PCR bias Toward the wild-type k-ras and p53 sequences: Implications for PCR detection of mutations and cancer diagnosis

PCR-based cancer diagnosis requires detection of rare mutations in k- ras, p53 or other genes. The assumption has been that mutant and wild-type sequences amplify with near equal efficiency, so that they are eventually present in proportions representative of the starting material. Work on factor IX suggests that this assumption is invalid for one case of near- sequence identity. To test the generality of this phenomenon and its relevance to cancer diagnosis, primers distant from point mutations in p53 and k-ras were used to amplify wild-type and mutant sequences from these genes. A substantial bias against PCR amplification of mutants was observed for two regions of the p53 gene and one region of k-ras. For k-ras and p53, bias was observed when the wild-type and mutant sequences were amplified separately or when mixed in equal proportions before PCR. Bias was present with proofreading and non-proofreading polymerase. Mutant and wild-type segments of the factor V, cystic fibrosis transmembrane conductance regulator and prothrombin genes were amplified and did not exhibit PCR bias. Therefore, the assumption of equal PCR efficiency for point mutant and wild-type sequences is invalid in several systems. Quantitative or diagnostic PCR will require validation for each locus, and enrichment strategies may be needed to optimize detection of mutants.

Influence of culture conditions on the molecular signature of mesenchymal stem cells

Cell based therapies require cells capable of self renewal and differentiation, and a prerequisite is the ability to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies is therefore an integral part of tissue engineering. Bone marrow is the most easily accessible source of mesenchymal stem cells (MSCs), and harbours two distinct populations of adult stem cells; namely hematopoietic stem cells (HSCs) and bone mesenchymal stem cells (BMSCs). Unlike HSCs, there are yet no rigorous criteria for characterizing BMSCs. Changing understanding about the pluripotency of BMSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to BMSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their in vitro study. Also, when BMSCs are cultured in vitro there is a loss of the in vivo microenvironment which results in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage number, characterized by the onset of senescence related changes. Accordingly, establishing protocols for generating large numbers of BMSCs without affecting their differentiation potential is necessary. The principal aims of this thesis were to identify potential molecular factors for characterizing BMSCs from osteoarthritic patients, and also to attempt to establish culture protocols favourable for generating large number of BMSCs, while at the same time retaining their proliferation and differentiation potential. Previously published studies concerning clonal cells have demonstrated that BMSCs are heterogeneous populations of cells at various stages of growth. Some cells are higher in the hierarchy and represent the progenitors, while other cells occupy a lower position in the hierarchy and are therefore more committed to a particular lineage. This feature of BMSCs was made evident by the work of Mareddy et al., which involved generating clonal populations of BMSCs from bone marrow of osteoarthritic patients, by a single cell clonal culture method. Proliferation potential and differentiation capabilities were used to group cells into fast growing and slow growing clones. The study presented here is a continuation of the work of Mareddy et al. and employed immunological and array based techniques to identify the primary molecular factors involved in regulating phenotypic characteristics exhibited by contrasting clonal populations. The subtractive immunization (SI) was used to generate novel antibodies against favourably expressed proteins in the fast growing clonal cell population. The difference between the clonal populations at the transcriptional level was determined using a Stem Cell RT2 Profiler TM PCR Array which focuses on stem cell pathway gene expression. Monoclonal antibodies (mAb) generated by SI were able to effectively highlight differentially expressed antigenic determinants, as was evident by Western blot analysis and confocal microscopy. Co-immunoprecipitation, followed by mass spectroscopy analysis, identified a favourably expressed protein as the cytoskeletal protein vimentin. The stem cell gene array highlighted genes that were highly upregulated in the fast growing clonal cell population. Based on their functions these genes were grouped into growth factors, cell fate determination and maintenance of embryonic and neural stem cell renewal. Furthermore, on a closer analysis it was established that the cytoskeletal protein vimentin and nine out of ten genes identified by gene array were associated with chondrogenesis or cartilage repair, consistent with the potential role played by BMSCs in defect repair and maintaining tissue homeostasis, by modulating the gene expression pattern to compensate for degenerated cartilage in osteoarthritic tissues. The gene array also presented transcripts for embryonic lineage markers such as FOXA2 and Sox2, both of which were significantly over expressed in fast growing clonal populations. A recent groundbreaking study by Yamanaka et al imparted embryonic stem cell (ESCs) -like characteristic to somatic cells in a process termed nuclear reprogramming, by the ectopic expression of the genes Sox2, cMyc and Oct4. The expression of embryonic lineage markers in adult stem cells may be a mechanism by which the favourable behaviour of fast growing clonal cells is determined and suggests a possible active phenomenon of spontaneous reprogramming in fast growing clonal cells. The expression pattern of these critical molecular markers could be indicative of the competence of BMSCs. For this reason, the expression pattern of Sox2, Oct4 and cMyc, at various passages in heterogeneous BMSCs population and tissue derived cells (osteoblasts and chondrocytes), was investigated by a real-time PCR and immunoflourescence staining. A strong nuclear staining was observed for Sox2, Oct4 and cMyc, which gradually weakened accompanied with cytoplasmic translocation after several passage. The mRNA and protein expression of Sox2, Oct4 and cMyc peaked at the third passage for osteoblasts, chondrocytes and third passage for BMSCs, and declined with each subsequent passage, indicating towards a possible mechanism of spontaneous reprogramming. This study proposes that the progressive decline in proliferation potential and multipotentiality associated with increased passaging of BMSCs in vitro might be a consequence of loss of these propluripotency factors. We therefore hypothesise that the expression of these master genes is not an intrinsic cell function, but rather an outcome of interaction of the cells with their microenvironment; this was evident by the fact that when removed from their in vivo microenvironment, BMSCs undergo a rapid loss of stemness after only a few passages. One of the most interesting aspects of this study was the integration of factors in the culture conditions, which to some extent, mimicked the in vivo microenvironmental niche of the BMSCs. A number of studies have successfully established that the cellular niche is not an inert tissue component but is of prime importance. The total sum of stimuli from the microenvironment underpins the complex interplay of regulatory mechanisms which control multiple functions in stem cells most importantly stem cell renewal. Therefore, well characterised factors which affect BMSCs characteristics, such as fibronectin (FN) coating, and morphogens such as FGF2 and BMP4, were incorporated into the cell culture conditions. The experimental set up was designed to provide insight into the expression pattern of the stem cell related transcription factors Sox2, cMyc and Oct4, in BMSCs with respect to passaging and changes in culture conditions. Induction of these pluripotency markers in somatic cells by retroviral transfection has been shown to confer pluripotency and an ESCs like state. Our study demonstrated that all treatments could transiently induce the expression of Sox2, cMyc and Oct4, and favourably affect the proliferation potential of BMSCs. The combined effect of these treatments was able to induce and retain the endogenous nuclear expression of stem cell transcription factors in BMSCs over an extended number of in vitro passages. Our results therefore suggest that the transient induction and manipulation of endogenous expression of transcription factors critical for stemness can be achieved by modulating the culture conditions; the benefit of which is to circumvent the need for genetic manipulations. In summary, this study has explored the role of BMSCs in the diseased state of osteoarthritis, by employing transcriptional profiling along with SI. In particular this study pioneered the use of primary cells for generating novel antibodies by SI. We established that somatic cells and BMSCs have a basal level of expression of pluripotency markers. Furthermore, our study indicates that intrinsic signalling mechanisms of BMSCs are intimately linked with extrinsic cues from the microenvironment and that these signals appear to be critical for retaining the expression of genes to maintain cell stemness in long term in vitro culture. This project provides a basis for developing an “artificial niche” required for reversion of commitment and maintenance of BMSC in their uncommitted homeostatic state.

Supportive-expressive group therapy for women with metastatic breast cancer: Improving access for Australian women through use of teleconference

While supportive-expressive group therapy (SEGT) has been found to be effective in significantly reducing distress associated with life-threatening illness, the challenge in Australia is to develop a means of providing supportive interventions to rural women who may be isolated both by the experience of illness and by geographical location. In this study an adaptation of SEGT was provided to women with metastatic breast cancer (n =21), who attended face-to-face or by telephone conference call. Participants showed significant gains on standardised measures of well-being, including a reduction in negative affect and an increase in positive affect, over a 12-month period. A reduction in intrusive and avoidant stress symptoms was also observed over 12 months; however, this difference was not significant. These outcomes suggest that SEGT, delivered in an innovative way within a community setting, may be an effective means of moderating the adverse effects of a diagnosis of metastatic breast cancer while improving access to supportive care for rural women. These results are considered exploratory, as the study did not include a matched control group.

Identification of area-level influences on regions of high cancer incidence in Queensland, Australia : a classification tree approach

Background: Strategies for cancer reduction and management are targeted at both individual and area levels. Area-level strategies require careful understanding of geographic differences in cancer incidence, in particular the association with factors such as socioeconomic status, ethnicity and accessibility. This study aimed to identify the complex interplay of area-level factors associated with high area-specific incidence of Australian priority cancers using a classification and regression tree (CART) approach. Methods: Area-specific smoothed standardised incidence ratios were estimated for priority-area cancers across 478 statistical local areas in Queensland, Australia (1998-2007, n=186,075). For those cancers with significant spatial variation, CART models were used to identify whether area-level accessibility, socioeconomic status and ethnicity were associated with high area-specific incidence. Results: The accessibility of a person’s residence had the most consistent association with the risk of cancer diagnosis across the specific cancers. Many cancers were likely to have high incidence in more urban areas, although male lung cancer and cervical cancer tended to have high incidence in more remote areas. The impact of socioeconomic status and ethnicity on these associations differed by type of cancer. Conclusions: These results highlight the complex interactions between accessibility, socioeconomic status and ethnicity in determining cancer incidence risk.

Developing the atlas of cancer in Queensland : methodological issues

Background: Achieving health equity has been identified as a major challenge, both internationally and within Australia. Inequalities in cancer outcomes are well documented, and must be quantified before they can be addressed. One method of portraying geographical variation in data uses maps. Recently we have produced thematic maps showing the geographical variation in cancer incidence and survival across Queensland, Australia. This article documents the decisions and rationale used in producing these maps, with the aim to assist others in producing chronic disease atlases. Methods: Bayesian hierarchical models were used to produce the estimates. Justification for the cancers chosen, geographical areas used, modelling method, outcome measures mapped, production of the adjacency matrix, assessment of convergence, sensitivity analyses performed and determination of significant geographical variation is provided. Conclusions: Although careful consideration of many issues is required, chronic disease atlases are a useful tool for assessing and quantifying geographical inequalities. In addition they help focus research efforts to investigate why the observed inequalities exist, which in turn inform advocacy, policy, support and education programs designed to reduce these inequalities.

Self-management of cancer treatment-related fatigue, nausea, vomiting and oral mucositis in Chinese cancer patients

Background: Treatment-related symptoms continue to place a significant burden on many cancer patients. Many side effects require patients to engage in a range of self-management actions. While some studies have explored self-management of treatment-related side effects in Western settings, very few studies were identified that described the self-management practices of cancer patients in China. Objective: The purposes of this study are to: (1) Investigate Chinese cancer patients. self-management behaviours in dealing with the fatigue, nausea/vomiting and oral mucositis that result from treatment, as well as the perceived effectiveness of these behaviours and related self-efficacy in performing them. (2) Explore factors influencing symptom self-management behaviours using the Cancer Symptom Self-management Framework based on Grey, Knafl and McCorkle.s (2006) self-management framework as a guide. Methods: This study was divided into two phases. Phase One consisted of the translation and modification of two instruments. The adaptation of these instruments to ensure applicability in the Chinese context was achieved through semi-structured interviews with six cancer patients, and content evaluation with eight experienced oncology nurses. A pilot study was conducted with nine cancer patients to trial the questionnaire set in the Chinese context. Based on the results of Phase One, Phase Two involved a cross-sectional survey of Chinese cancer patients undergoing cancer treatment using these instruments. A total of 277 chemotherapy patients with fatigue and/or nausea and vomiting, and 100 radiotherapy patients with oral mucositis were surveyed. Results: Participants in this study reported a variety of self-management behaviours to cope with fatigue, nausea, vomiting and oral mucositis. There are some consistencies as well disparities between strategies that are frequently used and those rated as effective. For fatigue self-management, participants were more likely to use strategies related to rest and sleep, while activity enhancement strategies were rated as achieving higher relief. For nausea and vomiting self-management, dietary modification and taking medication were most frequently used and rated as moderately effective. Psychological strategies were used by more than a third of participants and were rated as mildly effective. Some other infrequently used strategies, such as distraction by keeping busy and acupressure, were rated as moderately effective. For oral mucositis self-management, having soft, bland food and keeping the mouth moisturised were most frequently reported and they were rated as achieving moderate relief. A prescribed mouthwash was used by most but not all participants and brought moderate relief. In general, patients had low-to-moderate self-efficacy in nausea and vomiting self-management behaviours, moderate self-efficacy in fatigue self-management behaviours, and low-to-moderate self-efficacy in oral mucositis self-management behaviours. In terms of the factors influencing symptom self-management, different predictors were identified affecting engagement in fatigue, nausea/vomiting and oral mucositis self-management behaviours. Self-efficacy scores of different behaviours were consistently found to be a positive predictor of the relief level from corresponding behaviours, after controlling for other variables. Perceived social support from health care professionals was identified as an important factor influencing nausea and vomiting self-management behaviours, while neighbourhood support was important for fatigue self-management. In addition, symptom distress was identified as an important factor influencing nausea and vomiting self-management. Conclusion: Similar to reports from overseas, Chinese cancer patients initiate a wide range of self-management behaviours in response to treatment-related side effects. While some behaviours were reported to provide relief, many did not. Given these results, this study has a number of practical implications for health care professionals, particularly in relation to developing tailored self-management programs for fatigue, nausea, vomiting and oral mucositis. Additionally, this study suggests a number of theoretical implications and directions for future research. It is envisaged that these recommendations may pave the way for further studies understanding and promoting cancer symptom self-management in Chinese people affected by cancer.