Molecular genetics of bladder cancer: an update.

Urinary bladder cancer is a heterogeneous disease with tumors ranging from papillary non-invasive to solid muscle infiltrating high grade tumors. There are mainly three problems after initial management: recurrence, progression to higher stage and metastases. The respective risk is well known for each of the stages of the disease but not sufficiently for individual optimal risk assessments. The clinical need is initially to establish the correct risk irrespective of later treatment that is to find prognostic factors. Secondarily it is important to develop predictive factors for each specific therapy. With the advent of array-based molecular profiling it is possible to obtain a more complete picture of the cancer biology and thus hope to improve the prediction of risk. Today the microarray approach is implemented at DNA, RNA and protein level. Reported chromosomal alterations in low-grade papillary tumors are few and the most common are 9q and 9p deletions. Activation of the MAPK pathway through mutations of FGFR3, RAS or PI3K seems to be crucial in the genesis of these low malignant tumors. Muscle infiltrating bladder tumors typically have more genetic aberrations than non-muscle invasive cancers. Key genes are related to the p53 and RB pathways. Gene-expression signatures correlated to stage, CIS, progression and recurrence have been proposed but require further validation.

Identification of novel deletion breakpoints bordered by segmental duplications in the NF1 locus using high resolution array-CGH.

BACKGROUND: Segmental duplications flanking the neurofibromatosis type 1 (NF1) gene locus on 17q11 mediate most gene deletions in NF1 patients. However, the large size of the gene and the complexity of the locus architecture pose difficulties in deletion analysis. We report the construction and application of the first NF1 locus specific microarray, covering 2.24 Mb of 17q11, using a non-redundant approach for array design. The average resolution of analysis for the array is approximately 12 kb per measurement point with an increased average resolution of 6.4 kb for the NF1 gene. METHODS: We performed a comprehensive array-CGH analysis of 161 NF1 derived samples and identified heterozygous deletions of various sizes in 39 cases. The typical deletion was identified in 26 cases, whereas 13 samples showed atypical deletion profiles. RESULTS: The size of the atypical deletions, contained within the segment covered by the array, ranged from 6 kb to 1.6 Mb and their breakpoints could be accurately determined. Moreover, 10 atypical deletions were observed to share a common breakpoint either on the proximal or distal end of the deletion. The deletions identified by array-CGH were independently confirmed using multiplex ligation-dependent probe amplification. Bioinformatic analysis of the entire locus identified 33 segmental duplications. CONCLUSIONS: We show that at least one of these segmental duplications, which borders the proximal breakpoint located within the NF1 intron 1 in five atypical deletions, might represent a novel hot spot for deletions. Our array constitutes a novel and reliable tool offering significantly improved diagnostics for this common disorder.

IgG2a-mediated enhancement of antibody responses is dependent on FcRgamma+ bone marrow-derived cells.

Antibodies (Ab) administered in complex with antigens (Ag) have the capacity to regulate the out-coming specific immune response. Primary immunization with complexes of bovine serum albumin-2,4,6-trinitrophenyl (BSA-TNP) and immunoglobulin (Ig)G2a anti-TNP induced a significant enhancement of IgG1 and IgG2a BSA-specific Ab response compared to immunization with the Ag alone. Enhancement was absent in nude mice, demonstrating the requirement of T cells for this regulation. Secondary immunization with BSA alone in mice previously primed with BSA-TNP/IgG2a led to a dramatic increase of Ab production, showing that immune complexes are efficient inducers of immunological memory. IgG-mediated enhancement of Ab responses has previously been shown to be impaired in mice lacking FcgammaRI, FcgammaRIII and FcepsilonRI owing to gene targeting of the common FcRgamma subunit (FcRgamma-/-). Here we show that enhancement after immunization with BSA-TNP/IgG2a complexes is restored in irradiated FcRgamma-/- recipients transferred with wild-type (FcRgamma+/+) bone marrow (BM) cells. In contrast, no enhancement is seen in FcRgamma+/+ irradiated animals reconstituted with FcRgamma-/- BM cells. We conclude that IgG2a-mediated enhancement of Ab responses is dependent on the presence of FcgammaRI and/or FcgammaRIII on BM-derived cells and that the presence of these receptors on the radioresistant follicular dendritic cell is not essential.

IgE-mediated suppression of primary antibody responses in vivo.

The ability of immunoglobulin (Ig)G to feedback suppress antibody (Ab) responses is a well known property clinically used to prevent haemolytic disease of newborns. We recently found that IgG was able to suppress the primary Ab response to sheep red blood cells (SRBC) in mice lacking the known Fc-receptors for IgG. In addition, IgE and F(ab')2 fragments of IgG were able to suppress the response to SRBC in wild-type mice. These results suggested that the IgG-mediated suppression can take place independently of the IgG (Fc) portion and that masking of the epitopes is an important mechanism. In the present report we investigated whether the suppression caused by IgE is Fc-dependent. Monoclonal IgE anti-2,4,6-trinitrophenyl (TNP), administered with TNP-coupled SRBC (SRBC-TNP), can induce an efficient suppression in mice lacking FcgammaRI + RIII + FcepsilonRI (owing to the lack of the common gamma chain, FcRgamma), FcgammaRIIB or FcepsilonRII (CD23). Because the known IgE-binding receptors are FcepsilonRI, CD23, FcgammaRIIB and FcgammaRIII, the results suggest that also the IgE-mediated suppression can take place independently of the Fc-receptors. A slightly less efficient suppression in CD23-deficient animals, suggests a minor involvement of this receptor.

Non-autocrine, constitutive activation of Met in human anaplastic thyroid carcinoma cells in culture.

Activation of Met by its ligand HGF has been shown to elicit both mitogenic and motogenic responses in thyrocytes in vitro. In the present study we have investigated the expression of Met in human anaplastic thyroid carcinoma cells in culture. There was a variation in expression level and size of Met in the different cell lines; high Met expression was found in four cell lines, compared to non-neoplastic human thyrocytes. Treatment with glucoproteinase F showed that the size differences observed were due to variances in the degree of glycosylation. Interestingly, in cell lines with high expression of Met, the receptor proteins were found to be constitutively tyrosine phosphorylated. None of these cell lines expressed HGF mRNA, and addition of suramin did not affect the level of tyrosine phosphorylation of Met in unstimulated cells, suggesting the absence of autocrine stimulatory pathways. Furthermore, we did not observe MET gene amplification, activating mutations or phosphatase defects. The tyrosine phosphorylated receptors appeared functionally active since the receptors associated with the adaptor molecule Shc. In summary, we have found ligand-independent constitutively activated Met in four out of six anaplastic thyroid carcinoma cell lines.

Efficient IgG-mediated suppression of primary antibody responses in Fcgamma receptor-deficient mice.

IgG antibodies can suppress more than 99% of the antibody response against the antigen to which they bind. This is used clinically to prevent rhesus-negative (Rh-) women from becoming immunized against Rh+ erythrocytes from their fetuses. The suppressive mechanism is poorly understood, but it has been proposed that IgG/erythrocyte complexes bind to the inhibitory Fc receptor for IgG (FcgammaRIIB) on the B cell surface, thereby triggering negative signals that turn off the B cell. We show that IgG induces the same degree of suppression of the response to sheep erythrocytes in animals lacking the known IgG-binding receptors FcgammaRIIB, FcgammaRI + III, FcgammaRI + IIB + III, and FcRn (the neonatal Fc receptor) as in wild-type animals. Reinvestigation of the ability of F(ab')2 fragments to suppress antibody responses demonstrated that they were nearly as efficient as intact IgG. In addition, monoclonal IgE also was shown to be suppressive. These findings suggest that IgG inhibits antibody responses through Fc-independent mechanisms, most likely by masking of antigenic epitopes, thereby preventing B cells from binding and responding to antigen. In agreement with this, we show that T cell priming is not abolished by passively administered IgG. The results have implications for the understanding of in vivo regulation of antibody responses and Rh prophylaxis.