Claus Schildberg

One Step Nucleic Acid Amplification (OSNA) - a new method for lymph node staging in colorectal carcinomas

BackgroundAccurate histopathological evaluation of resected lymph nodes (LN) is essential for the reliable staging of colorectal carcinomas (CRC). With conventional sectioning and staining techniques usually only parts of the LN are examined which might lead to incorrect tumor staging. A molecular method called OSNA (One Step Nucleic Acid Amplification) may be suitable to determine the metastatic status of the complete LN and therefore improve staging.MethodsOSNA is based on a short homogenisation step and subsequent automated amplification of cytokeratin 19 (CK19) mRNA directly from the sample lysate, with result available in 30-40 minutes. In this study 184 frozen LN from 184 patients with CRC were investigated by both OSNA and histology (Haematoxylin & Eosin staining and CK19 immunohistochemistry), with half of the LN used for each method. Samples with discordant results were further analysed by RT-PCR for CK19 and carcinoembryonic antigen (CEA).ResultsThe concordance rate between histology and OSNA was 95.7%. Three LN were histology+/OSNA- and 5 LN histology-/OSNA+. RT-PCR supported the OSNA result in 3 discordant cases, suggesting that metastases were exclusively located in either the tissue analysed by OSNA or the tissue used for histology. If these samples were excluded the concordance was 97.2%, the sensitivity 94.9%, and the specificity 97.9%. Three patients (3%) staged as UICC I or II by routine histopathology were upstaged as LN positive by OSNA. One of these patients developed distant metastases (DMS) during follow up.ConclusionOSNA is a new and reliable method for molecular staging of lymphatic metastases in CRC and enables the examination of whole LN. It can be applied as a rapid diagnostic tool to estimate tumour involvement in LN during the staging of CRC.

Cdc2 as prognostic marker in stage UICC II colon carcinomas

PURPOSE Cyclin-dependent kinase 2 (cdc2) controls the G2-M checkpoint and, therefore, the entrance of cells into mitosis. It might play a crucial role during tumour progression in colon carcinomas (CCA). Thus, the prognostic value of cdc2 expression and connected markers relevant for proliferation and apoptosis has to be evaluated. EXPERIMENTAL DESIGN Punch biopsies from the tumour centre and the invasion front of 0.6mm diameter from 392 CCA stage UICC II-IV were integrated in 14 recipient paraffin blocks. After immunohistochemical staining for cdc2, p53, caspase 3 and ki-67, a present (+) and absent (-) scoring was performed in the tissue arrays. The logrank test was used to compare distant metastasis and cancer-related survival. Multivariate Cox regression analysis was done to identify independent prognostic factors for parameters with significant influence on cancer-related survival (CRS) and distant metastasis (DM). RESULTS The pT-category (p=0.007), nodal status (p<0.001), extramural venous infiltration (p<0.001) and lymphatic vessel invasion (p=0.003) were identified as independent histological parameters for CRS. Univariate analysis relating to stage UICC II-IV CCA showed caspase 3 in the tumour centre (p=0.047) to be a prognostic marker for CRS. In stage UICC II cdc2 (p=0.041) and caspase 3 in the invasion front (p=0.026) could be identified as independent prognostic factors for CRS and DM by multivariate analysis. CONCLUSIONS Cdc2 and caspase 3 could be identified as independent prognostic markers in stage UICC II CCA. They might be of value to select patients who should receive adjuvant treatment.

Gastric cancer patients less than 50 years of age exhibit significant downregulation of E-cadherin and CDX2 compared to older reference populations

PURPOSE There is an increasing need to identify molecular markers, which can be used to prognosticate patient populations in gastric cancer. Whereas a significant number have been identified, very few have been characterized in the context of their ability to discriminate between young and old age groups in which a survival difference clearly exists. MATERIAL/METHODS In this study, using immunohistochemistry, we evaluated three markers with proven involvement in gastric cancer. The p53 tumor suppressor, the cell adhesion glycoprotein epithelial cadherin (CDH1) and the caudal-related homeobox transcription factor (CDX2) all of these have important roles in the aetiopathogenesis and/or progression of gastric cancer. RESULTS After adjustments for TNM stage, tumor grade, histopathological characteristics (Lauren classification), we found significant differences in the expression of these proteins, particularly E-cadherin and CDX2 between young and elderly patients. However, these differences did not amount to a significant difference in survival. CONCLUSIONS This study demonstrates that the protein expression of p53, CDH1 and CDX2 significantly discriminates young patients with gastric cancer who have a better prognostic outlook from older patients, but this difference in expression does not contribute to a survival benefit.

Comparability of microarray data between amplified and non amplified RNA in colorectal carcinoma.

Microarray analysis reaches increasing popularity during the investigation of prognostic gene clusters in oncology. The standardisation of technical procedures will be essential to compare various datasets produced by different research groups. In several projects the amount of available tissue is limited. In such cases the preamplification of RNA might be necessary prior to microarray hybridisation. To evaluate the comparability of microarray results generated either by amplified or non amplified RNA we isolated RNA from colorectal cancer samples (stage UICC IV) following tumour tissue enrichment by macroscopic manual dissection (CMD). One part of the RNA was directly labelled and hybridised to GeneChips (HG-U133A, Affymetrix), the other part of the RNA was amplified according to the “Eberwine” protocol and was then hybridised to the microarrays. During unsupervised hierarchical clustering the samples were divided in groups regarding the RNA pre-treatment and 5.726 differentially expressed genes were identified. Using independent microarray data of 31 amplified vs. 24 non amplified RNA samples from colon carcinomas (stage UICC III) in a set of 50 predictive genes we validated the amplification bias. In conclusion microarray data resulting from different pre-processing regarding RNA pre-amplification can not be compared within one analysis.

Differences in the treatment of young gastric cancer patients: Patients under 50 years have better 5-year survival than older patients

PURPOSE In the literature, the manifestations of gastric cancer have been described based on all patients. In recent times, interest has focused on the subgroup of young patients. In the following analysis, the subgroup of young patients (< 50y) is compared with an older reference group (≥ 50y). MATERIAL AND METHODS Between 01.01.1995 and 31.12.2005, 482 patients with a previously untreated gastric cancer underwent surgery. Fifty-six patients in this group were under 50 years of age, and the remaining 367 patients constituted the reference group. All data were recorded prospectively and analyzed retrospectively from the clinical cancer registry of the University of Erlangen. RESULTS The analysis showed that the young patients had a similar tumor stage distribution. Diffuse tumor stages in the Laurén classification occurred significantly more often. The postoperative complication rate was similar, but the hospital mortality rate was significantly lower. The young patients had an obvious, but not significant, 5-year survival advantage in all tumor stages. CONCLUSIONS Younger patients can be operated on with greater confidence as they have a significantly lower hospital mortality rate. They exhibit markedly better 5-year survival at all tumor stages. According to our data, there is nothing to support the general belief that young patients have a poorer disease course. Further clinical and experimental studies are necessary to investigate this group more precisely.

Palliative first-line therapy with weekly high-dose 5-fluorouracil and sodium folinic acid as a 24-hour infusion (AIO regimen) combined with weekly irinotecan in patients with metastatic adenocarcinoma of the stomach or esophagogastric junction followed by secondary metastatic resection after downsizing

Summary Background The aim of this retrospective study was to evaluate the efficacy and safety of weekly high-dose 5-fluorouracil (5-FU)/folinic acid (FA) as 24-h infusion (AIO regimen) plus irinotecan in patients with histologically proven metastatic gastroesophageal adenocarcinoma (UICC stage IV). Material/Methods From 08/1999 to 12/2008, 76 registered, previously untreated patients were evaluable. Treatment regimen: irinotecan (80 mg/m2) as 1-h infusion followed by 5-FU (2000 mg/m2) combined with FA (500 mg/m2) as 24-h infusion (d1, 8, 15, 22, 29, 36, qd 57). Results Median age: 59 years; male/female: 74%/26%; ECOG ≤1: 83%; response: CR: 1%, PR: 16%, SD: 61%, PD: 17%, not evaluable in terms of response: 5%; tumor control: 78%; median OS: 11.2 months; median time-to-progression: 5.3 months; 1-year survival rate: 49%; 2-year survival rate: 17%; no evidence of disease: 6.6%; higher grade toxicities (grade 3/4): anemia: 7%, leucopenia: 1%, ascites: 3%, nausea: 3%, infections: 12%, vomiting: 9%, GI bleeding of the primary tumor: 4%, diarrhea: 17%, thromboembolic events: 4%; secondary metastatic resection after downsizing: 16 patients (21%), R-classification of secondary resections: R0/R1/R2: 81%/6%/13%, median survival of the 16 patients with secondary resection: 23.7 months. Conclusions Combined 5-FU/FA as 24-h infusion plus irinotecan may be considered as an active palliative first-line treatment accompanied by tolerable toxicity; thus offering an alternative to cisplatin-based treatment regimens. Thanks to efficient interdisciplinary teamwork, secondary metastatic resections could be performed in 16 patients. In total, the patients who had undergone secondary resection had a median survival of 23.7 months, whereas the median survival of patients without secondary resection was 10.1 months (p≤0.001).

Evidence for PTGER4,PSCA, and MBOAT7 as risk genes for gastric cancer on the genome and transcriptome level

Genetic associations between variants on chromosome 5p13 and 8q24 and gastric cancer (GC) have been previously reported in the Asian population. We aimed to replicate these findings and to characterize the associations at the genome and transcriptome level. We performed a fine‐mapping association study in 1926 GC patients and 2012 controls of European descent using high dense SNP marker sets on both chromosomal regions. Next, we performed expression quantitative trait locus (eQTL) analyses using gastric transcriptome data from 143 individuals focusing on the GC associated variants. On chromosome 5p13 the strongest association was observed at rs6872282 (P = 2.53 × 10−04) and on chromosome 8q24 at rs2585176 (P = 1.09 × 10−09). On chromosome 5p13 we found cis‐eQTL effects with an upregulation of PTGER4 expression in GC risk allele carrier (P = 9.27 × 10−11). On chromosome 8q24 we observed cis‐eQTL effects with an upregulation of PSCA expression in GC risk allele carrier (P = 2.17 × 10−47). In addition, we found trans‐eQTL effects for the same variants on 8q24 with a downregulation of MBOAT7 expression in GC risk allele carrier (P = 3.11 × 10−09). In summary, we confirmed and refined the previously reported GC associations at both chromosomal regions. Our data point to shared etiological factors between Asians and Europeans. Furthermore, our data imply an upregulated expression of PTGER4 and PSCA as well as a downregulated expression of MBOAT7 in gastric tissue as risk‐conferring GC pathomechanisms.

Is coagulation factor VIII a useful marker for colorectal carcinoma

Background and Objective Increased thromboembolic events are well known in patients suffering from malignant diseases. In the following pilot study, we investigated the usefulness of coagulation factor VIII (FVIII) as a possible prognostic marker in patients with colorectal carcinoma (CRC). Methods Plasma FVIII levels were measured in 79 patients with CRC, correlated with tumor characteristics, and compared with normal ranges of blood group (BG) 0 and BG A/AB/B and with 19 control patients. Results In CRC patients mean FVIII levels were elevated compared with controls (BG 0: p=0.283, BG A/AB/B: p=0.001) and normal ranges. Interestingly, mean FVIII levels varied significantly in different blood groups (p=0.002). UICC stage I CRC patients presented with mean FVIII plasma levels within normal ranges, whereas UICC stage II-IV CRC patients presented with elevated FVIII plasma levels. In BG A/AB/B a significantly elevated FVIII level was found in G2 compared with G1 tumors (p< 0.001). Patients with elevated carcinoembryonic antigen also showed significantly elevated FVIII levels (p=0.050). FVIII levels at time of surgery did not correlate with survival within the first 2 years following surgery. Conclusion In this pilot study, we demonstrated that FVIII plasma levels are elevated in patients with CRC and affected by T-stage and differentiation of the tumor. Whether FVIII is a clinical useful marker needs to be tested in a larger cohort.