Breast cancer paper

Recent Patents on DNA & Gene Sequences 2009, 3, 139-147 139 Recent Patents on Genes and Gene Sequences Useful for Developing Breast Cancer Detection Systems Divya Singh and Rekha Chaturvedi IPR Cell, Institute of Genomics & Integrative Biology, Delhi University North Campus, Mall Road, Delhi 110007, India Received: May 5, 2009; Accepted: May 18, 2009; Revised: May 21, 2009 Abstract: Breast cancer is the most prevalent disease and second leading cause of death among women in many countries. Mutations and consequent DNA damage in several genes such as oncogenes and tumour suppressor genes have been implicated with abnormal behavior of somatic cells, resulting into malignant growth termed cancer. DNA damage/changes in chromosomal DNA and failure of DNA repair mechanism are important factors for cancer causation. Aberrant behavior of cells leading to uncontrolled proliferation causing cancer is attributable to amplicon formation, large deletions in some genes, mutations, and recombination breakpoints etc. which cause aberrant gene expression. Several patented genes found to be associated with breast cancer, have been discussed, such as BRCA1, BRCA2, DAP kinase, MYH, BCSGs, BCW 2 and Id-2 etc. Several of the genes/markers associated with breast cancer can be considered as appropriate candidates for developing early detection systems/protocols for breast cancer. Keywords: Breast cancer, cell proliferation, mutations, altered gene expression, high risk, cancer diagnosis, genetic testing. INTRODUCTION Cancer is a fundamental artifact in cellular behavior that touches on many facets of molecular cell biology. The inherent components of cancer are mutation, competition and natural selection that operate within the population of somatic cells. Cancer cells are defined by two heritable properties that they and their progeny reproduce in nonconformity with the normal phenomena and invade and colonize the areas normally reserved for other cells. The combination of these properties makes cancers markedly dangerous [1, 2]. Cancer cells spread through the body by breaking through the basal laminas and establish the secondary areas of growth through a process called metastasis [3]. Metastatic tumours often secrete proteases that subsequently degrade the surrounding extracellular matrix [4]. Cancerous cells usually originate from a single primary tumour that arises in an identified organ that has undergone some heritable change that enables it to outrace its neighbours [5]. Several cancers with age-dependent incidences are diagnosed in the human population and four to seven rate-limiting, stochastic events are implicated for the same [6, 7]. Cancer development is guided by the accumulation of DNA changes in some of the approximately 40 000 chromosomal genes [8, 9]. Chromosomal numerical/ structural aberrations are common in solid tumours. Defects in DNA repair may lead to genome-wide genetic instability and it may drive the further progression of cancer [10]. Genomics and proteomics play a major role in the understanding, diagnosis, prognosis and potentially also treatment of cancer. Progressive series of genetic events occur in a single clone of cells because of alterations in a limited number of specific genes: the oncogenes and tumour *Address correspondence to this author at the IPR Cell, Institute of Genomics & Integrative Biology, Delhi University North Campus, Mall Road, Delhi 110007, India; Tel: (91) 11-27662691; Fax: (91) 11-27667471; E-mail: [email protected] 1872-2156/09 $100.00+.00 suppressor genes. The association of consistent chromosome aberrations with particular types of cancer has led to the identification of some of these genes and the elucidation of their mechanisms of action [9]. Breast cancer is one of the most common cancers among women. It accounts for 22% of all female cancers [11]. The estimated annual incidence of breast cancer worldwide is about one million cases. Breast tumours have been noted since the ancient era and were probably first described in the Edwin Smith surgical papyrus originating from Egypt at around 2500 B.C. [12]. A significant difference in the incidence rates of breast cancer has been observed between so-called low-risk areas such as the Far East, Africa and South America, and the high-risk areas North America and Northern Europe. Together, the USA and Europe roughly account for 16% of the worldwide population and 60% of the worldwide incidence of breast cancer [11, 13]. Breast cancer incidence increases in people who move from a region with low breast cancer incidence to other locations with higher breast cancer incidence. This effect is then passed to the next generation and the breast cancer risk of the native population is acquired by the descendents of the migrants within one or two generations [14, 15]. This underlines the crucial contribution of environmental factors to breast cancer risk. The etiology of breast cancer is extremely complex and its onset and progression is a multi step process resulting from a series of epigenetic, genetic and environmental factors [16]. Cells take highly variable paths to become malignant. Certain oncogenes and tumour suppressor genes can be mutated early in some tumour progression pathways and late in others [17]. As a consequence, the biological capabilities such as resistance to apoptosis, sustained angiogenesis, and unlimited replicative potential are acquired at different times during these various progressions. Accordingly, the particular sequence in which capabilities are acquired can vary widely, both among tumours of the same type and certainly between tumours of different types [18]. In higher organisms, mutations in somatic cells which affect critical genes that regulate cell © 2009 Bentham Science Publishers Ltd. 140 Recent Patents on DNA & Gene Sequences 2009, Vol. 3, No. 2 proliferation and survival are known to cause fatal cancers. Breakdown of the machinery that implements DNA damage greatly predisposes to cancer [19]. Nonetheless, even when cancer-causing mutations do occur, there exist potential mechanisms that check the expansion of affected cells by suppressing their growth and proliferation or triggering their apoptosis [20]. BREAST TUMOURS Breast cancer development shows stepwise progression from non-proliferative and mildly proliferative disease, through florid and a typical hyperplasia and finally to low and high grade ductal carcinoma in situ [16]. Four major molecular classes of breast cancer have emerged from several studies: luminal-A, luminal-B, basal-like, and human epidermal growth factor receptor (HER)-positive cancers [21-23]. The overall survival and chemotherapy sensitivity of the different molecular subgroups vary. Luminal-type cancers are mostly estrogen receptor (ER) positive, and patients with luminal-A cancers have the most favorable long-term survival (with endocrine therapy) compared with the other types, whereas basal-like and HER-2–positive tumours are more sensitive to chemotherapy [22, 24, 25]. The majority of invasive malignant breast tumours are represented by adenocarcinomas. These are derived from the mammary parenchymal epithelium, particularly cells of the terminal duct lobular unit. These tumours are characterized by invasion of adjacent tissues and a marked tendency to metastasize to distant sites, most commonly to the bones, lungs and pleurae, liver, adrenals, ovaries, skin and brain. The invasive breast carcinomas are routinely graded in histology based on an assessment of tubule/gland formation, nuclear pleomorphism and mitotic counts [26]. PREDISPOSITION DUE TO GENE MUTATIONS/ POLYMORPHISMS Etiology of breast cancer is quite complex where the host genetic factors play a key role. Therefore, it is important to evaluate the role of different biomarkers in breast cancer susceptibility for the better understanding of the disease etiology, which may contribute towards treatment and early detection of the cancer [27]. Up to 5-10% of all breast cancers are attributed to germ-line mutations in well characterized breast cancer susceptibility genes. Tumour necrosis factor (TNF) is critical to regulation of inflammation. Genetic variation in the promoter region of TNF has been associated with expression differences, and a range of autoimmune, infectious, and oncologic diseases [28]. It plays a pivotal role in cellular immunity of the host, thereby constituting important genetically determined host factors in breast carcinogenesis [28]. Breast cancer susceptibility is polygenic, that means, susceptibility is conferred by a large number of loci, each with a small effect on breast cancer risk [29]. CELLULAR RECEPTORS Three predominant families of factors, Estrogen receptor (ER) Progesterone receptor (PgR), Epidermal growth factor receptors (ErbB1/ErbB2), and their protein kinases, play a Singh and Chaturvedi fundamental role in development of breast cancers. Four ErbB receptors have been identified which include ErbB1 (HER1), ErbB2 (HER2), ErbB3 (HER 3) and ErbB4 (HER4). The deregulation of ErbB signaling pathway in cancer is caused due to aberrations that include gene amplification and gene mutation. Amplification of the HER2 gene results in strong over-expression of the receptor protein and, in turn, confers a growth advantage to the tumour cells. Herceptin, a monoclonal antibody targeting the HER2 protein, improves patient survival even in metastatic breast cancers and is regarded as a paradigm for the potential of a new generation of gene specific drugs [30] The presence of multiple copies of the HER-2 gene, and the corresponding over-expression of its protein, plays a pivotal role in the rapid growth of tumour cells in 25-30 percent of breast cancer patients. Many other factors that are present in different parts of a tumour cell, from the plasma membrane to the nucleus, participate in establishing a variety of states that characterize the phenotype of each breast tumour [31]. A predisposition for breast cancer is linked to BRCA1 that is a tumour-suppressor gene. GENES AND PROTEIN FACTORS ASSOCIATED WITH BREAST CANCER Several molecular targets comprising genes and protein factors have been found to be implicated with breast cancer. The breast cancer susceptibility gene 1 (BRCA1) gene encodes a protein that is involved in many nuclear processes related to transcription, chromatin remodeling and DNA repair [32]. BRCA1 has a role in conjunction with both H2A1 histone and K9 methylated histone H3 in the control of heterochromatin structure. It is considered as a suppressor of tumour, the absence of which could lead to a de-repressed form of heterochromatin, and this would activate the expression of some oncogenes in ovarian and breast cancers. Most of the cancers due to hereditary mutations involve BRCA1 and the close homologous BRCA2 gene, located on the long arms of chromosomes 17 and 13, respectively. Women carrying mutations in the BRCA1 or BRCA2 genes are subject to high risk of breast cancer development, being 7% in the cases due to alterations of these two genes [33, 34]. There are few other high-risk breast cancer susceptibility genes as well that include PTEN, TP53, LKB1/ STK11 and CDH1. PTEN represents phosphatase and tensin homolog gene. Mutations in the PTEN gene increase the risk of developing breast cancer as part of a rare inherited cancer syndrome called Cowden syndrome. Women carrying a PTEN-mutation have a 25-50% (2-4-fold) lifetime breast cancer risk. The majority of Cowden syndrome related breast cancers occur after the age of 30-35 years [35, 36]. The TP53 gene is located on chromosome 17p13.1, and encodes a protein involved in many overlapping cellular pathways that control cell proliferation and homeostasis, such as cell cycle, apoptosis and DNA-repair. The expression of the TP53 gene is activated in response to various stress signals, including DNA damage. Loss of TP53 function is thought to suppress a mechanism of protection against accumulating of genetic alterations [37]. Somatic mutations in TP53 are reported in 20-60% of human breast cancers [38]. The LKB1/STK11gene is located on chromosome 19p13.3 and encodes a transcript of ~1.3 kb, which acts as a tumour suppressor. Recent Patents on Breast Cancer Recent Patents on DNA & Gene Sequences 2009, Vol. 3, No. 2 141 Germline mutations in this serine/threonine kinase gene (LKB1/STK11) causes Peutz-Jeghers syndrome (PJS) [39]. An elevated risk of gastrointestinal malignancies, breast cancer, pancreas, ovary, uterus, cervix, lung and testicular cancers is recognized in patients with PJS [40-42]. The clinical features of PJS vary within and between families, especially with respect to cancer risk. Overall, the probability of developing cancer by age 65 is estimated to be about 50%. The risk of breast cancer by age 65 ranges between 29% and 54% [43, 44]. It is suggested that LKB1/STK11 can play the role of a tumour suppressor gene in sporadic breast cancer, and low expression of the LKB1/STK11 protein is significantly associated with a shorter survival [45]. The E-cadherin gene (CDH1) is located on chromosome 16q22.1. The mature protein product belongs to the family of cell-cell adhesion molecules and plays a fundamental role in the maintenance of cell differentiation and the normal architecture of epithelial tissues. Somatic CDH1 mutations are frequently found in infiltrating lobular breast cancer and in-situ lobular breast cancer (LCIS) in contrast to breast cancers of other histopathological subtype [46-48]. There are few other genes that are ‘low to moderate susceptibility’ genes. These include CHEK2, TGF 1, CASP8 and ATM genes. The CHEK2 gene is located on chromosome 22q12.1. The role of CHEK2 in breast cancer susceptibility was first suggested by the identification of the truncating mutation 1100delC, which eliminates kinase activity, in an individual with Li-Fraumeni syndrome (LFS) without a TP53 mutation. The possibility that this gene is only contributing to the breast cancer cases within LFS families rather than LFS per se has been raised [49]. The TGF 1-gene is located on chromosome 19q13.1. This gene contains seven exons and very large introns. TGF is a multifunctional peptide that controls proliferation, differentiation, and other functions in many cell types. To date, several somatic mutations that disrupt the TGF signalling pathway have been reported in human breast tumours [50-52]. The CASP8 gene is located on chromosome 2q33-34. This gene contains 13 exons and spans 51.2 kb. Because of the involvement in initiation of apoptosis, CASP8 acts as low-penetrance familial breast cancer susceptibility gene. Combined analysis of two different studies showed that one mis-sense variant (D302H) in CASP8 was associated with a reduced risk of breast cancer in a dose dependent manner [53]. Some individuals have inherent genetic mutations that predispose them to develop specific types of cancer. However, an individual cancer does not result from a single mutation. Rather it results from the accumulation of many mutations in genes that normally regulate cell division. The coexistence of gene-specific promoter hypermethylation and global genomic DNA hypomethylation is an epigenetic charac-teristic of cancer cells [57]. The global genomic DNA hypomethylation observed in cancer cells is a feature of malignancy because there is a loss in the methylation of repetitive sequences and parasitic elements of the genome. This has been linked to the chromosomal instability of cancer cells [58, 59]. The post-translational modifications of histones are also emerging as epigenetic processes that can explain the behaviour of cancer cells. The loss of the monoacetylated Lysine-16 (K16) and trimethylated Lysine20 (K20) residues of histone H4 appears in the early phase of cell transformation and increases with the progression of tumour [60, 61]. The ATM protein plays a central role in sensing and signaling the presence of DNA double-strand breaks. In the cell nucleus that is not irradiated, ATM is held inactive, which is dissociated by rapid intermolecular autophosphorylation after irradiation [54]. This initiates cellular ATM kinase activity, which has many substrates including the protein products of TP53, BRCA1 and CHEK2. Variations in ATM are involved in increasing breast cancer risk [55]. The ATM gene is located on chromosome 11q22-23. New breast cancer specific genes, often known as BCSGs, have now been identified for use in diagnosing, monitoring, staging, imaging and treating breast cancers. Breast cancer specific gene 1 (BCSG1), also referred as synuclein !, is the third member of a neuronal protein family synuclein. BCSG1 is not expressed in normal breast tissues but highly expressed in advanced infiltrating breast carcinomas [56]. PATENTED MOLECULAR TARGETS (GENES/ PROTEINS) FOR BREAST CANCER DETECTION Currently, the principal manner of identifying breast cancer is through detection of the presence of dense tumorous tissue. This may be accomplished to varying degrees of effectiveness by direct examination of the outside of the breast, or through mammography or other X-ray imaging methods [62]. The latter approach is cost intensive and, further, every time a mammogram is taken, the patient incurs a small risk of having a breast tumour induced by the ionizing properties of the radiation used during the test. In addition, the process is expensive and the subjective interpretations of a technician can lead to imprecision, e.g., one study showed major clinical disagreements for about one-third of a set of mammograms that were interpreted individually by a surveyed group of radiologists. Moreover, many women find that undergoing a mammogram is a painful experience [63]. Human breast tumours are diverse in their natural history and in the responsiveness to treatments. Variation in transcriptional programs accounts for much of the biological diversity of human cells and tumours. In each cell, signal transduction and regulatory systems transduce information from the cell's identity to its environmental status, thereby controlling the level of expression of every gene in the genome [64]. Mutations and consequent DNA damage in several genes such as oncogenes and tumour suppressor genes have been implicated with abnormal behavior of somatic cells, resulting into malignant growth termed as cancer which invade organs and proliferate very fast at the expense of normal cells and do not allow them to carry out their normal functions. DNA damage/changes in chromosomal DNA and failure of DNA repair defects are important factors for cancer causation. Genomics and proteomics play important role in understanding, diagnosis and prognosis of cancer. DIFFERENTIAL GENE EXPRESSION The patent literature predominantly encompasses comparison of expression of a marker gene or a panel of 142 Recent Patents on DNA & Gene Sequences 2009, Vol. 3, No. 2 Table 1. Singh and Chaturvedi Patented Molecular Targets (Gene/Protein) Associated with Breast Cancer Molecular Targets Patent/ Publication Number (Gene/ Polypeptide) HER2/neu (human epidermal receptor 2) Gene Location (NCBI) Chromosome Number US4968603 17 (17q11.2-q12; 17q21.1) ER (estrogen receptor markers) US7504222 5, 6 and 14 BCW2 US6649342 21 (21q22.12) c-mip WO2008086800 16 (16q23) CHEK2 US7407755 22 (22q12.1) NOD2 US7407755 16 (16q21) Id-1 US7429457 01 (1p13.3p13.1) Id-2 US7429457 09 (9p21) MELK WO2008023841 09 (9p13.2) cMYC WO2008000749 08 (8q24.21) ESR1 WO2008000749 06 (6q25.1) BRCA1 US20040058340 US7250497 17 (17q21) US7384743 BRCA2 US6051379 US7384743 13 (13q12.3) hMYH polypeptide US7470510 01 (1p34.3-p32.1) DAP-kinase 1 WO2005095980 09 (9q34.1) CA 125 marker US20080153177 19 (19p13.2) genes between control and breast cancer affected individuals. For example the invention titled ‘Gene Expression in Breast Cancer’, WO2004085621 of Polyak et al. [65] features nucleic acids encoding proteins that are expressed at a higher or a lower level in breast cancer cells than in normal breast cells or in a cell of one grade or stage of breast cancer than in a cell of another grade or stage of breast cancer. The invention also includes proteins encoded by the nucleic acids, vectors containing the nucleic acids, and cells containing the vectors. In another aspect, the invention features methods of diagnosing and treating breast cancers of various grades and stages. This invention relates to breast cancer, and more particularly to genes expressed in breast cancer cells. Ductal carcinoma in situ (DCIS) of the breast includes a heterogeneous group of pre-invasive breast tumours with a wide range of invasive potential. In order to initiate early aggressive treatment and avoid its frequent harsh side effects, where not needed, it is important that methods to distinguish between DCIS and invasive breast cancer and between different types of DCIS be developed. The invention is based on the inventors' discovery of differing patterns of gene expression in breast cancer cells versus normal cells, in DCIS cells versus invasive and/or metastatic breast cancer cells, and between different grades of DCIS. The invention includes methods of diagnosis, methods of treatment, nucleic acids corresponding to newly identified genes, polypeptides encoded by such genes, and methods of screening for gene expression. These assays in conjunction with other procedures are useful to diagnose breast cancer and/or identify the grade and/or stage of progression of a breast cancer. More specifically, the invention features a method of diagnosis and diagnostic kit. US 6649342 patent issued on 18th November 2003 also relates to the identification of expression profiles and the nucleic acids involved in breast cancer and the use of such expression profiles and nucleic acids in diagnosis and prognosis of breast cancer [66]. Additionally, methods and molecular targets (genes and their products) for therapeutic intervention in breast cancer are described. The present invention provides methods for screening for compositions which modulate breast cancer. Preferred embodiments of the expression profile gene as described herein include the sequence comprising BCW2 or a fragment thereof coding for BCMP, breast cancer modulating protein. BCW2 is up regulated in breast cancer tissue. The method further includes adding a drug candidate to the cell and determining the effect of the drug candidate on the expression profile of the gene. Death-associated protein (DAP) kinase is a novel multidomain calcium/calmodulin-regulated and cytoskeletal Recent Patents on Breast Cancer associated serine/threonine kinase mandatory for IFN(interferon gamma), TNF- (tumour necrosis factor alpha) and activated Fas-induced apoptotic cell death and detachment from the extracellular matrix, comprising modules such as ankyrin repeats mediating protein-to-protein interactions as well as a death domain. Invention WO2005095980 [67] discloses a method for prognosis of breast cancer comprising detecting DAP-kinase level of expression in breast cancer DAP-kinase protein strongly expressed in normal breast tissue and in human breast epithelial cells primary cultures cells and determining the percentage of cells expressing DAP-kinase or the change of DAP-kinase expression level compared to a control sample as a predictor of the outcome and survival. DAP-kinase protein was strongly expressed in normal breast tissue and in human breast epithelial cells primary cultures. The invention is aimed at a method for prognosis of breast cancer comprising detecting DAP-kinase level of expression in breast cancer cells and determining the percentage of cells expressing DAP-kinase. Loss of DAPkinase expression negatively correlates to survival and positively correlates to the probability of recurrence in a very significant manner. A percentage of cells expressing DAPkinase above 35%, 30%, 25% or 20% is indicative of a favorable prognosis. The method involves detection of DAPK expression in cells by immuno-histochemistry. It basically consists of staining tissue section with the antibodies against DAP-kinase. The invention is directed to the method as defined above comprising contacting breast cells with a DAP-K antibody, directly or indirectly labeled, detecting the signal and determining the ratio of cells expressing DAP-K. The kit can comprise the primers for specifically amplifying DAP-K from mRNA or cDNA, or such primers for performing q-RT-PCR for example and/or a DAP-K c-DNA array. The invention discloses diagnostic kit comprising the primers for specifically amplifying DAP kinase mRNA or cDNA. It also discloses a DAP-K antibody based diagnostic kit. Maternal Embryonic Leucine Zipper kinase (MELK) (GenBank Accession NO. NM 014791), is a cancer specific gene that has been found to be up-regulated in breast, bladder, and lung cancer, interacts with and phosphorylates Bcl-G, a pro-apoptotic member of the Bcl-2 family of proteins [68]. The invention WO2008023841 relates to the field of cancer treatment and prevention, particularly, to methods and kits for identifying agents useful in the treatment and prevention of cancer, more particularly breast, bladder and lung cancer, as well as methods and compositions for treating and preventing same. Immune complex kinase assays showed that Bcl-G was an ideal in vitro substrate for MELK kinase. Furthermore, the introduction of wild-type MELK was shown to rescue apoptosis induced by Bcl-G, whereas kinase-dead of MELK could not. These findings are consistent with the conclusion that the inhibition of Bcl-G by overexpression of MELK is likely to be involved in breast carcinogenesis through anti-apoptotic manners. The present method more particularly relates to the discovery that MELK, a cancer specific gene and its upregulation. Here the precise expression profiles of 81 breast tumours using a combination of laser-microbeam microdissection and a cDNA microarray consisting of 23,040 genes were examined. MELK gene was found to be Recent Patents on DNA & Gene Sequences 2009, Vol. 3, No. 2 143 significantly overexpressed in the great majority of breast cancer cases examined. The present inventors further identified MELK as a cancer-specific protein kinase, the down-regulation of which leads to growth suppression of breast cancer cells. The present invention also provides kits for screening for an agent useful in treating or preventing cancer. In one embodiment, such a kit includes: (a) a polypeptide having a Bcl-G-binding domain of a MELK polypeptide; (b) a polypeptide having a MELK-binding domain of a Bcl-G polypeptide; and (c) means to detect the interaction between the polypeptides. In a preferred embodient, the polypeptide having the Bcl-G-binding domain is a MELK polypeptide and the polypeptide having the MELK-binding domain is a Bcl-G polypeptide. GENE AMPLIFICATIONS/DELETIONS Amplification of genomic DNA is the result of a selection process aiming at facilitating tumour cell growth, e.g. by high level over expression of genes that otherwise would be growth rate limiting. Amplified genes, therefore, are likely to be vitally important for tumour cells and represent particular attractive targets for new gene specific therapies. In breast cancer, more than 30 regions of amplification have been detected by means of classical comparative genomic hybridization (CGH). Numerous important oncogenes have been identified within these amplicons, for example CMYC at chromosome 8q24, EGFR at 7p21, or CCNDl at Ilql3. However, it is assumed that the majority of genes which undergo amplification in breast malignancies have not yet been identified. Thus, there is a hope that other amplified genes can be used in diagnosis, estimation of prognosis and treatment of these diseases. Patent application WO2008000749 details an in vitro method of identifying a tumour resulting from a proliferative breast disease as responsive to anti-estrogen treatment [69]. Further, the invention relates to an in vitro method of identifying a candidate patient with a proliferative breast disease as suitable for anti-estrogen treatment. In a further aspect, the invention provides an in vitro method of identifying an individual with a non-cancerous proliferative breast disease who is at risk of developing breast cancer. The invention also provides kits for performing the above methods. Amplification of the ESR 1 gene located at 6q25.1 and encoding the alpha isoform of the estrogen receptor appears to be the most frequent gene amplification that is detectable in breast cancer. In the experiments conducted by the inventors, amplification of the ESRl gene was observed in 31% of the examined tumours. Even more importantly, the present invention provides evidence that amplification of the ESRl gene is correlated to an enhanced susceptibility of a tumour, such as a breast cancer, to anti-estrogen treatment, e.g. by administration of Tamoxifen. As a consequence, detection of ESRl amplification is of significant clinical relevance and may be used in diagnosis and estimation of prognosis and also as a tool for making decisions as to the specific treatment protocol to be used with a particular patient suffering from a proliferative breast disease such as breast cancer. Microarray technology allows for the measurement of the steady-state mRNA level of thousands of genes simultaneously thereby presenting a powerful tool for identifying 144 Recent Patents on DNA & Gene Sequences 2009, Vol. 3, No. 2 effects such as the onset, arrest, or modulation of uncontrolled cell proliferation. Two microarray technologies are currently in wide use. The first is cDNA arrays and the second is oligonucleotide arrays. Although differences exist in the construction of these chips, essentially all downstream data analysis and output are same. The product of these analyses are typically measurements of the intensity of the signal received from a labeled probe used to detect a cDNA sequence from the sample that hybridizes to a nucleic acid sequence at a known location on the microarray. Typically, the intensity of the signal is proportional to the quantity of cDNA, and thus mRNA, expressed in the sample cells. A large number of such techniques are available and useful. The US patent 7473526 discloses a method of prognosticating metastasis in a breast cancer patient that involves identifying differential modulation of each gene (relative to the expression of the same genes in a normal population) (portfolio of 28 genes/markers) in a combination of genes as well as kits for employing the method [70]. Usually, however, diseases are not easily diagnosed with molecular diagnostics for one particular gene. Multiple markers are often required and the number of such markers that may be included in an assay based on differential gene modulation can be large, even in the hundreds of genes. MUTATIONS/GENE VARIATIONS US20087470510 issued on December 30, 2008, features a human mutMYH polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques [71]. Invention also discloses methods for utilizing such polypeptide for preventing and/or treating diseases associated with a mutation in this gene. Diagnostic assays for identifying mutations in nucleic acid sequence encoding a polypeptide of the present invention and for detecting altered levels of the polypeptide for detecting diseases, for example, cancer, are also disclosed. Mutation in a hMYH gene of decreases the activity of the encoded hMYH protein. Presence or absence of a difference in a coding region in hMYHencoding nucleotide sequence, results in decreased binding of the encoded hMYH protein to a substrate containing an A/GO mispair, causing decreased glycosylase activity of the encoded hMYH protein. The mutation results in a frame shift in, or a truncation of, the coding region. Mutations of the BRCA1 gene in humans are associated with predisposition to breast and ovarian cancers. A large number of deleterious mutations in BRCA1 gene have been discovered. Genetic testing on patients to determine the presence or absence of such deleterious mutations has proven to be an effective approach in detecting predispositions to breast and ovarian cancers. Genetic testing is now commonly accepted as the most accurate method for diagnosing hereditary breast cancer and ovarian risk. The invention of Scholl et al., issued a US20077250497 in July 2007 assigned to Myriad Genetics, is based on the discovery of a number of large deletions in human BRCA1 gene in patients. Isolated BRCA1 nucleic acids (genomic DNAs, corres-ponding mRNAs and corresponding cDNAs) comprising one of the newly discovered genetic variants are disclosed in this patent [72]. These large deletions are deleterious and cause significant alterations in structure or Singh and Chaturvedi biochemical activities in the BRCA1 gene products expressed from mutant BRCA1 genes. Patients with such deletions in one of their BRCA1 genes are predisposed to, and thus have a significantly increased likelihood of, breast cancer and/or ovarian cancer. Therefore, these deletion variants are useful in genetic testing as markers for the prediction of predisposition to cancers, especially breast cancer and ovarian cancer, and in therapeutic applications for treating cancers. This invention also discloses a kit and methods which comprise detecting a deletion in the BRCA1 gene that can result from an unequal crossover event between specific repetitive sequences, commonly referred to as recombination breakpoints or regions. Women who carry the BRCA1 gene face a 10-fold increased risk of contracting the disease by age 70. US20040058340 of Dai, Hong Yue et al. published on March 25, 2004 relates to genetic markers whose expression is correlated with breast cancer [73]. Specifically, the invention provides sets of markers whose expression patterns can be used to differentiate clinical conditions associated with breast cancer, such as the presence or absence of the estrogen receptor ESR1, and BRCA1 and sporadic tumours, and to provide information on the likelihood of tumour distant metastases within five years of initial diagnosis. The invention also relates to methods of using these markers to distinguish these conditions and to kits containing ready-to-use microarrays and computer software for data analysis using the diagnostic, prognostic and statistical methods. BRCA2 gene, located on chromosome 13q12-q13, is thought to be a tumour suppressor gene associated with breast and ovarian cancer. Thus mutations which form an altered tumour suppressor or altered concentrations of tumour suppressor may be indicative of a higher succeptibility to certain cancers. The location of one or more mutations of the BRCA2 gene provides a promising approach to reduce the high incidence and mortality associated with breast and ovarian cancer through the early detection of women at high risk. Many mutations and normal polymorphisms have already been reported in the BRCA2 gene. A worldwide web site has been built to facilitate the detection and characterization of alterations in breast cancer susceptibility genes. Such mutations in BRCA2 can be accessed through the Breast Cancer Information Core. US20066051379 of Lescallett et al. on Cancer suscep-tibility mutations of BRCA2 discloses new mutations C2192G, 3772delTT, C5193G, 5374del4, 6495delGC, or 6909insG located at nucleotide numbers 2192, 3772, 5193, 5374, 6495 or 6909 of the published nucleotide sequence of BRCA2 gene [74]. It also provides a process for identifying a sequence variation in a BRCA2 polynucleotide sequence. The identification process includes allele specific sequencebased assays of known sequence variations. The methods can be used for efficient and accurate detection of a mutation in a test BRCA2 gene sample. The invention also provides a kit useful for detection of these mutations [74]. US7384743 of Arena et al., issued on June 10, 2008 discloses a method for analyzing a biological sample is performed by analyzing a biological sample for the presence of one or more mutations or polymorphisms in the BRCA1 and/or BRCA2 genes [75]. It particularly relates to a method for analyzing a biological sample from an African American Recent Patents on Breast Cancer Recent Patents on DNA & Gene Sequences 2009, Vol. 3, No. 2 145 woman for the presence of a polymorphism in BCRA1 gene, an adenine to guanine, transition at position 5217 in the BRCA1 gene (5217 A>G); and detecting the presence of a guanine at position 5217 of the BRCA1 gene. The method further comprises analyzing the sample for the presence of a cytosine to thymine transition at position 4959 in the BRCA1 gene (4959C>T). Breast cancer susceptibility gene 1 (BRCA1) is a tumour suppressor gene identified on the basis of its genetic linkage to familial breast cancers. US20087429457 issued on September 30, 2008 discloses a method for detection and prognosis of breast cancer and other types of cancer and discloses involvement of Id-1 (inhibitor of differentiation-1) and Id-2 (inhibitor of differentiation-2) genes and products as markers of epithelial cancer [80]. When expressed, Id-1 gene is a prognostic indicator that breast cancer cells are invasive and metastatic, whereas Id-2 gene is a prognostic indicator that breast cancer cells are localized and noninvasive in the breast tissue. US20087407755 of Lubinski et al. provides methods and kits for determining a predisposition and surveillance protocols for developing cancer of various sites including breast cancer due to specific mutation in at least one allele of CHEK2 gene and/or at least one allele of NOD2 gene and/or at least one allele of CDKN2A gene [76]. The invention published as WO2008086800 relates to a method for the detection of predisposition to a period of metastasis-free, recurrence-free and/or disease-free survival of individuals suffering from breast cancer [81]. This invention relates to methods and kits based on the presence or absence of the c-mip gene located on the human chromosome 16, wherein reduced amounts of c-mip gene, transcriptional product, or translational product is indicative of a period of metastasis-free, recurrence-free and/or diseasefree survival in an individual suffering from breast cancer. The invention also relates to a method for determining the treatment regime of an individual suffering from breast cancer. The invention also pertains to a method for classification of at least one tumour from an individual suffering from breast cancer. Furthermore a method for determining the prognosis for a period of metastasis-free, recurrence-free and/or disease-free survival in an individual suffering from breast cancer is disclosed. MOLECULAR RECEPTORS Human epidermal growth factor receptor-2 ("HER2") is a member of a class of molecules in growth stimulatory pathways, called growth factor receptors. 25% to 30% of women with breast cancer have been found to have amplification of this gene. HER2 expression was found to directly correlate to its degree of amplification. Moreover, the detection of amplification of the HER2 gene, as a measure of patient disease status and survivability, is further described in US4968603 [77]. Determination of HER-2 status is therefore a critical tool for selecting appro-priate therapeutic options. The invention disclosed in US20087504222 issued in 2009 [63], relates to ER (estrogen receptor) positive and ER negative markers associated with breast cancer as well as methods of assessing whether a patient is afflicted with breast cancer and methods of characterizing, monitoring and treating breast cancer. The markers are over-expressed in breast cancer cells compared to normal (i.e. non cancerous) cells. This invention therefore provides methods and reagents for the diagnosis, characterization, prognosis, monitoring, and treatment of breast cancer, including the identification of ER positive and ER negative breast tumours. ER negative cancers tend to recur sooner and show a different rate of recurrence in distant organ sites compared to ER positive tumours. In particular, the identified markers may be utilized to determine appropriate therapy, to monitor clinical therapy and human trials of a drug being tested for efficacy, and to develop new agents and therapeutic combinations. CA-125 is a useful tumour marker for ovarian cancer. In breast cancer overall 5-80% (median 30%) have been found to be positive for CA-125 in blood plasma [78]. This invention disclosed in US patent application US20080153177 published in 2008 relates to a diagnostic method for diagnosing the possible recurrence of breast cancer, wherein the presence of the monoclonal antibody CA-125 is monitored in a BRCA2 tumour sample, whereby any presence of CA-125 is indicative of metastazing BRCA2. The invention concerns a method wherein the breast cancer comprises BRCA2 germ line mutations. The invention also relates to the use of monoclonal antibody CA-125, or oregovo monoclonal antibody for the preparation of a therapeutic composition for the treatment of breast cancer of the BRCA2 type [79]. To conclude, genomics and proteomics play an important role in understanding, diagnosis, and prognosis of breast cancer. Breast cancer is the most prevalent disease and second leading cause of death among women in many countries. The identification of high-risk, individual genetic profiles and low penetrant, recessive cancer susceptibility genes are needed to provide strong prognostic and predictive markers directed towards a broad spectrum of patients. Several granted patents and pending patent applications disclose such nucleic acids corresponding to newly identified genes, polypeptides encoded by such genes, and methods of screening for gene expression. These assays in conjunction with other procedures are useful to diagnose breast cancer and/or identify the grade and/or stage of progression of a breast cancer. Development of detection protocols and kits for early detection of breast cancer is the need of the hour. Genetic testing on patients to determine the presence or absence of such deleterious mutations has proven to be an effective approach in detecting predispositions to breast cancer which is deemed to be a serious threat to woman health. A few genetic tests for patients with breast cancer are now commercially available, and from the status of ongoing research, it can be predicted that more tests will be available in near future. Further assessment of various cancer drug candidates on the expression profiles of various genes implicated in breast cancer for evolving therapeutics effective to inhibit recurrence is also very much needed. CURRENT & FUTURE DEVELOPMENTS The inventions discussed herein could be useful in development of diagnostic kits/systems and methods for early detection and prediction of prognosis of breast cancer. 146 Recent Patents on DNA & Gene Sequences 2009, Vol. 3, No. 2 ACKNOWLEDGEMENT Authors thank Dr Shantanu Chowdhury, Scientist, Institute of Genomics and Integrative Biology, Delhi, for reviewing the manuscript and helpful suggestions. Authors also acknowledge support from Council of Scientific and Industrial Research, New Delhi and Department of Science and technology (TIFAC). Singh and Chaturvedi [24] [25] [26] [27] CONFLICT OF INTEREST No conflict of interest pertaining to the authors exists regarding any of the patents and patent applications cited in this manuscript [28] [29] REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. Cancer as a micro evolutionary process. Mol Biol Cell 1994; 46: 12551272. Cairns J. Mutation selection and the natural history of cancer. Nature 1975; 255: 197-200. Fidler I, Hart I. Biological diversity in metastatic neoplasms: Origins and implications. Science 1982; 217: 998-1003. Stetler-Stevenson WG, Aznavoorian S, Liotta LA. Tumour cell interactions with the extracellular matrix during invasion and metastasis. Annu Rev Cell Biol 1993; 9: 541-574. Nowell PC. The clonal evolution of tumour cell populations. Science 1976; 194: 23-28. Armitage P, Doll R. The age distribution of cancer and a multistage theory of carcinogenesis. Br J Cancer 1954; 8: 1-12. Vogelstein B, Kinzler KW. The multistep nature of cancer. Trends Genet 1993; 9: 138-141. Baak JPA, Path FRC, Hermsen MAJA, Meijer G, Schmidt J, Janssen EAM. Genomics and proteomics in cancer. Eur J Cancer 2003; 39: 1199-1215. Soloman E, Borrow J, Goddard AD. Chromosome aberrations and cancers. Science 1991; 254: 1153-1160. Hartwell L. Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell 1992; 71: 543-546. Parkin DM, Pisani P, Ferlay J. Global cancer statistics. CA Cancer J Clin 1999; 49: 33-64. Breasted JH. The Edwin smith surgical papyrus. Chicago: University of Chicago Press 1930; 403-406. Parkin DM. International variation. Oncogene 2004; 23: 6329-6340 Ziegler RG, Hoover RN, Pike MC, et al. Migration patterns and breast cancer risk in Asian-American women. J Natl Cancer Inst 1993; 85: 1819-1827 Kliewer EV, Smith KR. Breast cancer mortality among immigrants in Australia and Canada. J Natl Cancer Inst 1995; 87: 1154-1161 Tsongalis GJ, Ricci A Jr. Breast cancer as a model of realistic challenges in pharmacogenomics. Clin Biochem 2003; 36(2): 8994. Knudson AG Jr. Mutation and cancer, statistical study of retinoblastoma. Proc Natl Acad Sci USA 1971; 68: 820-823. Mitelman F, Johansson B, Mandahl N, Mertens F. Clinical significance of cytogenetic findings in solid tumours. Cancer Genet Cytogenet 1997; 95: 1-8. Cahill DP, Kinzler KW, Vogelstein B, Lengauer C. Genetic instability and Darwinian selection in tumours. Trends Cell Biol 1999; 9: 57-60. Bishop JM. Molecular themes in oncogenesis. Cell 1991; 64: 235248. Perou CM, Sørlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature 2000; 17: 747-752. Sorlie T, Tibshirani R, Parker J, et al. Repeated observation of breast tumour subtypes in independent gene expression data sets. Proc Natl Acad Sci USA 2003; 8: 8418-8423. Sotiriou C, Neo SY, McShane LM, et al. Breast cancer classification and prognosis based on gene expression profiles from a population-based study. Proc Natl Acad Sci USA 2003; 2:1039310398. [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] Rouzier R, Perou CM, Symmans WF, et al. Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clin Cancer Res 2005; 15: 5678-5685. Sørlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumour subclasses with clinical implications. Proc Natl Acad Sci USA 2001; 11: 10869-10874. Oldenburg RA, Meijers-Heijboer H, Cornelisse CJ, Devilee P. Genetic susceptibility for breast cancer: How many more genes to be found? Clin Rev Oncol/Hematol 2007; 63: 125-149. Kohaar I, Tiwari P, Kumar R, Nasare, et al. Association of single nucleotide polymorphisms (SNPs) in TNF-LTA locus with breast cancer risk in Indian population. Breast Cancer Res Treat 2009; 114(2): 347-355. Gaudet MM, Egan KM, Lissowska J, et al. Genetic variation in tumour necrosis factor and lymphotoxin-alpha (TNF-LTA) and breast cancer risk. Hum Genet 2007; 121(3-4): 483-490. Pharoah PD, Antoniou A, Bobrow M, Zimmern RL, Easton DF, Ponder BA. Polygenic susceptibility to breast cancer and implications for prevention. Nat Genet 2002; 31(1): 33-36. Pegram MD, Pienkowski T, Northfelt DW, et al. Results of two open-label, multicenter phase II studies of docetaxel, platinum salts, and trastuzumab in HER2-positive advanced breast cancer. J Natl Cancer Inst 2004; 96: 759-769 Giancotti V. Breast cancer markers. Cancer Lett 2006; 243: 145159. Starita LM, Parvin JD, The multiple nuclear functions of BRCA1: Transcription, ubiquitination and DNA repair. Curr Opin Cell Biol 2003; 15: 345-350. Butcher DT, Mancini-DiNardo DN, Archer TK, Rodenhiser DI. DNA binding, sites for putative methylation boundaries in the unmethylated region of the BRCA1 promoter. Int J Cancer 2004; 111: 669-678. Calderon-Margalit R, Paltiel O. Prevention of breast cancer in women who carry BRCA1 or BRCA2 mutations: a critical review of literature. Int J Cancer 2004; 112: 357-364. Eng C. Genetics of Cowden syndrome: Through the looking glass of oncology. Int J Oncol 1998; 12: 701-710. Starink TM, van der Veen JP, Arwert F, et al. The Cowden syndrome: A clinical and genetic study in 21 patients. Clin Genet 1986; 29: 222-233. Pluquet O, Hainaut P. Genotoxic and non-genotoxic pathways of p53 induction. Cancer Lett 2001; 174: 1-15. de Jong MM, Nolte IM, Meerman GJT, et al. Genes other thanBRCA1 and BRCA2 involved in breast cancer susceptibility. J Med Genet 2002; 39: 225-242. Hemminki A, Tomlinson I, Markie D, et al. Localization of a susceptibility locus for Peutz-Jeghers syndrome to 19p using comparative genomic hybridization and targeted linkage analysis. Nat Genet 1997; 15: 87-90. Giardiello FM, Welsh SB, Hamilton SR, et al. Increased risk of cancer in the Peutz-Jeghers syndrome. N Engl J Med 1987; 316: 1511-1514. Spigelman AD, Murday V, Phillips RK. Cancer and the PeutzJeghers syndrome. Gut 1989; 30: 1588-1590. Boardman LA, Thibodeau SN, Schaid DJ, et al. Increased risk for cancer in patients with the Peutz-Jeghers syndrome. Ann Intern Med 1998; 128: 896-899. LimW, Hearle N, Shah B, et al. Further observations on LKB1/STK11 status and cancer risk in Peutz-Jeghers syndrome. Br J Cancer 2003; 89: 308-313. Giardiello FM, Brensinger JD, Tersmette AC, et al. Very high risk of cancer in familial Peutz-Jeghers syndrome. Gastroenterology 2000; 119: 1447-1453. Shen Z, Wen XF, Lan F, Shen ZZ, Shao ZM. The tumour suppressor gene LKB1 is associated with prognosis in human breast carcinoma. Clin Cancer Res 2002; 8: 2085-2090. Berx G, Cleton-Jansen AM, Strumane K, et al. E-cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extracellular domain. Oncogene 1996; 13: 1919-1925. Mastracci TL, Tjan S, Bane AL, O’Malley FP, Andrulis IL. Ecadherin alterations in atypical lobular hyperplasia and lobular carcinoma in situ of the breast. Mod Pathol 2005; 18: 741-751. Sarrio D, Moreno-Bueno G, Hardisson D, et al. Epigenetic and genetic alterations of APC and CDH1 genes in lobular breast cancer: Relationships with abnormal E-cadherin and catenin Recent Patents on Breast Cancer [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] expression and microsatellite instability. Int J Cancer 2003; 106: 208-215. Sodha N, Houlston RS, Bullock S, et al. Increasing evidence that germline mutations in CHEK2 do not cause Li- Fraumeni syndrome. Hum Mutat 2002; 20: 460-462. Lucke CD, Philpott A, Metcalfe JC, et al. Inhibiting mutations in the transforming growth factor beta type 2 receptor in recurrent human breast cancer. Cancer Res 2001; 61: 482-485. Chen T, Carter D, Garrigue-Antar L, Reiss M. Transforming growth factor beta type I receptor kinase mutant associated with metastatic breast cancer. Cancer Res 1998; 58: 4805-4810. Xie W, Mertens JC, Reiss DJ, et al. Alterations of Smad signaling in human breast carcinoma are associated with poor outcome: Atissue microarray study. Cancer Res 2002; 62: 497-505. MacPherson G, Healey CS, Teare MD, et al. Association of a common variant of the CASP8 gene with reduced risk of breast cancer. J Natl Cancer Inst 2004; 96: 1866-1869. Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 2003; 421: 499-506. Szabo CI, Schutte M, Broeks A, et al. Are ATM mutations 7271T G and IVS10-6T G really high-risk breast cancersusceptibility alleles? Cancer Res 2004; 64: 840-843. Ji H, Liu YE, Jia T, et al. Identification of a breast cancer-specific gene, BCSG1, by direct differential cDNA sequencing. Cancer Res 1997; 57 (4): 759-64. Weber M, Davies JJ, Wittig D, et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 2005; 37: 853-62. Yoder JA, Walshcp, Bestor TH. Cytosine methylation and the ecology of intragenomic parasites. Trends Genet 1997; 13: 335-40. Widschwendter M, Jiang G, Woods C, et al. DNA hypo-methylation and ovarian cancer biology. Cancer Res 2004; 64: 4472-80. Deng G, Nguyen A, Tanaka H, et al. Regional hypermethylation and global hypomethylation are associated with altered chromatin Recent Patents on DNA & Gene Sequences 2009, Vol. 3, No. 2 [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] 147 conformation and histone acetylation in colorectal cancer. Int J Cancer 2006; 118: 2999-3005. Fraga MF, Ballestar E, Villar-Garea A, et al. Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat Genet 2005; 37: 391-400. Jatoi I. Breast cancer screening. Am J Surg 1999; 177(6): 518-24. Ayers, M.D., Stec, J.D., Clark, A. Edwin, Hess, E., Hortobagyi, K.R., Gabriel N., Pusztai, G.N., Symmans, W.F., Bast, Jr., Robert C.: US20097504222 (2009). Perou CM, Sørlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature 2000; 17: 747-752. Polyak, K., Porter, D., Allinen, M.: WO2004085621 (2004). Mack, D., Gish, K.C.: US20036649342 (2003). Gompel, A., Rostene, W.: WO2005095980 (2005). Nakamura, Y., Katagiri, T., Nakatsuru, T.: WO2008023841 (2008). Sauter, G., Simon, R., Stahl, P., Holst F, A-Kuraya, K., Ruiz, C.: WO2008000749 (2008). Wang, Y.: US20097473526 (2009). Wei, Y.F.: US20087470510 (2008). Scholl, T., Hendrickson, B.C., Ward, B., Pruss.: US20077250497 (2007). Dai, H., He, Y., Linsley, P.S., Mao, M., Roberts, C.J., Vant, V., Laura, J., Vande, V., Marc, J., Bernards, R., Hart, A.A.M.: US20040058340 (2004). Lescallet, J.L., Lawrence, T., Allen, A.P., Olson, S.J., Thurber, D.B., White, M.B.: US2006051379 (2006). Arena, J.F., Baumbach-Reardon L, G.L., Ahearn, M.E.: US20087384743 (2008). Lubinski, J., Suchy, J., Kurzawski, G., Dbniak, T., Cybulski, C.: US20087407755 (2008). Slamon, D.J., McGuire; W.L.: US4968603 (1986). Hensley ML, Spriggs DR. Cancer screening: How good is good enough? J Clin Oncol 2004; 22: 4037-4039. Olsson, H.: US20080153177 (2008). Desprez, P.Y., Campisi, J.: US20087429457 (2008). Hansen, L.L., Overgaard, J.: WO2008086800 (2008).