The incidence of breast cancer is rising steadily in Sweden and the proportion of carcinoma in situ (CIS) has increased appreciably, most likely due to mammography screening. The aim of this study was twofold: (1) to examine risk factors for subsequent invasive breast carcinoma and breast cancer death after primary ductal carcinoma in situ (DCIS) and (2) to study the biology in the progress between in situ and invasive carcinoma. In a cohort-study based on 3,398 women with a primary CIS reported to the Swedish Cancer Registry (SCR) 1980-1992, women diagnosed in 1989-1992 ran a relative risk of 0.1 (CI 95%, 0.0-0.9) from dying of breast cancer as compared with women diagnosed in 1980-1982. Women in counties with mammography screening ran a relative risk of 0.2 (CI 95%, 0.0-2.1) for breast cancer death in comparison with women in non-screening counties. In a case-control study derived from all 4,661 women with primary CIS reported to the SCR 1960-1992, we investigated risk factors for subsequent invasive breast carcinoma (n=118) and breast cancer death (n=39). Large size and multifocality were found to increase the risk for breast cancer death. Postoperative radiotherapy and mastectomy lowered the risk for ipsilateral invasive cancer. The standardised incidence rates (SIR) for invasive breast cancer were estimated in the cohort from 1980-1992. The SIR after primary DCIS and primary lobular carcinoma in situ (LCIS) was 4.5 (CI 95%, 3.7-5.5) and 4.0 (CI 95%, 2.1-7.5), respectively. New histopathological classification systems for DCIS were evaluated in 195 women consecutively diagnosed with primary DCIS between 1986-1994. One group with highly differentiated lesions was defined with the EORTC classification system and had an excellent prognosis. Histopathological grade and expression of p53, c-erbB-2, Ki 67, hormone receptors, Bcl-2 and angiogenesis were compared in 626 women with either a pure DCIS, a small invasive carcinoma or a lesion with both an invasive and in situ component. When grade was taken into account, no change in tumour markers could be detected that signalled the progression from an in situ stage to invasiveness. All tumour markers correlated to grade and their distribution was very similar in the two components of mixed lesions.

This thesis focuses on the involvement of apoptosis and proliferation in the mechanisms of menstruation and hormonal replacement therapy, HRT, as well as in the mechanisms of progesterone therapy in endometrial carcinoma. The aim of the first study was to investigate endometrium for 4 days before and for 2 days during menstruation. In the epithelium, rapid increase in the apoptotic index, decreasing expression of estrogen receptor α (ER) and progesterone receptor (PR), and minimal proliferation were observed prior to menstruation. In the stroma, an increase in the expression of ER and PR and proliferation was seen before the final decrease, and increased apoptosis was seen during menstruation. Thus, apoptosis is involved in the remodeling of the endometrium during menstruation. Postmenopausal endometrium showed unaffected homeostasis, i.e. unchanged ratio between apoptotic index and Ki-67 index during substitution therapy. ER expression was decreased both in the epithelium and stroma, while PR showed some increase in receptor expression. The unchanged homeostasis contributes to endometrial safety during combined continuous HRT. Unchanged apoptosis and increasing proliferation were observed with increasing tumor grade in 29 patients with endometrioid endometrial carcinoma, which may contribute to greater aggression as tumor grade increases. Decreased proliferation was observed after medroxy-progesterone at 20 mg per day particularly in the foci of maximal proliferation in G1 and G2 tumors. The expression of ER was unchanged, while PR was decreased in the foci of maximal expression for PR in G1 and G2 tumors. Since high proliferation and PR expression also coexisted in the same foci, evaluated in G1 and G2 tumors, the effect of progesterone could be facilitated in these tumor groups. High expression of sex steroid receptors was also a predicting factor for good response to progesterone (= decrease in proliferation), while the amount of stroma could not predict that effect.

Interferons (IFNs) are a class of structurally related proteins first discovered to be produced by virus-infected cells. By now, several other inducing agents have been described. IFNs exert multiple effects on cells exemplified by the establishment of an antiviral state, inhibition of cell proliferation and alteration of different immune reactions. In the present thesis the inhibition of cellular growth concentrated on effects in the early cell cycle have been studied. The human glioma cell line 251 MG was found to be blocked in the S phase of the cell cycle upon addition of IFN both to exponentially growing and growth-factor depleted, synchronized cells. Thymidine kinase and DNA-polymerase activities were reduced in parallel with the S phase effect. 2-5 oligo Anucleotides transfected into glioma cells lead to inhibition of cell growth, exponentially growing cells being blocked in the S phase as during IFN treatment. In contrast, synchronized, restimulated cells were blocked in the cellcycle phase where they resided at the time of transfection. As 2-5 oligo A synthetase activity was induced in the middle of the Gl phase, these results might indicate that the kinetics of expression of oligonucleotides after IFN additiondetermines the type of cell cycle block obtained in differenttumor cells. IFN inhibited preferentially proteins originating from newly synthesized mRNA in Sw 3T3 cells, c-mvc did not seem to be included among these proteins. In both cell systems c-myc expression was unaltered after IFN treatment. In clone T1 selected from the the Sw 3T3 cell line , c-mvc expression was uncoupled to growth and seemed to be growth factor independent. The change in c-myc expression in clone T1 compared to SW 3T3 cells did not render the cells sensitive to IFN. Hence, c-myc regulation does not seem to be the mechanism by which IFN regulates cell growth in this system. The proliferation marker KI-67 antigen was shown not to be causatively involved in growth inhibition of IFN. The reduced levels of the antigen was proposed to be a secondary effect caused by the G0/G1 arrest. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1991, härtill 6 uppsatser</p> / digitalisering@umu

The natural history of localized prostate cancer is not fully understood. In most patients the tumor will never progress to a lethal disease, while a subset of patients will ultimately die of the disease. Efficient tools to separate indolent from lethal disease is currently lacking which means that many patients will be offered treatment without any benefit, but still be at risk of experiencing treatment related side effects. The aims of these studies were to get more insight into the natural history of untreated localized prostate cancer, to assess the prognostic value of established clinical parameters such as Gleason score, nuclear grade and tumor volume and, moreover, some new prognostic markers Ki-67, AMACR and MUC-1. We also aimed to study time trends in the detection of incidental tumors in Sweden. Patients with localized disease (n=223) and no initial treatment were followed for 21 years. Most patients had a favorable outcome. However, a subset of patients developed lethal disease even beyond 15 years of follow-up and these patients define the group that may benefit most from treatment with curative intent. Patients with poorly differentiated tumors experienced a 9 time higher risk of dying in prostate cancer. The studies on prognostic markers are based on a cohort of patients (n=253) with incidental prostate cancer detected by transurethral resection for presumed benign hyperplasia. All patients were left without initial treatment. Gleason grade, nuclear grade and tumor volume turned all out to be independent prognostic factors. MUC-1, AMACR and Ki-67 also carried prognostic information. However, after adjustment for Gleason grade, nuclear grade and tumor volume only MUC-1 and AMACR remained as statistically significant prognostic factors. When tested for sensitivity and specificity they all failed and, consequently, they seem to be of less value in daily practice for cancelling an individual patient regarding the choice of treatment. Time trends in incidental prostate tumors in Sweden were analyzed in a cohort of patients with prostate tumors detected by transurethral resection (TUR-P). Through linkage of the national registration number (NRN) with several registers, e.g. the Swedish Cancer Registry, the National Inpatient registry and the Cause of Death Registry we identified, during the period 1970 through 2003, in total 23288 patients with incidental prostate cancer, who constituted the study group. As comparison group we choose all patients diagnosed with prostate cancer between 1970-2003 excluding those with incidental cancer, in total 112204 patients. Our result confirms earlier findings that there has been a dramatic change over time in incidence of incidental prostate cancers in Sweden, which parallels the introduction of prostate specific antigen. We also found that the cumulative incidence of prostate cancer death is high in the incidental group, opposing earlier findings that incidental tumours are a non-lethal disease.

Background: Prostate Cancer (PC) is a common and highly variable disease. Using current diagnostic methods, the prostate specific antigen (PSA) blood test and histological grading of prostate tissue needle biopsies, it is often difficult to evaluate whether the patient has a PC that requires active treatment or not. The absolute majority of all 10,000 cases of PCs diagnosed annually in Sweden have tumours graded as Gleason score (GS) 6-7 and a PSA value in blood below 10. Many of these are harmless and can be left without active treatment and hence spared problematic post-therapy side-effects, others are highly malignant and require early diagnosis and treatment. Better prognostic markers are needed and the aim of this study was to evaluate prognostic markers and to test if these markers could identify patients with indolent tumours. Methods: We have studied tumour material from 419 men consecutively diagnosed with PC at transurethral resection (1975-1990). The majority of these patients (295) had no metastasis at diagnosis and was not given any curative treatment and only hormonal treatment upon symptoms from metastatic progression. Standard histological sections and tissue microarrays (TMA) from these tumours and surrounding normal prostate tissue were stained and evaluated for cell proliferation (Ki67), blood vessels (endoglin and von Willebrand factor, vWf) and the extracellular matrix component hyaluronan (HA). An orthotopic rat PC model was used to explore hyaluronan staining, hyaluronic acid synthase (HAS)-1 mRNA levels and the effect of local HA treatment on tumour growth. Results: Tumour cell proliferation (Ki67) and the density of intra-tumoural endoglin stained blood vessels were independent prognostic markers (i.e. they added prognostic information to the conventional prognostic markers; clinical stage and GS). None of the GS 6 patients with low staining for both Ki67 and endoglin died of PC within 15 years of follow-up. High HA staining in the tumour epithelium and stroma was a negative prognostic marker of cancer specific survival but they were not independent of GS. High HA staining and high vascular density in the stroma of the surrounding morphologically normal prostate were prognostic for short cancer specific survival. Implantation of tumour cells in the normal rat prostate resulted in an increase in HA and HAS-1 mRNA levels in the prostate tissue surrounding prostate tumours. Concurrently intra-prostatic injection of HA also stimulated tumour growth. Conclusions: By evaluating both tumour cell proliferation (Ki67) and vascular density, it is possible to identify patients with very low risk of cancer specific death in the absence of active treatment. Prostate tumours influence the surrounding non-malignant prostate tissue, for example they cause an increased angiogenesis and synthesis of hyaluronan. Such responses can possibly be used to diagnose PC and to evaluate PC aggressiveness.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Base teórica: doenças linfoproliferativas crônicas de linhagem B (DLPC-B) são neoplasias clonais que afetam linfócitos B maduros. A tirosina quinase de Bruton (do inglês Bruton’s tyrosine kinase, BTK) é uma proteína essencial para o desenvolvimento, diferenciação e sinalização nos linfócitos B. Ki-67 é uma proteína nuclear associada à proliferação celular. A avaliação de proteínas envolvidas nas vias de sinalização oncogênicas pode levar ao aprimoramento do diagnóstico, tratamento e definição de prognóstico das DLPC-B. Objetivo: avaliar a expressão de BTK e Ki-67 em linfócitos de portadores de DLPC-B. Métodos: para avaliação de BTK foi realizado um estudo transversal; foi avaliada a expressão de BTK em amostras de pacientes saudáveis e de pacientes com diagnóstico de DLPC-B. Para avaliação de Ki-67 foi realizado um estudo transversal. As amostras foram marcadas com CD45 FITC e CD19 APC para identificação dos linfócitos B. Após a lise das hemácias, foi realizada marcação citoplasmática de BTK PE e/ou Ki-67 PerCP-Cy5.5. O percentual de expressão e a intensidade de fluorescência média (IFM) dos marcadores avaliados foram determinados nos linfócitos B. A análise estatística foi realizada com testes de correlação de Pearson e Spearman entre BTK ou Ki-67 e as demais variáveis clínicas e laboratoriais, e ANOVA seguido por teste post hoc de Bonferroni para comparações entre grupos. Foi considerado resultado significante quando P < 0,05. Resultados: não foram observadas diferenças na expressão de BTK e não houve associação entre a expressão de BTK e as variáveis clínicas avaliadas. A expressão de Ki-67 foi maior nos grupos linfoma do manto, linfoma de Burkitt e linfoma difuso de grandes células B em relação aos demais; após análise multivariada, a IFM de Ki-67 foi associada à IFM de CD38. Conclusão: no presente trabalho, a expressão de BTK em DLPC-B foi similar a de linfócitos B normais e a expressão de Ki-67 foi maior nas DLPC-B com curso clínico mais agressivo. / Background: mature B-cell neoplasms (MBCN) are clonal neoplasms that affect mature B-cell lymphocytes. Bruton’s tyrosine kinase (BTK) is an essential protein for the development, differentiation and signaling in B-cell lymphocytes. Ki-67 is a nuclear protein associated to cellular proliferation. Evaluation of proteins involved in oncogenic signaling pathways can lead to improvement in the diagnosis, treatment and prognosis definition in MBCN. Objective: to evaluate the expression of BTK and Ki-67 in lymphocytes of MBCN patients using flow cytometry. Methods: a cross-sectional study was conducted for BTK assessment; BTK expression was assessed on healthy patients samples and MBCN samples. For evaluation of Ki-67 a cross-sectional study was conducted. Samples were stained with CD45 FITC and CD19 APC for identification of B-cell lymphocytes. After lysis of red blood cells, cytoplasmic staining of BTK PE and/or Ki-67 PerCP-Cy5.5 was performed. Percentage of expression and mean fluorescence intensity (MFI) of the markers were determined in B-cell lymphocytes. Statistical analysis was performed with Pearson and Spearman correlation tests between BTK and Ki-67 and the other clinical and laboratory variables, and ANOVA followed by post-hoc Bonferroni test for comparisons between groups. Results were considered significant when P < 0.05. Results: no differences in BTK expression were identified and there was no association between BTK expression and clinical variables evaluated. Ki-67 expression was higher in mantle cell lymphoma, Burkitt lymphoma and diffuse large B-cell lymphoma cases; after multivariate analysis, MFI of Ki-67 was associated with MFI of CD38. Conclusions: in this study, BTK expression in B-cell neoplasms was similar to that of normal B-cell lymphocytes and Ki-67 expression was higher in MBCN with more aggressive clinical courses.

Base teórica: doenças linfoproliferativas crônicas de linhagem B (DLPC-B) são neoplasias clonais que afetam linfócitos B maduros. A tirosina quinase de Bruton (do inglês Bruton’s tyrosine kinase, BTK) é uma proteína essencial para o desenvolvimento, diferenciação e sinalização nos linfócitos B. Ki-67 é uma proteína nuclear associada à proliferação celular. A avaliação de proteínas envolvidas nas vias de sinalização oncogênicas pode levar ao aprimoramento do diagnóstico, tratamento e definição de prognóstico das DLPC-B. Objetivo: avaliar a expressão de BTK e Ki-67 em linfócitos de portadores de DLPC-B. Métodos: para avaliação de BTK foi realizado um estudo transversal; foi avaliada a expressão de BTK em amostras de pacientes saudáveis e de pacientes com diagnóstico de DLPC-B. Para avaliação de Ki-67 foi realizado um estudo transversal. As amostras foram marcadas com CD45 FITC e CD19 APC para identificação dos linfócitos B. Após a lise das hemácias, foi realizada marcação citoplasmática de BTK PE e/ou Ki-67 PerCP-Cy5.5. O percentual de expressão e a intensidade de fluorescência média (IFM) dos marcadores avaliados foram determinados nos linfócitos B. A análise estatística foi realizada com testes de correlação de Pearson e Spearman entre BTK ou Ki-67 e as demais variáveis clínicas e laboratoriais, e ANOVA seguido por teste post hoc de Bonferroni para comparações entre grupos. Foi considerado resultado significante quando P < 0,05. Resultados: não foram observadas diferenças na expressão de BTK e não houve associação entre a expressão de BTK e as variáveis clínicas avaliadas. A expressão de Ki-67 foi maior nos grupos linfoma do manto, linfoma de Burkitt e linfoma difuso de grandes células B em relação aos demais; após análise multivariada, a IFM de Ki-67 foi associada à IFM de CD38. Conclusão: no presente trabalho, a expressão de BTK em DLPC-B foi similar a de linfócitos B normais e a expressão de Ki-67 foi maior nas DLPC-B com curso clínico mais agressivo. / Background: mature B-cell neoplasms (MBCN) are clonal neoplasms that affect mature B-cell lymphocytes. Bruton’s tyrosine kinase (BTK) is an essential protein for the development, differentiation and signaling in B-cell lymphocytes. Ki-67 is a nuclear protein associated to cellular proliferation. Evaluation of proteins involved in oncogenic signaling pathways can lead to improvement in the diagnosis, treatment and prognosis definition in MBCN. Objective: to evaluate the expression of BTK and Ki-67 in lymphocytes of MBCN patients using flow cytometry. Methods: a cross-sectional study was conducted for BTK assessment; BTK expression was assessed on healthy patients samples and MBCN samples. For evaluation of Ki-67 a cross-sectional study was conducted. Samples were stained with CD45 FITC and CD19 APC for identification of B-cell lymphocytes. After lysis of red blood cells, cytoplasmic staining of BTK PE and/or Ki-67 PerCP-Cy5.5 was performed. Percentage of expression and mean fluorescence intensity (MFI) of the markers were determined in B-cell lymphocytes. Statistical analysis was performed with Pearson and Spearman correlation tests between BTK and Ki-67 and the other clinical and laboratory variables, and ANOVA followed by post-hoc Bonferroni test for comparisons between groups. Results were considered significant when P < 0.05. Results: no differences in BTK expression were identified and there was no association between BTK expression and clinical variables evaluated. Ki-67 expression was higher in mantle cell lymphoma, Burkitt lymphoma and diffuse large B-cell lymphoma cases; after multivariate analysis, MFI of Ki-67 was associated with MFI of CD38. Conclusions: in this study, BTK expression in B-cell neoplasms was similar to that of normal B-cell lymphocytes and Ki-67 expression was higher in MBCN with more aggressive clinical courses.

Le CD81 est une molécule de surface appartenant à la superfamille des tetraspanines. Son rôle pronostique a été précédemment étudié dans les pathologies lymphoïdes, dont le myélome multiple où son expression est associée à un pronostic péjoratif. A ce jour, ce marqueur n'a pas été étudié dans les leucémies aiguës myéloïdes (LAM). Nous avons étudié l'expression membranaire du CD81 sur les blastes de LAM au diagnostic, son association aux autres caractéristiques des LAM et sa potentielle influence sur la survie des patients sur une cohorte de 134 patients traités par chimiothérapie intensive.Le CD81 a été retrouvé chez 92 patients sur 134 (69%). Les patients exprimant ce marqueur avaient une leucocytose initiale plus élevée (p=0.02) et présentaient une cytogénétique intermédiaire ou défavorable (p<0.001). L'expression du CD81 avait un impact négatif sur la survie des patients (survie sans évènements (EFS), survie globale (OS), survie sans rechute (RFS)) en analyse uni- (p<0.001) et multivariées (p=0.003, 0.002 and <0.001 respectivement).De plus, le CD81 avait un impact négatif sur l'OS des patients avec une mutation de NPM1 (p=0.01) et chez les patients du groupe cytogénétique favorable (p=0.002) selon la classification ELN.Les anomalies du cycle cellulaire étant associées à la chimiorésistance, la croissance tumorale et l'agressivité de la pathologie, nous avons étudié l'expression du Ki67 sur les blastes de LAM au diagnostic. Ainsi, 10 prélèvements médullaires de patients avec une faible expression du CD81 par les blastes (moins de 20% de positivité) et 10 prélèvements avec une forte expression du marqueur ont été étudiés. Nous avons pu démontrer une expression significativement inférieure du Ki67 sur les blastes CD81 positifs par rapport aux blastes CD81 négatifs (p<0.001), suggérant ainsi un rôle potentiel du CD81 dans le contrôle du cycle cellulaire. De plus, nous nous sommes intéressés au rôle du CD81 dans la chimiorésistance et sur les différentes voies de signalisation cellulaire en étudiant le profil d'expression génique.En conclusion, le CD81 semble être un nouveau marqueur pronostique des LAM ainsi qu'une cible potentielle de traitement de ces pathologies. / CD81 is a cell surface protein which belongs to the tetraspanin family. While in multiple myeloma its expression on plasma cells is associated with worse prognosis, this has not yet been explored in acute myeloid leukemia (AML). We measured membrane expression of CD81 on AML cells at diagnosis, evaluated its association with AML characteristics and its influence on patient outcome after intensive chemotherapy in a cohort of 134 patients. CD81 was detected in 92/134 (69%) patients. Patients with AML expressing CD81 had elevated leukocyte count (p=0.02) and were more likely classified as intermediate or adverse-risk by cytogenetics (p<0.001). CD81 expression had a negative impact on survival (event-free [EFS], overall [OS] and relapse-free survival [RFS]) in univariate (p<0.001) and in multivariate analyses (p=0.003, 0.002 and <0.001, respectively). CD81 has a negative impact on OS in patients with NPM1 mutation (p=0.01) and in favorable risk patients by European Leukemia Net (ELN) classification (p=0.002).Since aberrations in cell cycle signaling can cause drug resistance, tumor growth and aggressiveness we measured Ki67 on primary blast cells from AML patients. We considered 10 bone marrow samples from AML patients with either weak CD81 expression (less than 20% of blast cells) or 10 bone marrow samples with strong CD81 expression on blasts. We found a significant lower ki67 expression on blast cells from CD81 positive patients compared with those from CD81 negative patients (p<0.001), indicating a potential role of CD81 in cell cycle control. Furthermore, we investigated the role of CD81 in chemotherapy resistance and investigated potentially implicated signaling pathways by gene expression profiling.In conclusion, the cell surface marker CD81 may be a new prognostic marker for diagnostic risk classification and a new potential therapeutic target for drug development in AML.

The three-dimensional organization of the human genome is non-random in interphase cells. Heterochromatin is highly clustered at the nuclear periphery, adjacent to nucleoli, and near centromeres. These localizations are reshuffled during mitosis when the chromosomes are condensed, nucleoli disassembled, and the nuclear envelope broken down. After cytokinesis, heterochromatin is re-localized to the domains described above. However, the mechanisms by which this localization is coordinated are not well understood. This dissertation will present evidence showing that both CAF-1 p150 and Ki-67 regulate nuclear structure throughout the human cell cycle. Chromatin Assembly Factor 1 (CAF-1) is a highly conserved three-subunit protein complex which deposits histones (H3/H4)2 heterotetramers onto replicating DNA during S-phase of the cell cycle. The N-terminal domain of the largest subunit of CAF-1 (p150N) is dispensable for histone deposition, and instead regulates the localization of specific loci (Nucleolar-Associated Domains, or “NADs”) and several proteins to the nucleolus during interphase. One of the proteins regulated by p150N is Ki-67, a protein widely used as a clinical marker of cellular proliferation. Depletion of Ki-67 decreases the association of NADs to the nucleolus in a manner similar to that of p150. Ki-67 is also a fundamental component of the perichromosomal layer (PCL), a sheath of proteins that surrounds all condensed chromosomes during mitosis. A subset of p150 localizes to the PCL during mitosis, and depletion of p150 disrupts Ki-67 localization to the PCL. This activity was mapped to the Sumoylation Interacting Motif (SIM) within p150N, which is also required for the localization of NADs and Ki-67 to the nucleolus during interphase. Together, these studies indicate that p150N coordinates the three-dimensional arrangement of both interphase and mitotic chromosomes via Ki-67.