Colorectal cancer is one of the three most common malignant diseases in Sweden, with about 5,000 new cases each year. Thirty-five percent of these are rectal cancer, for which local recurrence after surgery has been a serious problem. The five-year survival rate in colorectal cancer has improved from about 40% in 1960 to 55% in 1995. Adjuvant chemotherapy of colon cancer, preoperative radiotherapy and improved surgical techniques in rectal cancer have contributed to the improved  results. To select patients best suited for pre- or postoperative therapy, we need indicators of both prognosis and response to therapy.

Using antibodies against cytokeratin, we found that 39% of patients with colorectal carcinoma that had penetrated the muscularis propria but without lymph-node metastases by routine light microscopy, had got micrometastases. Survival among patients with micrometastases was not significantly different from that among patients without such metastases.

We also identified subsets of tumour-infiltrating mononuclear cells and studied their pattern of distribution in relation to regressive tumour areas and Dukes class. Our interpretation is that the subsets of tumourinfiltrating mononuclear cells change with advancing Dukes class, indicating gradual deterioration of the local immune control.

We also investigated the interaction between p53, Ki-67, apoptosis and the outcome in rectal cancer with and without short-term preoperative radiotherapy. The expression of nuclear p53 protein seemed to be a significant predictive factor for local treatment failure after preoperative radiotherapy. Low tumour cell proliferation measured with Ki-67 in the preoperative biopsy correlated with improved local control and disease-free survival after preoperative radiotherapy.

High apoptotic index was associated with improved local control of rectal cancer even without pre-operative radiotherapy, whereas local control of tumours with low and intermediate apoptotic index was significantly improved by preoperative radiotherapy.

In conclusion, micrometastases in regional lymph nodes are an interesting phenomenon but with limited prognostic value. The subsets of tumour-infiltrating mononuclear cells change with advancing Dukes class, and its seems that the local immune control is gradually broken down. In rectal cancer, p53 expression, tumour proliferation measured with Ki-67 and apoptotic index seem to be interesting indicators of rectal cancer prognosis and response to preoperative radiotherapy.

OBJECTIVE: To evaluate the incidence and prognostic importance of micrometastatic disease in regional lymph nodes from Dukes' B colorectal carcinomas.

DESIGN: Retrospective study.

SETTING: University hospital, Sweden.

SUBJECTS: 100 patients operated on for primary colorectal carcinoma, classified as Dukes' B lesions.

INTERVENTIONS: The regional lymph nodes were re-examined immunohistochemically using monoclonal antibodies against cytokeratin.

OUTCOME MEASURES: Incidence and prognostic importance of micrometastases.

RESULTS: Micrometastases were found in 39% (39/100) of the patients. The number of positive cells in the lymph nodes examined varied from 1 to over 100. They appeared as single cells or small clusters of cells located within the capsule or in the peripheral sinus of the lymph node. At least three sections from each of three lymph nodes had to be examined to identify 95% of the patients with lymph node micrometastases. The outcome of the patients with micrometastases was not significantly different from that of patients with no epithelial cells in the lymph nodes.

CONCLUSION: Micrometastases in regional lymph nodes are a interesting phenomenon but clinically seem to be of only weak prognostic value.

BACKGROUND: Rectal carcinoma is common, with considerable local recurrence and death rates. Preoperative radiotherapy and refined surgical techniques can improve local control. The aim of this study was to investigate the interaction between apoptosis and the outcome of rectal carcinoma, with and without short-term preoperative radiotherapy.

METHODS: Specimens were from 162 patients from the Southeast Swedish Health Care region included in the Swedish Rectal Cancer Trial between 1987-1990. New sections from the paraffin blocks of the preoperative biopsies and the surgical specimens were examined for apoptosis using the terminal deoxynucleotidyl transferase mediated digoxigenin nick end labeling (TUNEL) method.

RESULTS: The mean percentage of apoptotic cells was 0.3% (0-4%) and 1.1% (0-14.5%) for the preoperative biopsy and the surgical specimen, respectively. The authors analyzed the surgical specimens from nonirradiated patients and divided them into three groups by apoptotic index (AI) as follows: 0%, 0-1%, and > 1%. A high AI was associated with a decreased local recurrence rate compared with an intermediate or a low AI (P = 0.024). There was no significant relation between AI and survival. There was a significant reduction in the local recurrence rate for irradiated patients compared with the nonirradiated in the low (P = 0.015) and intermediate (P = 0.038) AI groups. In the high AI group, there were few recurrences and no significant difference was observed between irradiated and nonirradiated patients. The relative risk of death from rectal carcinoma in Dukes A-C patients was not significantly decreased by radiotherapy, but, in the intermediate AI group, there was a trend (P = 0.08) in favor of the irradiated patients.

CONCLUSION: A high AI in rectal carcinoma indicated a decreased local recurrence rate.

BACKGROUND: Rectal carcinoma is a common malignancy, with a history of high local recurrence rates following surgery. In recent years. preoperative radiotherapy and refined surgical technique have improved local control rates.

AIM: To investigate the relationship between expression of nuclear p53 protein and the outcome in rectal carcinoma, with and without short-term preoperative radiotherapy.

MATERIAL: Specimens from 163 patients from the Southeast Swedish Health Care region included in the Swedish rectal cancer trial between 1987-1990.

METHOD: New sections from the paraffin blocks of the preoperative biopsy and the surgical specimen were examined immunohistochemically using a p53 antibody (PAb 1801).

RESULT: Expression of nuclear p53 protein was seen in 41% of the tumours. The p53 negative patients treated with preoperative radiotherapy had a significant reduction of local failure compared with the non-irradiated p53 negative patients (P = 0.0008). In contrast, p53 positive patients showed no benefit from preoperative radiotherapy. The interaction between p53 status and the benefit of radiotherapy was statistically significant (P = 0.018).

CONCLUSION: Expression of nuclear p53 protein in rectal carcinoma seems to be a significant predictive factor for local treatment failure after preoperative radiotherapy. Further investigations are necessary to select patients for preoperative treatment based on analysis of the preoperative biopsies.

BACKGROUND: Rectal cancer is a common malignancy, with significant local recurrence and death rates. Preoperative radiotherapy and refined surgical technique can improve local control rates and disease-free survival.

PURPOSE: To investigate the relationship between the tumor growth fraction in rectal cancer measured with Ki-67 and the outcome, with and without short-term preoperative radiotherapy.Method: Ki-67 (MIB-1) immunohistochemistry was used to measure tumor cell proliferation in the preoperative biopsy and the surgical specimen.

MATERIALS: Specimens from 152 patients from the Southeast Swedish Health Care region were included in the Swedish rectal cancer trial 1987-1990.

RESULTS: Tumors with low proliferation treated with preoperative radiotherapy had a significantly reduced recurrence rate. The influence on death from rectal cancer was shown only in the univariate analysis. Preoperative radiotherapy of tumors with high proliferation did not significantly improve local control and disease-free survival. The interaction between Ki-67 status and the benefit of radiotherapy was significant for the reduced recurrence rate (p = 0.03), with a trend toward improved disease-free survival (p = 0.08). In the surgery-alone group, Ki-67 staining did not significantly correlate with local recurrence or survival rates.

CONCLUSION: Many Ki-67 stained tumor cells in the preoperative biopsy predicts an increased treatment failure rate after preoperative radiotherapy of rectal cancer.

Young breast cancer patients have a decreased survival rate and it has been demonstrated that young age is an independent predictor of adverse prognosis. Overexpression of c-erbB-2 protein (also known as HER-2/neu) has been shown to be a prognostic indicator in breast cancer in general and especially among patients with axillary nodal metastases. The present study was initiated to determine the prognostic significance of c-erbB-2 protein overexpression in early onset breast cancer.

A population consisting of 110 young breast cancer patients, ≤ 36-year-old at diagnosis, was analyzed with immunohistochemical staining for c-erbB-2 protein.

Thirty patients (27%) were found to overexpress the c-erbB-2 protein. C-erbB-2 positivity was significantly associated with poor survival when all patients were included in the analysis (P = 0.002) and for patients with axillary nodal metastases (P = 0.0007). No such association was found for node-negative patients. Furthermore, the difference in prognosis in relation to c-erbB-2 among node-positive patients was maintained, when these were stratified in groups treated or not treated with adjuvant chemotherapy.

The study indicates that overexpression of c-erbB-2 protein is a strong prognostic factor in young breast cancer patients with axillary nodal metastases. Moreover, the adverse prognosis associated with c-erbB-2 overexpression in node-positive patients was observed whether or not the patients had received adjuvant chemotherapy.

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras incolorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P=0.004, HR 1.3) and overall survival (P=0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes' C cancers (failure-free survival, P=0.008, HR 1.5, overall survival P=0.02, HR 1.45) than in Dukes' B tumours (failure-free survival, P=0.46, HR 1.12, overall survival P=0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer. ⌐ 2001 Cancer Research Campaign.

Developing a reliable and quantitative assessment of the potential virulence of a malignancy has been a long-standing goal in clinical cytometry. DNA histogram analysis provides valuable information on the cycling activity of a tumor population through S-phase estimates; it also identifies nondiploid populations, a possible indicator of genetic instability and subsequent predisposition to metastasis. Because of conflicting studies in the literature, the clinical relevance of both of these potential prognostic markers has been questioned for the management of breast cancer patients. The purposes of this study are to present a set of 10 adjustments derived from a single large study that optimizes the prognostic strength of both DNA ploidy and S-phase and to test the validity of this approach on two other large multicenter studies. Ten adjustments to both DNA ploidy and S-phase were developed from a single node-negative breast cancer database from Baylor College (n = 961 cases). Seven of the adjustments were used to reclassify histograms into low-risk and high-risk ploidy patterns based on aneuploid fraction and DNA index optimum thresholds resulting in prognostic P values changing from little (P < 0.02) or no significance to P < 0.000005. Other databases from Sweden (n = 210 cases) and France (n = 220 cases) demonstrated similar improvement of DNA ploidy prognostic significance, P < 0.02 to P < 0.0009 and P < 0.12 to P < 0.002, respectively. Three other adjustments were applied to diploid and aneuploid S-phases. These adjustments eliminated a spurious correlation between DNA ploidy and S-phase and enabled them to combine independently into a powerful prognostic model capable of stratifying patients into low, intermediate, and high-risk groups (P < 0.000005). When the Baylor prognostic model was applied to the Sweden and French databases, similar significant patient stratifications were observed (P < 0.0003 and P < 0.00001, respectively). The successful transference of the Baylor prognostic model to other studies suggests that the proposed adjustments may play an important role in standardizing this test and provide valuable prognostic information to those involved in the management of breast cancer patients.

Background: Chemotherapy drug distribution varies greatly among individual patients. Therefore, we developed an individualised fluorouracil, epirubicin, cyclophosphamide (FEC) regimen to improve outcomes in patients with high-risk early breast cancer. We then did a randomised trial to compare this individually tailored FEC regimen with conventional adjuvant chemotherapy followed by consolidation with high-dose chemotherapy with stem-cell support. Methods: 525 women younger than 60 years of age with high-risk primary breast cancer were randomised after surgery to receive nine cycles of tailored FEC to haematological equitoxicity with granulocyte colony-stimulating factor (G-CSF) support (n=251), or three cycles of FEC at standard doses followed by high-dose chemotherapy with cyclophosphamide, thiotepa, and carboplatin (CTCb), and peripheral-blood stem-cell or bone-marrow support (n=274). Both groups received locoregional radiation therapy and tamoxifen for 5 years. The primary outcome measure was relapse-free survival, and analysis was by intention to treat. Findings: At a median follow-up of 34.3 months, there were 81 breast-cancer relapses in the tailored FEC group versus 113 in the CTCb group (double triangular method p=0.04). 60 deaths occurred in the tailored FEC group and 82 in the CTCb group (log-rank p=0.12). Patients in the CTCb group experienced more grade 3 or 4 acute toxicity compared with the tailored FEC group (p<0.0001). Two treatment-related deaths (0.7%) occurred in the CTCb group. Six patients in the tailored FEC group developed acute myeloid leukaemia and three developed myelodysplastic syndrome. Interpretation: Tailored FEC with G-CSF support resulted in a significantly improved relapse-free survival and fewer grade 3 and 4 toxicities compared with marrow-supported high-dose chemotherapy with CTCb as adjuvant therapy of women with high-risk primary breast cancer.

Breast cancer is the most common malignancy among women in the western world. Although the disease is rare in young women, it is one of the main causes of death at young age. The early onset breast cancer has demonstrated more aggressive pathological features than the late onset disease. These observations have raised the hypothesis that the biological background may differ between age categories.

Breast carcinogenesis is a micro-evolutionary process that requires accumulation of DNA-damage and other epigenetic changes that promote cell survival and proliferation. The complexity of the disease makes it difficult to identify specific risk factors. Nevertheless, a large and compelling body of epidemiological and experimental data suggests that the cumulative dose of oestrogen is one key factor in the aetiology. Also, substantial data indicates that oxidative stress, from phosphorylation or other metabolic processes, is involved in the development of breast cancer. In young women, there is a strong genetic influence of breast cancer risk and beside mutations in highly penetrant genes, polymorphisms in a number of crucial genes may modify an individual's risk. Such modifier genes, associated with a more modest risk and high prevalence in the population, may contribute to a large proportion of the disease in the population. Identification of such predisposing polymorphisms may be an important step forward in identifiying individuals at risk. In the present thesis genetic polymorphisms in four different genes and their relation to early onset breast cancer, were analysed.

In the first study, a polymorphism with a TaqI restriction site in the vitamin D₃ receptor (VDR) gene was studied. VDR and its ligand, 1,25(OH)₂D₃, have been suggested to be important factors for differentiation of the breast epithelium and may suppress mammary tumorigenesis. The presence of a TaqI restriction site has been shown to correlate with increased transcriptional activity and mRNA stability of VDR, as well as high serum levels of 1,25(OH)₂D₃ and this high receptor activity may be protective against breast cancer. In the present study VDR TaqI polymorphism did not predict risk of early onset breast cancer. However, the results indicate an association between lymph node metastasis and genotype. In the second study, a promoter polymorphism in the CYP17 gene, which may influence the oestrogen synthesis, has been analysed. The polymorphism was correlated to the risk of early onset breast cancer, and the risk increased in a dose dependent manner. The fmdings indicated also a trend for risk allele carriers to have ER-negative and large tumours. Oestrogens are metabolized to potentially carcinogenic catecholoestrogens, which could be inactivated by and O-methylation, catalysed by Catechol-O-methyltransferase (COMT). This gene contains a variant which encode for a protein with decreased activity and is therefore predicted to be a risk allele. In the third study, the investigation of allele frequencies of the polymorphic COMT gene did not show any epidemiological evidences of implication in breast cancer. Finally; increasing numbers of studies indicate an important role for MnSOD in a number of cancer cell types. A genetic variant of MnSOD results in a less efficient transport into the mitochondria which may lead to an insufficient scavenging of free radicals. In this study, the mitochondrial targeting polymorphism was associated with risk of breast cancer in young women.

In conclusion, genetic polymorphism in crucial genes may have impact on the risk of early onset breast cancer. Furthermore, some genotypes seems to influences the progression and outcome of the disease.

Purpose: Manganese superoxide dismutase (MnSOD) is one of the major enzymes implicated in the cellular defence against reactive oxygen species. Low expression of MnSOD has been observed in different cancer tissues and several reports have shown that overexpression of MnSOD inhibits growth in various human cancer cells. These observations suggest that MnSOD is involved in carcinogenesis. A polymorphism (Ala-9Val) in the mitochondrial targeting sequence (MTS) of the MnSOD gene has been proposed to affect protein localization and thereby influence cellular defence against superoxide radicals.

Methods: In the present case-control study, including 118 early onset breast cancer patients (≤36 years) and 174 age-matched controls, the MTS polymorphism and loss of heterozygosity (LOH) in the locus of MnSOD were analysed.

Results: We found that individuals with MnSOD^Val/Val and MnSOD^Val/Ala genotypes showed an increased risk of breast cancer (OR, 2.7; 95% CI, 2.2–5.5, p=0.01, OR, 3.0; 95%CI, 1.4–6.5, p=0.002). Moreover, 45% of the informative cases expressed allelic loss at the chromosomal locus of the MnSOD gene. No correlation was found between LOH and the genotype.

Conclusion: The present study suggests that MnSOD may be implicated in breast carcinogenesis in young women.

Purpose: Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis, which is crucial in cancer progression. We have previously shown that estradiol (E2) increases VEGF in breast cancer. Phytoestrogens are potential compounds in breast cancer prevention and treatment by poorly understood mechanisms. The main phytoestrogens in Western diet are lignans, and flaxseed is a rich source of the mammalian lignans enterodiol and enterolactone.

Experimental Design: In the present study, ovariectomized mice were treated with continuous release of E2. MCF-7 tumors were established and mice were fed with basal diet or 10% flaxseed, and two groups that were fed basal diet received daily injections with enterodiol or enterolactone (15 mg/kg body weight).

Results: We show that flaxseed, enterodiol, and enterolactone counteracted E2-induced growth and angiogenesis in solid tumors. Extracellular VEGF in vivo, sampled using microdialysis, in all intervention groups was significantly decreased compared with tumors in the basal diet group. Our in vivo findings were confirmed in vitro. By adding enterodiol or enterolactone, E2-induced VEGF secretion in MCF-7 cells decreased significantly without agonistic effects. The increased VEGF secretion by E2 in MCF-7 cells increased the expression of VEGF receptor-2 in umbilical vein endothelial cells, suggesting a proangiogenic effect by E2 by two different mechanisms, both of which were inhibited by the addition of lignans.

Conclusions: Our results suggest that flaxseed and its lignans have potent antiestrogenic effects on estrogen receptor-positive breast cancer and may prove to be beneficial in breast cancer prevention strategies in the future.

The urinary excretion of the hydroxylated DNA base 8-hydroxydeoxyguanosine (8-OHdG) and the lipid peroxidation product malondialdehyde (MDA) was monitored in 11 patients with hematological malignancies undergoing total body irradiation and high-dose chemotherapy preceding bone marrow transplantation. Nine patients showed a prompt increase in urinary 8-OHdG (8-25 times the initial baseline level) on days 0-7 after irradiation onset, the excretion then decreased during the aplastic period and increased again when engraftment took place (in 7 patients). A significant positive correlation was found between urinary 8-OHdG and whole blood leukocyte count, both on day 5 (p = .04, r = .72) and on day 22 (p = .009, r = .80) after irradiation onset. One patient who lacked the first peak of 8-OHdG excretion showed low blood leukocyte counts (less than 2×109/l) before therapy onset, this patient, however, later had a successful engraftment and then also showed considerable increases in both 8-OHdG excretion and leukocyte count. These observations suggest leukocytes play a part in the excretion of 8-OHdG after conditioning therapy preceding bone marrow transplantation. As opposed to the biphasic 8-OHdG excretion, the excretion of MDA showed a single peak appearing on days 11-19 after radiochemotherapy onset, i.e., during the period in which the patients suffered from cytopenia, mucositis, and other side effects of the treatment. It is suggested, therefore, that these clinical manifestations are associated with increased lipid peroxidation. Altogether, these findings illustrate the utility of serial urinary samples for monitoring oxidative stress due to conditioning therapy in clinical practice. They also demonstrate that different oxidative stress markers may behave quite differently regarding their appearance in the urine after whole-body oxidative stress.

Long-term exposure to oestrogens is a well-recognised risk factor for breast cancer, whereas little is known about the influence of polymorphisms of genes involved in oestrogen biosynthesis and metabolism. A candidate, containing a single bp polymorphism, T→C, (designated, A2 allele), might be the CYP17 gene, which codes for an enzyme involved in oestrogen synthesis. This polymorphism creates an additional Sp1-type promoter site (CCACC box), which has been shown to be associated with increased serum oestrogen levels. We performed a case-control study, to evaluate association of the CYP17 gene polymorphism with risk of breast cancer in young women (younger than 37 years). We found a statistically significant increased risk in carriers of at least 1 A2 allele [odds ratio (OR), 2.0; 95% confidence interval (CI), 1.1–3.5, p = 0.027], and a trend toward a gene-dose effect illustrated by a slightly higher risk for A2-homozygous subjects (OR, 2.8) than for heterozygous women (OR, 1.9). Furthermore, when we investigated the CYP17 genotype in relation to tumour characteristics, breast cancer patients with 1 or 2 A2 alleles tended to have lower oestrogen receptor levels (risk ratio, 0.70; CI, 0.41–1.2, p = 0.44). Our findings suggest that CYP17 gene polymorphism influences breast carcinogenesis in young women.

Oestrogen exposure has long been considered to be a main risk factor of breast cancer. More recently, interest has also focused on the possible carcinogenic influence from oestrogen metabolites, such as catechol oestrogens. O-methylation, catalysed by Catechol-O-Methyltransferase (COMT), is one pathway by which the potentially carcinogenic catechol oestrogens can be inactivated. The gene coding for COMT protein contains a single-nucleotide polymorphism (SNP), resulting in an amino acid shift Val Met, which has been shown to determine high- and low-activity configuration of the enzyme. We hypothesized that the low-activity allele, COMT^Met, may be implicated in early onset breast cancer. In the present case–control study, including 126 young breast cancer patients ( 36 years) and 117 healthy female blood donors, we analysed the association between COMT^Met genotype and risk of breast cancer. No significant difference in the frequency of low-/high-activity alleles was found between cases and controls, indicating that the polymorphism, as a single factor, may not contribute to breast carcinogenesis in young women.

Heparan sulphate (HS) represents a heterogeneous class of molecules on cell membranes and extracellular matrices. These molecules are involved in a variety of biological processes, including immune responses, through their binding and functional modulation of proteins. Recently a panel of HS-epitope-specific, human single chain antibodies have been generated by phage display, facilitating analysis of the structural heterogeneity of HS in relation to pathological conditions. In a pilot study a heterogeneous staining pattern in melanoma metastases was observed with one of the clones (EW4G1). Using a double-staining technique, the expression of this epitope was studied in 12 metastatic melanoma lesions in relation to the presence of a CD3 + cell infiltrate. Different staining patterns with EW4G1 were observed in the different lesions. The different staining patterns were associated with the presence and pattern of inflammation with CD3+ cells. A pronounced staining pattern of blood vessels with EW4G1 was associated with a more or less brisk presence of CD3+ cells, while a pronounced staining of tumour cells or tumour cell matrix or absence of staining with EW4G1 was associated with absence of CD3+ cells. These results suggest a dualistic role for HS in the recruitment and intratumoural migration of CD3+ cells, depending on the location of expression of its epitope recognized by EW4G1. Further characterization of the structural diversity of HS and its function in T-cell recruitment and migration is therefore warranted, since detailed understanding of this relation may provide new targets for therapeutic intervention, such that better homing and migration of T cells (in)to tumours might be achieved in immunologically based treatment strategies.

A large number of studies have indicated that specific immune reactivity plays a crucial role in the control of malignant melanoma. In this context, expression of MHC I, MHC II and B7 molecules by melanoma cells is seen as relevant for the immune response against the tumour. For a better understanding of the biological relevance of MHC II and B7 expression by tumour cells in metastatic melanoma, we studied the expression of these molecules in melanoma metastases in relation to the inflammatory response, regression of the tumour and survival from 27 patients treated with biochemotherapy (30 mg m-2 Cisplatin and 250 mg m-2 decarbazine (dimethyl-triazene-imidazole-carboxamide, DTIC) on days 1-3 i.v., and 107 IU IFN-a2b 3 days a week s.c., q. 28d). In 19 out of 27 lesions studied, we found expression of MHC II by the tumour cells, while only in one out of II tumour biopsies obtained from untreated metastatic melanoma patients, MHC II expression was detected. Expression of B7.1 and B7.2 by tumour cells was found in nine out of 24 and 19 out of 24 lesions, respectively. In all cases where B7.1 expression was found, expression of B7.2 by the tumour cells was also seen. In general, no or only few inflammatory cells positive for B7 were found. Expression of MHC II by tumour cells was positively correlated with the presence of tumour-infiltrating lymphocytes, regression of the lesion, and with time to progression (TTP) and overall survival (OS) of the patient. However, no significant correlation between B7.1 or B7.2 expression and regression of the tumour, TTP or OS was found. In light of other recent findings, these data altogether do support a role as biomarker for MHC II expression by tumour cells, however, its exact immunological pathomechanism(s) remain to be established. ⌐ 2003 Cancer Research UK.

Purpose: To evaluate the role of particularly interesting new cysteine-histidine-rich protein (PINCH) in corneal wound healing and early neovascularization and to assess the influence of granulocytes. Methods: A standardized corneal alkali wound was inflicted under general anaesthesia to the right eye of 14 New Zealand White rabbits. Seven of the rabbits received i.v. 5 mg/kg fucoidin every 2 hours to prevent granulocytes from entering the wound area. After 36 hours, the rabbits were killed, the corneas excised, fixed in 4% formaldehyde and embedded in paraffin. The sections were double-stained with antibodies against PINCH and with haematoxylin. Results: In the normal cornea and limbus, PINCH was weakly expressed in the corneal epithelium and in a wedge of the conjunctival stroma. In the wounded corneas, PINCH expression was seen in the frontline of repopulating endothelial and epithelial cells, and in active keratocytes. The vascular endothelium and the granulocytes expressed PINCH, as did the conjunctival epithelium. In the fucoidin-treated rabbits, PINCH expression was markedly reduced. The vascular endothelial cells and the few granulocytes did not express PINCH in these rabbits. Conclusions: PINCH is only slightly expressed in the normal cornea. A corneal wound induces PINCH expression in the repopulating cells, in the vascular endothelial cells of the limbus, in the limbal epithelium and in the granulocytes. Exclusion of granulocytes reduces expression of PINCH and there is no expression at all in the vascular endothelium. © 2007 The Authors Journal compilation 2007 Acta Ophthalmol Scand.

Background and Objectives. Thalidomide modulates the production of tumor necrosis factor (TNF-α). Soluble TNF receptors, TNFR p55 and TNFR p75, modify TNF-α activity. In this study, we explored the relation between soluble TNF receptors and outcome in patients with advanced multiple myeloma treated with thalidomide. Design and Methods. The levels of soluble TNF receptor p55 and p75 were assessed in serum from 34 myeloma patients with relapsed or refractory disease before starting thalidomide treatment. Serial measurements were performed for 16 patients in serum collected during treatment. Results. The pre-treatment serum level of soluble TNFR p55 in thalidomide responders was significantly lower than that in non-responders (median 1.75 ng/mL (range 1.19-2.84) vs. 2.79 ng/mL (1.36-5.51), p=0.004). The levels of p55 declined significantly during treatment. The levels of p75 showed the same pattern as p55, but the differences were not significant. The median survival of myeloma patients with pre-treatment levels of p55 < 2.79 ng/mL was 404 days, the median survival of patients with pre-treatment levels ≥ 2.79 ng/mL was shorter (65 days, log-rank test p=0.02). Interpretation and Conclusions. We conclude that soluble TNFR p55 is an adverse prognostic factor in myeloma patients with relapsed or refractory disease treated with thalidomide. Patients with a low pre-treatment level of this receptor have a better response rate and a longer overall survival.

Polymorphisms of GSTM1, GSTT1 and GSTP1 were examined in melanoma patients and tumor-free individuals. Relationships between the polymorphisms and tumor characteristics and pigment phenotypes of the patients were analyzed. There was no significant difference in GSTM1 null and GSTT1 null genotypes nor GSTP1 GG genotype between melanoma patients and controls. In melanoma patients, these polymorphisms were not correlated with early or later onset of melanomas or gender of the patients. Frequency of GSTM1 null genotype was higher in patients with melanoma >2.5 mm than in those with tumors <1.0 mm, and higher frequency was found in nodular melanoma than in the other tumor types. GSTP1 GG genotype was more often found in the patients with brown and mixed eye color or brown and black hair than those with blue and green eyes or blond hair. It is unlikely that polymorphisms of GSTM1, GSTT1 and GSTP1 are general risk factors for melanoma in the Swedish population. GSTM1 null genotype was correlated with Breslow thickness and tumor type, which might serve as an additional biomarker for a rapid tumor progression. GSTP1 GG increases risk for melanoma in the subgroup of individuals with dark eyes or hair.

Background and Objectives: The redox-regulatory protein thioredoxin has several functions including transcriptional regulation, and antioxidant, cytokine, and chemokine activities. We have previously shown that extracellular thioredoxin protects B-cell chronic lymphocytic leukemia (CLL) cells from apoptosis in vitro. In this study we were interested to determine whether thioredoxin is produced by cells surrounding the CLL cells in the in vivo microenvironment and whether this cell-derived thioredoxin has any leukemia growth-promoting effect in vitro. Design and Methods: Lymph nodes from CLL patients (n=25) were analyzed for thioredoxin expression by immunohistology. Stromal cells purified from the lymph nodes were analyzed for thioredoxin secretion at the single cell level using an ELIspot assay. The survival effect of the stromal-derived thioredoxin was tested by co-culturing stromal- and CLL cells with and without Fab-fragments of an anti-thioredoxin antibody. Results: The results indicated that the thioredoxin production correlated with the amount of proliferating cells and was mainly localized to the proliferation centers (pseudofollicles) in the CLL lymph nodes. The leukemia cells per se showed minimal thioredoxin levels, in contrast, stromal cells strongly expressed thioredoxin. Purified primary stromal cells, which secreted extracellular thioredoxin, significantly protected the CLL cells from undergoing apoptosis in 72 h co-cultures. Interestingly, this anti-apoptotic effect could be abrogated by addition of Fab-fragments of an anti- thioredoxin antibody. Interpretation and Conclusions: In conclusion, we have shown that stromal cells in the lymph node microenvironment produce thioredoxin and that the thioredoxin production is localized to the proliferation centers of the CLL lymph nodes. In addition, thioredoxin produced by purified stromal cells rescued CLL cells from apoptosis in vitro. ©2007 Ferrata Storti Foundation.

Combination chemotherapy may induce remission from acute myeloid leukemia (AML), but validated criteria for treatment of elderly are lacking. The remission intention (RI) rate for elderly patients, as reported to the Swedish Leukemia Registry, was known to be different when comparing the six health care regions, but the consequences of different management are unknown. The Leukemia Registry, containing 1672 AML patients diagnosed between 1997 and 2001, with 98% coverage and a median follow-up of 4 years, was completed with data from the compulsory cancer and population registries. Among 506 treated and untreated patients aged 70 -79 years with AML (non-APL), there was a direct correlation between the RI rate in each health region (range 36 -76%) and the two-year overall survival, with no censored observations (6 -21%) (χ2 for trend=11.3, P<0.001, r2=0.86, P<0.02, nonparametric). A 1-month landmark analysis showed significantly better survival in regions with higher RI rates (P=0.003). Differences could not be explained by demographics, and was found in both de novo and secondary leukemias. The 5-year survival of the overall population aged 70 -79 years was similar between the regions. Survival of 70 -79-year-old AML patients is better in regions where more elderly patients are judged eligible for remission induction. © 2006 Nature Publishing Group All rights reserved.

Lack of neonatal angiotensin II type I receptor (AT1) stimulation produces renal abnormalities characterized by papillary atrophy and impaired urinary concentrating ability, but the mechanisms involved are still unclear. DNA microarray was used to identify genes that are differentially expressed in renal medulla in response to neonatal treatment with AT 1 receptor antagonist losartan (30 mg/kg per d), which commenced within 24 h after birth. The data showed that losartan treatment for 48 h downregulated 68 genes, ~30% of which encode various components of cytoskeleton and cytoskeleton-associated proteins, extracellular matrix, and enzymes involved in extracellular matrix maturation or turnover. With the use of immunohistochemistry and Western immunoblot, the microarray data were confirmed and it was demonstrated that losartan suppressed renal expression of syndecan 2, a-smooth muscle actin, MHC class II, and leukocyte type 12-lipoxygenase by day 4. In addition, losartan inhibited medullary expression of integrin a6 and caused relocalization of integrins a6 and a3. Moreover, losartan inhibited cell proliferation in medullary tubules by day 9, as detected by Ki-67 immunostaining. This study provides new data supporting the contention that a lack of AT1 receptor stimulation results in abnormal matrix assembly, disturbed cell-cell and cell-matrix interactions, and subsequent abnormal tubular maturation. Moreover, regulation of the expression of leukocyte type 12-lipoxygenase and a-smooth muscle actin by the renin-angiotensin system in the immature kidney adds new knowledge toward the understanding of renal vascular development.

BACKGROUND. Colorectal cancer is one of the most common forms of cancer in the Western world. Staging based on histopathology is currently the most accurate predictor of outcome after surgery. Colorectal cancer is curable if treated at an early stage (stage I-III). However, for tumors in stages II and III there is a great need for tests giving more accurate prognostic information defining the patient population in need of closer follow-up and/or adjuvant therapy. Furthermore, tests that provide prognostic information preoperatively could provide a guide both for preoperative oncologic treatment and the surgical procedure. METHODS. Peripheral blood mononuclear cells (PBMCs) were isolated preoperatively, within a week before primary surgery, from 39 patients undergoing surgery for colorectal cancer. The PBMCs were cultured in vitro for 24 hours in the presence of autologous serum and lipopolysaccharide (LPS). Interleukin-6 (IL-6) production was measured with enzyme-linked immunosorbent assay (ELISA). Staging based on histopathology was performed in all patients. Patients were followed for at least 54 months. RESULTS. A production of >5000 pg/mL of IL-6 identified colorectal cancer patients with a poor prognosis. Eight out of 13 patients with >5000 pg/mL IL-6 died from cancer within the follow-up period, whereas no cancer-related deaths were recorded among 21 patients with 5000 pg/mL IL-6 or less. A multivariate Cox regression analysis, stratified for T- and N-stage, identified IL-6 production as an independent prognostic factor. CONCLUSIONS. IL-6 production in vitro by PBMC can predict survival after radical surgery for colorectal cancer. © 2007 American Cancer Society.

In vitro cell culture models that measure cytokine production can be of great value when analyzing regulatory mechanisms underlying various pathological conditions. However, testing the function of peripheral blood cells has to take into consideration that serum factors are likely to be of importance in maintaining their function. Interleukin-1 receptor antagonist (IL-1Ra) is a cytokine of key importance in immune regulation and is believed to be involved in numerous pathological processes, such as autoimmunity and cancer. We investigated the influence of normal, human serum on spontaneous production of IL-1Ra by human peripheral blood mononuclear cells (PBMC) in vitro. IL-1Ra production in vitro spanned over a wide range of concentrations, which could be attributed to a combined effect of both cellular parameters and properties of the serum used. The production of IL-1Ra in vitro could be correlated to the level of immobilized IgG, especially IgG1 and IgG3, which is adsorbed from the serum and bound to the tissue culture wells during culture. However, the amount of serum IgG adsorbed to the tissue culture wells could not necessarily be predicted based on the serum concentration of IgG. © 2006 Elsevier Ltd. All rights reserved.

In vitro cell culture models can be of great value in order to further analyze the regulatory mechanisms underlying the inappropriate function of the immune system in diseases such as autoimmunity and cancer. Cell culture conditions have to be well controlled in a way that they mirror the in vivo situation. The objective of this study was to compare tissue culture microtiter plates from different manufacturers with respect to their ability to support monokine production by human monocytes cultured in human serum. Tissue culture ware, made of polystyrene, undergoes treatment by the manufacturers to make the surface more suitable for culture of adherent cell populations. It is possible that quality differences in this treatment can lead to variations in protein binding properties and thereby influence the adherence and functional properties of monocytes. We measured spontaneous interleukin-1 receptor antagonist (IL-1ra) production by peripheral blood monocytes, cultured in human serum, in five different microtiter plates made for adherent cell culture. Culture in plates from two of the five manufacturers resulted in significantly lower amounts of secreted IL-1ra. IL-1ra release by human monocytes can be induced by adherent IgG cross-linking membrane receptors for the Fc part of IgG (Fc?R). We found that reduced IL-1ra production coincided with a reduced capacity for binding of serum IgG in one case. Furthermore, this brand of microtiter plate also displayed the lowest level of adsorption of human albumin. We conclude that the protein adsorption properties of the plastic tissue culture ware have to be taken into consideration when assessing monokine production by human monocytes in vitro.

Prolonged exposure to endogenous and exogenous sex steroids increases the risk of breast cancer but the mechanisms are poorly understood. Increased levels of circulating insulin-like growth factor-1 (IGF-1) and low levels of IGF binding protein are associated with increased risk of breast cancer suggesting that IGF-1 has to be in its free form to be biologically active. Little is known about sex steroid regulation of IGF-1 locally in the breast. In this study microdialysis was used to determine the local levels of free IGF-1 in normal human breast tissue in healthy female volunteers during the menstrual cycle. The results showed that the extracellular levels of free IGF-1 locally in the breast were doubled in the luteal phase, when estradiol and progesterone levels were elevated, compared with the follicular phase. In plasma, free IGF-1 levels also exhibited a cyclic variation but to a less extent. The increased local levels of the tree form of IGF-1 may promote proliferation in the breast epithelium. This could be important in sex steroid dependent breast cancer development.

Estrogen exposure is a major risk factor for breast cancer. Tissue estrogen originates from the ovaries but a significant portion is also produced by enzyme activity locally in the breast itself. How these enzymes are regulated is not fully understood. The extracellular space, where the metabolic exchange and cell interactions take place, reflects the environment that surrounds the epithelium but there has been no previous study of hormone concentrations in this compartment. In the present study microdialysis was used to measure extracellular estrogen concentrations in breast tissue and abdominal subcutaneous fat in 12 healthy women in vivo. It was found that women with high plasma progesterone levels had significant increased levels of estradiol in breast tissue compared with fat tissue (breast tissue 168 ± 6 pM, subcutaneous fat, 154 ± 5 pM, P<0.05), whereas women with low plasma progesterone exhibited no difference. Moreover, there was a significant correlation between local breast tissue estradiol and plasma progesterone levels (r=0.709, P<0.01). There was no difference in estrone sulphate in breast and fat tissue regardless of progesterone levels. Estrone was not detectable. The results in this study suggest that progesterone may be one regulator in the local conversion of estrogen precursors into potent estradiol in normal breast tissue. © 2005 Society for Endocrinology.

Changes in the microenvironment are important in the development of cancer and further tumor growth. Although landmark discoveries have been made regarding genetic alterations in cancer at a cellular level very little is known about protein regulation in the extracellular space. In the microenvironment many growth factors are activated at a post-translational level by interactions of different cell types such as epithelial cells, fibroblasts, adipose cells, and immune cells. The extracellular space is the bioactive site for the majority of growth factors and increased knowledge of protein activation in this compartment is of utmost importance for our comprehension of tumor biology. Microdialysis is a minimally invasive technique, which enables sampling of molecules in the extracellular space. It is applicable in human cancer as well as in experimental tumors. This review describes microdialysis, its application and the up to date literature of microdialysis for detection of growth factors in cancer with special emphasis on breast cancer.

Purpose: Angiogenesis is crucial in tumor development and progression. Ovarian hormones regulate angiogenesis in the reproductive tract but very little is known about its regulation in the normal breast. Sex steroids play an important role in breast cancer development by poorly understood mechanisms. Vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) are potent stimulators of angiogenesis. Both VEGF and FGF-2 function in autocrine/ paracrine pathways and there is a major contribution of bioactive proteins by a posttranslational activation of sequestered molecules in the extracellular space. A direct measurement of these molecules in the extracellular compartment is, therefore, needed. Experimental Design: In this study, microdialysis was used to measure extracellular VEGF and FGF-2 in normal human breast tissue in situ in 11 premenopausal and 5 postmenopausal women. Results: Significantly higher level of VEGF in breast tissue of premenopausal women was found. Plasma as well as local estradiol and breast tissue VEGF exhibited significant correlations, whereas progesterone had no correlation with breast VEGF. FGF-2 did not correlate with either estradiol or progesterone. Conclusion: The result suggests that estradiol is a more potent regulator of free VEGF levels than progesterone in the normal breast. The control of free FGF-2 seems to be independent of sex steroids in the breast. Estrogen induction of free extracellular VEGF may be one mechanism involved in sex steroid - dependent breast carcinogenesis. © 2005 American Association for Cancer Research.

Angiogenesis is essential for normal function in the female reproductive tract and a prerequisite for growth and metastasis of solid tumors. Several factors, both inducers and inhibitors, play essential roles in the regulation of the angiogenic process. Exposure to sex steroids increases the risk of breast cancer but the mechanisms are poorly understood and the importance of angiogenesis in breast carcinogenesis is undefined. In the female reproductive tract ovarian hormones tightly regulate angiogenesis. The breast is also a target organ for sex steroids but very little is known about sex steroid effects on angiogenesis in normal breast tissue and breast cancer. In this review several regulators of angiogenesis, and their relation to sex steroids, in breast tissue are discussed. Increased knowledge in this area is of utmost importance for future therapeutic treatment options and for breast cancer prevention. © Springer 2005.

The lysosomal protease Catliepsin D (Cath D) is associated with increased invasiveness and metastasis in breast cancer. Both estrogen and tamoxifen have been reported to increase Cath D, which seems to contradict the efficacy of tamoxifen as an adjuvant for estrogen dependent breast cancer. Cath D is bioactive in the extracellular space but very little is known about hormonal regulation of secreted Cath D in vivo. In this study we used microdialysis to sample the extracellular fluid in estrogen receptor positive MCF-7 tumors in nude mice. We show that tamoxifen in combination with estradiol decreased secreted Cath D compared with estradiol treatment only in solid tumors in situ. Cell culture of MCF-7 cells revealed that estradiol and tamoxifen increased intracellular proteolytic activity of Cath D in a similar fashion whereas secretion of Cath D was increased by estradiol and inhibited by tamoxifen. Immunofluorescence showed that estradiol located Cath D to the cell surface, while tamoxifen accumulated Cath D to dense lysosomes in perinuclear regions. Moreover, tamoxifen increased the intracellular transporter of Cath D, the mannose 6-phosphate/IGF-II receptor (M6P/IGF2R). In contrast, estradiol decreased the levels of this receptor. Thus, secretion of Cath D is hormone dependent and may be mediated by altered expression of the M6P/IGF2R. Our results highlight the importance of measurements of proteins in all compartments where they are biological active and show that microdialysis is a viable technique for sampling of Cath D in vivo.

Angiogenesis is essential for tumor growth and metastasis and an important prognostic factor in breast cancer. VEGF, a key factor for angiogenesis, has been correlated with tumor vessel density in breast cancer. Estrogen, another crucial factor in breast cancer, stimulates VEGF, and an ERE in the VEGF gene has been defined. VEGF is bioactive in the extracellular fluid, where it becomes available to endothelial cells. Whether E2 affects VEGF levels in the extracellular fluid is not known. We show, using intratumoral microdialysis in vivo, that E2 treatment increased tumor extracellular levels of VEGF in an estrogen-dependent breast cancer model. Moreover, extracellular levels of VEGF in the tumor showed a strong correlation with total tumor VEGF, contrary to plasma levels of VEGF. Ninety-three percent of measured VEGF in the extracellular fluid in the tumor was tumor-derived, while only 45% of VEGF in circularing plasma originated from the tumor. We conclude that E2 increases extracellular VEGF and that microdialysis is a sensitive method for measurement of local VEGF production in vivo. Our results have potential application to the assessment of tumor characteristics in vivo in human tumors for individualized cancer therapy.

The majority of breast cancers are oestrogen dependent and although current treatment strategies have improved, approximately 50% of the patients will develop metastasis. New treatments that result in long-term systemic immunity are therefore being developed. We have previously shown that adenoviral gene transfer of B7-I/IL-2 to murine breast cancer induces a high rate of complete turnout regression and systemic immunity. Since oestrogens not only affect breast cancer but also have been shown to modulate immune function and secretion of immune-regulatory cytokines, we explored whether administration of oestradiol altered the immune response induced by an adenoviral vector expressing B7-I/IL-2. An oestrogen-dependent murine breast cancer tumour was used in ovariectomised mice, supplemented either oestradiol or placebo. We report the somewhat unexpected finding that intratumoral injection of adenovirus expressing B7-I/IL-2 induces complete turnout regression in 76% of oestradiol-supplemented mice, while only 18% of the tumours regressed in the oestrogen-depleted group. Cured mice in both groups exhibited a similar CTL response against the tumour antigen. However, intratumoral IFN-? levels, 2 days after B7-I/IL-2 injection, were significantly higher in mice treated with oestradiol compared to placebo. This may be one mechanism explaining the higher response rate of tumours in oestradiol-replenished mice.

Oxidative stress and alterations in the antioxidative defense system may be involved in carcinogenesis. We have previously shown that the levels of glutathione (GSH) in vivo in both breast tissue and subcutaneous fat were higher in the luteal phase compared with the follicular phase, suggesting an overall increase in GSH. This result was confirmed in the present study. Moreover, we exposed normal breast tissue in vivo, breast epithelial cells in vitro, and hepatocytes in culture to ovarian hormones. We found that local perfusion with estradiol, using microdialysis, in normal human breast tissue did not alter the local GSH levels in vivo. In vitro, treatment with estradiol and progesterone of normal human breast epithelial cells did not alter GSH levels. However, levels of GSH in hepatocytes were after 8 h estradiol exposure initially decreased, 76.6 ± 5% of control cells, p < .05, whereas 20 h exposure more than doubled GSH, 209 ± 26% compared with control cells, p < .01. Progesterone had no additional effect. Exposure of hepatocytes to estradiol increased the cellular content of γ-glutamylcysteine synthetase, the rate-limiting enzyme in GSH synthesis. In conclusion we suggest that estradiol affects the GSH homeostasis mainly by effects on hepatocytes, whereas local production in the breast is unaffected by estradiol.

[No abstract available]

Background: Several studies have shown that microsatellite instability (MSI) is related to favourable survival in colorectal cancer patients but there are controversial results. Tumour suppressor gene RIZ is a susceptible mutational target of MSI. However, its clinicopathological significance has not been investigated. We investigated the prognostic significance of MSI in Swedish colorectal cancer patients and the clinicopathological significance of RIZ mutations. Patients and methods: We analysed 438 colorectal adenocarcinomas for MSI by microsatellite analysis. Among them, 29 MSI and 28 microsatellite stable (MSS) tumours were examined for RIZ mutations by DNA sequencing. Results: MSI (13% of 438 cases) was not associated with survival (rate ratio=0.97, 95% confidence interval =0.57-1.64, P=0.90), although it was related to proximal tumour (P <0.001), poor differentiation and mucinous carcinomas (P <0.001), multiple tumours (P=0.01) and negative/weak expression of hMLH1 (P=0.03). RIZ mutations were detected in 31% of 29 MSI tumours but in none of the 28 MSS tumours. The mutations were related to female (P=0.01), proximal tumour (P=0.01), stage B (P=0.01) and poor differentiation (P=0.047). Conclusions: MSI was not a prognostic factor in the Swedish patients included in this study. Clinicopathological variables associated with RIZ mutations might be a consequence of the MSI characteristics.

Objective: The nucleoporin Nup88 is overexpressed in a series of human malignancies, however, its clinicopathological significance has not been studied. Our aims were to analyze Nup88 expression in normal mucosa, primary tumors and metastases from colorectal cancer patients and further to identify relationships of Nup88 expression with clinicopathological and other factors.

Materials and Methods: Using immunohistochemistry, we investigated Nup88 expression in 198 primary colorectal tumors, 96 normal mucosa samples and 35 lymph node metastases.

Results: The results showed that the intensity of Nup88 expression increased from the normal mucosa to the primary tumors (p < 0.0001) and tended to increase from the primary tumors to the metastases (p = 0.15). Both primary tumors and metastases presented stronger expression in the invasive margin and vascular-invaded areas. Nup88 expression was positively related to distal tumor location (p = 0.01), infiltrative growth pattern (p = 0.04) and higher proliferative activity (p = 0.04) and reversely to the grade of differentiation (p = 0.02) and apoptosis (p = 0.049). Strong expression of Nup88 predicted a worse outcome in the patients with distal tumors during the follow-up period of up to 3 years (p = 0.02).

Conclusions: It seems that overexpression of Nup88 was involved in the tumorigenesis and aggressiveness of colorectal cancers, and Nup88 may be used as a prognostic factor in patients with distal tumors.

Colorectal cancer is a serious health problem in Western societies. In Sweden it is the second most common malignancy among females, and the third in males, and the third leading cause of cancer related death in both sexes. To date it is known that several biological pathways can lead to colorectal cancer. An enhanced understanding of the molecular and cellular events occurring within these pathways should ultimately result in better tailored preventive, diagnostic and therapeutic approaches. An important goal is to identify cancer predisposing genetic variants that would make genetic screening of at-risk individuals possible, and to find parametres which could define homogenous subgroups associated with a certain prognosis.

Impaired apoptotic processes have been implicated in colorectal tumourigenesis. In order to explore the significance of apoptosis in relation to proliferative activity, and other clinicopatological data including prognosis, apoptotic rates were measured by TUNEL in 158 colorectal tumours. A significant increase of apoptotic rate was observed with advancing tumour stage, from Dukes' A to D. Apoptotic rates were further significantly associated with the proliferative activity in the tumour, indicated by the proliferating cell nuclear antigen (PCNA). Thus it seems that apoptotic levels in the tumour might reflect tumour progression, and further that the processes of apoptosis and proliferation are coupled. Apoptosis was not related to p53, bcl-2 or K-ras, and did not confer any additional prognostic information.

APC has been referred to as a "gatekeeper" in the progression of colorectal cancer. It is in a key position to regulate the balance between cell adhesion and migration and may also be involved in proliferation and apoptosis. The two germ line APC variants I1307K and E1317Q have been associated with increased colorectal cancer risk in certain populations, but have yet never been investigated in Swedish colorectal cancer patients. We screened J 94 colorectal cancer patients with and without a family history of colorectal cancer by DNA sequencing analysis, but did not find any of the variants. We conclude that I1307K and E1317Q are rare or absent in this population and should not confer any increased colorectal cancer risk in the Swedish population.

The microsatellite instability pathway accounts for the majority of tumours in hereditary non-polyposis colorectal cancer (HNPCC) patients and about 15% of sporadic colorectal cancer cases. Inactivated mismatch repair (MMR) genes leads to an increased mutation rate, especially within repetitive DNA sequencies called microsatellites which are prone to replication errors, a phenomenon referred to as microsatellite instability (MSI). Several genes with microsatellites within their coding regions are targeted by MSI, and are possibly involved in tumourigenesis, including the repair gene MBD4 which is involved in maintaining the integrity of genomic methylation patterns. We aimed to determine the microsatellite status of 201 colorectal tumours using the marker Bat 26 in order to evaluate the relationship of MSI and clinicopathological variables including survival. We further aimed to investigate whether MBD4 is a MSI target gene in colorectal cancer, and to search for mutational associations with clinicopathological factors to reveal eventual phenotypical effects. Surprisingly, MSI was not associated with survival, as opposed to many previous studies, suggesting that the prognostic significance and the factors associated with it demands further investigation. MBD4 seemed to be a target of MSI, since the mutations were restricted only to MSI tumours. The clinicopathological variables associated with the MBD4 mutated tumours were likely the reflection of MSI features aquired prior to the MBD4 mutation, including right colon location and mucinous histology. The significance of these mutations remains to be determined in a largermaterial.

Colorectal cancer is a disease that is associated with default in the balance of apoptotic regulation. In the present study apoptosis was examined in 158 colorectal adenocarcinomas using the terminal deoxynucleotidyl transferase mediated digoxigenin nick end labeling (TUNEL) method. The median apoptotic index (AI) was 0.95% (range 0-6. 68%). Eighty-two tumours exhibited AI 0.95%. We revealed a positive correlation between apoptosis and proliferation determined as the expression of proliferating cell nuclear antigen (PCNA, p=0.002). The frequency of apoptosis increased from Dukes' stage A, B, C to D (p=0.01). No correlations were found between apoptosis and the patients' sex, age, tumour location, growth pattern, differentiation, prognosis, bcl-2, p53 or K-ras. Our findings suggest that we should further investigate the relationship between apoptosis and cellular proliferative activity in colorectal cancer to evaluate whether this might provide additional information in the selection of patients for effective adjuvant therapy.

Recently, a germ line mutation of the APC gene, I1307K, was discovered in a subset of Ashkenazi jews. The mutation involves an amino acid exchange and creates a tract consisting of eight contiguous adenosine residues believed to cause hypermutability in this region. Another germ line missense variant, E1317Q, not restricted to a certain ethnic population, could functionally alter the protein. These APC variants have been linked with increased colorectal cancer risk in several studies. However, they have not yet been investigated in Swedish colorectal cancer patients. Thus, our aim was to investigate the prevalence of I1307K and E1317Q in Swedish colorectal cancer patients in order to determine if these genetic variants are important predisposing factors to colorectal cancer in this population. To this end, sequence analysis was carried out of the APC gene in order to identify any I1307K and E1317Q variants in 106 unselected cases and 88 hereditary/familial colorectal cancer cases including 22 cases of hereditary non-polyposis colorectal cancer (HNPCC) fulfilling the Amsterdam criteria. Out of a total of 194 cases examined, we did not find any variants. It seems that these alterations are rare or absent in the Swedish population.