CIP2An rooli syövän synnyssä

Worldwide and notably in the developed countries, cancer is an increasing cause of morbidity and mortality, being the second most common cause of death after ischemic heart disease. Now and in the future new cancer cases need to be diagnosed earlier. Prognostic factors may be helpful in recognizing and handling those patients who need more aggressive therapy, and it is also desirable to predict treatment response accurately. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein predominantly expressed in malignant tissues and inhibiting protein phosphatase 2A (PP2A) activity; it is a promising target for cancer therapy. The aim of this thesis was to evaluate the prognostic role of CIP2A in solid cancers, and for this purpose to explore expression of CIP2A, and investigating regulation of CIP2A in order to gain insight into signalling pathways leading to alteration in prognosis. Patients diagnosed with gastric, serous ovarian, tongue, or colorectal cancer at Helsinki University Central Hospital were included. Tumour tissue microarrays assembled from specimens from these patients were prepared and stained immunohistochemically for CIP2A protein expression. Associations with clinicopathologic parameters and other biomarkers were explored, and survival analyses were done according to the Kaplan-Meier method. Study of the role of CIP2A in intracellular signalling in vitro involved gastric, ovarian, and tongue cancer cell lines. We found CIP2A to be highly expressed in gastric, ovarian, tongue, and colorectal cancer specimens. CIP2A was associated with clinicopathologic parameters characterizing an aggressive disease, namely advanced stage, high grade, p53 immunopositivity, and high proliferation index. CIP2A led to recognition of gastric, ovarian, and tongue cancer patients with poor prognosis, however, with a cancer type-specific cut-off level for prognostic significance. In tongue cancer, it served as an independent prognostic marker. In contrast, in colorectal cancer, CIP2A provided no prognostic value. In cancer cell lines, CIP2A was highly expressed at both protein and mRNA levels, and promoted cell proliferation and anchorage-independent growth. In gastric cancer, we demonstrated with a MYCER construct in mouse embryo fibroblasts that activation of MYC led to increased CIP2A mRNA expression, and hence we suggested that a positive feedback mechanism between CIP2A and MYC may potentiate and prolong the oncogenic activity of these proteins. We demonstrated in ovarian cancer an association between CIP2A and EGFR protein overexpression and EGFR gene amplification. In ovarian and tongue cancer cells we showed that depletion of EGFR downregulates CIP2A expression. In conclusion, high CIP2A expression occurred frequently among patients with aggressive disease. CIP2A may serve as a prognostic marker in gastric, ovarian, and tongue cancer and thus may help in tailoring therapy for cancer patients. The positive feedback mechanism between CIP2A and MYC, as well as the positive regulation of CIP2A by EGFR, are a few signalling pathways regulating and regulated by CIP2A. These and other mechanisms need to be studied further, however. CIP2A is a potential target for therapy, and its potential role as predictive marker and as a tumour marker in serum requires exploration.Högt uttryck av onkoproteinet cancerous inhibitor of protein phosphatase 2A (CIP2A) i tumörvävnad ses ofta hos cancerpatienter som har en aggressiv form av cancer. CIP2A kan förutspå en sämre prognos i magcancer, tungcancer och äggstockscancer och det kan därför möjligtvis vara till nytta för att skräddarsy behandlingen för dessa patienter. I undersökningen identifierades en positiv feedback-mekanism mellan CIP2A och onkoproteinet MYC som är känt för att stimulera celldelning. Denna och andra regleringsmekanismer av CIP2A kan användas för att i framtiden rikta in specifik terapi mot tumörer som påvisar CIP2A-proteinet. Därtill gav avhandlingsarbetet skäl till att i fortsättningen utreda huruvida CIP2A kunde fungera som en tumörmarkör för att upptäcka cancer. Cancer är en av de vanligaste sjukdomsgrupperna i västvärlden och den näst vanligaste dödsorsaken efter iskemisk hjärtsjukdom. För att förbättra prognosen blir det allt viktigare att tidigare diagnosticera cancerfall och skräddarsy behandlingen. Prognostiska faktorer kan vara gynnsamma för att identifiera de patienter som behöver mer aggressiv behandling. CIP2A förekommer främst i cancervävnader och fungerar genom att inhibera protein fosfatas 2A (PP2A) och därmed stabilisera onkoproteinet MYC. Målsättningen med avhandlingen var att utreda CIP2As prognostiska roll hos cancerpatienter samt att studera hur regleringen av CIP2A-proteinet eventuellt kunde påverka en avvikande prognos. Vävnadsprover från patienter med magcancer, äggstockscancer, tungcancer och tjock- och ändtarmscancer samlades och färgades immunhistokemiskt för CIP2A proteinexpression. Associationer med vanliga kliniskpatologiska faktorer och andra biomarkörer studerades och därtill gjordes överlevnadsanalyser. CIP2As roll i intracellulär signallering studerades även i mag-, ovarie- och tungcancercellinjer. I magcancer-, äggstockscancer- och tungcancerpatienter fungerade högt uttryck av CIP2A-proteinet som prognostisk faktor, men däremot inte i tjock- och ändtarmscancerpatienter. Styrkan av uttrycket varierade i de olika cancerformerna och därför bör gränsvärdet för CIP2A-positivitet i tumörvävnad vid bedömning av prognos slås fast specifikt för varje enskild cancerform.Syöpäproteiini cancerous inhibitor of protein phosphatase 2A :n (CIP2A) vahva ilmentyminen kasvainkudoksessa voi kuvata huonoa ennustetta mahasyöpä-, kielisyöpä- ja munasarjasyöpäpotilailla ja voi siten mahdollisesti olla hyödyksi hoidon räätälöimisessä näille potilaille. Tässä väitöstutkimuksessa löydettiin positiivinen yhteys CIP2An ja syöpäsolujen kasvun säätelyssä tärkeän MYC-onkoproteiinin välillä, joka voisi siten olla lupaava syöpähoitojen kohde CIP2A-positiivisissa kasvaimissa. Lisäksi löydettiin viitteitä siitä, että olisi hyödyllistä tutkia CIP2An ilmentymää myös kasvainmerkkiaineena syövän löytämisessä ja toteamisessa. Syöpätaudit aiheuttavat suuren osan sairastavuudesta ja ovat toiseksi yleisin kuolinsyy länsimaissa iskeemisen sydänsairauden jälkeen. Potilaan ennusteen parantamiseksi on tärkeää todeta syöpä sekä löytää ennusteellisia tekijöitä jotka voivat olla hyödyksi tehokkaampaa hoitoa vaativien potilaiden tunnistamisessa. CIP2A-proteiini ilmentyy lähinnä syöpäkudoksissa ja estää proteiinifosfataasi 2An (PP2A) toimintaa stabiloiden siten MYC-onkoproteiinia. Tämän väitöstutkimuksen tavoitteena oli selvittää miten CIP2A toimii ennusteellisena tekijänä syöpäpotilailla ja tutkia miten CIP2A-proteiinin säätely mahdollisesti vaikuttaa ennusteeseen. Mahasyöpä-, munasarjasyöpä-, kielisyöpä- ja paksu- ja peräsuolisyöpäpotilailta kerättiin kudosnäytteitä, jotka värjättiin immunohistokemiallisesti CIP2An ilmentymisen osoittamiseksi. CIP2An yhteyttä kliinispatologisiin tekijöihin ja muihin biomarkkereihin tutkittiin ja lisäksi analysoitiin CIP2A-ilmentymisen yhteyttä eloonjäämiseen. CIP2An merkitystä solunsisäisessä signaloinnissa tutkittiin maha-, munasarja- ja kielisyöpäsolulinjoissa. CIP2A-proteiinin vahva ilmentyminen kuvasi huonoa ennustetta mahasyöpä-, munasarjasyöpä- ja kielisyöpäpotilailla, ei kuitenkaan paksu- ja peräsuolisyöpäpotilailla. Ennusteellisen merkityksen saavuttamiseksi tarvittiin jokaiselle syöpäkudokselle oma raja-arvo CIP2An ilmentymisen suhteen. Tuloksien yleistäminen muihin syöpätauteihin on siten haastavaa ja tulevaisuudessa tuleekin arvioida CIP2An ennusteellista arvoa syöpäkohtaisesti

Epidermal growth factor receptor and other tissue biomarkers in gastrointestinal cancers

Personalised medicine has plays an increasing role in the treatment of cancer. New therapeutic molecules are being developed, but their compatibility for each patient has to be tested before starting the treatment by examining the appropriate tissue biomarkers expressed in the tumour. These biomarkers can be utilised not only in treatment selection but also in predicting treatment efficacy and patient survival. They can also be used to classify tumours into specific molecular subtypes that have distinct characteristics related to tumour behaviour, response to cancer treatments and prognosis of the patients. In order to implement these classifications in clinical practice, instead of time-consuming sequencing-based methods, the methods have to be simple enough and easy to interpret. Gastrointestinal cancers are among the most prevalent malignancies and often lead to death. Monoclonal antibodies against the epidermal growth factor receptor (EGFR) can be used in the treatment of RAS wild-type metastatic colorectal cancer. It has been shown that in addition to RAS mutation testing, determining the EGFR gene copy number (GCN) of the tumours can aid in selecting the patients likely to benefit from the antibody treatment. In oesophagogastric cancer, EGFR GCN has not yet been shown to have a predictive role, although the overexpression of HER2, which belongs to the same receptor family as EGFR, is used as a biomarker to predict response to anti-HER2 antibody treatment. In this thesis, the prevalence of EGFR amplification was observed to be at a similar level with the prevalence of HER2 amplification specifically among the intestinal-type oesophagogastric adenocarcinomas from 220 patients. This implies that it might be useful to examine whether EGFR GCN analysis could function as a biomarker predicting anti-EGFR treatment response in the intestinal-type tumours. In addition, in this thesis, tissue microarray was used to detect the different molecular subtypes of oesophagogastric cancers from 244 patients by staining methods applicable to clinical practice. Comparative studies detecting EGFR GCN in primary colorectal tumours and their metastases are scarce. In this thesis, corresponding primary and metastatic tumours from 80 patients were examined. EGFR GCN was observed to decrease between the primary and metastatic tumours during anti-EGFR treatment but to remain stable or even increase among patients not receiving treated with anti-EGFR antibodies. This EGFR GCN change may be relevant regarding the clinical response to anti-EGFR treatment. Preoperative chemoradiotherapy can be used in the treatment of rectal cancer patients to enable a complete resection of the tumour or reduce the risk of local recurrence. However, treatment response among patients is variable. Thus, a suitable biomarker could be helpful in predicting response or stratifying patients into separate treatment groups according to their prognosis. In this thesis, CIP2A expression was examined in rectal cancer tissue samples from 210 patients. Low CIP2A expression level was observed to associate with a better response among patients treated with long-course chemoradiotherapy. Affirming results were obtained in cell culture studies, where the suppression of CIP2A expression was observed to render the cells more sensitive to irradiation than the cells with normal CIP2A expression.Epidermaalinen kasvutekijäreseptori ja muita kudosbiomarkkereita ruoansulatuskanavan syövissä Yksilöllistetty lääketiede on yhä merkittävämmässä osassa myös syöpätautien hoidossa. Syövän hoitoon kehitetään jatkuvasti uusia lääkemolekyylejä, joiden soveltuvuus kullekin potilaalle on ennen hoidon aloitusta selvitettävä kasvaimen ilmentämien molekulaaristen biomarkkereiden avulla. Biomarkkereita voidaan käyttää paitsi hoitojen valitsemisen apuna niin myös syövän käyttäytymisen ja potilaiden ennusteen arvioimiseen. Biomarkkereiden avulla tietty syöpätyyppi voidaan myös luokitella molekulaarisiin alatyyppeihin, joilla on toisistaan poikkeavia kasvaimen käyttäytymiseen, ennusteeseen ja hoitovasteisiin liittyviä ominaisuuksia. Molekulaaristen luokittelun käytännön soveltamiseen tarvitaan kuitenkin riittävän yksinkertaisia ja helposti tulkittavissa olevia menetelmiä aikaa vievien sekvennointitutkimusten sijaan. Ruoansulatuskanavan syövät ovat yleisimpiä pahanlaatuisia kasvaimia ja johtavat usein kuolemaan. Levinneen suolistosyövän hoidossa voidaan käyttää epidermaaliseen kasvutekijäreseptoriin (EGFR) kohdistuvaa vasta-ainehoitoa, mikäli kasvaimessa ei ole osoitettavissa Ras-geenimutaatiota. Aikaisemmin on todettu, että Ras-geenitestin ohella EGFR:n geenikopiomäärän selvittämisen avulla voidaan valikoida pelkkää geenitestiä paremmin hoidosta todennäköisesti hyötyvät potilaat. Maha- ja ruokatorvisyövässä suurentuneen EGFR:n geenikopiomäärän ennusteellista merkitystä ei kuitenkaan ole vielä pystytty osoittamaan, vaikka samaan reseptoriperheeseen kuuluvan HER2:n geenimonistuman tiedetään ennustavan siihen kohdistuvan vasta-ainehoidon tehoa. Väitöstutkimuksessa EGFR:n geenimonistuman yleisyyden todettiin 220 potilaan aineistossa olevan nimenomaan intestinaalisen alatyypin maha- ja ruokatorvisyövissä HER2:n monistuman tasoa, joten sen selvittämisestä voisi olla hyötyä tutkittaessa EGFR-vasta-ainehoidon tehoa maha-ja ruokatorvisyövän hoidossa. Lisäksi maha- ja ruokatorvisyöpien molekulaaristen alatyyppien tunnistamista selvitettiin 244 potilaan näytteistä koostetun kudosmikrosirun avulla, ja tunnistamisen todettiin onnistuvan myös kliiniseen diagnostiikkaan soveltuvien menetelmien avulla. EGFR:n geenikopiomäärää alkuperäisen paksusuolikasvaimen ja sen lähettämän etäpesäkkeen välillä vertailevia tutkimuksia on tehty niukasti. Väitöstutkimuksessa verrattiin 80 potilaan primaari- ja metastaattisia kasvaimia. EGFR:n kopiolukumäärän todettiin pienenevän vasta-ainehoitoa saaneilla potilailla mutta pysyvän samana tai jopa suurentuvan muuta hoitoa saaneiden potilaiden etäpesäkkeissä. Kopiolukumäärän muutoksella saattaisi olla merkitystä vasta-ainehoidon tehon kannalta. Peräsuolipotilaiden hoidossa voidaan käyttää ennen leikkausta annettavaa kemosädehoitoa, jonka avulla pyritään mahdollistamaan kasvaimen täydellinen poisto. Osa potilaista jää kuitenkin ilman merkittävää hoitovastetta tai saa haitallisia sivuvaikutuksia, joten soveltuvasta biomarkkerista voisi olla hyötyä hoidon kohdentamisen parantamisessa. CIP2A:n ilmentymistä tutkittiin 210 potilaan peräsuolisyöpänäytteissä, ja matalan ilmenemistason todettiin olevan yhteydessä parempaan vasteeseen pitkän kemosädehoidon saaneilla potilailla. Samansuuntainen tulos saatiin syöpäsoluviljelmien sädetyskokeissa, joissa sädetyksen todettiin estävän enemmän CIP2A:ta ilmentämättömien kuin sitä normaalisti ilmentävien solujen kasvua

Spectrum of autoantibody response to tumour associated antigens in normal population

Early detection and diagnosis of cancer has a significant impact on cancer specific mortality as shown in randomised screening trials for breast, colon and lung cancer. However, current screening tests have limitations as they reduce cancer specific mortality for breast, colon and lung cancer by only 24%, 16% and 20% respectively, and partly as a result cancer continues to be one of the common causes of death in the developed world(Aberle et al, 2011; Hardcastle et al, 1996; Larsson et al, 1996). One of the other problems associated with current screening tests is patient compliance (mammography, colonoscopy and CT) (Jonnalagadda et al, 2012; Maurer, 1995; Pooler et al, 2012). Diagnosing cancers with a blood test by identifying tumour associated antibodies in serum is a novel method which may allow the identification of early stage cancers and hopefully it would have greater patient acceptability. These tumour associated antibodies represent an indirect amplified signal generated as a response by the immune system to tumour associated antigens secreted early on in development of cancer. One of the common limitations of many autoantibody studies is the selection of appropriate controls - or the lack of such. One common problem is the use of limited number of normal individuals without cancer as controls, the data from which may not be representative of the normal population as a whole (Stockert et al, 1998). In addition not only the numbers of controls are often incorrect but also the age of the controls. Many studies report using ‘blood donors’ as controls(Guy et al, 1981) and clearly for most tumour types this involves both a younger population and also a relatively health population which may not always be reflective of the individuals to be screened (e.g. compare heavy smokers). We hypothesised that autoantibody response to cancer associated antigens may alter with demographics (age, sex, and smoking) and the aim of our study was to identify the spectrum of response of tumour associated antigens in a range of demographic groups within the normal population of the East Midlands. EarlyCDT-Lung™ is a simple commercial blood test which is reported to aid the early detection of lung cancer. The technology was initially developed in the laboratories of the Division of Breast Surgery and subsequently underwent further development by the university spinout company, Oncimmune. EarlyCDT-Lung initially measured autoantibodies (AAbs) to six cancer associated antigens (p53, NY-ESO-1, CAGE, GBU4-5, Annexinl, and SOX2) and was reported to identify up to 40% of lung cancers, at both at eariy- and late-stage disease(Boyle et al, 2011; Chapman et al, 2012; Chapman et al, 2011; Lam et al, 2011; Macdonald et al, 2012a; Macdonald et al, 2012b; Murray et al, 2010).The initial technical (Murray et al 2010) and clinical (Boyle et a;l 2011; Lam et al 2011) validation studies matched high risk individuals to every lung cancer patient. Controls were individually matched to a patient with lung cancer by age, gender and smoking history. As the 6 antigen panel had been developed and validated (Boyle et al, 2011; Murray et al, 2010) it was decided to proceed to assess the level of autoantibodies across the population and in particular to look at differences by gender and different decades of life. We used a semi-automated Enzyme linked immunosorbent assays (ELISA) to run the serum samples collected from individuals with no previous history of cancer. Informed consent was taken prior to a detailed health questionnaire and then a blood sample by standard venipunture. The information acquired in the questionnaire included age, gender, smoking history, any autoimmune disease and family history of cancer. Serum samples used in this thesis were collected from 2065 individuals. Male to female ratio was 1: 2.6(566:1487). Ratio of smoker versus ex-smokers versus non-smoker was 1: 2.4: 3.8 (285:672:1096). There was a fall in the number of smokers with increasing decade of life. The proportion of smokers and ex-smokers versus non-smokers remained approximately the same in both genders. Almost half the patients (964) had family history of some form of cancer. One hundred and eighty six subjects (9%) had personal history of autoimmune disease. Analysis of autoantibody levels revealed a small but steady increase with increasing age for 4 out of the 6 antigens (p53, NY-ESO-1, CAGE and GBU4- 5). Except for CAGE, there was no significant difference in mean optical densities between males and females. For CAGE, when analysis of variance was used to adjust for run differences, there was no significant difference in mean optical densities between males and females. Autoantibody response to all 6 cancer related antigens were consistently low in smokers. The rise in autoantibody response was more in the ex-smoker group compared to the other two groups suggesting the possibility of rebound effect when smoking is stopped. It reached statistical significance except in case of NY-ESO-1. Age matched analysis were done, and the differences were statistically significant for p53, GBU 4-5 and Annexinl. To explore further the "rebound" hypothesis further, the year of quitting for ex-smokers were extracted from the database. Any association between AAb levels and time lapse since quitting might provide support to this hypothesis. Very little difference was seen for most antigens back to 1970, but decades before that there was an observed increase in the mean AAb level for all antigens except SOX2. Further work would be required to establish such a rebound effect. Family history and history of autoimmune disease did not have a significant impact on autoantibody levels. Analysis of autoantibody levels in a large cohort of the normal population of the East Midlands revealed that age has a small but significant influence on the serum levels of certain autoantibodies to cancer related antigens. However, this could be confounded by the fact that incidence of cancer also increases with age, and would need further investigation and in particular longer followup of patients who have given blood in this research study to see which individuals have developed cancer

Hereditary colorectal cancer: A clinical and molecular genetic study

The general aim of this thesis was to advance our understanding of the genetics of colorectal cancer. The specific aims were 1) to assess the psychological impact of familial adenomatous polyposis and patient attitude to predictive DNA testing, 2) to assess the prognostic value of allele loss in colorectal cancer and 3) to assess the effectiveness of a family cancer clinic for the targeted screening of colorectal cancer. Attitudes to predictive DNA testing and the psychological impact of familial adenomatous polyposis (FAP) were documented in 62 affected adults. In the majority of cases, FAP, appeared to have a fairly minimal impact on the everyday life of the patient. However, in a significant minority (20%), a diagnosis of FAP had a devastating effect on psychological well being. Factors which might be important include a previous unpleasant experience with an ileostomy, a history of cancer death within the family, a poor understanding of the mode of transmission of FAP and a perceived delay in diagnosis. With respect to patient views on prenatal testing and termination of pregnancy for FAP, fifteen (24%) of those questioned stated that they would proceed to termination of pregnancy if a prenatal test indicated that the unborn baby was affected. Six (10%) of people who had refrained from having children for fear of passing on the polyposis gene felt that the arrival of prenatal testing would enable them to consider planning a family. The majority of patients (93%) said that they would like their children tested by DNA analysis at birth or infancy, but felt that 10-12 years was the most appropriate time to discuss the diagnosis with the child. The frequency of allele loss at the APC, P53, Nm23 and DCC gene loci was investigated in a panel of 63 colorectal cancer specimens by Southern blotting analysis and correlation with prognosis studied. Sixty-percent of the specimens studied, exhibited allele loss for at least one genetic marker ( 52% at the P53 locus, 38% at the APC locus and 36% at the DCC locus). No allele loss was found at the Nm 23 locus. Univariate analysis found that allele loss on chromosome 17p was related to prognosis ( P < 0.02), although a multivariate analysis, including other accepted prognostic indicators for colorectal cancer, failed to support this association. The results of screening individuals referred to the Family Cancer Clinic at St Mark's Hospital, London, are described. Colonoscopy was performed in 644 asymptomatic individuals (from 436 families) with a family history of colorectal cancer over a six year period. Families were subdivided according to family history and sixty nine (15.8%) of the families fulfilled the Amsterdam criteria for the hereditary nonpolyposis colorectal cancer syndromes (HNPCC). Seven cases of colorectal cancer were diagnosed at an average age of 49 years; six at Dukes' stage A and one at stage C; four in HNPCC families. One hundred and forty four (22.4%) subjects had one or more adenomas. The prevalence of adenomas in the subjects from Amsterdam criteria families was 34 of 127 (26.8%) compared with 110 of 517 (21.3%) in non - Amsterdam criteria families. Men were twice as likely to develop adenomas as women, and the prevalence of adenomas increased in both sexes with age; the odds ratio (O.R.) increasing approximately two-fold for each decade (p or = 2 versus 1) affected by colorectal cancer or adenomas was a highly significant independent variable associated with a an increased risk of adenoma development. As part of this project, a very large kindred, St Marks' Family 96, was identified, which appears to have an autosomal dominant predisposition to an atypical polyposis syndrome and colorectal cancer. This syndrome has been called the "Hereditary Mixed Polyposis Syndrome" (HMPS) by the author. Affected individuals usually present in the fourth decade with symptomatic polyps or cancer. Although adenomatous and hyperplastic polyps occur in affected members, the characteristic lesion is an atypical juvenile polyp. Some individuals have developed polyps of more than one type, and individual polyps may have mixed histological features. Typically, fewer than 15 polyps are found at colonoscopy, and there is no extracolonic disease associated with the development of polyps. St Mark's Family 96 consist of 10 second generation, 35 third generation, 63 fourth generation and 42 fifth generation individuals. All surviving members are derived from the third, fourth and fifth generations, and updated clinical information has been obtained in 71 patients over the age of 21 years. Thirty three members (13 females, 19 males) are known to have developed either colorectal cancer or polyps. A genetic linkage study was performed on this family using 77 genetic markers spanning the genome. Data did not support linkage to the APC locus or any of the loci responsible for HNPCC. The most positive LOD score (0.69) was obtained with the marker D6S44 which maps to chromosome 6p21-qter. Although the gene responsible for HMPS was not localised within the time period of this project, a subsequent linkage study found significant two-point LOD scores for linkage between HMPS and the D6S283 locus. Analysis of recombinants and multipoint linkage analysis suggests that the gene responsible for HMPS lies in a 4-cM interval containing the D8S283 locus and flanked by markers D6S468 and D6S301