is part of an Aß/Dkk1 neurotoxic pathway. Knockdown of clusterin in primary neurons reduced Aß toxicity and DKK1 upregulation and, conversely, Aß increased intracellular clusterin and decreased clusterin protein secretion, resulting in the p53-dependent induction of DKK1. To further elucidate how the clusterin-dependent induction of Dkk1 by Aß mediates neurotoxicity, we measured the effects of Aß and Dkk1 protein on whole-genome expression in primary neurons, finding a common pathway suggestive of activation of wnt-planar cell polarity (PCP)-c-Jun N-terminal kinase (JNK) signalling leading to the induction of genes including EGR1 (early growth response-1), NAB2 (Ngfi-A-binding protein-2) and KLF10 (Krüppel-like factor-10) that, when individually silenced, protected against Aß neurotoxicity and/or tau phosphorylation. Neuronal overexpression of Dkk1 in transgenic mice mimicked this Aß-induced pathway and resulted in age-dependent increases in tau phosphorylation in hippocampus and cognitive impairment. Furthermore, we show that this Dkk1/wnt-PCP-JNK pathway is active in an Aß-based mouse model of AD and in AD brain, but not in a tau-based mouse model or in frontotemporal dementia brain. Thus, we have identified a pathway whereby Aß induces a clusterin/p53/Dkk1/wnt-PCP-JNK pathway, which drives the upregulation of several genes that mediate the development of AD-like neuropathologies, thereby providing new mechanistic insights into the action of Aß in neurodegenerative diseases.

Whole-genome sequencing reveals complex mechanisms of intrinsic resistance to BRAF inhibition.

BACKGROUND: BRAF is mutated in ∼42% of human melanomas (COSMIC. http://www.sanger.ac.uk/genetics/CGP/cosmic/) and pharmacological BRAF inhibitors such as vemurafenib and dabrafenib achieve dramatic responses in patients whose tumours harbour BRAF(V600) mutations. Objective responses occur in ∼50% of patients and disease stabilisation in a further ∼30%, but ∼20% of patients present primary or innate resistance and do not respond. Here, we investigated the underlying cause of treatment failure in a patient with BRAF mutant melanoma who presented primary resistance. METHODS: We carried out whole-genome sequencing and single nucleotide polymorphism (SNP) array analysis of five metastatic tumours from the patient. We validated mechanisms of resistance in a cell line derived from the patient's tumour. RESULTS: We observed that the majority of the single-nucleotide variants identified were shared across all tumour sites, but also saw site-specific copy-number alterations in discrete cell populations at different sites. We found that two ubiquitous mutations mediated resistance to BRAF inhibition in these tumours. A mutation in GNAQ sustained mitogen-activated protein kinase (MAPK) signalling, whereas a mutation in PTEN activated the PI3 K/AKT pathway. Inhibition of both pathways synergised to block the growth of the cells. CONCLUSIONS: Our analyses show that the five metastases arose from a common progenitor and acquired additional alterations after disease dissemination. We demonstrate that a distinct combination of mutations mediated primary resistance to BRAF inhibition in this patient. These mutations were present in all five tumours and in a tumour sample taken before BRAF inhibitor treatment was administered. Inhibition of both pathways was required to block tumour cell growth, suggesting that combined targeting of these pathways could have been a valid therapeutic approach for this patient.

Whole-exome sequencing and imaging genetics identify functional variants for rate of change in hippocampal volume in mild cognitive impairment.

Whole-exome sequencing of individuals with mild cognitive impairment, combined with genotype imputation, was used to identify coding variants other than the apolipoprotein E (APOE) ε4 allele associated with rate of hippocampal volume loss using an extreme trait design. Matched unrelated APOE ε3 homozygous male Caucasian participants from the Alzheimer's Disease Neuroimaging Initiative (ADNI) were selected at the extremes of the 2-year longitudinal change distribution of hippocampal volume (eight subjects with rapid rates of atrophy and eight with slow/stable rates of atrophy). We identified 57 non-synonymous single nucleotide variants (SNVs) which were found exclusively in at least 4 of 8 subjects in the rapid atrophy group, but not in any of the 8 subjects in the slow atrophy group. Among these SNVs, the variants that accounted for the greatest group difference and were predicted in silico as 'probably damaging' missense variants were rs9610775 (CARD10) and rs1136410 (PARP1). To further investigate and extend the exome findings in a larger sample, we conducted quantitative trait analysis including whole-brain search in the remaining ADNI APOE ε3/ε3 group (N=315). Genetic variation within PARP1 and CARD10 was associated with rate of hippocampal neurodegeneration in APOE ε3/ε3. Meta-analysis across five independent cross sectional cohorts indicated that rs1136410 is also significantly associated with hippocampal volume in APOE ε3/ε3 individuals (N=923). Larger sequencing studies and longitudinal follow-up are needed for confirmation. The combination of next-generation sequencing and quantitative imaging phenotypes holds significant promise for discovery of variants involved in neurodegeneration.

Genome-wide association with MRI atrophy measures as a quantitative trait locus for Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with considerable evidence suggesting an initiation of disease in the entorhinal cortex and hippocampus and spreading thereafter to the rest of the brain. In this study, we combine genetics and imaging data obtained from the Alzheimer's Disease Neuroimaging Initiative and the AddNeuroMed study. To identify genetic susceptibility loci for AD, we conducted a genome-wide study of atrophy in regions associated with neurodegeneration in this condition. We identified one single-nucleotide polymorphism (SNP) with a disease-specific effect associated with entorhinal cortical volume in an intron of the ZNF292 gene (rs1925690; P-value=2.6 × 10(-8); corrected P-value for equivalent number of independent quantitative traits=7.7 × 10(-8)) and an intergenic SNP, flanking the ARPP-21 gene, with an overall effect on entorhinal cortical thickness (rs11129640; P-value=5.6 × 10(-8); corrected P-value=1.7 × 10(-7)). Gene-wide scoring also highlighted PICALM as the most significant gene associated with entorhinal cortical thickness (P-value=6.7 × 10(-6)).

Assessing the health behaviors of Texas college students.

The authors of this study examined the health behaviors of Texas college students, using a telephone survey to assess 1,408 randomly selected students enrolled in 23 institutions of higher education. Eighty-nine percent of those surveyed had consumed alcohol at least once, and nearly one third considered themselves regular smokers. More than 81% reported they had had sexual intercourse at least once, and one fourth of the sexually active men had had more than 10 partners. However, of the 1,148 students who were sexually active, only 40.1% reported using a condom at last intercourse. Almost 59% of the students surveyed had never been taught about HIV or AIDS in any of their college classes. Given the large sample size and the random methods employed, the authors suggest that the results of this study can be used to establish a baseline of information regarding health behaviors of college students that can be extrapolated to college populations across the country.

Using the Youth Risk Behavior Survey to compare risk behaviors of Texas High School and college students.

This study used the Youth Risk Behavior Survey (YRBS) to assess selected health behaviors of Texas high school and college students. The YRBS was administered during 1993 in paper and pencil form to 6,015 high school students representing 329 classrooms from 78 school districts. A total of 1,408 college students representing 23 college and universities were surveyed by telephone in 1993 using a modified version of YRBS. Texas college students reported a higher percentage who had experienced sexual intercourse (82% versus 55.4%), but Texas high school students reported a younger age of first sexual intercourse. High school students also initiated alcohol consumption at a younger age, although college students were more likely to binge drink (33.5% versus 31%). Regular cigarette use also was higher among college students (25.4% versus 19.3%), but was initiated at a younger age by high school students. Study results indicate that health education programs must begin much earlier than during the high school years. Due to early initiation of negative health behaviors, emphasis must be placed on abstinence and risk-reduction techniques for both populations.

Effect of rifabutin on disseminated Mycobacterium avium infections in thymectomized, CD4 T-cell-deficient mice.

Disseminated Mycobacterium avium infection is the major cause of bacteremia in patients with acquired immunodeficiency syndrome. We present here a new animal model of this disease, thymectomized C57BL/6 mice that were intravenously infused with monoclonal antibody to selectively deplete CD4+ T cells. The increased susceptibility of such animals to M. avium infection is comparable to that of C57BL/6 beige mice and thus may provide a viable alternative to the latter model. Further, using representative strains of acquired immunodeficiency syndrome-associated M. avium (serotypes 1, 4, and 8 and a rough isolate), we show that the course of such infections in thymectomized, CD4-deficient mice can be markedly restrained and in some cases the infections can be sterilized by treatment over a 120-day period with a regimen containing 40 mg of the new antimycobacterial agent rifabutin per kg (body weight).

Dissemination of enteric Mycobacterium avium infections in mice rendered immunodeficient by thymectomy and CD4 depletion or by prior infection with murine AIDS retroviruses.

This study shows that infection of mice with the murine AIDS virus LP-BM5 or Du5H profoundly depressed the capacity of splenic T cells from these animals to respond to the T-cell mitogen phytohemagglutinin or concanavalin A or to alloantigens. Similar effects were also observed if mice were thymectomized and then infused with monoclonal anti-CD4 antibody (TxCD4- mice). When such mice were infected intravenously with Mycobacterium avium, growth of the infection was markedly exacerbated in the TxCD4- mice or in mice given murine AIDS virus 2 months earlier. In view of these data, we then investigated whether such treatments might cause dissemination of M. avium following enteric implantation of bacteria into the mouse cecum; this route was chosen in an attempt to model events in AIDS patients, in which the gut appears to be one of the major portals of M. avium infection. In this model, the entry and hematogenous dissemination of four clinical isolates of M. avium were monitored against time and found to be accelerated and enhanced in T-cell-deficient mice. In view of this finding, these novel approaches for enteric infection that use immunodeficient mice are presented as potential new models for the evaluation of immunotherapy and chemotherapy in a setting that bears some similarity to events believed to occur in AIDS patients.

Metronidazole therapy in mice infected with tuberculosis.

The capacity of metronidazole to inhibit the growth of Mycobacterium tuberculosis was tested in in vitro and in vivo mouse models. In vitro addition of metronidazole to cultures of infected bone marrow-derived macrophages had no effect, nor did it increase the reduction in bacterial load due to isoniazid. In vivo, metronidazole did not reduce bacterial numbers in the lungs of aerosol-infected mice during the active stage of the disease, during a phase of containment, or after prolonged isoniazid therapy (Cornell model). A small but significant reduction was seen if metronidazole therapy was given during an established chronic disease state 100 days after aerosol administration. These data indicate that under most conditions M. tuberculosis organisms are not in a metabolic state in which they are susceptible to the action of metronidazole and, hence, that this drug would be of limited clinical value.

Animal and cell-culture models for the study of mycobacterial infections and treatment.

Emerging problems with the treatment of infections caused by Mycobacterium avium and Mycobacterium tuberculosis require the development of new models, both in vitro and in vivo, in which new chemotherapeutic and immunotherapeutic approaches can be tested. In this brief review, the use of cell culture models, in which drugs can be tested for their capacity to inhibit mycobacterial growth within the infected host macrophage, and new models in vivo in which drugs and/or cytokines can be tested in infected mice are discussed. In this latter case, new emerging mouse models include animals with engineered gene disruptions, in which severely disseminated infections can be produced, thus mimicking events in severely immunocompromised human patients.

Capacity of Mycobacterium avium isolates to grow well or poorly in murine macrophages resides in their ability to induce secretion of tumor necrosis factor.

The results of this study show that clinical isolates of Mycobacterium avium fall into two categories in terms of their capacity to grow within murine bone marrow-derived macrophage cultures: those that grow progressively and those that are incapable of growing within such cells. Members of the first category were invariably of the smooth-transparent colonial type, while most of the second were of the smooth-doomed type. In addition, this paper shows that although all isolates induced tumor necrosis factor (TNF) secretion by host cells to some extent, this production was always delayed in isolates that subsequently grew well in the host cells. This observation, coupled with the demonstration that the growth of the latter isolates was inhibited by the exogenous addition of TNF, leads us to hypothesize that the ability of a given isolate to somehow avoid host macrophage TNF production early during the course of the infection is a key factor in the pathogenesis of the disease.

Low-dose aerosol infection model for testing drugs for efficacy against Mycobacterium tuberculosis.

As a paradigm for chronic infectious diseases, tuberculosis exhibits a variety of clinical presentations, ranging from primary pulmonary tuberculosis to reactivation tuberculosis and cavitary disease. To date, the animal models used in evaluating chemotherapy of tuberculosis have been high-dose intravenous models that mimic the disseminated forms of the disease. In the present study, we have used a low-dose aerosol exposure model which we feel better reflects newly diagnosed tuberculosis in patients converting to tuberculin positivity. As appropriate examples of chemotherapy, four rifamycins (rifampin, rifabutin, rifapentine, and KRM-1648) were tested, first in an in vitro murine macrophage model and then in the low-dose aerosol infection model, for their activity against Mycobacterium tuberculosis. In both models, KRM-1648 had the highest level of activity of the four compounds. In the infected-lung model, rifabutin, rifapentine, and KRM-1648 all had sterilizing activity when given orally at 5 mg/kg of body weight per day. When given at 2.5 mg/kg/day, KRM-1648 had the highest level of activity of the four drugs, reducing the bacterial load by 2.7 logs over 35 days of therapy.