MICROSATELLITE INSTABILITY AND DISEASE

Abstract: A method for diagnosing disease or predisposition to disease, including cancer and premature ageing, is achieved by determining whether an individual has microsatellite instability (MSI). Also variously described are methods for directing therapy regimes and a kit for diagnosis of individuals susceptible to disease.

Description: MICROSATELLITE INSTABILITY AND DISEASE This invention relates to microsatellite instability and genetic diagnosis of disease, including cancer or predisposition to cancer, in particular HNPCC, and to methods and apparatus therefor. Present diagnostic techniques that determine whether an individual is at risk of developing cancer are generally based upon analysis of family history, lifestyle or prompt diagnosis of cancer or tumour in its early stages. These techniques are, however, unreliable and do not give a definitive assessment of an individual's particular predisposition. Family history is often an indicator of hereditary predisposition to cancer and possibly even the types of cancer, but the vagaries of inheritance mean that some cancers might skip a generation, making prediction uncertain. Furthermore, if the individual concerned has a small family or is not familiar with their family history this mode of prediction of predisposition to cancer is unavailable to the clinician. Screening for predisposition may involve invasive techniques such as biopsy or internal examination, eg. colonoscopy. These techniques are often painful and have limited reliability as they rely on tumours or abnormal cells having already formed and thus being observable. Tumours that are large enough to be seen by such techniques are made up of many thousands of cells and already have a well established blood supply. Accurate diagnosis of the type of cancer that an individual is suffering from or is predisposed to is generally dependent upon histopathological analysis of tumours removed from the individual. Morphologic or genetic analysis of the tumour tissue allows the type of tumour to be diagnosed and may indicate future risk to the individual of contracting such a cancer again. Individuals who possess a germline mutation in a particular gene might show a predisposition to a particular type of cancer but this might only be evident on analysis of a tumour taken from the susceptible tissue. It would accordingly be of great advantage to be able to isolate a sample of tissue from an individual who has a perceived level of risk and be able, genetically, to determine from this sample whether they do indeed have a predisposition to particular types of cancer. It would be of further advantage for the tissue sample to be taken non-invasively and from non-cancerous tissue. It would be of yet further advantage for the genetic analysis to be a diagnostic technique that is relatively quick and highly reliable. Errors in DNA replication can occur if the fidelity of replication is low and is particularly noticeable in regions where a short DNA sequence is repeated many times, for example in the tandem repeats of 2-5 base pair sequences scattered at regular intervals throughout the genome; these repeats are known as microsatellites. Individuals who possess a malfunctioning DNA mismatch repair system are subject to much higher rates of mutation and are often prone to diseases such as cancer. Such individuals are said to be positive for the replication error phenotype (RER + ), characterised by the presence of microsatellite instability (MSI) in cancerous cells. MSI is easily identified by the presence of additional bands on gel electrophoresis of Polymerase Chain Reaction (PCR) products, representing the multiple novel tumour alleles. RER + individuals may possess a germline mutation in DNA mismatch repair genes, and there are at present six known genes whose products are believed to be involved in DNA mismatch repair; they are MSH2, MSH3, MLH1, MSH6, PMS1 and PMS2. Germline mutations in some of these genes have been identified in patients suffering from a number of cancers. Most notably, mutations in MSH2, MLH1, PMS1 and MSH6 have been linked to incidence of hereditary non-polyposis colorectal cancer (HNPCC) with most familial forms of this disease being linked to mutations affecting MSH2 or MLH1. HNPCC is linked predominantly to high incidence of cancers of the colon and uterus, although cancers of the kidney, ovaries, stomach and pancreas are also linked to HNPCC. At present in order positively to diagnose an individual as being of the RER + phenotype it is necessary to sequence all of the DNA mismatch repair genes and thereby identify a mutation in one of the genes. This procedure is time consuming, expensive and is only around 60 to 70% accurate. Furthermore, such a procedure is only successful in instances where the mutation is in one of the known mismatch repair genes, if the mutation was in an as yet undiscovered gene it would go undetected. It would therefore be advantageous to provide an alternative means for identifying individuals with an RER + phenotype. HNPCC is a dominantly inherited condition that is believed to be responsible for up to 1 5% of all cases of colorectal cancer. Most colon tumours in these HNPCC families arise before the age of 50 and are right sided. MSI has also been detected in up to 20% of endometrial tumours; up to 20% of prostate cancers; up to 26% of breast tumours; around 20% of gliomas (brain tumours); and up to 30% of gastric tumours. Amongst all the tumours found in HNPCC kindreds, 92% have a RER + phenotype (Liu et al 1 996) . However, the incidence of polyp formation in the epithelia of the colon (a good indicator of the presence of abnormal cells) is no greater in individuals with HNPCC than in the normal population. Although the rates of polyp formation in HNPCC and normal individuals are the same, the number of polyps that will go on to become cancerous in HNPCC individuals is greater than 70%, whereas in the normal population the figure is in the region of 5%. Recent work by Aaltonen et al (Aaltonen et al. 1 998) has diagnosed individuals with HNPCC by testing tissue from excised colorectal tumours. This work also investigates the feasibility of genetic screening for the disease using the presence of MSI in the colon tumour tissue as an indicator of damage to DNA mismatch repair genes and then subsequently sequencing the DNA mismatch repair genes to identify the mutation. The study states that the criteria for identification of those individuals who should be tested for the RER + phenotype are: a family history of colorectal or endometrial cancer at an age of less than 50 and a personal history of colorectal or endometrial cancer. Indeed, a similar regime of testing has been described in Helland et al (1 997), in which excised endometrial and cervical cancer tissue was tested for the presence of MSI . Overall instability was identified in 30% of the endometrial cancers and 6% of the cervical cancers. Thus the Aaltonen et al and Helland et al systems of screening are effective at diagnosing predisposition to further cancers only in individuals who have already contracted colorectal, endometrial or cervical cancers. Often the analyses are performed on archival tissue that may be years old and thus the actual value of the diagnosis is diminished as the patient may have developed cancer, or even died in the meantime. At present the diagnosis of HNPCC in a family is made on the basis of the 'Amsterdam criteria' (Vasen et al, 1 991 ); the presence of at least 3 histologically verified cases of colorectal cancer in at least 2 generations, one being diagnosed before age 50 and one being a first degree relative of the other two, with familial adenomatous polyposis (APC) excluded. With respect to skin tumours, MSI is an unusual finding and there are only a few reports of RER + phenotype in the skin: it has been demonstrated in rare sebaceous tumours and actinic keratoses (dysplastic skin lesion) in one patient from a Muir- Torre kindred (Quinn et al 1 994) and in a small percentage of malignant melanomas or metastatic tumours (Peris et al 1 995, Tomlinson et al 1 996, Walker et al 1 994, Richetta et al 1 997) . It is an object of the present invention to provide a more reliable means of diagnosing cancer or a predisposition to cancer. It is a further object to diagnose cancer or predisposition to cancer by genetic analysis of a benign or malignant tumour in one tissue and correlating it to a predisposition to cancer in another tissue. It is yet a further object of the invention to correlate genetic analysis of benign and malignant tumours in easily accessible tissue to a predisposition to cancer in a different tissue. It is still a further object to provide a means for diagnosis of individuals predisposed to cancer and then to place susceptible individuals on a regime of screening and/or prophylactic therapy to prevent onset or progression of cancer. Accordingly, the present invention relies on a correlation of MSI present in benign or malignant tumours in one tissue with a predisposition to disease in another tissue. A first aspect of the invention provides a method of diagnosing disease or predisposition to disease in a first tissue, comprising identifying microsatellite instability (MSI) in a second tissue, different from the first. It is thus an advantage that the invention enables diagnosis in relation to a relatively inaccessible tissue to be obtained from a tissue that is more convenient to sample. Cancers in non-surface tissue, such as cancers of internal organs, the gut and its many subdivisions for example, as well as diagnosis of predisposition to cancers therein, may thus be diagnosed using, say, skin samples. Preferably, a diagnosis of disease or predisposition to disease in tissues other than skin is carried out using a skin sample. This sample is suitably taken from a skin tumour, and samples from both benign and cancerous lesions are suitable, though, generally, benign tumours are more plentiful and therefore preferred. Archived tissue can also be used. With fresh samples, a skin shave is convenient, under local anaesthetic for comfort of the patient. Lesions of the skin suitable for analysis according to the method of the invention typically include moles; papules; naevi; ; seborrhoeic warts/keratoses; solar keratoses; disseminated actinic porokeratoses; solar lentigines; basal cell carcinoma; squamous cell carcinoma; melanoma; sebaceous carcinoma; and sebaceous adenoma, although, virtually any abnormal growth of cells in the skin can be suitably used in the method of the invention Screening for predisposition to HNPCC in members of families with a history of colon cancer is presently done in a number of ways. Individuals are subject to regular examination by colonoscopy and pelvic ultrasound. However, these prior art methods only successfully identify tumours that have already formed and are large enough to detect. Moreover, colonoscopy is a particularly invasive technique and carries with it a risk of perforation of the colon which is potentially life threatening. Therefore, if such invasive methods were reserved only for those individuals positively identified as being RER + , as determined from a skin shave for example, then unnecessary screening could be alleviated. Genetic screening is also an option, this involves sequencing the DNA mismatch repair genes and analysing the sequence data for evidence of mutation. This method is unreliable, time consuming and costly as it involves the sequencing of large genes and in the past has shown an accuracy of no more than 60% in identifying mutations. For a large scale screening programme, sequencing of these DNA mismatch repair genes would not be an efficient option to adopt. Furthermore direct sequencing would not pick up mutations in distant enhancers or repressors of the gene, or in other genes that serve to regulate the expression of the DNA mismatch repair genes or regulate the activity of their protein products. It is therefore an advantage of the present invention that genetic screening of individuals for cancer or a predisposition to cancer is achieved by correlating MSI in the skin to a number of internal cancers associated with HNPCC, thus facilitating screening and reducing the risks associated with present methods which are invasive and/or unreliable. Individuals who are identified as having the RER + phenotype and also having an RER + family would typically undergo regular colonoscopy and pelvic ultrasound investigations as well as analysis of blood CA1 25 level. They may also be subject to further genetic analysis, such as mutation analysis in known DNA mismatch repair genes and screening for the RER + phenotype would be offered to other members of the family. If an individual is identified as being RER- but is from an RER + family then they are considered to be of low risk and could be spared from having to undergo colonoscopy or pelvic ultrasound on a regular basis. Similarly an RER- individual from an unknown family background could also be exempted from such a screening programme. A specific embodiment of the invention provides a method for diagnosis of hereditary non-polyposis colorectal cancer (HNPCC) , or predisposition to HNPCC. This diagnosis may be carried out by identifying MSI in non-colorectal tissue; preferably by identifying MSI in a benign or malignant skin tumour. A further embodiment of the present invention provides for diagnosis of disease or predisposition to disease in a tissue selected from (a) stomach, uterine, kidney, renal, cervix, endometrium, pancreas, brain and ovarian tissue, comprising identifying MSI in a tissue (b), different from any (a) tissue. Tissue samples suitable for use in the method of the invention are conveniently obtained via biopsy of a benign or malignant tumour. Other means of obtaining samples of suitable tissue typically include, for example, cells isolated from a cervical smear; colorectal cells deposited on stool; and oral epithelial cells taken from a cheek scrape or mouth wash. A second aspect of the invention provides method of diagnosing disease or predisposition to disease comprising:- a) obtaining a biopsy sample of a first tissue; b) extracting DNA from said tissue; c) amplifying a specific region of the DNA by PCR; and d) analysing the products of the PCR reaction; wherein identification of MSI in the first tissue provides diagnosis of disease or predisposition to disease in a second tissue, different from the first. A preferred embodiment provides for diagnosis of cancer or predisposition to cancer. A further preferred embodiment provides for diagnosis of HNPCC or predisposition to HNPCC, with test tissue conveniently obtained from a skin tumour, preferably by a skin shave. A further embodiment of the present invention provides for a method for diagnosis of predisposition to skin photodamage and/or premature skin ageing. In an example of the invention in use, described in more detail below, individuals are characterised for their predisposition to photodamage and/or premature ageing (e.g. by exposure to solar radiation) by identifying the presence of MSI. Individuals who possess the RER + phenotype also often have significantly higher numbers of cutaneous lesions, such as solar lentigines and stucco keratoses. They can appear to be much older than their chronological age would suggest, in part due to an increased rate of skin atrophy. Although, solar U.V. associated photodamage accounts for some of the apparent ageing process in normal adults, individuals with MSI tend to age much more quickly, and are at a much higher risk of developing malignant skin cancers as a result of solar photodamage. The present invention thereby provides for the implementation of chemopreventative treatments for individuals with MSI. Such treatments typically include topical administration of medicaments that comprise the vitamins A and/or C, which can reduce the effects of photodamage over a lifetime. It is therefore an advantage of the present invention that it provides a means for identifying individuals with increased risk of photodamage by determining whether they are positive for MSI. Accordingly, it is an advantage of the present invention that it provides a method of diagnosis of or predisposition to a number of different diseases and conditions in an individual via analysis of tissue taken from a single benign tumour. It is of particular note that the tissue sample can be taken both from accessible tissue such as skin, or even from excised tumour tissue that has undergone histological procedures, thus reducing for the individual the psychological trauma that can be associated with undergoing repeated biopsy procedures. A third aspect of the invention is a kit, comprising:- a) PCR primers for the amplification of microsatellite containing regions of the genome; and b) positive and negative controls; wherein, in use of the kit, identification of MSI in patient DNA by comparison of the product of the PCR reaction to the controls provides diagnosis of disease or predisposition to disease. In preferred embodiments of the invention the kit is suitable for diagnosis of or predisposition to cancer, skin photodamage and premature skin ageing. The kit suitably comprises at least two or more sets of PCR primers. In yet a further embodiment of the kit PCR primer pairs are selected from the group consisting of D1 S214 and D 1 S201 ; D6S305 and D6S262; D9S1 62 and D9S1 71 ; D9S1 60 and D9S1 76; D 10S1 85 and D1 0S573; D1 1 S910 and D1 1 S29; D 1 3S1 55 and D 13S 1 70; D 1 7S796 and D 1 7S520; D 1 7S785 and D 1 7S579; and D18S70 and D 1 8S34. An affirmative identification of MSI typically relies upon instability in the PCR products of at least three gene markers, and the kit preferably includes at least three marker PCR primer pairs. A preferred embodiment of the invention provides a kit comprising all or a combination of the specified PCR primer pairs. In use of a specific embodiment of the invention, described in further detail below, PCR primers are used to diagnose HNPCC or predisposition thereto. A fourth aspect of the invention provides a method of cancer therapy which includes:- screening an individual for cancer or predisposition to cancer; and if such a cancer or predisposition to cancer is identified, treating that individual to prevent, reduce or delay the onset of cancer, wherein a cancer or predisposition to cancer in a first tissue is correlated to MSI in a second tissue, different from the first. An individual who is identified as having MSI in one tissue and identified as having a predisposition to colon cancer is optionally placed upon a preventative or prophylactic regime of therapy. One such prophylactic regime of therapy may include treatment with inhibitors of the enzyme, cyclooxygenase 2 (COX2) which is expressed at elevated levels in 90% of colon cancers. Such inhibitors typically include aspirin, aspirin-like compounds, sulindac™ and other non-steroidal anti inflammatory drugs (NSAIDS), these therapies are described with particular reference to HNPCC individuals in Ruschoff et al (1 998) . A fifth aspect of the invention provides a method of predicting response to anti- cancer therapy, by determining whether there is MSI in skin. There is evidence that RER + tumours of the colon are less aggressive than other types of colon cancers and whilst they respond well to some types of chemotherapy, they respond less well to others. Thus, in a further embodiment of the present invention, identification of MSI in the skin leads to the choice of particular chemotherapeutic drugs for the treatment of associated colon tumours. The invention, therefore, provides a means of predicting whether an individual may be more responsive to certain chemotherapy agents, regimens or dosages. In a specific embodiment of the invention, a skin shave is taken from an individual that comprises a seborrhoeic wart. The skin shave is fixed, sectioned and the sections mounted onto microscope slides using techniques commonly known to the art. The sections are examined under a light microscope and tissue that is identified as being tumour tissue is microdissected away from normal tissue. DNA is extracted from the microdissected tumour tissue using standard techniques. A PCR is performed using the extracted DNA and primers known to encompass regions of the genome that include microsatellite repeats. The primers may include some or all of the group comprising: D 1 S214 and D 1 S201 ; D6S305 and D6S262; D9S1 62 and D9S1 71 : D9S1 60 and D9S 1 76; D 1 0S1 85 and D 1 0S573; D 1 1 S910 and D1 1 S29; D1 3S1 55 and D1 3S1 70; D1 7S796 and D17S520; D1 7S785 and D1 7S579; and D 1 8S70 and D 1 8S34. The products of each PCR reaction are run on a gel and analysed. The presence of a ladder like pattern of bands on the gel denotes multiple alleles in the microsatellite region and corresponds to MSI and thus an RER + phenotype. The RER + phenotype is correlated with a predisposition to cancer in tissue other than skin; particularly cancer of the HNPCC kind. In a further embodiment of the invention PCR primers are selected to allow amplification of other regions of the genome that also contain microsatellite repeats. It is generally considered that at least two and as many as eight different regions should be amplified in order to correctly diagnose the presence of MSI and thus predisposition to cancer. However, it is possible to only amplify a single region of the genome and make the diagnosis from the PCR products of the reaction. In yet a further embodiment of the invention, PCR primers are selected that encompass loci including microsatellite repeats close to or within known tumour suppressor genes, thereby allowing for the simultaneous diagnosis of loss of heterozygosity (LOH) at known suppressor gene loci. In a specific use of the invention, a PCR based technique has been used, and found to be reliable and reproducible in detecting MSI in common and easily accessible skin lesions. Any technique for recognition of MSI is however appropriate. In two patients identification of MSI in benign cutaneous lesions lead directly to investigation of germline mutations in the most commonly mutated mismatch repair genes (hMSH2, hMLHD . Mutations were detected in the families of the two patients, although, one patient was only identified as belonging to a "HNPCC like" family once the RER + phenotype was detected in the skin. This patient was aware that she had a strong family of cancers but had not been referred to the dermatology clinic for that purpose. The benign cutaneous lesions which demonstrate MSI, such as seborrhoeic warts, solar keratoses and naevi are very common in the general population. They are conveniently removed under local anaesthetic and can be readily identified both clinically and histologically. The skin biopsy for seborrhoeic warts or solar keratoses is relatively non invasive with a 3 to 5 mm skin sample shaven under local anaesthesia. Archival tissue in paraffin blocks can also be used so that previous skin lesions or any other non cutaneous dysplastic or malignant lesions can also be studied. MSI is present throughout tumour DNA and any panel of DNA markers may be used. In the specific embodiment described below, we selected DNA markers for tumour suppressor genes as this allows for the detection of LOH at these loci. For melanoma and melanocytic lesions, previous studies have shown that the most common loci for LOH are 9p, 10q, 6q and 1 1 q (Healy et al 1 996) . Whilst for solar keratoses, frequent LOH at 1 7p, 1 7q,9q and 13p have previously been reported and these loci harbour several tumour suppressor genes. LOH in solar keratoses in the study reported here was detected in lesions. The detection of LOH in benign and dysplastic skin lesions has already been extremely valuable for the discovery of two genes involved in skin carcinogenesis: p1 6 or CDKN2 on chromosome 9p and Patch on chromosome 9q. The study of genetic alterations in the skin is a powerful tool as skin lesions are easily accessible and often numerous. This allow for the detection of non random chromosome loss as more than 5 or 6 skin lesions can sometimes be sampled. By selecting skin lesions in patients belonging to family cancer syndromes, the chance of detecting non random chromosome loss which may play a role in the solid tumours susceptibility is much higher. No studies have yet investigated genetic changes in benign skin lesions in patients from family cancer syndromes. The identification, according to the invention, of MSI in benign skin lesions of patients belonging to "HNPCC like" families has important clinical implications as the PCR based technique can be used in clinical practice to screen potentially affected individuals in these families. Hitherto, potentially affected members of HNPCC families were offered colonoscopy at 3 to 5 year intervals, pelvic ultrasound and urine cytology. The detection of DNA mismatch repair gene mutations in these families is expensive and laborious and is not, as yet, used in day to day clinical practice. Furthermore, genotyping in clinical practice has poor predictive value as the failure rate for mutation detection can be as high as 40% (Liu et al 1 996) . The invention thus is of advantage in providing simplified and more reliable genetic diagnosis of cancer and predisposition thereto. A sixth aspect of the invention provides for a method of predicting increased risk of cancer in an immunosuppressed individual by determining presence of MSI in said individual. Such a method comprises phenotyping individuals for the RER + phenotype, when the individuals are immunosuppressed or likely to undergo immunosuppressive therapy, in order to diagnose predisposition to cancer. Preferably, the individuals are screened for MSI before they undergo immunosuppressive therapy. The inventors have found that RER + individuals who are immunosuppressed have a greater chance of developing aggressive tumours, such as the rare skin cancers: sebaceous carcinomas. In an example of the invention described in more detail below, a group of renal transplant recipients (RTRs) with aggressive skin cancers were identified as having MSI. It is believed that the combination of an RER + phenotype with the immunosuppression therapy that necessarily follows organ transplantation increases the risk of developing cancer and in some cases even hastens the onset of particularly aggressive tumours. The cancers that are most typically associated with this phenomenon are found in the skin and include squamous cell carcinomas. In an embodiment of the invention individuals are screened prior to undergoing immunosuppression therapy in order to identify those individuals with MSI, thereby allowing prophylactic treatment to be administered - suitable treatment typically includes oral administration of retinoids - or possibly the adjustment or avoidance altogether of the immunosuppression therapy. Immunosuppression therapy typically includes treatment with pharmaceutical agents such as cyclosporin, azathioprine, prednisolone, chlorambucil or cyclophosphamide. Immunosuppression may also typically be due to a clinical condition, a disease or as a result of chemotherapy, although exposure to any agent that is capable of reducing immune function is likely to place individuals with MSI at increased risk of developing cancer. In an example of the invention in use MSI located in a first tissue diagnoses increased risk of cancer in a second tissue different from the first tissue. Typically the first tissue (a) and said second tissue (b) are selected from the group consisting of stomach, colon, uterine, kidney, renal, cervix, endometrium, pancreas, skin, brain and ovarian tissue. Immunosuppressant therapies are often employed for organ transplant recipients or for individuals suffering from autoimmune diseases, such as rheumatoid arthritis, and inflammatory conditions such as psoriasis. Thus, by screening individuals for MSI according to the method of the invention (e.g. by biopsy of a benign skin tumour), prior to placing them on a course of immunosuppressants, the clinician is able to identify whether the individual is predisposed to internal cancers as well as to development of aggressive cancers aggravated by the prescribed therapy. A seventh aspect of the invention provides for use of means for identification of MSI in manufacture of a means of diagnosis of disease or predisposition to disease. It is preferred that the disease is either cancer, skin photodamage or premature skin ageing. In a specific embodiment of the invention the disease is HNPCC. In further preferred embodiments of the invention PCR primers are provided that enable identification of MSI in a first tissue taken from individuals and thus diagnosis of or predisposition to cancer in a second tissue different from the first tissue. Such tissue typically includes skin tumours or internal solid tumours, either of a benign or malignant type. There now follows a description of the specific embodiments of the invention, illustrated by drawings, in which:- Fig. 1 shows the Atypical Mole Syndrome phenotype; multiple large irregularly pigmented naevi; Fig . 2 shows a typical lesion suitable for use in the method of the invention; Fig. 3 shows MSI in cases 1 and 2 at the locus D1 3S1 55 (1 3q); Fig. 4 shows loss of heterozygosity in squamous cell carcinomas at the locus D9S1 76 (9q); Example 1 Inclusion criteria Patients were seen in the family cancer clinic in the dermatology department at the Royal London Hospital and were recruited from the surgery, gastroenterology and gynaecology departments at the Royal London Hospital. Other families were referred from other specialised family cancer clinics at Imperial Cancer Research Units at Northwick Park and Guy's Hospital. For the selection criteria, all patients with a family history strongly suggestive of HNPCC were selected and the strict Amsterdam criteria were not necessarily fulfilled. Other family cancer syndromes with skin cancers or relevant skin lesions (solar keratoses, dysplastic naevi and seborrhoeic warts) were also included in the study. A detailed questionnaire, family tree, peripheral blood sample and full skin phenotype were collected by two trained dermatologists, with biopsy of benign or malignant skin lesions for histological examination and DNA extraction. Histological reports were available for review prior to microdissection, and any lesions with doubtful histology were reviewed by a dermatopathologist. DNA Extraction and Analysis Archival paraffin-fixed material is microdissected from unstained sections to enrich for tumour tissue. DNA was extracted from blood, fresh and archived tissue using standard techniques. PCR-Based Microsatellite Analysis Primer pairs were chosen on the basis of loci of known tumour suppressor genes and published work. All primer pairs used (Research Genetics, Huntsville, US) had a heterozygosity score of > 80%. Microsatellite analysis for MSI and LOH in skin lesions was performed using primer pairs as follows; Chromosome arm Primers used 1 p D1 S21 4 D 1 S201 6q D6S305 D6S262 9p D9S162 D9S171 9q D9S160 D9S176 10q D10S185 D10S573 11q D11S910 D11S29 13q D13S155 D13S170 17p D17S796 D17S520 17q D17S785 D17S579 18q D18S70 D18S34 The Polymerase Chain reaction (PCR) protocol is as follows: 1 cycle of 3 minutes at 95 ° C, 1 minute at 55 ° C, 1 minute at 72 ° C, 29 cycles of 1 minute at 95 ° C, 1 minute at 55 ° C, 1 minute at 72 ° C, 1 cycle of 1 cycle of 3 minutes at 95 ° C, 1 minute at 55 ° C, 10 minutes at 72 ° C. PCR is carried out in a volume of 10 μ\ with 1 -2 //I DNA using a Robocycler, using primers (Research Genetics, Huntsville, US) end-labelled with 33 y ATP (Amersham UK). PCR products were mixed with an equal volume of loading buffer, denatured for at least 2 minutes at 95 ° C and run on 5% polyacrylamide gels at 55 ° C in 1 X TBE buffer, before fixing in 10% methanol and acetic acid. Dried gels were exposed for 24-48 hours with phosphor screens before visualisation on a Storm 840 Molecular Dynamics Imager. Mutation Detection Detection of mutations in peripheral blood was carried out at the Oxford ICRF Unit using single strand conformational polymorphism analysis (SSCP) to investigate the 1 9 exons of hMLH 1 and the 1 6 exons of hMSH2. Each exon of hMSH2 and hMLH1 was amplified specifically using PCR and detected using standard silver staining. A detailed description of mutations detection in DNA mismatch repair genes has been published elsewhere (Beck et al 1 997, Human Genetics) Results PCR- Based Microsatellite Analysis To date, 1 20 skin lesions (malignant and benign) from 43 patients from 23 well documented cancer syndrome families with pedigrees strongly suggestive of either HNPCC or other family cancer syndromes were recruited. Screening was carried out using 8 microsatellite markers. Microsatellite instability Two patients to date consistently show major MSI in a variety of skin lesions (benign and malignant) using a range of markers (Table 1 ) . The first has a personal history of breast cancer and sebaceous adenomata and epitheliomata, and a strong family history of cancers of the ovary, stomach and kidney (Pedigree 1 AM) . The other has a personal history of cancers of the breast and uterus, a sibling with colon cancer and 5 second degree relatives with malignancies, one with three primaries (Pedigree 2 IM) . Fig. 3 shows the MSI with slippage on gel electrophoresis in patients 1 and 2 respectively using the 1 3q (D 1 3S1 55) marker. Loss of Heterozyqositv LOH has been detected in 2 squamous cell carcinomas from a patient with a family history of melanoma, breast carcinoma and osteosarcoma, using a 9q marker (D9S176) (see Fig. 4) and a 1 7q marker (D1 7S785) (not shown) . Another patient with a history of multiple basal cell carcinomas and a family history of ovarian and breast carcinoma showed LOH in a basal cell carcinoma using a 1 7p marker(D 1 7S796). Mismatch repair gene mutations Mutation detection in the most commonly mutated DNA mismatch repair genes (hMSH2 and hMLH D have been carried out in five families who had a family history highly suggestive of HNPCC. Apart from bowel tumours, most of these families also presented with multiple atypical naevi with or without melanoma. Blood DNA from 3 of the 5 patients showed variants in hMSH2, although none of the variants detected was predicted to affect protein function. MSI was not detected in the skin lesions of the three patients in whom hMSH2 variants have been shown. Thus the invention provides diagnosis of cancer or predisposition to cancer by identification of MSI, and finds clinical application, inter alia, in in vitro screening and test kits. Table 1 Example 2 Clinical Features Nine lesions from 9 individuals were identified and subsequently confirmed as sebaceous carcinomas after detailed histological review. One of these patients (EG) has previously been recognised as fulfilling the criteria for Muir-Torre Syndrome (MTS); he had a past history of multiple primary colorectal adenocarcinomas, multiple sebaceous adenomas and a cutaneous squamous cell carcinoma (SCC); his father had both colorectal and laryngeal carcinomas and his brother had died from a brain tumour as had his sister and a niece. The remaining 8 patients were not known to have MTS prior to the diagnosis of sebaceous carcinoma and were therefore initially regarded as 'sporadic' sebaceous carcinomas. One striking finding was that four individuals (44.4%) were renal transplant recipients (RTRs) receiving immunosuppression with azathioprine, prednisolone and cyclosporin (DD, JA, HA) or azathioprine and prednisolone only (BM) . The average age of the RTRs was 60.5 years, and in all cases the sebaceous carcinoma arose an average of 1 1 .25 years (range 3-28 years) after transplantation. The cause of end stage renal failure differed in all 4 patients. All of the RTRs except one (DD) had previously had multiple non-melanoma skin cancers (NMSCs) and were on prophylactic systemic retinoids to reduce the frequency of occurrence of these malignancies, many of which had proved poorly differentiated and/or clinically aggressive. For example, patient JG, had a number of SCCs and basal cell carcinomas (BCCs) including a highly aggressive recurrent spindle cell SCC of the scalp which required extensive surgery and post operative radiotherapy. In 2 RTRs (JG, HA), there were also other clinical features suggestive of possible MTS. Although neither had visceral cancers of the Lynch ll/HNPCC spectrum, nor strictly fulfilled the Amsterdam Criteria for family history of cancer, JG had a carcinoid tumour of the gastric antrum removed 4 years prior to receiving a renal transplant, and HA had previously had a single benign colonic polyp removed and his mother had died of a colorectal adenocarcinoma aged 55 years. Of the 4 remaining patients, none was immunosuppressed and none had a family history of cancer or a personal history of visceral cancer. However, 2 had a past history of skin cancer; patient CV had a malignant melanoma of the ear and patient EC had a previous SCC. These 4 immunocompetent patients had an older average age of 73.8 years compared with the RTRs. Microsatellite analysis One of the tumours could not be analysed because of insufficient tumour cell content in the paraffin embedded material. Of the remaining 8 lesions, 3 showed evidence of MSI. Two of the MSI positive tumours were from RTRs (JG, DD), and one was from a patient with a previous diagnosis of MTS (EG) . Although one of the MSI positive patients (JG) had other features in keeping with MTS, the second RTR with suggestive features (HA) did not display MSI. The additional RTR tumour from patient KCH also demonstrated a similar degree of MSI. Although there are no internationally agreed criteria for the designation of MSI in extra-colonic tissue, criteria developed for the classification of MSI in the colon designate the degree of instability as high (MSI-H) if multiple novel alleles represent at ≥ 40% of loci examined (Boland, 1 998), as was the case in all 3 of the tumours displaying MSI in this study. LOH was not identified with any of the microsatellite markers in any of the tumours examined. Histopathologγ There were no correlations between the histology of the tumours and the presence of immunosuppression and/or MTS phenotype. Furthermore, there were no clear correlations between the presence of MSI and tumour pathology. We have retrospectively investigated an unselected series of sebaceous carcinomas presenting over a 1 0 year period, analysing both clinicopathological characteristics and assessing each tumour for the presence of MSI. Particularly noteworthy were the observations that: ( 1 ) Four of the 9 (44.4%) sebaceous carcinomas occurred in RTRs after the introduction of immunosuppression: (2) In 2 of the RTRs there were additional clinical features consistent with MTS: (3) Large band shifts at multiple loci characteristic of MTS/Lynch type II syndrome were observed in 3 individuals, one with known MTS and 2 of whom were RTRs; in a fifth RTR lesion obtained from another centre, a similar degree of MSI was observed: (4) Of the 4 immunocompetent patients none displayed MSI. Example 3 HNPCC patients with RER + in skin lesions demonstrated a cutaneous phenotype characterised by the presence of multiple pigmented macules or plaques on sun exposed areas of skin, particularly the dorsum of the hands, face, forearms and lower legs. Collectively, these lesions had features of flat seborrhoeic keratoses, disseminated superficial actinic pororkeratosis (DSAP) lesions or stucco keratoses, although the characteristics of individual lesions were not in keeping with these diagnoses. These individuals had a markedly sun-damaged phenotype, far in excess of the damage expected for the patients' age and reported ultraviolet light exposure. In addition to the numerous well demarcated pigmented lesions observed, these patients had the elastosis and the generalised hyperpigmentation usually seen in elderly individuals with a lifetime of sun exposure, unexpected in most UK inhabitants in their fourth and fifth decades of life. No epidermal dysplasia or histological evidence of human papillomavirus infection was noted in any lesion. Four individuals from three cancer prone families showed widespread microsatellite instability in their benign, non-sebaceous cutaneous lesions (Table 2). Three of the individuals demonstrating MSI in their skin lesions were from two families which fulfilled the 'Amsterdam criteria' for HNPCC. However, only one of these families had already been characterised as an HNPCC pedigree prior to attending the dermatology clinic. The other proband was only recognised as belonging to an HNPCC/MTS pedigree after taking a full family history in the Dermatology clinic. The presence of sebaceous adenomas confirmed the diagnosis of MTS. All skin lesions biopsied from the three probands from the two HNPCC families showed MSI. No malignant cutaneous or extracutaneous lesions were available from these cases for analysis of genetic alterations. Cases 1 , 2 and 3 were all notable for their cutaneous phenotype of apparent severe actinic damage in the absence of a history of excess sun exposure, with multiple pale brown macules and plaques over the sun exposed areas of arms, face and lower legs. For case 4 there are insufficient details available to fulfil the Amsterdam criteria although it is possible that she is indeed a member of an HNPCC family. Her cutaneous phenotype was very striking, with multiple pigmented macules and plaques on sun exposed areas of the skin, giving the appearance of severe premature actinic damage, although she did not have a history of excess UV exposure. She, however had two cancer primaries - a breast cancer and a transitional carcinoma of the renal pelvis, which is a tumour characteristic of the HNPCC spectrum. She also had a family history of colon cancer affecting her brother. Her parents also died of cancer, although the source of the tumours could not be confirmed. Table 2 NP - No product NT - Not tested * - MSI detected in a minimum of one marker for chromosome arm listed Markers used listed by chromosome arm: 9p - D9S171, D9S162, D9S174 17p - D17S796 9q - D9S160, D9S176 13q - D13S155 1p - D1S201, D1S214 10q - D10S185 References Aaltonen LA et al. Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease. The New England Journal of Medicine 1 998; 338(21 ) : 1481 -1487 Aaltonen LA et al. Replication errors in benign and malignant tumours from hereditary non polyposis colorectal cancer patients. Cancer Research 1 994;54: 1 645-1 648 Akiyama Y, Sato H, Yamada T et al. Germline mutation of the hMSH6/GTBP gene in an atypical hereditary non polyposis colorectal cancer. Cancer research 1 997;57:3920-3932. Bataille V, Grulich A, Sasieni P et al. Association between naevi, solar keratoses and melanoma: a case-control study of melanoma in New South Wales, Australia. 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Claims: CLAIMS: 1 . A method of diagnosing disease or predisposition to disease in a first tissue, comprising identifying microsatellite instability (MSI) in a second tissue, different from the first. 2. A method according to Claim 1 , for diagnosis of cancer or predisposition to cancer. 3. A method according to Claim 1 or 2 for diagnosis of disease or predisposition to disease in tissue other than skin, comprising identifying MSI in skin. 4. A method according to Claim 3, comprising identifying MSI in a skin tumour. 5. A method according to Claim 4, comprising identifying MSI in a benign skin tumour. 6. A method according to any of Claims 3 to 5, for diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) , or predisposition to HNPCC. 7. A method according to Claims 1 or 2 for diagnosis of disease or predisposition to disease in colorectal tissue, comprising identifying MSI in non-colorectal tissue. 8. A method according to Claim 7, comprising identifying MSI in a skin tumour. 9. A method according to Claim 1 or 2, for diagnosis of disease or predisposition to disease in a tissue selected from (a) stomach, uterine, kidney, renal, cervix, endometrium, pancreas, brain and ovarian tissue, comprising identifying MSI in a tissue (b), different from any (a) tissue. 10. A method according to Claim 9, comprising identifying MSI in a skin tumour. 1 1 . A method of diagnosing disease or predisposition to disease comprising, a) obtaining a biopsy sample of a first tissue; b) extracting DNA from said tissue; c) amplifying a specific region of the DNA by PCR; and d) analysing the products of the PCR reaction; wherein identification of MSI in the first tissue provides diagnosis of disease or predisposition to disease in a second tissue, different from the first. 1 2. A method according to Claim 1 1 , for diagnosis of cancer or predisposition to cancer. 1 3. A method according to Claim 1 2, for diagnosis of HNPCC or predisposition to HNPCC. 14. A method according to any of Claims 1 1 to 1 3 wherein the tissue is obtained from a skin tumour. 1 5. A method according to Claim 1 4, comprising obtaining the tissue by a skin shave. 1 6. A method for diagnosis of an individual who is predisposed to skin photodamage and/or premature skin ageing, comprising identifying MSI in a tissue of the individual. 1 7. A kit for diagnosis of disease or predisposition to disease comprising, a) PCR primers for amplification of microsatellite-containing regions of the genome; and b) positive and negative controls; wherein, in use, identification of MSI in patient DNA by comparison of the products of the PCR reaction to the controls provides diagnosis of disease or predisposition to disease. 18. A kit according to Claim 17, wherein said disease is cancer. 19. A kit according to Claim 18 for diagnosis of HNPCC or predisposition to HNPCC. 20. A kit according to Claim 17, wherein said disease is skin photodamage and/or premature skin ageing. 21. A kit according to any of Claim 17 to 20, comprising at least two or more sets of PCR primers. 22. A kit according to any of Claims 17 to 21, wherein PCR primer pairs are selected from the group consisting of D1S214 and D1S201; D6S305 and D6S262; D9S162 and D9S171: D9S160 and D9S176; D10S185 and D10S573; D11S910and D11S29; D13S155 and D13S170; D17S796and D17S520; D17S785 and D17S579; and D18S70 and D18S34. 23. A kit according to Claim 22, comprising all or a combination of the specified PCR primer pairs. 24. A method of cancer therapy comprising:- screening an individual for cancer or predisposition to cancer; and if such a cancer or predisposition to cancer is identified, treating that individual to prevent, reduce or delay the onset of cancer, wherein a cancer or predisposition to cancer in a first tissue is correlated to MSI in a second tissue, different from the first. 25. A method according to Claim 24, wherein cancer or predisposition to cancer in colorectal tissue is correlated with MSI in non-colorectal tissue. 26. A method according to Claim 25, wherein cancer in colorectal tissue is correlated with MSI in skin tumours. 27. A method according to Claim 26, wherein cancer or predisposition to cancer in non-skin tissue is correlated with MSI in skin tissue. 28. A method according to Claim 24 wherein cancer or predisposition to cancer in a tissue selected from (a) stomach, uterine, kidney, renal, cervix, endometrium, pancreas, skin, brain and ovarian tissue, is correlated with MSI in a tissue (b), being different from (a) . 29. A method of predicting response to anti-cancer therapy, comprising determining whether there is MSI in skin. 30. A method of predicting increased risk of cancer in an immunosuppressed individual by determining presence of MSI in said individual. 31 . A method according to claim 30, wherein MSI located in a first tissue diagnoses increased risk of cancer in a second tissue different from the first tissue. 32. A method according to claim 31 wherein said first tissue (a) and said second tissue (b) are selected from the group consisting of stomach, colon, uterine, kidney, renal, cervix, endometrium, pancreas, skin, brain and ovarian tissue, and said second tissue (b) is different from (a) . 33. Use of means for identification of MSI in manufacture of a means of diagnosis of disease or predisposition to disease. 34. Use according to claim 33 wherein the disease is cancer. 35. Use according to Claim 34 of PCR primers for identification of MSI in tumours of non-colorectal tissue, in manufacture of means for the diagnosis of cancer or predisposition to cancer in colorectal tissue. 36. Use according to Claim 34 of PCR primers for the identification of MSI in skin tumours, in manufacture of means for diagnosis of cancer or predisposition to cancer in non-skin tissue. 37. Use according to Claim 34 of PCR primers for identification of MSI in a test tissue, in manufacture of a means for diagnosis of cancer or predisposition to cancer in target tissue, said target tissue being different from said test tissue. 38. Use according to Claim 37 wherein said target tissue is selected from stomach, uterine, kidney, renal, endometrium, pancreas, brain and ovarian tissue. 39. Use according to claim 33 wherein the disease is skin photodamage and premature skin ageing. 40. Use according to Claim 39 of PCR primers for identification of MSI in skin in manufacture of a means for diagnosis of skin photodamage and/or premature skin ageing.

Inventors: BATAILLE VERONIQUE ALICE, BATAILLE, VERONIQUE, ALICE
Applicants: BATAILLE VERONIQUE ALICE, GEMINI RESEARCH LIMITED, BATAILLE, VERONIQUE, ALICE
Application year: 1999
Main classification: C12Q1
IPC codes: C12Q1