<p>Malt lymphoma is a malignant disease that can arise in a variety of extra nodal sites. Previous studies indicate that tumour arise from more mature B-cells.</p><p>Our purpose was to examine the presence of clonality and somatic hypermutation of immunoglobulin (IgVн) of MALT lymphomas.</p><p>Paraffin-embedded tumour samples from13 MALT lymphoma were subjected to rearrangement analysis, by using PCR, heteroduplex gels and sequence analysis.</p><p>Successful amplification was seen in 10/13 cases and sequences of IgVн genes were obtained in 6/13, all of them were mutated. The percentage of mutation compared to germline sequences was 1,1% to 8,6% monoclonal rearrangemang. It was demonstrated that 5 of 7 clones were derived from the Vн3 family, 2 from Vн1 and 1 from the Vн 4 family.</p>

A novel technology for protein detection, proximity ligation, has been developed along with improved methods for in situ synthesis of DNA microarrays. Proximity ligation enables a specific and quantitative transformation of proteins present in a sample into nucleic acid sequences. As pairs of so-called proximity probes bind the individual target protein molecules at distinct sites, these reagents are brought in close proximity. The probes consist of a protein specific binding part coupled to an oligonucleotide with either a free 3’- or 5’-end capable of hybridizing to a common connector oligonucleotide. When the probes are in proximity, promoted by target binding, then the DNA strands can be joined by enzymatic ligation. The nucleic acid sequence that is formed can then be amplified and quantitatively detected in a real-time monitored polymerase chain reaction. This convenient assay is simple to perform and allows highly sensitive protein detection. Parallel analysis of multiple proteins by DNA microarray technology is anticipated for proximity ligation and enabled by the information carrying ability of nucleic acids to define the individual proteins. Assays detecting cytokines using SELEX aptamers or antibodies, monoclonal and polyclonal, are presented in the thesis. Microarrays synthesized in situ using photolithographic methods generate impure products due to damaged molecules and interrupted synthesis. Through a molecular inversion mechanism presented here, these impurities may be removed. At the end of synthesis, full-length oligonucleotides receive a functional group that can then be made to react with the solid support forming an arched structure. The 3’-ends of the oligonucleotides are then cleaved, removing the impurities from the support and allowing the liberated 3’-hydroxyl to prime polymerase extension reactions from the inverted oligonucleotides. The effect of having pure oligonucleotides probes compared to ones contaminated with shorter variants was investigated in allele specific hybridization reactions. Pure probes were shown to have greater ability to discriminate between matched and singly mismatched targets at optimal hybridization temperatures.

The mature vascular system is composed of a network of blood vessels organized into arteries, capillaries, and veins. The vessels are composed of endothelial cells surrounded by smooth muscle cells and embedded in a specialized basement membrane. The demand for oxygen during embryonal development regulates vessel formation through a process denoted vasculogenesis. These primitive vessels are further remodeled through proliferation, sprouting and migration of endothelial cells in a process denoted angiogenesis. Vasculogenesis and angiogenes are regulated by growth factors, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). To study vasculogenesis and angiogenesis, we employed differentiating embryonal stem cells (embryoid bodies). Vascularization of embryoid bodies follows a vascular pattern highly reminiscent of the in vivo pattern, leading to expression of a set of endothelial cell markers. Treatment of the embryoid bodies with different angiogenic growth factors led to distinct vascular morphologies. Expression of VEGF receptor-2 was an absolute demand for proper vascular development. PDGF-BB was shown to be potent in regulating vascular plexus formation in embryoid bodies. PDGF-BB induced capillary formation by promoting endothelial cell migration and differentiation. Hypoxia is a powerful inducer of angiogenic growth factors, such as VEGF-A, leading to angiogenesis. Hypoxia treatment induced an extensive vascular network that covered the entire embryoid body. Hypoxia-induced vascularization still occurred when VEGF receptor function was blocked, indicating that other pathway than VEGF/VEGF receptors may be critical for hypoxia-driven vessel formation. Heparan sulfated proteoglycans (HSPGs) are present in the vascular basement membrane and are known to modulate angiogenic growth factor effects on endothelial cells in normal and pathological conditions such as tumor growth and formation of metastases. We employed heparin as an HSPG equivalent to show that PDGF-BB stimulation of PDGF a-receptor phosphorylation was augmented by heparin, resulting in increased mitogen activated protein kinase (MAPK) and protein kinase B PKB/Akt activation, and enhanced cellular migration towards PDGF-BB.

Malignant primary brain tumors, gliomas, often overexpress both platelet-derived growth factor (PDGF) ligands and receptors providing an autocrine and/or paracrine boost to tumor growth. Glioblastoma multiforme (GBM) is the most frequent glioma. Its aggressive and infiltrative growth renders it extremely difficult to treat. Median survival after diagnosis is currently only 14 months. The present thesis describes the use of retroviral tagging to identify candidate cancer-causing genes that cooperate with PDGF in brain tumor formation. Newborn mice were injected intracerebrally with a Moloney murine leukemia retrovirus carrying the sis/PDGF-B oncogene and a replication competent helper virus. Brain tumors with many characteristics of human glioblastomas developed after 13-42 weeks. Analysis of proviral integrations in the brain tumors identified almost 70 common insertion sites (CISs). These CISs were named brain tumor loci and harbored known but also putative novel cancer-causing genes. An array with over 15000 unique cDNAs was used to screen for differentially expressed genes in the mouse brain tumors compared to normal brain. Known tumor genes and markers of immature cells were upregulated in the tumors. Short latency tumors were further distinguished as fast growing and GBM-like. Long latency tumors resembled slow-growing oligodendrogliomas and contained significantly less integrations as compared to short latency tumors. The gene Prkg2, encoding the cGMP-dependent protein kinase II, was targeted by insertions in two brain tumors. Overexpression of Prkg2 in human glioma cell lines led to a reduction in colony formation, cell proliferation and migration. A glioma cell line expressing markers of immature stem cells showed loss of cell adhesion, G1 cell cycle arrest and decreased activation of the survival signaling protein Akt upon stimulation with a cGMP analog that activates the Prkg2 protein. The present thesis shows that proviral tagging may be a useful tool in the search for candidate glioma genes.

Early Effects of Castration Therapy in Non-malignant and Malignant Prostate Tissue BACKGROUND. Androgen ablation, the standard treatment for advanced prostate cancer, results in increased apoptosis, decreased cell proliferation and subsequent involution of the prostate gland. The mechanisms behind these responses are largely unknown, but effects in the prostatic epithelium are believed to be mediated by primary changes in the stroma. The purpose of this thesis was to investigate short-term cellular effects of castration-induced prostate tissue involution in mice and humans. METHODS. Prostate tissue factors affected by castration were investigated using cDNA-arrays, micro-dissection, RT-PCR, immunohistochemistry and Western blot analysis. The effects of local insulin-like growth factor-1 (IGF-1) administration were investigated in intact and castrated mice. Non-malignant and malignant epithelial and stromal cells were micro-dissected from human prostate biopsies taken before and within two weeks after castration treatment from patients with advanced prostate cancer. These tissue compartments were analyzed by RT-PCR and/or immunohistochemistry for IGF-1, IGF-1 receptor, androgen receptor (AR) and prostate specific antigen (PSA) expression. Treatment-induced changes in these factors were related to apoptosis and proliferation as well as to clinical data and cancer specific survival. RESULTS. Similar to our observations in mouse ventral prostate (VP), non-malignant and malignant human prostate tissues responded with increased epithelial cell apoptosis and decreased proliferation after androgen withdrawal. Also, the PSA mRNA levels were reduced within the first days after therapy both in non-malignant and malignant human prostate epithelial cells. However, neither of these changes was related to subsequent nadir serum PSA or to survival. Locally injected IGF-1 increased epithelial cell proliferation and vascular volume in intact but not in castrated mice. IGF-1 was found to be mostly, but not exclusively, expressed in the stroma, and it decreased rapidly after castration in both humans and mice. This decrease was, however, largely absent in prostate tumor stroma, and tumor stroma cells showed lower pre-treatment levels of AR than stroma surrounding normal epithelial glands. Furthermore, decreased levels of IGF-1 mRNA in the non-malignant and tumor stroma cells, and in tumor epithelial cells in response to castration, were associated with high levels of apoptosis in epithelial cells after therapy. CONCLUSIONS. In the prostate, IGF-1 may be an important mediator of stroma-epithelial cell interaction that is involved in castration-induced epithelial and vascular involution. Moreover, reduced AR in the tumor stroma may play an important role in prostate cancer progression towards androgen-independency, resulting in inadequate IGF-1 reduction and apoptosis induction in response to castration. Most primary tumors initially respond to castration with markedly decreased PSA synthesis and cell proliferation, and moderately increased apoptosis. Death due to metastatic disease is, however, still common, despite primary tumor regression. This may suggest that tumor cells in metastases respond differently to treatment than primary tumor cells, probably influenced by a different and possibly androgen-independent stroma. Further studies should test the hypothesis that the effect of castration therapy can be enhanced by simultaneous blocking of IGF-1 signaling.

Binder-based assays are employed throughout the life sciences. Powerful signal amplification techniques have enabled detection of very rare molecule species diluted in simple buffers. Unspecific binding of primary binders leads to increased background in more complex samples. By requiring two recognition events, ligation-based molecular analyses provide highly specific detection of biomolecules in complex samples. We developed a highly multiplexed padlock-ligation assay targeting signature sequences in the hemagglutinin and neuraminidase genes. From a panel of 77 avian influenza isolates of all major serotypes, 97% were genotyped correctly in accordance with previous classifications by classical diagnostic methods (Paper I). Alternative splicing is an important mechanism expanding the proteome. Current analysis techniques fail to provide sequences of complete transcripts beyond the read length of sequencing instruments. We devised and implemented a strategy to compress the sequence information contained in the splicing pattern of a transcript into the presence or absence of sequence-blocks. We demonstrate that this assay yields information about the splicing patterns in thousands of transcripts from cellular cDNA (Paper II). Expression changes of mucin proteins and glycosylation structures are frequently observed from the early stages of cancer development. Expression of mucin 2 and sialyl-Tn are common features of intestinal metaplasia and gastric cancer, and are known to co-locate. Here we have developed an in situ proximity ligation assay (PLA) directed against mucin 2 and sialyl-Tn. Our study on intestinal metaplasia and gastric cancer tissue sections identified mucin 2 as a major carrier of sialyl-Tn in these conditions, and demonstrated how conveniently glycosylation of proteins can be studied by in situ PLA (Paper III). This thesis shows how the dual recognition requirement of ligation-based assays can be employed to detect target molecules with high specificity, to analyze several sequence features of nucleic acids or to study the proximity of two antigens in situ.

BACKGROUND: Prostate cancer is the most common cancer among Swedish men. For patients with metastatic prostate cancer the standard therapy is castration, a treatment that initially provides symptomatic relief but unfortunately is not curative. New therapeutic targets for advanced prostate cancer are therefore needed.  Prostate cancers are composed of tumor epithelial cells as well as many non-epithelial cells such as cancer associated fibroblasts, blood vessels and inflammatory cells.  Many components of the tumor microenvironment such as tumor associated macrophages and angiogenesis have been shown to stimulate tumor progression. This thesis aims to explore mechanisms by which the local environment influences prostate tumor growth and how such mechanisms could be targeted for treatment. MATERIALS AND METHODS: We have used animal models of prostate cancer, in vitro cell culture systems and clinical materials from untreated prostate cancer patients with long follow up. Experiments were evaluated with stereological techniques, immunohistochemistry, western blotting, quantitative real-time PCR, PCR arrays and laser micro dissection. RESULTS: We found that the presence of a tumor induces adaptive changes in the surrounding non-malignant prostate tissue, and that androgen receptor negative prostate tumor cells respond to castration treatment with temporarily reduced growth when surrounded by normal castration-responsive prostate tissue. Further, we show that macrophages are important for prostate tumor growth and angiogenesis in the tumor and in the surrounding non-malignant tissue. In addition, the angiogenesis inhibitor Pigment epithelium-derived factor (PEDF) was found  to be down-regulated in metastatic rat and human prostate tumors. Over-expression of PEDF inhibited experimental prostate tumor growth, angiogenesis and metastatic growth and stimulated macrophage tumor infiltration and lymphangiogenesis. PEDF was found to be down-regulated by the prostate microenvironment and tumor necrosis factor (TNF) α. CONCLUSIONS: Our studies indicate that not only the nearby tumor microenvironment but also the surrounding non-malignant prostate tissue are important for prostate tumor growth. Both the tumor and the surrounding non-malignant prostate were characterized by increased angiogenesis and inflammatory cell infiltration. Targeting the surrounding prostate tissue with castration, targeting tumor associated macrophages, or targeting the vasculature directly using inhibitors like PEDF were all shown to repress prostate tumor growth and could prove beneficial for patients with advanced prostate cancer.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease where a significant proportion of patients will develop an aggressive disease. Today, the mutational status of the immunoglobulin heavy variable (IGHV) genes is one of the strongest prognostic markers in CLL, where unmutated IGHV genes correlate with poor outcome. In addition, IGHV3-21 gene usage is associated with poor prognosis independent of mutational status. Recently, several genes were shown to be differently expressed between IGHV mutated and unmutated CLL and were suggested as prognostic markers. The aim of this thesis was to examine the applicability of these RNA-based prognostic markers in CLL. In papers I and II, the prognostic significance of LPL and TCL1A mRNA expression in CLL was investigated in 140 and 144 patients, respectively. High expression was found to be associated with inferior clinical outcome for both markers. However, CLL cases with mutated IGHV3-21 genes displayed low levels of LPL expression, indicating that LPL cannot identify this poor-risk patient group. In contrast, high TCL1A expression was detected in all IGHV3-21 cases. To elucidate the functionality of LPL in CLL, LPL lipase activity was measured in 33 cases. The lipase activity was found to be invariably low, implying an alternative function for LPL in CLL. In paper III, a comprehensive analysis of five RNA-based markers (LPL, TCL1A, ZAP70, CLLU1 and MCL1) was performed in 252 CLL patients. All RNA-based markers except MCL1 predicted clinical outcome, with LPL being the strongest. Moreover, LPL expression independently predicted overall survival when adjusted for established markers. All of the RNA-based markers added additional prognostic information to established markers, e.g. high LPL expression predicted an inferior outcome in patients with mutated IGHV genes or good-risk cytogenetics. For clinical application, over time stability of prognostic markers is crucial. In paper IV, the expression of LPL, TCL1A, ZAP70 and MCL1 was investigated in samples taken at diagnosis and at a follow-up of seven years in 104 CLL patients. LPL was found to be the most stable marker, displaying high correlation between the sequential samples, whereas ZAP70 and MCL1 varied significantly. TCL1A expression increased at follow-up, which may indicate disease progression as TCL1A promotes cell survival. In summary, this thesis highlights the applicability of RNA-based markers in CLL prognostication, both as single markers or in combination with established markers. In particular, LPL was shown to be the strongest RNA-based marker in terms of prognostic strength and stability.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia among the adult population in western countries and accounts for 30-40% of all leukemias. With survival time ranging from months to decades, the clinical course of individual CLL patients is highly variable. This heterogeneity and in the end the need for means to identify the patients with less favorable disease has encouraged the search for biomarkers that can predict the prognosis. Telomeres are repetitive structures protecting the chromosomal endings and shorten at each cell division. Telomere length (TL) has been indicated as a prognostic factor both in hematological malignancies and solid tumors. In B-CLL, TL is associated with mutation status of the immunoglobulin heavy chain variable (IGHV) gene and with clinical course. In the present thesis the main aim was to evaluate TL as a biomarker in B-CLL using a quantitative PCR-based method for TL determination. In paper I, TL was shown to be a prognostic factor for stage A and stage B/C patients, whereas IGHV mutation status predicted outcome only in stage A patients. Moreover, IGHV mutated CLL cases were subdivided by TL into two groups with different prognosis, a subdivision not seen for unmutated cases. Interestingly, the IGHV-mutated group with short telomeres had en overall survival close to that of the unmutated cases. Thus, a combination of IGHV mutation status and telomere length gave an improved subclassification of CLL identifying previously unrecognized patient groups with different outcomes. TL correlates with cellular origin of B-cell malignancies in relation to the germinal center (GC). In paper II different B-cell lymphoma/leukemia subtypes were analyzed. Shortest telomeres were found in IGHV unmutated CLLs, differing significantly from IGHV mutated cases. Contrary to this, mantle cell lymphomas (MCL) demonstrated similar TL regardless of IGHV mutation status. TL differed significantly between GC-like and non-GC-like diffuse large B-cell lymphomas (DLBCL) and follicular lymphomas (FL) had shorter telomeres than GC-like DLBCL. Hairy cell leukemias, which display Ig gene intraclonal heterogeneity, had longer telomeres than FLs and non-GC-DLBCL, but shorter than GC-DLBCL. In conclusion, TL seemed not to simply correlate with GC origin. Paper III presents a B-CLL cohort assessed for TL, genomic aberrations, IGHV mutation status, CD38 and ZAP-70 expression. An inverse correlation existed between TL and IGHV homology, CD38 and ZAP-70 expression. The presence of genomic aberrations was similar among patients regardless of TL. In contrast, 13q deletion, a favorable biomarker, was more frequent in patients with long telomeres, while 11q and 17p deletions (markers of less favorable outcome) were more frequent in the subgroup with short telomeres. In paper IV a large group of mainly indolent CLL cases from a population based cohort was studied again showing an association between TL and prognosis, especially in “good” prognosis cases as defined by other biomarkers. Multivariate analysis indicated a strong connection between IGHV mutation status, lipoprotein lipase (LPL) expression and TL. A comparison of TL in diagnostic and follow up samples demonstrated a significant correlation, and also in the follow samples TL constituted a significant biomarker for survival.

Tumor growth and progression not only depend on properties of the malignant cells but are strongly influenced by the tumor microenvironment. The tumor stroma consists of various cell types such as inflammatory cells, endothelial cells and fibroblasts, which can either inhibit or promote tumor growth. Consequently, therapeutic targeting of the tumor stroma is increasingly recognized as an important tool to fight cancer. Two particularly important processes that contribute to the pathology of most types of tumors are angiogenesis and inflammation. In order to target these processes specifically and efficiently, it is fundamental to identify and understand the factors and signaling pathways involved. This thesis initially describes the multiple functions of the small heat shock protein αB-crystallin in the tumor microenvironment. αB-crystallin was first identified in a screen of proteins specifically up-regulated in endothelial cells forming vessel-like structures. We found that αB-crystallin is expressed in a subset of tumor vessels and promotes angiogenesis by inhibiting endothelial apoptosis, suggesting that targeting of αB-crystallin might inhibit angiogenesis and thereby decrease tumor growth. However, we also discovered an important role of αB-crystallin in regulation of inflammatory processes. We show that αB-crystallin increases the surface levels of E-selectin, an important leukocyte-endothelial adhesion molecule. Thereby, αB-crystallin may alter leukocyte recruitment to inflamed tissues such as the tumor stroma. In addition, we found that αB-crystallin is expressed in immature myeloid cells that accumulate in the periphery and at the tumor site during tumor development. Importantly, lack of αB-crystallin resulted in increased accumulation of immature myeloid cells, which might increase tumor associated inflammation. Finally, through combining laser microdissection of vessels from human tissue and microarray analysis, we identified a gene expression signature specifically associated with vessels in high grade glioma. Blood vessels in malignant glioma are highly abnormal and contribute to the pathology of the disease. Thus, knowledge about the molecular set-up of these vessels might contribute to the development of future vascular normalizing treatments.