Helmut E. Gabbert

Prevalence and Heterogeneity of KRAS, BRAF, and PIK3CA Mutations in Primary Colorectal Adenocarcinomas and Their Corresponding Metastases

Purpose: Epidermal growth factor receptor (EGFR) antibody therapy is established in patients with wild-type KRAS colorectal carcinoma; however, up to 50% of these patients do not respond to this therapy. To identify the possible causes of this therapy failure, we searched for mutations in different EGFR-dependent signaling proteins and analyzed their distribution patterns in primary tumors and corresponding metastases. Experimental Design: Tumor tissues, macrodissected from tumor centers, invasion fronts (n = 100), lymph nodes (n = 55), and distant metastases (n = 20), respectively, were subjected to DNA extraction and mutation analysis of KRAS, BRAF, and PIK3CA. Results: Activating mutations were detected in 41% (KRAS), 7% (BRAF), and 21% (PIK3CA) of the primary tumors. By comparing tumor centers and invasion fronts, the intratumoral heterogeneity of KRAS, BRAF, and PIK3CA mutations was observed in 8%, 1%, and 5% of primary tumors, respectively. Heterogeneity between primary tumors and lymph node metastases was found in 31% (KRAS), 4% (BRAF), and 13% (PIK3CA) of the cases. Heterogeneity between primary tumors and distant metastases was present in two patients (10%) for KRAS and one patient for PIK3CA (5%), but not for BRAF. Discordant results between primary tumors and metastases could markedly be reduced by testing the additional tumor samples. Conclusions: Failure of EGFR antibody therapy in patients with wild-type KRAS colorectal cancer may result from activating BRAF or PIK3CA mutations and false-negative sequencing results caused by intratumoral heterogeneity. Due to the particularly high rates of heterogeneity between primary tumors and lymph node metastases, the latter are least suitable for diagnostic mutation analysis. Clin Cancer Res; 16(3); 790–9

HER2 expression in gastric cancer: Rare, heterogeneous and of no prognostic value – conclusions from 924 cases of two independent series

Background: Patients with gastric cancer (GC) have a poor survival and biologicals such as Trastuzumab have not been used routinely in these patients. Existing data on HER2 expression and its clinical relevance in GC are still limited and controversial. Methods: HER2 expression was investigated by immunohistochemistry in 418 GC from Germany and 506 GC from England. Results were compared to clinicopathological parameters and patient survival. Results: Less than 10% of all GC showed HER2 expression in more than 5% of tumour cells and 91% of these were intestinal type GC. In both series, no relationship was found between HER2 expression, patient survival or TNM stage. Marked intratumoural heterogeneity was noted. Conclusions: This is the largest study to date demonstrating in two independent series that HER2 expression is not related to gastric cancer patient prognosis and that only a very small subgroup of intestinal type GC may potentially respond to HER2 targeting therapy. Due to prominent intratumoural heterogeneity of HER2 expression in GC, HER2 testing in endoscopic biopsies before treatment will be prone to false negative results.

Immunohistochemical Demonstration of Enzymatically Modified Human LDL and Its Colocalization With the Terminal Complement Complex in the Early Atherosclerotic Lesion

Treatment of low density lipoprotein (LDL) with degrading enzymes transforms the molecule to a moiety that is micromorphologically indistinguishable from lipoproteinaceous particles that are present in atherosclerotic plaques, and enzymatically modified LDL (E-LDL), but not oxidized LDL (ox-LDL), spontaneously activates the alternative complement pathway, as do lesion lipoprotein derivatives. Furthermore, because E-LDL is a potent inducer of macrophage foam cell formation, we propose that enzymatic degradation may be the key process that renders LDL atherogenic. In this article, we report the production of two murine monoclonal antibodies recognizing cryptic epitopes in human apolipoprotein B that become exposed after enzymatic attack on LDL. One antibody reacted with LDL after single treatment with trypsin, whereas recognition by the second antibody required combined treatment of LDL with trypsin and cholesterol esterase. In ELISAs, both antibodies reacted with E-LDL produced in vitro and with lesion complement activator derived from human atherosclerotic plaques, but they were unreactive with native LDL or ox-LDL. The antibodies stained E-LDL, but not native LDL or ox-LDL, that had been artificially injected into arterial vessel walls. With the use of these antibodies, we have demonstrated that early human atherosclerotic coronary lesions obtained at autopsy as well as lesions examined in freshly explanted hearts always contain extensive extracellular deposits of E-LDL. Terminal complement complexes, detected with a monoclonal antibody specific for a C5b-9 neoepitope, colocalized with E-LDL within the intima, which is compatible with the proposal that subendothelially deposited LDL is enzymatically transformed to a complement activator at the earliest stages in lesion development.

Prognostic value of E‐cadherin expression in 413 gastric carcinomas

E‐cadherin is Ca2+‐dependent intercellular adhesion molecule known to exert an invasion‐suppressor function. In the present study, E‐cadherin expression was immunohistochemically investigated in a retrospective series of 413 RO‐resected gastric carcinomas using the monoclonal antibody (MAb) 5H9. Of these cases, 108 tumors revealed a preserved E‐cadherin expression similar to that of normal gastric mucosa. In 95 tumors, E‐cadherin expression was moderately reduced and in 86 tumors highly reduced. In 124 tumors, no or only a very weak dotted expression could be detected. There was a significant correlation between the degree of E‐cadherin expression and the grade of tumor differentiation, as well as with histological type according to the Laurén and the WHO classifications. In contrast, no correlation could be demonstrated between E‐cadherin expression and the prognostic parameters depth of invasion, lymph node involvement and vascular invasion. As shown by univariate Cox regression analysis, patients with E‐cadherin‐positive tumors had significantly better 3‐ and 5‐year survival rates than patients with E‐cadherin‐negative tumors. This prognostic impact remained present in a multivariate Cox regression analysis, including the prognostic parameters pT category, pN category and vascular invasion.

Expression of different survivin variants in gastric carcinomas: first clues to a role of survivin-2B in tumour progression

Survivin is a novel member of the inhibitor of apoptosis family and determines the susceptibility of tumour cells to pro-apoptotic stimuli. Recently, we identified two novel alternative splice variants of survivin, differing in their anti-apoptotic properties: whereas the anti-apoptotic potential of survivin-ΔEx3 is preserved, survivin-2B has lost its anti-apoptotic potential and may act as a naturally occurring antagonist of survivin. Because the in vivo expression of these alternative splice variants has not been explored so far, we analysed gastric carcinomas of different histological subtypes, grades and stages. Since no antibodies are currently available to determine the novel splice variants, quantitative reverse transcriptase polymerase chain reaction was performed, using RNA samples obtained from 30 different gastric carcinomas. Polymerase chain reactions products were quantified by densitometric evaluation. We found that all gastric carcinomas, irrespective of their histological types, grades or stages, express survivin-ΔEx3, survivin-2B and survivin, the latter being the dominant transcript. Comparing the disease stages I+II with III+IV, expression of survivin and survivin-ΔEx3 remained unchanged. In contrast, a significant (P=0.033) stage-dependent decrease in the expression of survivin-2B became evident. Our study demonstrates for the first time the expression of alternative splice variants in gastric carcinomas and provides a first clue to a role of survivin-2B in tumour progression.

p53 protein expression and prognosis in squamous cell carcinoma of the esophagus.

Background. The p53 gene product is known to regulate cell growth and proliferation. Whereas the wild‐type p53 protein suppresses cell growth, the mutated p53 protein acts as an oncogene. Mutations in the p53 gene usually result in p53 protein stabilization and accumulation; so that the gene product can be detected by immunohistochemistry. Recently, the immunohistochemical detection of the p53 protein was associated with prognosis in breast, colorectal, and other types of cancer. However, its prognostic role in esophageal cancer remains to be elucidated.

Mechanisms of tumor metastasis: cell biological aspects and clinical implications.

Abstract The clinical course – and hence the prognosis – of patients suffering from malignant tumors are essentially determined by the capability of tumor cells to metastasize. During the past decade knowledge about genetic aberrations, as well as molecular and cell biological mechanisms which are involved in the regulation of tumor metastasis, has dramatically increased and consequently led to the development of new theoretical and experimental strategies in cancer treatment. The objective of this review is not only to give an overview about the principal cell biological and molecular mechanisms of tumor metastasis, but also to discuss potential therapeutical options resulting from this knowledge.

Distinct in vivo expression patterns of survivin splice variants in renal cell carcinomas

Survivin, a novel member of the inhibitor of apoptosis protein (IAP) family, reduces the susceptibility of tumor cells to proapoptotic stimuli, thereby promoting tumor cell survival during tumor progression and treatment with anticancer drugs. Recently, we identified 2 novel alternative splice variants of survivin, survivin‐2B and survivin‐ΔEx3, which differ in their antiapoptotic properties. Survivin‐2B has lost its antiapoptotic potential and may act as a naturally occurring antagonist of antiapoptotic survivin and survivin‐ΔEx3. Because the in vivo expression of these splice variants in human cancer has not been analyzed so far, 57 renal cell carcinomas (RCCs) were explored using quantitative reverse transcriptase polymerase chain reaction. We found that all RCCs express survivin‐ΔEx3, survivin‐2B and survivin, the latter being the dominant transcript. When we compared early and intermediate stages with late stages of clear cell RCCs, no significant changes in the expression levels of survivin and survivin‐ΔEx3 became evident. However, a significant decrease was observed for the mRNA ratio between survivin‐2B and survivin in late tumor stages (p = 0.036). Chromophilic/papillary RCCs, which are known to be less aggressive than clear cell RCCs, did not show significantly lower expression levels of antiapoptotic survivin and survivin‐ΔEx3, compared with stage‐adjusted clear cell RCCs. Our study demonstrates for the first time in vivo expression of functionally different survivin variants and suggests a role of these survivin splice variants in the progression and clinical behavior of human RCCs.

Molecular characterization of commonly used cell lines for bone tumor research: A trans-European EuroBoNet effort

Usage of cancer cell lines has repeatedly generated conflicting results provoked by differences among subclones or contamination with mycoplasm or other immortal mammalian cells. To overcome these limitations, we decided within the EuroBoNeT consortium to characterize a common set of cell lines including osteosarcomas (OS), Ewing sarcomas (ES), and chondrosarcomas (CS). DNA fingerprinting was used to guarantee the identity of all of the cell lines and to distinguish subclones of osteosarcoma cell line HOS. Screening for homozygous loss of 38 tumor suppressor genes by MLPA revealed deletion of CDKN2A as the most common event (15/36), strictly associated with absence of the CDKN2A (p16) protein. Ten cell lines showed missense mutations of the TP53 gene while another set of nine cell lines showed mutations resulting in truncation of the TP53 protein. Cells harboring missense mutations expressed high levels of nuclear TP53, while cell lines with nonsense mutations showed weak/absent staining for TP53. TP53wt cell lines usually expressed the protein in 2–10% of the cells. However, seven TP53wt osteosarcomas were negative for both mRNA and protein expression. Our analyses shed light on the correlation between immunohistochemical and genetic data for CDKN2A and TP53, and confirm the importance of these signaling pathways. The characterization of a substantial number of cell lines represents an important step to supply research groups with proven models for further advanced studies on tumor biology and may help to make results from different laboratories more comparable.

Histopathology of the gastroesophageal junction: a study on 36 operation specimens.

The entire gastroesophageal junction of 36 patients who had been operated for squamous cell carcinoma of the upper or middle esophagus was examined. Hematoxylin and eosin-stained slides were evaluated by two pathologists for the following histologic details: minimal and maximal length of cardiac mucosa (CM) and oxyntocardiac mucosa (OCM, mixture of cardiac and fundic glands), degree of inflammation in CM and OCM, and presence of intestinal metaplasia or pancreatic metaplasia. Sections of gastric corpus mucosa were evaluated for the presence of gastritis and Helicobacter pylori infection; sections of esophageal squamous epithelium were evaluated for the presence of reflux esophagitis. CM was present in the entire circumference of the gastroesophageal junction in 20 cases, in parts of the circumference in 15 cases, and entirely absent in one case. The maximal length per case ranged between 1 and 15 mm (median 5 mm). OCM was circumferentially present in 22 cases and partially present in 14 cases. The maximal length ranged between 2 and 24 mm (median 7 mm). Locations of CM/OCM over submucosal esophageal glands or squamous epithelium-lined ducts, both indicating a location in the esophagus, were found in eight cases (22%) and in four cases (11%), respectively. In 18 cases (50%) intestinal metaplasia was present in CM/OCM; pancreatic metaplasia was found in 22 cases (61%). A statistically not significant trend for increase of minimal length of CM, OCM, and the sum of both was found in the presence of gastroesophageal reflux disease. Neither the presence of intestinal metaplasia nor of pancreatic metaplasia in CM/OCM was correlated with gastroesophageal reflux disease. In conclusion, the high variability in length, the frequent occurrence of intestinal metaplasia and pancreatic metaplasia, and the frequent extension into the esophagus suggest that CM/OCM is a dynamic structure that probably mirrors the influence of underlying gastroesophageal diseases. Because of the short length and incomplete circumferential extension of CM/OCM, future endoscopic–bioptic investigations will probably have to be based on more extensive sampling of the gastroesophageal junction.