Fibroblast growth factor receptor (FGFR) alterations in squamous differentiated bladder cancer: a putative therapeutic target for a small subgroup.

Although drugable fibroblast growth factor receptor (FGFR) alterations in squamous cell carcinomas (SCC) of various entities are well known, little is known about FGFR modifications in squamous differentiated bladder cancer. Therefore, our study evaluated FGFR1-3 alterations as a putative therapeutic target in this subgroup. We analyzed 73 squamous differentiated bladder cancers (n = 10 pT2, n = 55 pT3, n = 8 pT4) for FGFR1-3 protein expression, FGFR1-3 copy number variations, FGFR3 chromosomal rearrangements (fluorescence in situ hybridization (FISH)) and FGFR3 mutations (SNapShot analysis). Only single cases displayed enhanced protein expression, most frequently FGFR3 overexpression (9.4% (6/64)). FISH showed no amplifications of FGFR1, 2 or 3. Break apart events were only slightly above the cut off in 12.1% (8/66) of cases and no FGFR3-TACC3 rearrangements could be proven by qPCR. FGFR3 mutations (p.S249C) were found in 8.5% (6/71) of tumors and were significantly associated with FGFR3 protein overexpression (p < 0.001), and unfavourable clinical outcome (p = 0.001). Our findings are consistent with the results of the TCGA data set for the "squamous-like" subtype of bladder cancer (n = 85), which revealed reduced overall expression of FGFR1 and FGFR2 in tumors compared to normal tissue, while expression of FGFR3 remained high. In the TCGA "squamous-like" subtype FGFR3 mutations were found in 4.9% and correlated with high FGFR3 RNA expression. Mutations of FGFR1 and FGFR2 were less frequent (2.4% and 1.2%). Hence, our comprehensive study provides novel insights into a subgroup of squamous differentiated bladder tumors that hold clues for novel therapeutic regimens and may benefit from FGFR3-targeted therapies

Diagnostic and Prognostic Implications of FGFR3(high)/Ki67(high) Papillary Bladder Cancers

Prognostic/therapeutic stratification of papillary urothelial cancers is solely based upon histology, despite activated FGFR3-signaling was found to be associated with low grade tumors and favorable outcome. However, there are FGFR3-overexpressing tumors showing high proliferation-a paradox of coexisting favorable and adverse features. Therefore, our study aimed to decipher the relevance of FGFR3-overexpression/proliferation for histopathological grading and risk stratification. N = 142 (n = 82 pTa, n = 42 pT1, n = 18 pT2-4) morphologically G1-G3 tumors were analyzed for immunohistochemical expression of FGFR3 and Ki67. Mutation analysis of FGFR3 and TP53 and FISH for FGFR3 amplification and rearrangement was performed. SPSS 23.0 was used for statistical analysis. Overall FGFR3(high)/Ki67(high) status (n = 58) resulted in a reduced Delta mean progression-free survival (PFS) (p < 0.01) of 63.92 months, and shorter progression-free survival (p < 0.01;mean PFS: 55.89 months) in pTa tumors (n = 50). FGFR3(mut)/TP53(mut) double mutations led to a reduced Delta mean PFS (p < 0.01) of 80.30 months in all tumors, and FGFR3(mut)/TP53(mut) pTa tumors presented a dramatically reduced PFS (p < 0.001;mean PFS: 5.00 months). Our results identified FGFR3(high)/Ki67(high) papillary pTa tumors as a subgroup with poor prognosis and encourage histological grading as high grade tumors. Tumor grading should possibly be augmented by immunohistochemical stainings and suitable clinical surveillance by endoscopy should be performed

Diagnostic and Prognostic Implications of FGFR3high/Ki67high Papillary Bladder Cancers

Prognostic/therapeutic stratification of papillary urothelial cancers is solely based upon histology, despite activated FGFR3-signaling was found to be associated with low grade tumors and favorable outcome. However, there are FGFR3-overexpressing tumors showing high proliferation—a paradox of coexisting favorable and adverse features. Therefore, our study aimed to decipher the relevance of FGFR3-overexpression/proliferation for histopathological grading and risk stratification. N = 142 (n = 82 pTa, n = 42 pT1, n = 18 pT2-4) morphologically G1–G3 tumors were analyzed for immunohistochemical expression of FGFR3 and Ki67. Mutation analysis of FGFR3 and TP53 and FISH for FGFR3 amplification and rearrangement was performed. SPSS 23.0 was used for statistical analysis. Overall FGFR3high/Ki67high status (n = 58) resulted in a reduced ∆mean progression-free survival (PFS) (p < 0.01) of 63.92 months, and shorter progression-free survival (p < 0.01; mean PFS: 55.89 months) in pTa tumors (n = 50). FGFR3mut/TP53mut double mutations led to a reduced ∆mean PFS (p < 0.01) of 80.30 months in all tumors, and FGFR3mut/TP53mut pTa tumors presented a dramatically reduced PFS (p < 0.001; mean PFS: 5.00 months). Our results identified FGFR3high/Ki67high papillary pTa tumors as a subgroup with poor prognosis and encourage histological grading as high grade tumors. Tumor grading should possibly be augmented by immunohistochemical stainings and suitable clinical surveillance by endoscopy should be performed

ALA- and ALA-hexylester-induced protoporphyrin IX fluorescence and distribution in multicell tumour spheroids

Synthesis of protoporphyrin IX (PpIX) in intact murine mammary cancer cell spheroids is reported from optical sections obtained using a laser scanning confocal fluorescence microscope. EMT6 spheroids 275–350 μ m in diameter were incubated in 0.1–15 mM aminolevulinic acid (ALA) or 0.001–2 mM ALA-hexylester (h-ALA) to test the ability of both pro-drugs to diffuse into the spheroids and induce PpIX production. Spheroids incubated with ALA show significant fluorescence nonuniformity for all concentrations, with the outermost cells exhibiting greater porphyrin fluorescence. Comparable levels of fluorescence throughout the optical section are achieved with approximately 100-fold lower h-ALA concentrations, indicating that the interior cells maintain esterase activity and porphyrin synthesis and that h-ALA diffuses efficiently to the spheroid interior. Fluorescence gradients are less pronounced with h-ALA incubation, in part because of apparent saturation of esterase activity in the spheroid perimeter. Proliferating (Ki67 positive) and quiescent cell populations exhibit remarkably different h-ALA concentration dependencies. The incubation concentration resulting in maximum fluorescence with ALA is 10 mM, while the optimal concentration for h-ALA is 200-fold lower at 0.05 mM. Exceeding these optimal concentrations for both pro-drugs leads to an overall loss of fluorescence. © 2001 Cancer Research Campaign http://www.bjcancer.co

Comparative genomic profiling of glandular bladder tumours

Abstract Primary glandular bladder tumours (bladder adenocarcinoma [BAC], urachal adenocarcinoma [UAC], urothelial carcinoma with glandular differentiation [UCg]) are rare malignancies with histological resemblance to colorectal adenocarcinoma (CORAD) in the majority of this subgroup. Definite case numbers are very low, molecular data are limited and the pathogenesis remains poorly understood. Therefore, this study was designed to complement current knowledge by in depth analysis of BAC (n = 12), UAC (n = 13), UCg (n = 11) and non-invasive glandular lesions (n = 19). In BAC, in addition to known alterations in TP53, Wnt, MAP kinase and MTOR pathway, mutations in SMAD4, ARID1A and BRAF were identified. Compared to published data on muscle invasive bladder cancer (BLCA) and CORAD, UCg exhibited frequent “urothelial” like alterations while BAC and UAC were characterised by a more “colorectal” like mutational pattern. Immunohistochemically, there was no evidence of DNA mismatch repair deficiency or PD-L1 tumour cell positivity in any sample. Depending on the used antibody 0–45% of BAC, 0–30% of UCg and 0% UAC cases exhibited PD-L1 expressing tumour associated immune cells. A single BAC (9%, 1/11) showed evidence of ARID1A protein loss, and two cases of UCg (20%, 2/10) showed loss of SMARCA1 and PBRM1, respectively. Taken together, our data suggest at least in part involvement of similar pathways driving tumourigenesis of adenocarcinomas like BAC, UAC and CORAD independent of their tissue origin. Alterations of TERT and FBXW7 in single cases of intestinal metaplasia further point towards a possible precancerous character in line with previous reports

Frequent expression loss of Inter-alpha-trypsin inhibitor heavy chain (ITIH) genes in multiple human solid tumors: A systematic expression analysis

<p>Abstract</p> <p>Background</p> <p>The inter-alpha-trypsin inhibitors (ITI) are a family of plasma protease inhibitors, assembled from a light chain – bikunin, encoded by <it>AMBP </it>– and five homologous heavy chains (encoded by <it>ITIH1</it>, <it>ITIH2</it>, <it>ITIH3</it>, <it>ITIH4</it>, and <it>ITIH5</it>), contributing to extracellular matrix stability by covalent linkage to hyaluronan. So far, ITIH molecules have been shown to play a particularly important role in inflammation and carcinogenesis.</p> <p>Methods</p> <p>We systematically investigated differential gene expression of the <it>ITIH </it>gene family, as well as <it>AMBP </it>and the interacting partner <it>TNFAIP6 </it>in 13 different human tumor entities (of breast, endometrium, ovary, cervix, stomach, small intestine, colon, rectum, lung, thyroid, prostate, kidney, and pancreas) using cDNA dot blot analysis (Cancer Profiling Array, CPA), semiquantitative RT-PCR and immunohistochemistry.</p> <p>Results</p> <p>We found that <it>ITIH </it>genes are clearly downregulated in multiple human solid tumors, including breast, colon and lung cancer. Thus, <it>ITIH </it>genes may represent a family of putative tumor suppressor genes that should be analyzed in greater detail in the future. For an initial detailed analysis we chose <it>ITIH2 </it>expression in human breast cancer. Loss of <it>ITIH2 </it>expression in 70% of cases (n = 50, CPA) could be confirmed by real-time PCR in an additional set of breast cancers (n = 36). Next we studied ITIH2 expression on the protein level by analyzing a comprehensive tissue micro array including 185 invasive breast cancer specimens. We found a strong correlation (p < 0.001) between ITIH2 expression and estrogen receptor (ER) expression indicating that ER may be involved in the regulation of this ECM molecule.</p> <p>Conclusion</p> <p>Altogether, this is the first systematic analysis on the differential expression of <it>ITIH </it>genes in human cancer, showing frequent downregulation that may be associated with initiation and/or progression of these malignancies.</p