Expression of the glioma-associated oncogene homolog (GLI) 1 in human breast cancer is associated with unfavourable overall survival.

BACKGROUND: The transcription factor GLI1, a member of the GLI subfamily of Krüppel-like zinc finger proteins is involved in signal transduction within the hedgehog pathway. Aberrant hedgehog signalling has been implicated in the development of different human tumour entities such as colon and lung cancer and increased GLI1 expression has been found in these tumour entities as well. In this study we questioned whether GLI1 expression might also be important in human breast cancer development. Furthermore we correlated GLI1 expression with histopathological and clinical data to evaluate whether GLI1 could represent a new prognostic marker in breast cancer treatment. METHODS: Applying semiquantitative realtime PCR analysis and immunohistochemistry (IHC) GLI1 expression was analysed in human invasive breast carcinomas (n = 229) in comparison to normal human breast tissues (n = 58). GLI1 mRNA expression was furthermore analysed in a set of normal (n = 3) and tumourous breast cell lines (n = 8). IHC data were statistically interpreted using SPSS version 14.0. RESULTS: Initial analysis of GLI1 mRNA expression in a small cohort of (n = 5) human matched normal and tumourous breast tissues showed first tendency towards GLI1 overexpression in human breast cancers. However only a small sample number was included into these analyses and values for GLI1 overexpression were statistically not significant (P = 0.251, two-tailed Mann-Whitney U-test). On protein level, nuclear GLI1 expression in breast cancer cells was clearly more abundant than in normal breast epithelial cells (P = 0.008, two-tailed Mann-Whitney U-test) and increased expression of GLI1 protein in breast tumours significantly correlated with unfavourable overall survival (P = 0.019), but also with higher tumour stage (P < 0.001) and an increased number of tumour-positive axillar lymph nodes (P = 0.027). Interestingly, a highly significant correlation was found between GLI1 expression and the expression of SHH, a central upstream molecule of the hedgehog pathway that was previously analysed on the same tissue microarray. CONCLUSION: Our study presents a systematic expression analysis of GLI1 in human breast cancer. Elevated levels of GLI1 protein in human breast cancer are associated with unfavourable prognosis and progressive stages of disease. Thus GLI1 protein expression measured e.g. by an IHC based scoring system might have an implication in future multi-marker panels for human breast cancer prognosis or molecular sub typing. The highly significant correlation between SHH and GLI1 expression characterises GLI1 as a potential functional downstream target of the hedgehog signalling pathway in human breast cancer as well. Furthermore, our study indicates that altered hedgehog signalling may represent a key disease pathway in the progression of human breast cancer.

Enhancer of the rudimentary gene homologue (ERH) expression pattern in sporadic human breast cancer and normal breast tissue.

BACKGROUND: The human gene ERH (Enhancer of the Rudimentary gene Homologue) has previously been identified by in silico analysis of four million ESTs as a gene differentially expressed in breast cancer. The biological function of ERH protein has not been fully elucidated, however functions in cell cycle progression, pyrimidine metabolism a possible interaction with p21(Cip1/Waf1) via the Ciz1 zinc finger protein have been suggested. The aim of the present study was a systematic characterization of ERH expression in human breast cancer in order to evaluate possible clinical applications of this molecule. METHODS: The expression pattern of ERH was analyzed using multiple tissue northern blots (MTN) on a panel of 16 normal human tissues and two sets of malignant/normal breast and ovarian tissue samples. ERH expression was further analyzed in breast cancer and normal breast tissues and in tumorigenic as well as non-tumorigenic breast cancer cell lines, using quantitative RT-PCR and non-radioisotopic in situ hybridization (ISH). RESULTS: Among normal human tissues, ERH expression was most abundant in testis, heart, ovary, prostate, and liver. In the two MTN sets of malignant/normal breast and ovarian tissue,ERH was clearly more abundantly expressed in all tumours than in normal tissue samples. Quantitative RT-PCR analyses showed that ERH expression was significantly more abundant in tumorigenic than in non-tumorigenic breast cancer cell lines (4.5-fold; p = 0.05, two-tailed Mann-Whitney U-test); the same trend was noted in a set of 25 primary invasive breast cancers and 16 normal breast tissue samples (2.5-fold; p = 0.1). These findings were further confirmed by non-radioisotopic ISH in human breast cancer and normal breast tissue. CONCLUSION: ERH expression is clearly up-regulated in malignant as compared with benign breast cells both in primary human breast cancer and in cell models of breast cancer. Since similar results were obtained for ovarian cancer, ERH overexpression may be implicated in the initiation and/or progression of certain human malignancies. Further studies on large breast cancer tissue cohorts should determine whether ERH could function as a prognostic factor or even a drug target in the treatment of human breast cancer.

Association of platelet-derived growth factor receptor alpha mutations with gastric primary site and epithelioid or mixed cell morphology in gastrointestinal stromal tumors.

Most gastrointestinal stromal tumors (GISTs) carry activating mutations of the KIT gene encoding the receptor tyrosine kinase KIT. In a previous study we were able to show an association between the lack of KIT mutations (wild-type GISTs) and the presence of a significant epithelioid tumor component. A very recent study described the occurrence of PDGFRalpha mutations in KIT wt GISTS. Therefore, we studied a panel of 87 GISTs for mutations in the hot spot regions of the PDGFRalpha gene with single strand conformation polymorphism analysis and sequencing and correlated the PDGFRalpha status with pathomorphological data. We detected 20 cases with exon 18 mutations but none with exon 12 mutations. The mutations were located in the second kinase domain of PDGFRalpha with 16 point mutations, and four larger deletions of 9 to 12 bp. All cases with mutations in the PDGFRalpha gene revealed wild-type KIT in common regions of mutation, ie, exons 9 and 11. Most interestingly, the occurrence of PDGFRalpha mutations was significantly associated with a higher frequency of epithelioid or mixed morphology (18 of 20 cases, P < 0.0001) and gastric location (all cases, P = 0.0008). Our data indicate that GISTs represent distinctive entities, differing in genetic, biological, and morphological features.

Current diagnostic methods of HER-2/neu detection in breast cancer with special regard to real-time PCR.

In this study we compare different diagnostic methods to measure HER-2/neu gene amplification in breast cancer with special regard to real-time polymerase chain reaction. Fifty specimens of breast cancer were analyzed, and the use of laser-assisted microdissection prior to PCR was investigated. A total of 38 of 50 cases showed HER-2/neu overexpression in immunohistochemistry. In the 2+ scored group, 2 of 23 cases turned out to be amplified after FISH analysis and 14 of 15 cases in the 3+ group were amplified. Of the 16 amplified cases, 3 initially were measured as nonamplified by real-time PCR but showed amplification after laser capture microdissection. One case showed amplification by PCR but turned out to have only one copy of chromosome 17 by FISH. All 0 or 1+ scored cases were measured as nonamplified both by FISH and PCR. Initial concordance rate between FISH and PCR was 92% and could be increased to 98% using laser-assisted microdissection. FISH and PCR showed high diagnostic accuracy and concordance while immunohistochemistry overestimates amplification within the 2+ scored group. Therefore, either FISH or PCR should be applied in cases scored 2+ by immunohistochemistry. Diagnostic accuracy of PCR can be increased using laser-assisted microdissection.

Insights from a whole cystectomy specimen--association of primary small cell carcinoma of the bladder with transitional cell carcinoma in situ.

Small cell carcinoma of the urinary bladder is a rare entity known as an aggressive tumor. As it is rarely associated with transitional cell carcinoma in situ but more commonly with invasive transitional cell carcinoma, its origin is not well understood. We report a case of small cell carcinoma with coexisting transitional cell carcinoma in situ, where histologic mapping and parallel immunohistochemical and molecular analyses (TP53 mutation analyses, loss of heterozygosity) were performed. Immunohistochemical characterization (synaptophysin, thyroid transcription factor-1, chromogranin A, neuron specific enolase, CD56, CK, CK7, CK20, CD44v6, and p53) emphasized the morphology. Identical point mutations of TP53 were identified in invasive small cell carcinoma and transitional cell carcinoma in situ. No loss of heterozygosity of microsatellite markers D3S3050, D9S303, D9S304, D9S171, D9S775, D9S1748, D9S1751, D17S786, D17S918, and TP53alu was found in either component. We provide, for the first time, molecular evidence for the development of invasive small cell carcinoma out of transitional cell carcinoma in situ.

Deletion of Trp-557 and Lys-558 in the juxtamembrane domain of the c-kit protooncogene is associated with metastatic behavior of gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) typically express high levels of the Kit-receptor. The majority of GISTs carry mutations in the c-kit protooncogene clustering in exon 11. The significance of c-kit mutations for the biological behavior of GISTs is still under discussion. We evaluated 55 sporadic GISTs with available follow-up data for c-kit mutations in the juxtamembrane domain and detected mutations in 35 cases (63.6%). We found a mutational hotspot in codons 557 (tryptophan) and 558 (lysine) preferentially in histomorphologically malignant tumors. In the group of GISTs carrying c-kit mutations, 16 of 21 malignant, but only 3 of 8 benign GISTs and 3 of 6 lesions with uncertain malignant potential, carried mutations of Trp-557 and/or Lys-558. We investigated whether mutations in these 2 amino acids had an impact on biological behavior. Trp-557 and/or Lys-558 were mutated in all 15 metastatic GISTs carrying c-kit mutations but only in a minority of nonmetastatic tumors. A combined deletion of Trp-557 and Lys-558 occurred exclusively in 8 metastatic GISTs. We conclude that in addition to histomorphological evaluation determination of mutations in exon 11 may be an additional parameter for predicting the metastatic risk of GISTs and may be important for the decision that patients will need close clinical follow-up or further adjuvant treatment with kit antagonists.