Evidence-based guidelines for interpreting change scores for the European Organisation for the Research and Treatment of Cancer Quality of Life Questionnaire Core 30

Aim: To use published literature and experts' opinion to investigate the clinical meaning and magnitude of changes in the Quality of Life (QOL) of groups of patients measured with the European Organisation for the Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30). Methods: An innovative method combining systematic review of published studies, expert opinions and meta-analysis was used to estimate large, medium, and small mean changes over time for QLQ-C30 scores. Results: Nine hundred and eleven papers were identified, leading to 118 relevant papers. One thousand two hundred and thirty two mean changes in QOL over time were combined in the meta-analysis, with timescales ranging from four days to five years. Guidelines were produced for trivial, small, and medium size classes, for each subscale and for improving and declining scores separately. Estimates for improvements were smaller than respective estimates for declines. Conclusions: These guidelines can be used to aid sample size calculations and interpretation of mean changes over time from groups of patients. Observed mean changes in the QLQ-C30 scores are generally small in most clinical situations, possibly due to response shift. Careful consideration is needed when planning studies where QOL changes over time are of primary interest; the timing of follow up, sample attrition, direction of QOL changes, and subscales of primary interest are key considerations. (C) 2012 Elsevier Ltd. All rights reserved.

Cancer cell proliferation is inhibited by specific modulation frequencies

BACKGROUND: There is clinical evidence that very low and safe levels of amplitude-modulated electromagnetic fields administered via an intrabuccal spoon-shaped probe may elicit therapeutic responses in patients with cancer. However, there is no known mechanism explaining the anti-proliferative effect of very low intensity electromagnetic fields. METHODS: To understand the mechanism of this novel approach, hepatocellular carcinoma (HCC) cells were exposed to 27.12 MHz radiofrequency electromagnetic fields using in vitro exposure systems designed to replicate in vivo conditions. Cancer cells were exposed to tumour-specific modulation frequencies, previously identified by biofeedback methods in patients with a diagnosis of cancer. Control modulation frequencies consisted of randomly chosen modulation frequencies within the same 100 Hz-21 kHz range as cancer-specific frequencies. RESULTS: The growth of HCC and breast cancer cells was significantly decreased by HCC-specific and breast cancer-specific modulation frequencies, respectively. However, the same frequencies did not affect proliferation of nonmalignant hepatocytes or breast epithelial cells. Inhibition of HCC cell proliferation was associated with downregulation of XCL2 and PLP2. Furthermore, HCC-specific modulation frequencies disrupted the mitotic spindle. CONCLUSION: These findings uncover a novel mechanism controlling the growth of cancer cells at specific modulation frequencies without affecting normal tissues, which may have broad implications in oncology. British Journal of Cancer (2012) 106, 307-313. doi:10.1038/bjc.2011.523 www.bjcancer.com Published online 1 December 2011 (C) 2012 Cancer Research UK

High-throughput sequencing of small RNA transcriptomes reveals critical biological features targeted by microRNAs in cell models used for squamous cell cancer research

Abstract Background The implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery. Results Ten miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer. Conclusions Critical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.

Characterization of a Novel Oral Glucocorticoid System and Its Possible Role in Disease

Synthetic corticosteroids are used widely for the treatment of a variety of diseases of the mouth. However, little is known as to whether the oral mucosa is able to modulate the local concentration of active corticosteroids or to produce steroids de novo. This has important clinical implications, because tissue-specific regulation of glucocorticoids is a key determinant of the clinical efficacy of these drugs. In the present study, we show that oral fibroblasts and keratinocytes expressed ACTH receptor (MC2R), glucocorticoid receptor (GR), and 11 beta-hydroxysteroid dehydrogenases (11 beta-HSDs). Unlike keratinocytes, fibroblasts lacked 11 beta-HSD2 and could not effectively deactivate exogenously administered cortisol. However, both cell types were able not only to activate cortisone into the active form cortisol, but also to synthesize cortisol de novo following stimulation with ACTH. 11 beta-HSD2, the enzyme controlling cortisol deactivation, exhibited different patterns of expression in normal (squamous epithelium and salivary glands) and diseased oral mucosa (squamous cell carcinoma and mucoepidermoid carcinoma). Blocking of endogenous cortisol catabolism in keratinocytes with the 11 beta-HSD2 inhibitor 18 beta-glycyrrhetinic acid mimicked the effect of exogenous administration of hydrocortisone and partially prevented the detrimental effects induced by pemphigus vulgaris sera. Analysis of the data demonstrates that a novel, non-adrenal glucocorticoid system is present in the oral mucosa that may play an important role in disease.

Germline DNA copy number variation in familial and early-onset breast cancer

Introduction: Genetic factors predisposing individuals to cancer remain elusive in the majority of patients with a familial or clinical history suggestive of hereditary breast cancer. Germline DNA copy number variation (CNV) has recently been implicated in predisposition to cancers such as neuroblastomas as well as prostate and colorectal cancer. We evaluated the role of germline CNVs in breast cancer susceptibility, in particular those with low population frequencies (rare CNVs), which are more likely to cause disease." Methods: Using whole-genome comparative genomic hybridization on microarrays, we screened a cohort of women fulfilling criteria for hereditary breast cancer who did not carry BRCA1/BRCA2 mutations. Results: The median numbers of total and rare CNVs per genome were not different between controls and patients. A total of 26 rare germline CNVs were identified in 68 cancer patients, however, a proportion that was significantly different (P = 0.0311) from the control group (23 rare CNVs in 100 individuals). Several of the genes affected by CNV in patients and controls had already been implicated in cancer. Conclusions: This study is the first to explore the contribution of germline CNVs to BRCA1/2-negative familial and early-onset breast cancer. The data suggest that rare CNVs may contribute to cancer predisposition in this small cohort of patients, and this trend needs to be confirmed in larger population samples.

Frequent downregulation of the transcription factor Foxa2 in lung cancer through epigenetic silencing

Purpose: We sought to determine the mechanisms of downregulation of the airway transcription factor Foxa2 in lung cancer and the expression status of Foxa2 in non-small-cell lung cancer (NSCLC). Methods: A series of 25 lung cancer cell lines were evaluated for Foxa2 protein expression, FOXA2 mRNA levels, FOXA2 mutations, FOXA2 copy number changes and for evidence of FOXA2 promoter hypermethylation. In addition, 32 NSCLCs were sequenced for FOXA2 mutations and 173 primary NSCLC tumors evaluated for Foxa2 expression using an immunohistochemical assay. Results: Out of the 25 cell lines, 13 (52%) had undetectable FOXA2 mRNA. The expression of FOXA2 mRNA and Foxa2 protein were congruent in 19/22 cells (p = 0.001). FOXA2 mutations were not identified in primary NSCLCs and were infrequent in cell lines. Focal or broad chromosomal deletions involving FOXA2 were not present. The promoter region of FOXA2 had evidence of hypermethylation, with an inverse correlation between FOXA2 mRNA expression and presence of CpG dinucleotide methylation (p < 0.0001). In primary NSCLC tumor specimens, there was a high frequency of either absence (42/173, 24.2%) or no/low expression (96/173,55.4%) of Foxa2. In 130 patients with stage I NSCLC there was a trend towards decreased survival in tumors with no/low expression of Foxa2 (HR of 1.6, 95%CI 0.9-3.1; p = 0.122). Conclusions: Loss of expression of Foxa2 is frequent in lung cancer cell lines and NSCLCs. The main mechanism of downregulation of Foxa2 is epigenetic silencing through promoter hypermethylation. Further elucidation of the involvement of Foxa2 and other airway transcription factors in the pathogenesis of lung cancer may identify novel therapeutic targets. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Subgroup-specific structural variation across 1,000 medulloblastoma genomes

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4 alpha. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-beta signalling in Group 3, and NF-kappa B signalling in Group 4, suggest future avenues for rational, targeted therapy.

Vitamin or mineral supplement intake and the risk of head and neck cancer: pooled analysis in the INHANCE consortium

To investigate the potential role of vitamin or mineral supplementation on the risk of head and neck cancer (HNC), we analyzed individual-level pooled data from 12 casecontrol studies (7,002 HNC cases and 8,383 controls) participating in the International Head and Neck Cancer Epidemiology consortium. There were a total of 2,028 oral cavity cancer, 2,465 pharyngeal cancer, 874 unspecified oral/pharynx cancer, 1,329 laryngeal cancer and 306 overlapping HNC cases. Odds ratios (OR) and 95% confidence intervals (CIs) for self reported ever use of any vitamins, multivitamins, vitamin A, vitamin C, vitamin E, and calcium, beta-carotene, iron, selenium and zinc supplements were assessed. We further examined frequency, duration and cumulative exposure of each vitamin or mineral when possible and stratified by smoking and drinking status. All ORs were adjusted for age, sex, race/ethnicity, study center, education level, pack-years of smoking, frequency of alcohol drinking and fruit/vegetable intake. A decreased risk of HNC was observed with ever use of vitamin C (OR = 0.76, 95% CI = 0.590.96) and with ever use of calcium supplement (OR = 0.64, 95% CI = 0.420.97). The inverse association with HNC risk was also observed for 10 or more years of vitamin C use (OR = 0.72, 95% CI = 0.540.97) and more than 365 tablets of cumulative calcium intake (OR = 0.36, 95% CI = 0.160.83), but linear trends were not observed for the frequency or duration of any supplement intake. We did not observe any strong associations between vitamin or mineral supplement intake and the risk of HNC.

Diet and the risk of head and neck cancer: a pooled analysis in the INHANCE consortium

We investigated the association between diet and head and neck cancer (HNC) risk using data from the International Head and Neck Cancer Epidemiology (INHANCE) consortium. The INHANCE pooled data included 22 case-control studies with 14,520 cases and 22,737 controls. Center-specific quartiles among the controls were used for food groups, and frequencies per week were used for single food items. A dietary pattern score combining high fruit and vegetable intake and low red meat intake was created. Odds ratios (OR) and 95% confidence intervals (CI) for the dietary items on the risk of HNC were estimated with a two-stage random-effects logistic regression model. An inverse association was observed for higher-frequency intake of fruit (4th vs. 1st quartile OR = 0.52, 95% CI = 0.43-0.62, p (trend) < 0.01) and vegetables (OR = 0.66, 95% CI = 0.49-0.90, p (trend) = 0.01). Intake of red meat (OR = 1.40, 95% CI = 1.13-1.74, p (trend) = 0.13) and processed meat (OR = 1.37, 95% CI = 1.14-1.65, p (trend) < 0.01) was positively associated with HNC risk. Higher dietary pattern scores, reflecting high fruit/vegetable and low red meat intake, were associated with reduced HNC risk (per score increment OR = 0.90, 95% CI = 0.84-0.97).

RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old) FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR) for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4) was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old") samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of extracted RNA, although it produced similar quality RNA to other protocols. If a chosen protocol fails to extract biologically useful RNA from a given sample in a first attempt, another attempt and then another protocol should be tried before excluding the case from molecular analysis.

Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

Under homeostatic conditions, a proportion of senescent CXCR4(hi) neutrophils home from the circulation back to the bone marrow, where they are phagocytosed by bone marrow macrophages. In this study, we have identified an unexpected role for the anti-inflammatory molecule annexin A1 (AnxA1) as a critical regulator of this process. We first observed that AnxA1(-/-) mice have significantly increased neutrophil numbers in their bone marrow while having normal levels of GM and G colony-forming units, monocytes, and macrophages. Although AnxA1(-/-) mice have more neutrophils in the bone marrow, a greater proportion of these cells are senescent, as determined by their higher levels of CXCR4 expression and annexin V binding. Consequently, bone marrow neutrophils from AnxA1(-/-) mice exhibit a reduced migratory capacity in vitro. Studies conducted in vitro also show that expression of AnxA1 is required for bone marrow macrophages, but not peritoneal macrophages, to phagocytose apoptotic neutrophils. Moreover, in vivo experiments indicate a defect in clearance of wild-type neutrophils in the bone marrow of AnxA1(-/-) mice. Thus, we conclude that expression of AnxA1 by resident macrophages is a critical determinant for neutrophil clearance in the bone marrow.-Dalli, J., Jones, C. P., Cavalcanti, D. M., Farsky, S. H., Perretti, M., Rankin, S. M. Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow. FASEB J. 26, 387-396 (2012). www.fasebj.org

Dose-dependent change in biomarkers during neoadjuvant endocrine therapy with fulvestrant: results from NEWEST, a randomized Phase II study

NEWEST (Neoadjuvant Endocrine Therapy for Women with Estrogen-Sensitive Tumors) is the first study to compare biological and clinical activity of fulvestrant 500 versus 250 mg in the neoadjuvant breast cancer setting. We hypothesized that fulvestrant 500 mg may be superior to 250 mg in blocking estrogen receptor (ER) signaling and growth. A multicenter, randomized, open-label, Phase II study was performed to compare fulvestrant 500 mg (500 mg/month plus 500 mg on day 14 of month 1) versus fulvestrant 250 mg/month for 16 weeks prior to surgery in postmenopausal women with ER+ locally advanced breast cancer. Core biopsies at baseline, week 4, and surgery were assessed for biomarker changes. Primary endpoint: change in Ki67 labeling index (LI) from baseline to week 4 determined by automated computer imaging system (ACIS). Secondary endpoints: ER protein expression and function; progesterone receptor (PgR) expression; tumor response; tolerability. ER and PgR were examined retrospectively using the H score method. A total of 211 patients were randomized (fulvestrant 500 mg: n = 109; 250 mg: n = 102). At week 4, fulvestrant 500 mg resulted in greater reduction of Ki67 LI and ER expression versus 250 mg (-78.8 vs. -47.4% [p < 0.0001] and -25.0 vs. -13.5% [p = 0.0002], respectively [ACIS]); PgR suppression was not significantly different (-22.7 vs. -17.6; p = 0.5677). However, H score detected even greater suppression of ER (-50.3 vs. -13.7%; p < 0.0001) and greater PgR suppression (-80.5 vs. -46.3%; p = 0.0018) for fulvestrant 500 versus 250 mg. At week 16, tumor response rates were 22.9 and 20.6% for fulvestrant 500 and 250 mg, respectively, with considerable decline in all markers by both ACIS and H score. No detrimental effects on endometrial thickness or bone markers and no new safety concerns were identified. This provides the first evidence of greater biological activity for fulvestrant 500 versus 250 mg in depleting ER expression, function, and growth.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

The Aurora A and B kinases are up-regulated in bone marrow-derived chronic lymphocytic leukemia cells and represent potential therapeutic targets

Background The malignant B cells in chronic lymphocytic leukemia receive signals from the bone marrow and lymph node microenvironments which regulate their survival and proliferation. Characterization of these signals and the pathways that propagate them to the interior of the cell is important for the identification of novel potential targets for therapeutic intervention. Design and Methods We compared the gene expression profiles of chronic lymphocytic leukemia B cells purified from bone marrow and peripheral blood to identify genes that are induced by the bone marrow microenvironment. Two of the differentially expressed genes were further studied in cell culture experiments and in an animal model to determine whether they could represent appropriate therapeutic targets in chronic lymphocytic leukemia. Results Functional classification analysis revealed that the majority of differentially expressed genes belong to gene ontology categories related to cell cycle and mitosis. Significantly up-regulated genes in bone marrow-derived tumor cells included important cell cycle regulators, such as Aurora A and B, survivin and CDK6. Down-regulation of Aurora A and B by RNA interference inhibited proliferation of chronic lymphocytic leukemia-derived cell lines and induced low levels of apoptosis. A similar effect was observed with the Aurora kinase inhibitor VX-680 in primary chronic lymphocytic leukemia cells that were induced to proliferate by CpG-oligonucleotides and interleukin-2. Moreover, VX-680 significantly blocked leukemia growth in a mouse model of chronic lymphocytic leukemia. Conclusions Aurora A and B are up-regulated in proliferating chronic lymphocytic leukemia cells and represent potential therapeutic targets in this disease.

Trypanosoma cruzi Adjuvants Potentiate T Cell-Mediated Immunity Induced by a NY-ESO-1 Based Antitumor Vaccine

Immunological adjuvants that induce T cell-mediate immunity (TCMI) with the least side effects are needed for the development of human vaccines. Glycoinositolphospholipids (GIPL) and CpGs oligodeoxynucleotides (CpG ODNs) derived from the protozoa parasite Trypanosoma cruzi induce potent pro-inflammatory reaction through activation of Toll-Like Receptor (TLR) 4 and TLR9, respectively. Here, using mouse models, we tested the T. cruzi derived TLR agonists as immunological adjuvants in an antitumor vaccine. For comparison, we used well-established TLR agonists, such as the bacterial derived monophosphoryl lipid A (MPL), lipopeptide (Pam3Cys), and CpG ODN. All tested TLR agonists were comparable to induce antibody responses, whereas significant differences were noticed in their ability to elicit CD4(+) T and CD8(+) T cell responses. In particular, both GIPLs (GTH, and GY) and CpG ODNs (B344, B297 and B128) derived from T. cruzi elicited interferon-gamma (IFN-gamma) production by CD4(+) T cells. On the other hand, the parasite derived CpG ODNs, but not GIPLs, elicited a potent IFN-gamma response by CD8(+) T lymphocytes. The side effects were also evaluated by local pain (hypernociception). The intensity of hypernociception induced by vaccination was alleviated by administration of an analgesic drug without affecting protective immunity. Finally, the level of protective immunity against the NY-ESO-1 expressing melanoma was associated with the magnitude of both CD4+ T and CD8+ T cell responses elicited by a specific immunological adjuvant.