Multiple primary cancers associated with Merkel cell carcinoma in Queensland, Australia, 1982-2011

The relatively high incidence of Merkel cell carcinoma (MCC) in Queensland provides a valuable opportunity to examine links with other cancers. A retrospective cohort study was performed using data from the Queensland Cancer Registry. Standardized incidence ratios (SIRs) were used to approximate the relative risk of being diagnosed with another primary cancer either following or prior to MCC. Patients with an eligible first primary MCC (n=787) had more than double the expected number of subsequent primary cancers (SIR=2.19, 95% confidence interval (CI)=1.84–2.60; P<0.001). Conversely, people who were initially diagnosed with cancers other than MCC were about two and a half times more likely to have a subsequent primary MCC (n=244) compared with the general population (SIR=2.69, 95% CI=2.36–3.05; P<0.001). Significantly increased bi-directional relative risks were found for melanoma, lip cancer, head and neck cancer, lung cancer, myelodysplastic diseases, and cancer with unknown primary site. In addition, risks were elevated for female breast cancer and kidney cancer following a first primary MCC, and for subsequent MCCs following first primary colorectal cancer, prostate cancer, non-Hodgkin lymphoma, or lymphoid leukemia. These results suggest that several shared pathways are likely for MCC and other cancers, including immunosuppression, UV radiation, and genetics.

Pro-matrix metalloproteinase-2 transfection increases orthotopic primary growth and experimental metastasis of MDA-MB-231 human breast cancer cells in nude mice

The ability to activate pro-matrix metalloproteinase (pro-MMP)-2 via membrane type-MMP is a hallmark of human breast cancer cell lines that show increased invasiveness, suggesting that MMP-2 contributes to human breast cancer progression. To investigate this, we have stably transfected pro-MMP-2 into the human breast cancer cell line MDA-MB-231, which lacks MMP-2 expression but does express its cell surface activator, membrane type 1-MMP. Multiple clones were derived and shown to produce pro-MMP-2 and to activate it in response to concanavalin A. In vitro analysis showed that the pro-MMP-2-transfected clones exhibited an increased invasive potential in Boyden chamber and Matrigel outgrowth assays, compared with the parental cells or those transfected with vector only. When inoculated into the mammary fat pad of nude mice, each of the MMP-2-tranfected clones grew faster than each of the vector controls tested. After intracardiac inoculation into nude mice, pro-MMP-2-transfected clones showed a significant increase in the incidence of metastasis to brain, liver, bone, and kidney compared with the vector control clones but not lung. Increased tumor burden was seen in the primary site and in lung metastases, and a trend toward increased burden was seen in bone, however, no change was seen in brain, liver, or kidney. This data supports a role for MMP-2 in breast cancer progression, both in the growth of primary tumors and in their spread to distant organs. MMP-2 may be a useful target for breast cancer therapy when refinement of MMP inhibitors provides for MMP-specific agents.

CDKN2A mutation in a non-FAMMM kindred with cancers at multiple sites results in a functionally abnormal protein

The CDKN2A gene encodes p16 (CDKN2A), a cell-cycle inhibitor protein which prevents inappropriate cell cycling and, hence, proliferation. Germ-line mutations in CDKN2A predispose to the familial atypical multiple-mole melanoma (FAMMM) syndrome but also have been seen in rare families in which only 1 or 2 individuals are affected by cutaneous malignant melanoma (CMM). We therefore sequenced exons 1alpha and 2 of CDKN2A using lymphocyte DNA isolated from index cases from 67 families with cancers at multiple sites, where the patterns of cancer did not resemble those attributable to known genes such as hMLH1, hMLH2, BRCA1, BRCA2, TP53 or other cancer susceptibility genes. We found one mutation, a mis-sense mutation resulting in a methionine to isoleucine change at codon 53 (M531) of exon 2. The individual tested had developed 2 CMMs but had no dysplastic nevi and lacked a family history of dysplastic nevi or CMM. Other family members had been diagnosed with oral cancer (2 persons), bladder cancer (1 person) and possibly gall-bladder cancer. While this mutation has been reported in Australian and North American melanoma kindreds, we did not observe it in 618 chromosomes from Scottish and Canadian controls. Functional studies revealed that the CDKN2A variant carrying the M531 change was unable to bind effectively to CDK4, showing that this mutation is of pathological significance. Our results have confirmed that CDKN2A mutations are not limited to FAMMM kindreds but also demonstrate that multi-site cancer families without melanoma are very unlikely to contain CDKN2A mutations.

An MMP13-selective inhibitor delays primary tumor growth and the onset of tumor-associated osteolytic lesions in experimental models of breast cancer

We investigated the effects of the matrix metalloproteinase 13 (MMP13)-selective inhibitor, 5-(4-{4-[4-(4-fluorophenyl)-1,3-oxazol-2-yl]phenoxy}phenoxy)-5-(2-methoxyethyl) pyrimidine-2,4,6(1H,3H,5H)-trione (Cmpd-1), on the primary tumor growth and breast cancer-associated bone remodeling using xenograft and syngeneic mouse models. We used human breast cancer MDA-MB-231 cells inoculated into the mammary fat pad and left ventricle of BALB/c Nu/Nu mice, respectively, and spontaneously metastasizing 4T1.2-Luc mouse mammary cells inoculated into mammary fat pad of BALB/c mice. In a prevention setting, treatment with Cmpd-1 markedly delayed the growth of primary tumors in both models, and reduced the onset and severity of osteolytic lesions in the MDA-MB-231 intracardiac model. Intervention treatment with Cmpd-1 on established MDA-MB-231 primary tumors also significantly inhibited subsequent growth. In contrast, no effects of Cmpd-1 were observed on soft organ metastatic burden following intracardiac or mammary fat pad inoculations of MDA-MB-231 and 4T1.2-Luc cells respectively. MMP13 immunostaining of clinical primary breast tumors and experimental mice tumors revealed intra-tumoral and stromal expression in most tumors, and vasculature expression in all. MMP13 was also detected in osteoblasts in clinical samples of breast-to-bone metastases. The data suggest that MMP13-selective inhibitors, which lack musculoskeletal side effects, may have therapeutic potential both in primary breast cancer and cancer-induced bone osteolysis.

CDKN2A/p16 is inactivated in most melanoma cell lines

The CDKN2A gene maps to chromosome 9p21-22 and is responsible for melanoma susceptibility in some families. Its product, p16, binds specifically to CDK4 and CDK6 in vitro and in vivo, inhibiting their kinase activity. CDKN2A is homozygously deleted or mutated in a large proportion of tumor cell lines and some primary tumors, including melanomas. The aim of this study was to investigate the involvement of CDKN2A and elucidate the mechanisms of p16 inactivation in a panel of 60 cell lines derived from sporadic melanomas. Twenty-six (43%) of the melanoma lines were homozygously deleted for CDKN2A, and an additional 15 (25%) lines carried missense, nonsense, or frameshift mutations. All but one of the latter group were shown by microsatellite analysis to be hemizygous for the region of 9p surrounding CDKN2A. p16 was detected by Western blotting in only five of the cell lines carrying mutations. Immunoprecipitation of p16 in these lines, followed by Western blotting to detect the coprecipitation of CDK4 and CDK6, revealed that p16 was functionally compromised in all cell lines but the one that carried a heterozygous CDKN2A mutation. In the remaining 19 lines that carried wild-type CDKN2A alleles, Western blot analysis and immunoprecipitation indicated that 11 cell lines expressed a wild-type protein. Northern blotting was performed on the remaining eight cell lines and revealed that one cell line carried an aberrantly sized RNA transcript, and two other cell lines failed to express RNA. The promoter was found to be methylated in five cell lines that expressed CDKN2A transcript but not p16. Presumably, the message seen by Northern blotting in these cell lines is the result of cross-hybridization of the total cDNA probe with the exon 1beta transcript. Microsatellite analysis revealed that the majority of these cell lines were hemi/homozygous for the region surrounding CDKN2A, indicating that the wild-type allele had been lost. In the 11 cell lines that expressed functional p16, microsatellite analysis revealed loss of heterozygosity at the markers immediately surrounding CDKN2A in five cases, and the previously characterized R24C mutation of CDK4 was identified in one of the remaining 6 lines. These data indicate that 55 of 60 (92%) melanoma cell lines demonstrated some aberration of CDKN2A or CDK4, thus suggesting that this pathway is a primary genetic target in melanoma development.

Spatio-temporal modelling of ultrafine particle number concentration

This thesis developed semi-parametric regression models for estimating the spatio-temporal distribution of outdoor airborne ultrafine particle number concentration (PNC). The models developed incorporate multivariate penalised splines and random walks and autoregressive errors in order to estimate non-linear functions of space, time and other covariates. The models were applied to data from the "Ultrafine Particles from Traffic Emissions and Child" project in Brisbane, Australia, and to longitudinal measurements of air quality in Helsinki, Finland. The spline and random walk aspects of the models reveal how the daily trend in PNC changes over the year in Helsinki and the similarities and differences in the daily and weekly trends across multiple primary schools in Brisbane. Midday peaks in PNC in Brisbane locations are attributed to new particle formation events at the Port of Brisbane and Brisbane Airport.

The epidemiology of basal cell carcinoma

Basal cell carcinoma (BCC) is a skin cancer of particular importance to the Australian community. Its rate of occurrence is highest in Queensland, where 1% to 2% of people are newly affected annually. This is an order of magnitude higher than corresponding incidence estimates in European and North American populations. Individuals with a sun-sensitive complexion are particularly susceptible because sun exposure is the single most important causative agent, as shown by the anatomic distribution of BCC which is in general consistent with the levels of sun exposure across body sites. A distinguishing feature of BCC is the occurrence of multiple primary tumours within individuals, synchronously or over time, and their diagnosis and treatment costs contribute substantially to the major public health burden caused by BCC. A primary knowledge gap about BCC pathogenesis however was an understanding of the true frequency of multiple BCC occurrences and their body distribution, and why a proportion of people do develop more than one BCC in their life. This research project sought to address this gap under an overarching research aim to better understand the detailed epidemiology of BCC with the ultimate goal of reducing the burden of this skin cancer through prevention. The particular aim was to document prospectively the rate of BCC occurrence and its associations with constitutional and environmental (solar) factors, all the while paying special attention to persons affected by more than one BCC. The study built on previous findings and recent developments in the field but set out to confirm and extend these and propose more adequate theories about the complex epidemiology of this cancer. Addressing these goals required a new approach to researching basal cell carcinoma, due to the need to account for the phenomenon of multiple incident BCCs per person. This was enabled by a 20 year community-based study of skin cancer in Australians that provided the methodological foundation for this thesis. Study participants were originally randomly selected in 1986 from the electoral register of all adult residents of the subtropical township of Nambour in Queensland, Australia. On various occasions during the study, participants were fully examined by dermatologists who documented cumulative photodamage as well as skin cancers. Participants completed standard questionnaires about skin cancer-related factors, and consented to have any diagnosed skin cancers notified to the investigators by regional pathology laboratories in Queensland. These methods allowed 100% ascertainment of histologically confirmed BCCs in this study population. 1339 participants had complete follow-up to the end of 2007. Statistical analyses in this thesis were carried out using SAS and SUDAAN statistical software packages. Modelling methods, including multivariate logistic regressions, allowed for repeated measures in terms of multiple BCCs per person. This innovative approach gave new findings on two levels, presented in five chapters as scientific papers: 1. Incidence of basal cell carcinoma multiplicity and detailed anatomic distribution: longitudinal study of an Australian population The incidence of people affected multiple times by BCC was 705 per 100,000 person years compared to an incidence rate of people singly affected of 935 per 100,000 person years. Among multiply and singly affected persons alike, site-specific BCC incidence rates were far highest on facial subsites, followed by upper limbs, trunk, and then lower limbs 2. Melanocytic nevi and basal cell carcinoma: is there an association? BCC risk was significantly increased in those with forearm nevi (Odds Ratios (OR) 1.43, 95% Confidence Intervals (CI) 1.09-1.89) compared to people without forearm nevi, especially among those who spent their time mainly outdoors (OR 1.6, 95%CI 1.1-2.3) compared to those who spent their time mainly indoors. Nevi on the back were not associated with BCC. 3. Clinical signs of photodamage are associated with basal cell carcinoma multiplicity and site: a 16-year longitudinal study Over a 16-year follow-up period, 58% of people affected by BCC developed more than one BCC. Among these people 60% developed BCCs across different anatomic sites. Participants with high numbers of solar keratoses, compared to people without solar keratoses, were most likely to experience the highest BCC counts overall (OR 3.3, 95%CI 1.4-13.5). Occurrences of BCC on the trunk (OR 3.3, 95%CI 1.4-7.6) and on the limbs (OR 3.7, 95%CI 2.0-7.0) were strongly associated with high numbers of solar keratoses on these sites. 4. Occurrence and determinants of basal cell carcinoma by histological subtype in an Australian community Among 1202 BCCs, 77% had a single growth pattern and 23% were of mixed histological composition. Among all BCCs the nodular followed by the superficial growth patterns were commonest. Risk of nodular and superficial BCCs on the head was raised if 5 or more solar keratoses were present on the face (OR 1.8, 95%CI 1.2-2.7 and OR 4.5, 95%CI 2.1-9.7 respectively) and similarly on the trunk in the presence of multiple solar keratoses on the trunk (OR 4.2, 95%CI 1.5-11.9 and OR 2.2, 95%CI 1.1-4.4 respectively). 5. Basal cell carcinoma and measures of cumulative sun exposure: an Australian longitudinal community-based study Dermal elastosis was more likely to be seen adjacent to head and neck BCCs than trunk BCCs (p=0.01). Severity of dermal elastosis increased on each site with increasing clinical signs of cutaneous sun damage on that site. BCCs that occurred without perilesional elastosis per se, were always found in an anatomic region with signs of photodamage. This thesis thus has identified the magnitude of the burden of multiple BCCs. It does not support the view that people affected by more than one BCC represent a distinct group of people who are prone to BCCs on certain body sites. The results also demonstrate that BCCs regardless of site, histology or order of occurrence are strongly associated with cumulative sun exposure causing photodamage to the skin, and hence challenge the view that BCCs occurring on body sites with typically low opportunities for sun exposure or of the superficial growth pattern are different in their association with the sun from those on typically sun-exposed sites, or nodular BCCs, respectively. Through dissemination in the scientific and medical literature, and to the community at large, these findings can ultimately assist in the primary and secondary prevention of BCC, perhaps especially in high-risk populations.

Hybrid multi-channel cooperative spectrum sensing to satisfy channel target

Spectrum sensing of multiple primary user channels is a crucial function in cognitive radio networks. In this paper we propose an optimal, sensing resource allocation algorithm for multi-channel cooperative spectrum sensing. The channel target is implemented as an objective and constraint to ensure a pre-determined number of empty channels are detected for secondary user network operations. Based on primary user traffic parameters, we calculate the minimum number of primary user channels that must be sensed to satisfy the channel target. We implement a hybrid sensing structure by grouping secondary user nodes into clusters and assign each cluster to sense a different primary user channels. We then solve the resource allocation problem to find the optimal sensing configuration and node allocation to minimise sensing duration. Simulation results show that the proposed algorithm requires the shortest sensing duration to achieve the channel target compared to existing studies that require long sensing and cannot guarantee the target.

A dynamic in vivo model of epithelial-to-mesenchymal transitions in circulating tumor cells and metastases of breast cancer

Epithelial-to-mesenchymal transition (EMT) processes endow epithelial cells with enhanced migratory/invasive properties and are therefore likely to contribute to tumor invasion and metastatic spread. Because of the difficulty in following EMT processes in human tumors, we have developed and characterized an animal model with transplantable human breast tumor cells (MDA-MB-468) uniquely showing spontaneous EMT events to occur. Using vimentin as a marker of EMT, heterogeneity was revealed in the primary MDA-MB-468 xenografts with vimentin-negative and vimentin-positive areas, as also observed on clinical human invasive breast tumor specimens. Reverse transcriptase-PCR after microdissection of these populations from the xenografts revealed EMT traits in the vimentin-positive zones characterized by enhanced 'mesenchymal gene' expression (Snail, Slug and fibroblast-specific protein-1) and diminished expression of epithelial molecules (E-cadherin, ZO-3 and JAM-A). Circulating tumor cells (CTCs) were detected in the blood as soon as 8 days after s.c. injection, and lung metastases developed in all animals injected as examined by in vivo imaging analyses and histology. High levels of vimentin RNA were detected in CTCs by reverse transcriptase-quantitative PCR as well as, to a lesser extent, Snail and Slug RNA. Von Willebrand Factor/vimentin double immunostainings further showed that tumor cells in vascular tumoral emboli all expressed vimentin. Tumoral emboli in the lungs also expressed vimentin whereas macrometastases displayed heterogenous vimentin expression, as seen in the primary xenografts. In conclusion, our data uniquely demonstrate in an in vivo context that EMT occurs in the primary tumors, and associates with an enhanced ability to intravasate and generate CTCs. They further suggest that mesenchymal-to-epithelial phenomena occur in secondary organs, facilitating the metastatic growth.

Supporting the needs of public health decision-makers and review authors in the UK

Introduction Systematic reviews, through the synthesis of multiple primary research studies, can be powerful tools in enabling evidence-informed public health policy debate, development and action. In seeking to optimize the utility of these reviews, it is important to understand the needs of those using them. Previous work has emphasized that researchers should adopt methods that are appropriate to the problems that public health decision-makers are grappling with, as well as to the policy context in which they operate.1,2 Meeting these demands poses significant methodological challenges for review authors and prompts a reconsideration of the resources, training and support structures available to facilitate the efficient and timely production of useful, comprehensive reviews. The Cochrane Public Health Group (CPHG) was formed in 2008 to support reviews of complex, upstream public health topics. The majority of CPHG authors are from the UK, which has historically been at the forefront of efforts to promote the production and use of systematic reviews of research relevant to public health decision-makers. The UK therefore provides a suitably mature national context in which to examine (i) the current and future demands of decision-makers to increase the use, value and impact of evidence syntheses; (ii) the implications this has for the scope and methods of reviews and (iii) the required action to build and support capacity to conduct such reviews.

Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA

We report that tumor cells devoid of their mitochondrial genome (mtDNA) show delayed tumor growth and that tumor formation is associated with acquisition of mtDNA from host cells. This leads to partial recovery of mitochondrial function in cells derived from primary tumors grown from cells without mtDNA and a shorter lag in tumor growth. Cell lines from circulating tumor cells showed further recovery of mitochondrial respiration and an intermediate lag to tumor growth, while cells from lung metastases exhibited full restoration of respiratory function and no lag in tumor growth. Stepwise assembly of mitochondrial respiratory supercomplexes was correlated with acquisition of respiratory function. Our findings indicate horizontal transfer of mtDNA from host cells in the tumor microenvironment to tumor cells with compromised respiratory function to re-establish respiration and tumor-initiating efficacy. These results suggest a novel pathophysiological process for overcoming mtDNA damage and support the notion of high plasticity of malignant cells.

Genome-wide analysis in a murine Dnmt1 knockdown model identifies epigenetically silenced genes in primary human pituitary tumors

DNA methylation at promoter CpG islands (CGI) is an epigenetic modification associated with inappropriate gene silencing in multiple tumor types. In the absence of a human pituitary tumor cell line, small interfering RNA-mediated knockdown of the maintenance methyltransferase DNA methyltransferase (cytosine 5)-1 (Dnmt1) was used in the murine pituitary adenoma cell line AtT-20. Sustained knockdown induced reexpression of the fully methylated and normally imprinted gene neuronatin (Nnat) in a time-dependent manner. Combined bisulfite restriction analysis (COBRA) revealed that reexpression of Nnat was associated with partial CGI demethylation, which was also observed at the H19 differentially methylated region. Subsequent genome-wide microarray analysis identified 91 genes that were significantly differentially expressed in Dnmt1 knockdown cells (10% false discovery rate). The analysis showed that genes associated with the induction of apoptosis, signal transduction, and developmental processes were significantly overrepresented in this list (P < 0.05). Following validation by reverse transcription-PCR and detection of inappropriate CGI methylation by COBRA, four genes (ICAM1, NNAT, RUNX1, and S100A10) were analyzed in primary human pituitary tumors, each displaying significantly reduced mRNA levels relative to normal pituitary (P < 0.05). For two of these genes, NNAT and S100A10, decreased expression was associated with increased promoter CGI methylation. Induced expression of Nnat in stable transfected AtT-20 cells inhibited cell proliferation. To our knowledge, this is the first report of array-based "epigenetic unmasking" in combination with Dnmt1 knockdown and reveals the potential of this strategy toward identifying genes silenced by epigenetic mechanisms across species boundaries.

Factors influencing quality of life in patients with benign primary brain tumors : prior to and following surgery

Goals: Few studies have repeatedly evaluated quality of life and potentially relevant factors in patients with benign primary brain tumor. The purpose of this study was to explore the relationship between the experience of the symptom distress, functional status, depression, and quality of life prior to surgery (T1) and 1 month post-discharge (T2). ---------- Patients and methods: This was a prospective cohort study including 58 patients with benign primary brain tumor in one teaching hospital in the Taipei area of Taiwan. The research instruments included the M.D. Anderson Symptom Inventory, the Functional Independence Measure scale, the Hospital Depression Scale, and the Functional Assessment of Cancer Therapy-Brain.---------- Results: Symptom distress (T1: r=−0.90, p<0.01; T2: r=−0.52, p<0.01), functional status (T1: r=0.56, p<0.01), and depression (T1: r=−0.71, p<0.01) demonstrated a significant relationship with patients' quality of life. Multivariate analysis identified symptom distress (explained 80.2%, Rinc 2=0.802, p=0.001) and depression (explained 5.2%, Rinc 2=0.052, p<0.001) continued to have a significant independent influence on quality of life prior to surgery (T1) after controlling for key demographic and medical variables. Furthermore, only symptom distress (explained 27.1%, Rinc 2=0.271, p=0.001) continued to have a significant independent influence on quality of life at 1 month after discharge (T2).---------- Conclusions: The study highlights the potential importance of a patient's symptom distress on quality of life prior to and following surgery. Health professionals should inquire about symptom distress over time. Specific interventions for symptoms may improve the symptom impact on quality of life. Additional studies should evaluate symptom distress on longer-term quality of life of patients with benign brain tumor.