Silke Lassmann

Aurora kinase A messenger RNA overexpression is correlated with tumor progression and shortened survival in head and neck squamous cell carcinoma.

Purpose: Aurora kinase A (AURKA/STK15/BTAK) encodes a serine/threonine kinase associated with chromosomal distribution and its up-regulation induces chromosomal instability, thereby leading to aneuploidy and cell transformation in several types of cancer. In this study, we investigated the role of AURKA in head and neck squamous cell carcinoma (HNSCC). Experimental Design: The mRNA expression levels of AURKA were compared in tumor tissues of 66 HNSCC patients with those in corresponding normal squamous epithelium by real-time quantitative reverse transcriptase-PCR. In addition, the association between AURKA mRNA and protein expression, centrosome abnormalities, and aneuploidy was studied in a subset of cases (n = 34). All molecular variables were correlated to histomorphologic findings and clinical follow-up data of the patients. Results: AURKA mRNA up-regulation was significantly associated with tumor stage and the occurrence of regional lymph node, as well as distant metastasis (P < 0.0001 for all). Similarly, a correlation was found for protein expression and the occurrence of regional lymph node (P = 0.0183) and distant metastasis (P = 0.03). The mRNA was positively associated with protein expression (P = 0.003) and centrosome abnormalities (P = 0.03). Cox regression analysis revealed that AURKA mRNA up-regulation correlated with disease-free survival of the patients (P = 0.03) as well as shorter overall survival (P < 0.001). Conclusions: We conclude that the up-regulation of AURKA mRNA may play a critical role in the tumor progression of HNSCC and provides useful information as a prognostic factor for HNSCC patients.

Array CGH identifies distinct DNA copy number profiles of oncogenes and tumor suppressor genes in chromosomal- and microsatellite-unstable sporadic colorectal carcinomas

DNA copy number changes represent molecular fingerprints of solid tumors and are as such relevant for better understanding of tumor development and progression. In this study, we applied genome-wide array comparative genomic hybridization (aCGH) to identify gene-specific DNA copy number changes in chromosomal (CIN)- and microsatellite (MIN)-unstable sporadic colorectal cancers (sCRC). Genomic DNA was extracted from microdissected, matching normal colorectal epithelium and invasive tumor cells of formalin-fixed and paraffin-embedded tissues of 22 cases with colorectal cancer (CIN = 11, MIN = 11). DNA copy number changes were determined by aCGH for 287 target sequences in tumor cell DNAs, using pooled normal DNAs as reference. aCGH data of tumor cell DNAs was confirmed by fluorescence in situ hybridization (FISH) for three genes on serial tissues as those used for aCGH. aCGH revealed DNA copy number changes previously described by metaphase CGH (gains 7, 8q, 13q, and 20q; losses 8p, 15q, 18q, and 17p). However, chromosomal regions 20q, 13q, 7, and 17p were preferentially altered in CIN-type tumors and included DNA amplifications of eight genes on chromosome 20q (TOP1, AIB1, MYBL2, CAS, PTPN1, STK15, ZNF217, and CYP24), two genes on chromosome 13q (BRCA2 and D13S25), and three genes on chromosome 7 (IL6, CYLN2, and MET) as well as DNA deletions of two genes on chromosome 17p (HIC1 and LLGL1). Finally, additional CIN-tumor-associated DNA amplifications were identified for EXT1 (8q24.11) and MYC (8q24.12) as well as DNA deletions for MAP2K5 (15q23) and LAMA3 (18q11.2). In contrast, distinct MIN-tumor-associated DNA amplifications were detected for E2F5 (8p22–q21.3), GARP (11q13.5–q14), ATM (11q22.3), KAL (Xp22.3), and XIST (Xq13.2) as well as DNA deletions for RAF1 (3p25), DCC (18q21.3), and KEN (21q tel). aCGH revealed distinct DNA copy number changes of oncogenes and tumor suppressor genes in CIN- and MIN-type sporadic colorectal carcinomas. The identified candidate genes are likely to have distinct functional roles in the carcinogenesis and progression of CIN- and MIN-type sporadic CRCs and may be involved in the differential response of CIN- and MIN-type tumor cells to (adjuvant) therapy, such as 5-fluorouracil.

STAT3 mRNA and protein expression in colorectal cancer: effects on STAT3-inducible targets linked to cell survival and proliferation

Aims: To evaluate mRNA and protein expression of signal transducers and activators of transcription (STAT)3 in colorectal carcinomas (CRCs) and to define the association of STAT3 activity with the STAT3-inducible targets cyclin D1, survivin, Bcl-xl and Mcl-1. Materials and methods: Matching serial sections of normal colonic epithelium and invasive CRCs (n = 32) were subjected to quantitative reverse transcriptase polymerase chain reaction specific to STAT3, cyclin D1, survivin, Bcl-xl and Mcl-1, as well as immunohistochemistry. For STAT3 immunohistochemistry, two antibodies, recognising unphosphorylated (UP-) and phosphorylated (tyr705, P-) STAT3 were used. Ki-67 (MIB-1) staining was included as a proliferation marker. Results: Compared with normal colonic epithelium, UP-STAT3 and P-STAT3 (p = 0.023 and 0.006) protein expression and expression of its associated targets cyclin D1, survivin and Bcl-xl were significantly (all p<0.001) increased in carcinoma. In carcinomas, STAT3 (p = 0.019) and Bcl-xl (p = 0.001) mRNAs were correlated with lymph node status. Moreover, nuclear P-STAT3 protein expression (active state) was associated with the expression of its target genes Bcl-xl (p = 0.038) and survivin (p = 0.01) as well as with Ki-67 (p = 0.017). By contrast, cytoplasmic UP-STAT was significantly linked to Bcl-xl mRNA (p = 0.024) and protein (p = 0.001) as well as to cytoplasmic survivin protein expression (p = 0.019). Conclusion: Both inactive (UP-STAT3) and active (P-STAT3) STAT3 proteins are markedly increased in invasive CRCs. This is associated with Bcl-xl and survivin induction, increased proliferation and lymph node metastasis. This study therefore provides the basis for further examination of the prognostic or predictive value of these molecular markers in CRC.

Quantification of CK20 gene and protein expression in colorectal cancer by RT-PCR and immunohistochemistry reveals inter- and intratumour heterogeneity.

Cytokeratin 20 (CK20) is an epithelial protein expressed almost exclusively in the gastrointestinal (GI) tract and is widely used as immunohistochemical marker for routine diagnosis. In contrast, CK20 gene expression is not an established marker for the classification of tumours and the detection of disseminated cancer cells in colorectal cancer. Recently, real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) has provided the means for reproducible and quantitative investigation of molecular markers. This report directly compares CK20 mRNA and protein expression in serial sections of archival, formalin‐fixed, paraffin‐embedded (FFPE) colorectal adenocarcinomas. CK20 expression was detected by immunohistochemistry (IHC) in 60/63 (95.2%) cases, by conventional RT‐PCR in 58/60 (96.7%) and by quantitative RT‐PCR using the LightCycler® (LightCycler® is a trademark of a Member of the Roche Group) System in 29/32 (90.6%) microdissected cases, one case yielding variable results. Despite the high detection rate of all three techniques, marked heterogeneity of CK20 expression was seen between different cases and also within individual cases. CK20 expression profiles were not related to particular histopathological features of the tumours. A good correlation (r = 0.8964) was found between CK20 mRNA and protein expression by comparing quantitative RT‐PCR with IHC in 32 cases. This was also true for selected heterogeneous tumour cells within individual cases. Both RT‐PCR and IHC are therefore valuable tools for CK20 detection in colorectal adenocarcinoma, with real‐time RT‐PCR providing supplementary quantitative information. This suggests a promising supportive role for quantitative RT‐PCR in molecular pathology. Copyright

Predictive Value of Aurora-A/STK15 Expression for Late Stage Epithelial Ovarian Cancer Patients Treated by Adjuvant Chemotherapy

Purpose: To investigate the expression and regulation of the centrosomal kinase Aurora-A/STK15 (AURKA) in epithelial ovarian cancers and to determine the prognostic and predictive value of this marker for patients with late stage epithelial ovarian cancer treated by distinct adjuvant chemotherapies. Experimental Design: Archival resection specimens of epithelial ovarian cancers (n = 115) and nonneoplastic ovaries (n = 28) were analyzed for AURKA mRNA and protein expression by microdissection and quantitative reverse transcriptase-PCR and immunohistochemistry. AURKA DNA copy numbers were measured by fluorescence in situ hybridization in 37 cases. Statistical evaluation was done with respect to clinicopathologic variables, disease-free survival, and overall survival. Results: AURKA mRNA expression was significantly elevated in cancers (P < 0.001) and correlated with AURKA protein expression (P = 0.0134). Overexpression of AURKA protein was detected in 68 of 107 (63.5%) cases and was linked with increased AURKA DNA copy numbers (P = 0.0141) and centromere 20 aneusomy (P = 0.0137). Moreover, AURKA overexpression was associated with improved overall survival in optimal debulked patients receiving taxol/carboplatin therapy (n = 43, P = 0.018). Finally, in an exploratory approach, patients receiving non–taxane-based therapy, AURKA overexpression was predictive for worse overall survival (n = 30, P = 0.049). Conclusions: AURKA overexpression is seen in the majority of late stage epithelial ovarian cancers, most likely due to increased AURKA DNA copy numbers and/or chromosome 20 aneusomy. Importantly, AURKA overexpression may differentially affect taxane and non–taxane-based adjuvant therapy responses. The study sheds new light on AURKA expression and regulation in epithelial cancers in vivo and specifically shows its value as a clinically relevant marker and as a potential therapeutic target per se.

Significance of HER2 low-level copy gain in Barrett's cancer: implications for fluorescence in situ hybridization testing in tissues.

Purpose: HER2 may be a relevant biomarker in Barretts cancer. We compared three HER2 laboratory methods, standard fluorescence in situ hybridization (FISH), image-based three-dimensional FISH in thick (16 μm) sections, and immunohistochemistry, to predict patient outcome. Experimental Design: Tissue microarray sections from 124 Barretts cancer patients were analyzed by standard FISH on thin (4 μm) sections and by image-based three-dimensional FISH on thick (16 μm) sections for HER2 and chromosome-17, as well for p185HER2 by immunohistochemistry. Correlations with clinical and follow-up data were examined. Results: Only three-dimensional FISH on thick (16 μm) sections revealed HER2 gene copy gain to be associated with increased disease-specific mortality (relative risk, 2.1; 95% confidence interval, 1.06-4.26; P = 0.033). In contrast, standard FISH on thin (4 μm) sections and immunohistochemistry failed to predict clinical outcome. Low-level gain of HER2 occurred frequently in Barretts cancer (≥2.5-4.0 HER2 copies, 59.7%; HER2-to-chromosome-17 ratio, ≥1.1-2.0; 61.2%) and defined a subpopulation for patient outcome as unfavorable as HER2 gene amplification [disease-free survival, P = 0.017 (HER2 copies)]. This low-level group was neither definable by standard FISH nor immunohistochemistry. No prognostic significance was found for chromosome-17 aneusomy. Conclusions: Low-level copy gains of HER2 define a biologically distinct subpopulation of Barretts cancer patients. Importantly, these subtle copy number changes are not reliably detected by standard FISH in thin (4 μm) tissue sections, highlighting a thus far unrecognized weakness in HER2 FISH testing. These results should be taken into account for accurate evaluation of biomarkers by FISH and for HER2 FISH testing in tissue sections.

Application of BIOMED-2 Primers in Fixed and Decalcified Bone Marrow Biopsies Analysis of Immunoglobulin H Receptor Rearrangements in B-Cell Non-Hodgkin's Lymphomas

The detection of clonality in lymphomas was recently improved by the BIOMED-2 approach, but analysis of fixed tissues is limited. Here, we adapted the BIOMED-2 protocol for examining immunoglobulin H (IgH) receptor rearrangements in fixed, decalcified bone marrow biopsies (BMBs) for clonality analysis in B-cell non-Hodgkins lymphomas (B-NHL). The study included 111 decalcified BMBs (12 formalin fixed and 99 glutardialdehyde fixed), with B-NHL (n = 85), T-NHL (n = 8), or reactive infiltrates (n = 18). Initially, IgH FRIII polymerase chain reaction (PCR) analysis of crude DNA extracts from 75 glutardialdehyde-fixed BMBs (B-NHLs) using a standard seminested PCR resulted in clonal peaks in 46 of 75 (61.3%) BMBs compared with 19 of 70 (27.1%) for the original BIOMED-2 protocol. Modifications to both DNA extraction and PCR reaction improved the detection rate to 26 of 36 (72.2%) for BIOMED-2 primers, including 10 of 15 (66.7%) cases not detected by our standard IgH analysis. Moreover, introducing the same modifications for analysis of the FRII region by BIOMED-2 primers revealed clonal peaks in 19 of 36 (52.8%) B-NHLs compared with 5 of 70 (7.1%) for the original BIOMED-2 protocol. Together, analysis of FRII and FRIII regions by the modified BIOMED-2 protocol increased the detection rate to 31 of 36 (86.1%), particularly for BMBs with histological evidence of follicular lymphoma (FRIII, 70%; FRII, 90%). In summary, this study provides an improved protocol for detection of clonality by IgH-specific BIOMED-2 primers in fixed, decalcified bone marrow biopsies.

A novel approach for reliable microarray analysis of microdissected tumor cells from formalin-fixed and paraffin-embedded colorectal cancer resection specimens.

We present a novel approach for microarray analysis of RNA derived from microdissected cells of routinely formalin-fixed and paraffin-embedded (FFPE) cancer resection specimens. Subsequent to RNA sample preparation and hybridization to standard GeneChips (Affymetrix), RNA samples yielded 36.43 ± 9.60% (FFPE), 49.90 ± 4.43% (fresh-frozen), and 53.9% (cell line) present calls. Quality control parameters and Q-RT-PCR validation demonstrated reliability of results. Microarray datasets of FFPE samples were informative and comparable to those of fresh-frozen samples. A systematic measurement difference of differentially processed tissues was eliminated by a correction step for comparative unsupervised data analysis of fresh-frozen and FFPE samples. Within FFPE samples, unsupervised clustering analyses clearly distinguished between normal and malignant tissues as well as to further separate tumor samples according to histological World Health Organization (WHO) subtypes. In summary, our approach represents a major step towards integration of microarrays into retrospective studies and enables further investigation of the relevance of microarray analysis for clinico-pathological diagnostics.

Thymidine phosphorylase, dihydropyrimidine dehydrogenase and thymidylate synthase mRNA expression in primary colorectal tumors—correlation to tumor histopathology and clinical follow-up

AimEvaluation of thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), and thymidylate synthase (TS) mRNA levels in formalin-fixed, and paraffin-embedded tissues of patients with colorectal cancer and their prognostic and/or predictive value.Materials and methodsTotal RNA was isolated from microdissected, formalin-fixed, and paraffin-embedded tissues (controls and tumor) and subjected to quantitative RT-PCR (QRT-PCR) in the LightCycler system. Resulting mRNA levels correlated to tumor histology (n=102) and the clinical follow-up in patients treated by resection alone (n=40) and by resection plus adjuvant 5-FU-based chemotherapy (n=52).ResultsCorrelation to histopathological parameters revealed a significant association between tumor stage and the TP mRNA level (T and N category and UICC) as well as the TP:DPD (T and N category and UICC) and TS:DPD (T category) ratio. In addition, tumor differentiation was correlated to the TS mRNA level and the TS:DPD ratio. Finally, the TS:DPD ratio was a prognostic marker for overall survival in patients receiving resection alone (p=0.032). Moreover, a high TP:DPD ratio (>8.1; p=0.002) and, marginally, low DPD (<8.2; p=0.05) mRNA levels significantly correlated to disease-free survival.ConclusionWe present a novel, standardized approach for TP, DPD, and TS mRNA quantification in archival tissue specimens and applied this to a large series of primary colorectal tumors. Correlations to histopathological parameters and clinical follow-up revealed an association of TP, DPD and TS mRNA expression patterns with tumor stage and suggested new prognostic and predictive markers for patients with colorectal cancer.

Occurrence of multipolar mitoses and association with Aurora-A/-B kinases and p53 mutations in aneuploid esophageal carcinoma cells

BackgroundAurora kinases and loss of p53 function are implicated in the carcinogenesis of aneuploid esophageal cancers. Their association with occurrence of multipolar mitoses in the two main histotypes of aneuploid esophageal squamous cell carcinoma (ESCC) and Barretts adenocarcinoma (BAC) remains unclear. Here, we investigated the occurrence of multipolar mitoses, Aurora-A/-B gene copy numbers and expression/activation as well as p53 alterations in aneuploid ESCC and BAC cancer cell lines.ResultsA control esophageal epithelial cell line (EPC-hTERT) had normal Aurora-A and -B gene copy numbers and expression, was p53 wild type and displayed bipolar mitoses. In contrast, both ESCC (OE21, Kyse-410) and BAC (OE33, OE19) cell lines were aneuploid and displayed elevated gene copy numbers of Aurora-A (chromosome 20 polysomy: OE21, OE33, OE19; gene amplification: Kyse-410) and Aurora-B (chromosome 17 polysomy: OE21, Kyse-410). Aurora-B gene copy numbers were not elevated in OE19 and OE33 cells despite chromosome 17 polysomy. Aurora-A expression and activity (Aurora-A/phosphoT288) was not directly linked to gene copy numbers and was highest in Kyse-410 and OE33 cells. Aurora-B expression and activity (Aurora-B/phosphoT232) was higher in OE21 and Kyse-410 than in OE33 and OE19 cells. The mitotic index was highest in OE21, followed by OE33 > OE19 > Kyse-410 and EPC-hTERT cells. Multipolar mitoses occurred with high frequency in OE33 (13.8 ± 4.2%), followed by OE21 (7.7 ± 5.0%) and Kyse-410 (6.3 ± 2.0%) cells. Single multipolar mitoses occurred in OE19 (1.0 ± 1.0%) cells. Distinct p53 mutations and p53 protein expression patterns were found in all esophageal cancer cell lines, but complete functional p53 inactivation occurred in OE21 and OE33 only.ConclusionsHigh Aurora-A expression alone is not associated with overt multipolar mitoses in aneuploid ESCC and BAC cancer cells, as specifically shown here for OE21 and OE33 cells, respectively. Additional p53 loss of function mutations are necessary for this to occur, at least for invasive esophageal cancer cells. Further assessment of Aurora kinases and p53 interactions in cells or tissue specimens derived from non-invasive dysplasia (ESCC) or intestinal metaplasia (BAC) are necessary to disclose a potential causative role of Aurora kinases and p53 for development of aneuploid, invasive esophageal cancers.