Tumor-suppressor gene promoter hypermethylation in saliva of head and neck cancer patients

Head and neck squamous cell carcinoma (HNSCC) accounts for a bulk of the oral and laryngeal cancers, the majority (70%) of which are associated with smoking and excessive drinking, major known risk factors for the development of HNSCC. In contrast to reports that suggest an inverse relationship between smoking and global DNA CpG methylation, hypermethylation of promoters of a number of genes was detected in saliva collected from patients with HNSCC. Using a sensitive methylation-specific polymerase chain reaction (MSP) assay to determine specific methylation events in the promoters of RASSF1A, DAPK1, and p16 genes, we demonstrate that we can detect tumor presence with an overall accuracy of 81% in the DNA isolated from saliva of patients with HNSCC (n = 143) when compared with the DNA isolated from the saliva of healthy nonsmoker controls (n = 31). The specificity for this MSP panel was 87% and the sensitivity was 80%(with a Fisher exact test P < .0001). In addition, the test panel performed extremely well in the detection of the early stages of HNSCCs, with a sensitivity of 94% and a specificity of 87%, and a high. concordance value of 0.8, indicating an excellent overall agreement between the presence of HNSCC and a positive MSP panel result. In conclusion, we demonstrate that the promoter methylation of RASSF1A, DAPK1, and p16 MSP panel is useful in detecting hypermethylation events in a noninvasive manner in patients with HNSCC.

The tumour-promoting receptor tyrosine kinase, EphB4, regulates expression of Integrin-β8 in prostate cancer cells

Background The EphB4 receptor tyrosine kinase is overexpressed in many cancers including prostate cancer. The molecular mechanisms by which this ephrin receptor influences cancer progression are complex as there are tumor-promoting ligand-independent mechanisms in place as well as ligand-dependent tumor suppressive pathways. Methods We employed transient knockdown of EPHB4 in prostate cancer cells, coupled with gene microarray analysis, to identify genes that were regulated by EPHB4 and may represent linked tumor-promoting factors. We validated target genes using qRT-PCR and employed functional assays to determine their role in prostate cancer migration and invasion. Results We discovered that over 500 genes were deregulated upon EPHB4 siRNA knockdown, with integrin β8 (ITGB8) being the top hit (29-fold down-regulated compared to negative non-silencing siRNA). Gene ontology analysis found that the process of cell adhesion was highly deregulated and two other integrin genes, ITGA3 and ITGA10, were also differentially expressed. In parallel, we also discovered that over-expression of EPHB4 led to a concomitant increase in ITGB8 expression. In silico analysis of a prostate cancer progression microarray publically available in the Oncomine database showed that both EPHB4 and ITGB8 are highly expressed in prostatic intraepithelial neoplasia, the precursor to prostate cancer. Knockdown of ITGB8 in PC-3 and 22Rv1 prostate cancer cells in vitro resulted in significant reduction of cell migration and invasion. Conclusions These results reveal that EphB4 regulates integrin β8 expression and that integrin β8 plays a hitherto unrecognized role in the motility of prostate cancer cells and thus targeting integrin β8 may be a new treatment strategy for prostate cancer.

Runs of homozygosity and a cluster of vulvar cancer in young Australian Aboriginal women

Objective A cluster of vulvar cancer exists in young Aboriginal women living in remote communities in Arnhem Land, Australia. A genetic case–control study was undertaken involving 30 cases of invasive vulvar cancer and its precursor lesion, high-grade vulvar intraepithelial neoplasia (VIN), and 61 controls, matched for age and community of residence. It was hypothesized that this small, isolated population may exhibit increased autozygosity, implicating recessive effects as a possible mechanism for increased susceptibility to vulvar cancer. Methods Genotyping data from saliva samples were used to identify runs of homozygosity (ROH) in order to calculate estimates of genome-wide homozygosity. Results No evidence of an effect of genome-wide homozygosity on vulvar cancer and VIN in East Arnhem women was found, nor was any individual ROH found to be significantly associated with case status. This study found further evidence supporting an association between previous diagnosis of CIN and diagnosis of vulvar cancer or VIN, but found no association with any other medical history variable. Conclusions These findings do not eliminate the possibility of genetic risk factors being involved in this cancer cluster, but rather suggest that alternative analytical strategies and genetic models should be explored.

Guaiac versus immunochemical tests: Faecal occult blood test screening for colorectal cancer in a rural community

Objective: To describe patient participation and clinical performance in a colorectal cancer (CRC) screening program utilising faecal occult blood test (FOBT). Methods: A community-based intervention was conducted in a small, rural community in north Queensland, 2000/01. One of two FOBT kits – guaiac (Hemoccult-ll) or immunochemical (Inform) – was assigned by general practice and mailed to participants (3,358 patients aged 50–74 years listed with the local practices). Results: Overall participation in FOBT screening was 36.3%. Participation was higher with the immunochemical kit than the guaiac kit (OR=1.9, 95% Cl 1.6-2.2). Women were more likely to comply with testing than men (OR=1.4, 95% Cl 1.2-1.7), and people in their 60s were less likely to participate than those 70–74 years (OR=0.8, 95% Cl 0.6-0.9). The positivity rate was higher for the immunochemical (9.5%) than the guaiac (3.9%) test (χ2=9.2, p=0.002), with positive predictive values for cancer or adenoma of advanced pathology of 37.8% (95% Cl 28.1–48.6) for !nform and 40.0% (95% Cl 16.8–68.7) for Hemoccult-ll. Colonoscopy follow-up was 94.8% with a medical complication rate of 2–3%. Conclusions: An immunochemical FOBT enhanced participation. Higher positivity rates for this kit did not translate into higher false-positive rates, and both test types resulted in a high yield of neoplasia. Implications: In addition to type of FOBT, the ultimate success of a population-based screening program for CRC using FOBT will depend on appropriate education of health professionals and the public as well as significant investment in medical infrastructure for colonoscopy follow-up.

Genetic Basis of Pituitary Adenoma Predisposition

Much of the global cancer research is focused on the most prevalent tumors; yet, less common tumor types warrant investigation, since A rare disorder is not necessarily an unimportant one . The present work discusses a rare tumor type, the benign adenomas of the pituitary gland, and presents the advances which, during the course of this thesis work, contributed to the elucidation of a fraction of their genetic background. Pituitary adenomas are benign neoplasms of the anterior pituitary lobe, accounting for approximately 15% of all intracranial tumors. Pituitary adenoma cells hypersecrete the hormones normally produced by the anterior pituitary tissue, such as growth hormone (GH) and prolactin (PRL). Despite their non-metastasizing nature, these adenomas can cause significant morbidity and have to be adequately treated; otherwise, they can compromise the patient s quality of life, due to conditions provoked by hormonal hypersecretion, such as acromegaly in the case of GH-secreting adenomas, or due to compressive effects to surrounding tissues. The vast majority of pituitary adenomas arise sporadically, whereas a small subset occur as component of familial endocrine-related tumor syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 is caused by germline mutations in the MEN1 tumor suppressor gene (11q13), whereas the majority of CNC cases carry germline mutations in the PRKAR1A gene (17q24). Pituitary adenomas are also encountered in familial settings outside the context of MEN1 and CNC, but unlike in the latter syndromes, their genetic background until recently remained elusive. Evidence in previous literature supported the notion that a tumor suppressor gene on 11q13, residing very close to but still distinct from MEN1, causes genetic susceptibility to pituitary tumors. The aim of the study was to identify the genetic cause of a low penetrance form of Pituitary Adenoma Predisposition (PAP) in families from Northern Finland. The present work describes the methodological approach that led to the identification of aryl hydrocarbon receptor interacting protein (AIP) as the gene causing PAP. Combining chip-based technologies (SNP and gene expression arrays) with traditional gene mapping methods and genealogy data, we showed that germline AIP mutations cause PAP in familial and sporadic settings. PAP patients were diagnosed with mostly adenomas of the GH/PRL-secreting cell lineage. In Finland, two AIP mutations accounted for 16% of all patients diagnosed with GH-secreting adenomas, and for 40% of patients being younger than 35 years of age at diagnosis. AIP is suggested to act as a tumor suppressor gene, a notion supported by the nature of the identified mutations (most are truncating) and the biallelic inactivation of AIP in the tumors studied. AIP has been best characterized as a cytoplasmic interaction partner of aryl hydrocarbon receptor (AHR), also known as dioxin receptor, but it has other partners as well. The mechanisms that underlie AIP-mediated pituitary tumorigenesis are to date largely unknown and warrant further investigation. Because AIP was identified in the genetically homogeneous Finnish population, it was relevant to examine its contribution to PAP in other, more heterogeneous, populations. Analysis of pituitary adenoma patient series of various ethnic origins and differing clinical settings revealed germline AIP mutations in all cohorts studied, albeit with low frequencies (range 0.8-7.4%). Overall, PAP patients were typically diagnosed at a young age (range 8-41 years), mainly with GH-secreting adenomas, without strong family history of endocrine disease. Because many PAP patients did not display family history of pituitary adenomas, detection of the condition appeared challenging. AIP immunohistochemistry was tested as a molecular pre-screening tool on mutation-positive versus mutation-negative tumors, and proved to be a potentially useful predictor of PAP. Mutation screening of a large cohort of colorectal, breast, and prostate tumors did not reveal somatic AIP mutations. These tumors, apart from being the most prevalent among men and women worldwide, have been associated with acromegaly, particularly colorectal neoplasia. In this material, AIP did not appear to contribute to the pathogenesis of these common tumor types and other genes seem likely to play a role in such tumorigenesis. Finally, the contribution of AIP in pediatric onset pituitary adenomas was examined in a unique population-based cohort of sporadic pituitary adenoma patients from Italy. Germline AIP mutations may account for a subset of pediatric onset GH-secreting adenomas (in this study one of seven GH-secreting adenoma cases or 14.3%), and appear to be enriched among young (≤25 years old) patients. In summary, this work reveals a novel tumor susceptibility gene, namely AIP, which causes genetic predisposition to pituitary adenomas, in particular GH-secreting adenomas. Moreover, it provides molecular tools for identification of individuals predisposed for PAP. Further elaborate studies addressing the functional role of AIP in normal and tumor cells will hopefully expand our knowledge on endocrine neoplasia and reveal novel cellular mechanisms of pituitary tumorigenesis, including potential drug targets.

Molecular basis of colorectal cancer predisposition

Colorectal cancer (CRC) is one of the most frequent malignancies in Western countries. Inherited factors have been suggested to be involved in 35% of CRCs. The hereditary CRC syndromes explain only ~6% of all CRCs, indicating that a large proportion of the inherited susceptibility is still unexplained. Much of the remaining genetic predisposition for CRC is probably due to undiscovered low-penetrance variations. This study was conducted to identify germline and somatic changes that contribute to CRC predisposition and tumorigenesis. MLH1 and MSH2, that underlie Hereditary non-polyposis colorectal cancer (HNPCC) are considered to be tumor suppressor genes; the first hit is inherited in the germline and somatic inactivation of the wild type allele is required for tumor initiation. In a recent study, frequent loss of the mutant allele in HNPCC tumors was detected and a new model, arguing against the two-hit hypothesis, was proposed for somatic HNPCC tumorigenesis. We tested this hypothesis by conducting LOH analysis on 25 colorectal HNPCC tumors with a known germline mutation in the MLH1 or MSH2 genes. LOH was detected in 56% of the tumors. All the losses targeted the wild type allele supporting the classical two-hit model for HNPCC tumorigenesis. The variants 3020insC, R702W and G908R in NOD2 predispose to Crohn s disease. Contribution of NOD2 to CRC predisposition has been examined in several case-control series, with conflicting results. We have previously shown that 3020insC does not predispose to CRC in Finnish CRC patients. To expand our previous study the variants R702W and G908R were genotyped in a population-based series of 1042 Finnish CRC patients and 508 healthy controls. Association analyses did not show significant evidence for association of the variants with CRC. Single nucleotide polymorphism (SNP) rs6983267 at chromosome 8q24 was the first CRC susceptibility variant identified through genome-wide association studies. To characterize the role of rs6983267 in CRC predisposition in the Finnish population, we genotyped the SNP in the case-control material of 1042 cases and 1012 controls and showed that G allele of rs6983267 is associated with the increased risk of CRC (OR 1.22; P=0.0018). Examination of allelic imbalance in the tumors heterozygous for rs6983267 revealed that copy number increase affected 22% of the tumors and interestingly, it favored the G allele. By utilizing a computer algorithm, Enhancer Element Locator (EEL), an evolutionary conserved regulatory motif containing rs6983267 was identified. The SNP affected the binding site of TCF4, a transcription factor that mediates Wnt signaling in cells, and has proven to be crucial in colorectal neoplasia. The preferential binding of TCF4 to the risk allele G was showed in vitro and in vivo. The element drove lacZ marker gene expression in mouse embryos in a pattern that is consistent with genes regulated by the Wnt signaling pathway. These results suggest that rs6983267 at 8q24 exerts its effect in CRC predisposition by regulating gene expression. The most obvious target gene for the enhancer element is MYC, residing ~335 kb downstream, however further studies are required to establish the transcriptional target(s) of the predicted enhancer element.

Mode of action of antitumour antibiotics: Spectrophotometric studies on the interaction of chromomycin A3 with DNA and chromatin of normal and neoplastic tissue

The binding of chromomycin A3, an antitumour antibiotic, to various DNA and chromatin isolated from mouse and rat liver, mouse fibrosarcoma and Yoshida ascites sarcoma cells was studied spectrophotometrically at 29°C in 10−2 M Tris-HCl buffer, pH 8.0, containing small amounts of MgCl2 (4.5 · 10−5−25 · 10−5 M). An isobestic point at 415 nm was observed when chromomycin A3 was gradually titrated with Image and its spectrum shifted towards higher wavelength. The rates and extent of these spectral changes were found to be dependent on the concentration of Mg2+. The change in absorbance at 440 nm was used to calculate apparent binding constant (Ka p M−1) and sites per nucleotide (n) from Scatchard plots for various DNA and chromatins. As expected, values of n for chromatin (0.06–0.10) were found to be lower than that found for corresponding DNA (0.10–0.15). Apparently no such correlation exists between binding constants (Ka p M−1 · 10−4) of DNA (6.4–11.2) and of chromatin (3.1–8.3), but Ka p M−1 of chromatin isolated from mouse fibrosarcoma and Yoshida ascites sarcoma are 1.5–3 times higher than that found for mouse and rat liver chromatin. These differences may be taken to indicate structural difference in nucleoprotein complexes caused by neoplasia. The relevance of this finding to tumour suppressive action of chromomycin A3 is discussed.

Structure-Function Studies of GDNF Family Ligand-RET signalling

Glial cell line-derived neurotrophic factor (GDNF) and its family members neurturin (NRTN), artemin (ARTN) and persephin (PSPN) are growth factors, which are involved in the development, differentiation and maintenance of many neuron types. In addition, they function outside of the nervous system, e.g. in the development of kidney, testis and liver. GDNF family ligand (GFL) signalling happens through a tetrameric receptor complex, which includes two glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor (GFRα) molecules and two RET (rearranged during transfection) receptor tyrosine kinases. Each of the ligands binds preferentially one of the four GFRα receptors: GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The signal is then delivered by RET, which cannot bind the GFLs on its own, but can bind the GFL-GFRα complex. Under normal cellular conditions, RET is only phosphorylated on the cell surface after ligand binding. At least the GDNF-GFRα1 complex is believed to recruit RET to lipid rafts, where downstream signalling occurs. In general, GFRαs consist of three cysteine-rich domains, but all GFRα4s except for chicken GFRα4 lack domain 1 (D1). We characterised the biochemical and cell biological properties of mouse PSPN receptor GFRα4 and showed that it has a significantly weaker capacity than GFRα1 to recruit RET to the lipid rafts. In spite of that, it can phosphorylate RET in the presence of PSPN and contribute to neuronal differentiation and survival. Therefore, the recruitment of RET to the lipid rafts does not seem to be crucial for the biological activity of all GFRα receptors. Secondly, we demonstrated that GFRα1 D1 stabilises the GDNF-GFRα1 complex and thus affects the phosphorylation of RET and contributes to the biological activity. This may be important in physiological conditions, where the concentration of the ligand or the soluble GFRα1 receptor is low. Our results also suggest a role for D1 in heparin binding and, consequently, in the biodistribution of released GFRα1 or in the formation of the GFL-GFRα-RET complex. We also presented the crystallographic structure of GDNF in the complex with GFRα1 domains 2 and 3. The structure differs from the previously published ARTN-GFRα3 structure in three significant ways. The biochemical data verify the structure and reveal residues participating in the interactions between GFRα1 and GDNF, and preliminarily also between GFRα1 and RET and heparin. Finally, we showed that, the precursor of the oncogenic MEN 2B (multiple endocrine neoplasia type 2) form of RET gets phosphorylated already during its synthesis in the endoplasmic reticulum (ER). We also demonstrated that it associates with Src homology 2 domain-containing protein (SHC) and growth factor receptor-bound protein (GRB2) in the ER, and has the capacity to activate several downstream signalling molecules.

Structure and function of GDNF receptor alpha splice variants

The growth factors of the glial cell line-derived neurotrophic factor (GDNF) family consisting of GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN), are involved in the development, differentiation and maintenance of many types of neurons. They also have important functions outside the nervous system in the development of kidney, testis and thyroid gland. Each of these GFLs preferentially binds to one of the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptors α (GFRα). GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The GFLs in the complex with their cognate GFRα receptors all bind to and signal through the receptor tyrosine kinase RET. Alternative splicing of the mouse GFRα4 gene yields three splice isoforms. These had been described as putative GPI-anchored, transmembrane and soluble forms. My goal was to characterise the function of the different forms of mouse GFRα4. I firstly found that the putative GPI-anchored GFRα4 (GFRα4-GPI) is glycosylated, membrane-bound, GPI-anchored and interacts with PSPN and RET. We also showed that mouse GFRα4-GPI mediates PSPN-induced phosphorylation of RET, promotes PSPN-dependent neuronal differentiation of the rat pheochromocytoma cell line PC6-3 and PSPN-dependent survival of cerebellar granule neurons (CGN). However, although this receptor can mediate PSPN-signalling and activate RET, GFRα4-GPI does not recruit RET into lipid rafts. The recruitment of RET into lipid rafts has previously been thought to be a crucial event for GDNF- and GFL-mediated signalling via RET. I secondly demonstrated that the putative transmembrane GFRα4 (GFRα4-TM) is indeed a real transmembrane GFRα4 protein. Although it has a weak binding capacity for PSPN, it can not mediate PSPN-dependent phosphorylation of RET, neuronal differentiation or survival. These data show that GFRα4-TM is inactive as a receptor for PSPN. Surprisingly, GFRα4-TM can negatively regulate PSPN-mediated signalling via GFRα4-GPI. GFRα4-TM interacts with GFRα4-GPI and blocks PSPN-induced phosphorylation of RET, neuronal differentiation as well as survival. Taken together, our data show that GFRα4-TM may act as a dominant negative inhibitor of PSPN-mediated signaling. The most exciting part of my work was the finding that the putative soluble GFRα4 (GFRα4-sol) can form homodimers and function as an agonist of the RET receptor. In the absence of PSPN, GFRα4-sol can promote the phosphorylation of RET, trigger the activation of the PI-3K/AKT pathway, induce neuronal differentiation and support the survival of CGN. Our findings are in line with a recent publication showing the GFRα4-sol might contribute to the inherited cancer syndrome multiple endocrine neoplasia type 2. Our data provide an explanation to how GFRα4-sol may cause or modify the disease. Mammalian GFRα4 receptors all lack the first Cys-rich domain which is present in other GFRα receptors. In the final part of my work I have studied the function of this particular domain. I created a truncated GFRα1 construct lacking the first Cys-rich domain. Using binding assays in both cellular and cell-free systems, phosphorylation assays with RET, as well as neurite outgrowth assays, we found that the first Cys-rich domain contributes to an optimal function of GFRα1, by stabilizing the interaction between GDNF and GFRα1.

Obstetric and gynaecological aspects of HIV infection

The purpose of the present study was to examine the outcome of pregnancies among HIV-infected women in Helsinki, use of the levonorgestrel-releasing intrauterine system (LNG-IUS) among HIV-infected women and the prevalence and risk factors of cytological and histologically proven cervical lesions in this population. Between 1993 and 2003 a total of 45 HIV-infected women delivered 52 singleton infants. HIV infection was diagnosed during pregnancy in 40% of the mothers. Seventeen of the mothers received antiretroviral (ARV) medication prior to pregnancy and in 34 cases, the medication was started during pregnancy. A good virological response (i.e. HIV RNA load <1000/mL during the last trimester) to ARV medication was achieved in 36/40 (90%) of the patients in whom HI viral load measurements were performed. Of the infants, 92% were born at term, and their mean (±SD) birth weight was 3350±395 g. The Caesarean section rate was low, 25%. All newborns received ARV medication and none of the infants born to mothers with pre-delivery diagnosis of maternal HIV infection were infected. The safety and advantages of the LNG-IUS were studied prospectively (n=12) and retrospectively (n=6). The LNG-IUS was well tolerated and no cases of PID or pregnancy were noted. Menstrual bleeding was reduced significantly during use of the LNG-IUS; this was associated with a slight increase in haemoglobin levels. Serum oestradiol concentrations remained in the follicular range in all subjects. The key finding was that genital shedding of HIV RNA did not change after the insertion of the LNG-IUS. The mean annual prevalence of low-grade squamous intraepithelial lesions (SIL) was 15% and that of high-grade SIL was 5% among 108 systematically followed HIV-infected women during 1989 2003. A reduced CD4 lymphocyte count was associated with an increased prevalence of SIL, whereas duration of HIV infection, use of ARV medication and HI viral load were not. The cumulative risk of any type of SIL was 17% after one year and 48% after five years among patients with initially normal Pap smears. The risk of developing SIL was associated with young age and a high initial HI viral load. During the follow-up 51 subjects (n=153) displayed cervical intraepithelial neoplasia (CIN), (16% CIN1 and 18% CIN 2-3). Only one case of cancer of the uterine cervix was detected. Pap smears were reliable in screening for CIN. Both nulliparity (p<0.01) and bacterial vaginosis (p<0.04) emerged as significant risk factors of CIN. In conclusion, a combination of universal antenatal screening and multidisciplinary management allows individualized treatment and prevents vertical transmission of HIV. Use of the LNG-IUS is safe among HIV-infected women and cervicovaginal shedding of HIV RNA is not affected by use of the LNG-IUS. The risk of cervical pre-malignant lesions is high among HIV-infected women despite systematic follow-up.

Preterm Birth and Surgical Treatment of the Uterine Cervix

Cervical cancer is the second most common cancer among women globally. Most, probably all cases, arise through a precursor, cervical intraepithelial neoplasia (CIN). Effective cytological screening programmes and surgical treatments of precancerous lesions have dramatically reduced its prevalence and related mortality. Although these treatments are effective, they may have adverse effects on future fertility and pregnancy outcomes. The aim of this study was to evaluate the effects of surgical treatment of the uterine cervix on pregnancy and fertility outcomes, with the focus particularly on preterm birth. The general preterm birth rates and risk factors during 1987–2005 were studied. Long-term mortality rates of the treated women were studied. In this study, information from The Medical Birth Register (MBR), The Hospital Discharge Register (HDR), The Cause-of-Death Register (CDR), and hospital records were used. Treatments were performed during 1987–2003 and subsequent deliveries, IVF treatments and deaths were analyzed. The general preterm birth rate in Finland was relatively stable, varying from 5.1% to 5.4% during the study period (1987 to 2005), although the proportion of extremely preterm births had decreased substantially by 12%.The main risk factor as regards preterm birth was multiplicity, followed by elective delivery (induction of delivery or elective cesarean section), primiparity, in vitro fertilization treatment, maternal smoking and advanced maternal age. The risk of preterm birth and low birth weight was increased after any cervical surgical treatment; after conization the risk of preterm birth was almost two-fold (RR 1.99, 95% CI 1.81– 2.20). In the conization group the risk was the highest for very preterm birth (28–31 gestational weeks) and it was also high for extremely preterm birth (less than 28 weeks). In this group the perinatal mortality was also increased. In subgroup analysis, laser ablation was not associated with preterm birth. When comparing deliveries before and after Loop conization, we found that the risk of preterm birth was increased 1.94-fold (95% CI 1.10–3.40). Adjusting for age, parity, or both did not affect our results. Large or repeat cones increased the risk of preterm birth when compared with smaller cones, suggesting that the size of the removed cone plays a role. This was corroborated by the finding that repeat treatment increased the risk as much as five-fold when compared with the background preterm birth rate. We found that the proportion of IVF deliveries (1.6% vs. 1.5%) was not increased after treatment for CIN when adjusted for year of delivery, maternal age, or parity. Those women who received both treatment for CIN and IVF treatment were older and more often primiparous, which explained the increased risk of preterm birth. We also found that mortality rates were 17% higher among women previously treated for CIN. This excess mortality was particularly seen as regards increased general disease mortality and alcohol poisoning (by 13%), suicide (by 67%) and injury death (by 31%). The risk of cervical cancer was high, as expected (SMR 7.69, 95% CI 4.23–11.15). Women treated for CIN and having a subsequent delivery had decreased general mortality rate (by -22%), and decreased disease mortality (by -37%). However, those with preterm birth had increased general mortality (SMR 2.51, 95% CI 1.24–3.78), as a result of cardiovascular diseases, alcohol-related causes, and injuries. In conclusion, the general preterm birth rate has not increased in Finland, as in many other developed countries. The rate of extremely preterm births has even decreased. While other risk factors of preterm birth, such as multiplicity and smoking during pregnancy have decreased, surgical treatments of the uterine cervix have become more important risk factors as regards preterm birth. Cervical conization is a predisposing factor as regards preterm birth, low birth weight and even perinatal mortality. The most frequently used treatment modality, Loop conization, is also associated with the increased risk of preterm birth. Treatments should be tailored individually; low-grade lesions should not be treated at all among young women. The first treatment should be curative, because repeat treatments are especially harmful. The proportion of IVF deliveries was not increased after treatment for CIN, suggesting that current treatment modalities do not strongly impair fertility. The long-term risk of cervical cancer remains high even after many years post-treatment; therefore careful surveillance is necessary. In addition, accidental deaths and deaths from injury were common among treated women, suggesting risk-taking behavior of these women. Preterm birth seems be associated with extremely high mortality rates, due to cardiovascular, alcohol-related and injury deaths. These women could benefit from health counseling, for example encouragement in quitting smoking.

Matrix metalloproteinases as biomarkers in premalignant and malignant tumors of the human skin

The incidence of non-melanoma skin cancer is increasing worldwide. Basal cell carcinoma followed by squamous cell carcinoma and malignant melanoma are the most frequent skin tumors. Immunosuppressed patients have an increased risk of neoplasia, of which non-melanoma skin cancer is the most common. Matrix metalloproteinases (MMPs) are proteolytic enzymes that collectively are capable of degrading virtually all components of the extracellular matrix. MMPs can also process substrates distinct from extracellular matrix proteins and influence cell proliferation, differentiation, angiogenesis, and apoptosis. MMP activity is regulated by their natural inhibitors, tissue inhibitors of metallopro-teinases (TIMPs). In this study, the expression patterns of MMPs, TIMPs, and certain cancer-related molecules were investigated in premalignant and malignant lesions of the human skin. As methods were used immunohistochemisty, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR) from the cell cultures. Our aim was to evaluate the expression pattern of MMPs in extramammary Paget's disease in order to find markers for more advanced tumors, as well as to shed light on the origin of this rare neoplasm. Novel MMPs -21, -26, and -28 were studied in melanoma cell culture, in primary cutaneous melanomas, and their sentinel nodes. The MMP expression profile in keratoacanthomas and well-differentiated squamous cell carcinomas was analyzed to find markers to differentiate benign keratinocyte hyperproliferation from malignantly transformed cells. Squamous cell carcinomas of immunosuppressed organ transplant recipients were compared to squamous cell carcinomas of matched immunocompetent controls to investigate the factors explaining their more aggressive nature. We found that MMP-7 and -19 proteins are abundant in extramammary Paget's disease and that their presence may predict an underlying adenocarcinoma in these patients. In melanomas, MMP-21 was upregulated in early phases of melanoma progression, but disappeared from the more aggressive tumors with lymph node metastases. The presence of MMP-13 in primary melanomas and lymph node metastases may relate to more aggressive disease. In keratoacanthomas, the expression of MMP-7 and -9 is rare and therefore should raise a suspicion of well-differentiated squamous cell carcinomas. Furthermore, MMP-19 and p16 were observed in benign keratinocyte hyperproliferation of keratoacanthomas, whereas they were generally lost from malignant keratinocytes of SCCs. MMP-26 staining was significantly stronger in squamous cell carcinomas and Bowen s disease samples of organ transplant recipients and it may contribute to the more aggressive nature of squamous cell carcinomas in immunosuppressed patients. In addition, the staining for MMP-9 was significantly stronger in macrophages surrounding the tumors of the immunocompetent group and in neutrophils of those patients on cyclosporin medication. In conclusion, based on our studies, MMP-7 and -19 might serve as biomarkers for more aggressive extramammary Paget's disease and MMP-21 for malignant transformation of melanocytes. MMP -7, -9, and -26, however, could play an important role in the pathobiology of keratinocyte derived malignancies.

Integrative functional genomics analysis of sustained polyploidy phenotypes in breast cancer cells identifies an oncogenic profile for GINS2

Aneuploidy is among the most obvious differences between normal and cancer cells. However, mechanisms contributing to development and maintenance of aneuploid cell growth are diverse and incompletely understood. Functional genomics analyses have shown that aneuploidy in cancer cells is correlated with diffuse gene expression signatures and that aneuploidy can arise by a variety of mechanisms, including cytokinesis failures, DNA endoreplication and possibly through polyploid intermediate states. Here, we used a novel cell spot microarray technique to identify genes with a loss-of-function effect inducing polyploidy and/or allowing maintenance of polyploid cell growth of breast cancer cells. Integrative genomics profiling of candidate genes highlighted GINS2 as a potential oncogene frequently overexpressed in clinical breast cancers as well as in several other cancer types. Multivariate analysis indicated GINS2 to be an independent prognostic factor for breast cancer outcome (p = 0.001). Suppression of GINS2 expression effectively inhibited breast cancer cell growth and induced polyploidy. In addition, protein level detection of nuclear GINS2 accurately distinguished actively proliferating cancer cells suggesting potential use as an operational biomarker.