Fritz Wrba

EGFR/KRAS/BRAF Mutations in Primary Lung Adenocarcinomas and Corresponding Locoregional Lymph Node Metastases

Purpose: The epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated with different frequencies in non–small cell lung cancer and mutations predict clinical response to EGFR inhibitors. The present study compared the mutational status of EGFR, KRAS, and BRAF in primary tumors with the one in corresponding lymph node metastases. Experimental Design: Direct bidirectional sequencing of EGFR gene exons 18 to 21, KRAS gene codons 12/13 and 61 to 68, and BRAF exon 15 was done on 96 paired samples of primary lung adenocarcinomas and corresponding locoregional lymph node metastases. In addition, comparative genomic hybridization analyses in two pairs of corresponding primary and metastatic tumor samples with discordant EGFR mutation status were done. Results: Mutations in EGFR, KRAS, and BRAF were observed in 7 (7%), 36 (38%), and 2 (2%) patients, respectively. Interestingly, KRAS mutations were observed in two patients with an EGFR mutation. Mutations in primary tumors and lymph node metastases were identical in 1 of 7 (14%) patients in case of EGFR and 11 of 36 (31%) patients in case of KRAS. One patient harbored different KRAS mutations in primary and corresponding metastatic tumors. Comparative genomic hybridization analysis revealed similar patterns of chromosomal changes, strongly supporting a common clonal origin of primary tumors and metastases. Conclusions: The possibility of differences in the mutational status of EGFR, KRAS, BRAF between primary tumors and corresponding lymph node metastases should be considered whenever these mutations are used for the selection of patients for EGFR-directed tyrosine kinase inhibitor therapy.

Cetuximab, gemcitabine, and oxaliplatin in patients with unresectable advanced or metastatic biliary tract cancer: a phase 2 study

BACKGROUND Patients with biliary tract cancer have a poor prognosis, and, until recently, no standard palliative chemotherapy has been defined. We aimed to investigate the efficacy and safety of cetuximab in combination with gemcitabine and oxaliplatin (GEMOX) for first-line treatment of biliary tract cancer. METHODS From Oct 1, 2006, to July 26, 2008, patients with unresectable locally advanced or metastatic biliary tract cancer were sequentially enrolled and treated at one centre in Austria. All patients received intravenous infusions of 500 mg/m(2) cetuximab on day 1, 1000 mg/m(2) gemcitabine on day 1, and 100 mg/m(2) oxaliplatin on day 2, every 2 weeks for 12 cycles. The primary outcome was overall response rate. Analysis was by intention to treat. Adverse reactions were assessed according to National Cancer Institute Common Toxicity Criteria. The study is completed and registered with ClinicalTrials.gov, number NCT01216345. FINDINGS 30 patients with median age of 68 years (IQR 62-73) were enrolled and included in the analysis. Objective response occurred in 19 patients (63%; 95% CI 56·2-69·8), of whom three (10%; 3·2-16·8) achieved complete response, and 16 (53%; 46·2-59·8) achieved partial response. Nine patients underwent potentially curative secondary resection after major response to therapy. Grade 3 adverse events were recorded in 13 patients: skin rash (n=4), peripheral neuropathy (n=4), thrombocytopenia (n=3), nausea (n=1), diarrhoea (n=1), and neutropenia (n=1); no grade 4 adverse events were recorded. INTERPRETATION Cetuximab plus GEMOX was well tolerated and had encouraging antitumour activity, leading to secondary resection in a third of patients. These findings warrant further study of cetuximab plus GEMOX in a large randomised trial.

Current value of intraoperative sonography during surgery for hepatic neoplasms

Noninvasive liver imaging has developed rapidly resulting in increased accuracy for detecting primary and secondary hepatic tumors. Intraoperative ultrasonography (IOUS) was commonly considered to be the gold standard for liver staging, but the current value of IOUS is unknown in view of more sophisticated radiologic tools. The purpose of this prospective study was to evaluate the impact of IOUS on the treatment of 149 patients undergoing liver surgery for malignant disease (colorectal metastasis, 61 patients; hepatoma, 52 patients; other hepatic malignant tumors, 36 patients). The sensitivities of computed tomography (CT), helical CT, magnetic resonance imaging, and IOUS in patients with colorectal metastases were 69.2%, 82.5%, 84.9%, and 95.2% in a segment-by-segment analysis; in patients with hepatoma their sensitivities were 76.9%, 90.9%, 93.0%, and 99.3%; and in patients with other hepatic malignancies they were 66.7%, 89.6%, 93.3%, and 98.9%, respectively. Additional malignant lesions (AMLs) were first detected by inspection and palpation in 20 patients (13.4%). In another 18 patients (12.1%) IOUS revealed at least one AML. Overall, the findings obtained only by IOUS changed the surgical strategy in 34 cases (22.8%). It was concluded that IOUS, having undergone some refinement as well, still has immense diagnostic value in hepatectomy candidates. Frequently avoiding palliative liver resection and occasionally disproving unresectability as assessed by preoperative imaging, IOUS still has a significant impact on surgical decision making and should still be considered the gold standard.RésuméOn a récemment assisté à une amélioration importante dans la précision de la détection des tumeurs primitives et secondaires du foie par l’imagerie non-invasive. L’échographie peropératoire (EPO) a été considérée comme l’examen de référence («gold standard») dans le Staging du foie, mais la valeur de l’EPO est discutée à présent en raison de l’apparition d’investigations radiologiques plus sophistiquées. Le but de cette étude prospective a été d’évaluer l’impacte de l’EPO au cours d’une résection hépatique pour maladie maligne chez 149 patients (métastases d’origine colorectale: 61 patients; carcinome hépatocellulaire: 52 patients; autres tumeurs hépatiques malignes: 36 patients). Chez les patients ayant des métastases d’origine colorectale, la sensibilité de la tomodensitométrie simple (TDM), de la tomodensitométrie hélicoïdale (TDMh), de la résonance magnétique (RM) et l’EPO a été de 69.2%, 82.5%, 84.9% et 95.2% dans une analyse du foie segment par segment. Chez les patients porteurs de carcinome hépatocellulaire, la sensibilité de ces différentes méthodes était, respectivement, de 76.9%, 90.9%, 93% et 99.3%; chez les patients ayant d’autres tumeurs malignes du foie, la sensibilité était, respectivement, de 66.7%, 89.6%, 93.3% et 98.9%. D’autres lésions malignes ont été détectées à l’inspection et à la palpation chez 20 patients (13.4%). De plus, chez 18 autres patients (12.1%), l’EPO a décelé au moins une lésion maligne supplémentaire. Globalement, les données obtenues par l’EPO ont changé la stratégie chirurgicale dans 34 cas (22.8%). On conclue que l’EPO, grâce à quelques raffinements, a toujours une immense valeur diagnostique pour les candidats à l’hépatectomie. En évitant à certains patients une résection palliative, et en permettant, de temps à autre, une résection jugée impossible par les investigations préopératoires, l’EPO garde un impacte significatif sur la décision chirurgicale et devrait continuer à être le «gold standard».ResumenEl rápido desarrollo de los métodos no invasivos ha conferido una mayor precisión diagnóstica de los tumores hepáticos tanto primarios como secundarios. La ecografÍa intraoperatoria (IOUS) se consideró como el mejor método diagnóstico para la estadificación hepática, pero en la actualidad, su valor está en entredicho ante los nuevos y sofisticados estudios radiológicos. El objetivo de este estudio prospectivo fue evaluar el valor de la IOUS en el tratamiento quirúrgico de 149 pacientes con neoplasias malignas de hÍgado (metástasis colorrectales n=61; hepatomas n=52; otros tumores hepáticos malignos n=36). El análisis secuencial segmentario demostró en pacientes con metástasis colorrectales una sensibilidad para la tomografÍa axial o helicoidal computarizada (CT y hCT) del 69.2% y 82.5%, para la resonancia magnética nuclear (MR) del 84.9% y para la IOUS del 95.2%. En pacientes con hepatomas la sensibilidad de estos métodos fue del 76.9%, 90.9%, 93% y 98.9%. En 20 pacientes (13.4%) lesiones malignas adicionales (AML) se diagnosticaron por inspección y palpación. En otros 18 pacientes (12.1%) la IOUS fue capaz de detectar al menos una AML. En 34 casos (22.8%) el conjunto de hallazgos obtenidos exclusivamente con la IOUS propició un cambio de estrategia quirúrgica. En conclusión, la IOUS, con sus recientes mejoras, sigue teniendo un inmenso valor diagnóstico para aquellos que son candidatos a una hepatectomÍa. Previene, con frecuencia, las resecciones hepáticas paliativas y, ocasionalmente, contradice el diagnóstico de irresecabilidad obtenido con otros medios diagnósticos. La IOUS sigue teniendo un importante valor a la hora de establecer una decisión quirúrgica y continúa siendo el mejor método diagnóstico.

Detection of EpCAM positive and negative circulating tumor cells in metastatic breast cancer patients

Abstract Background. Immunomagnetic EpCAM based methods are used to enrich circulating tumor cells (CTCs) in metastatic breast cancer (mBC) patients. EpCAM negative CTCs may be missed. We addressed the question of the reliability of an EpCAM dependent assay to enrich CTCs. Methods. To elucidate this issue, our study has been designed to assess two different CTC enrichment technologies (i) in EpCAM positive (+) and EpCAM negative cell lines and (ii) in mBC patients in dependency on their respective EpCAM expression. These two technologies encompass one anti-EpCAM immunomagnetic enrichment technology, MACS HEA MicroBeads® (MACS), and one EpCAM independent density centrifugation method, OncoQuick® plus (OQ+). Furthermore, the coherence between EpCAM expression in the primary tumor tissue of mBC patients and the CTC detection rates in the corresponding patients is analyzed. Results. (i) MACS recovered significantly more EpCAM (+) than EpCAM (−) tumor cells (p < 0.001) in spiked blood samples. With OQ+ no significantly different recovery rates between EpCAM (+) and EpCAM (−) tumor cells (p = 0.796) were detected. (ii) In mBC patients MACS yielded a significantly higher (p = 0.024) detection rate of EpCAM (+) CTCs. No statistically significant difference (p = 0.070) was found concerning the EpCAM status-based detection rate of CTCs by OQ+. (iii) CTC detection rates are independent of the primary tumors’ EpCAM expression. Conclusions. EpCAM (−) CTCs can not be detected by immunomagnetic EpCAM dependent enrichment methods. EpCAM independent enrichment technologies seem to be superior to detect the entire CTC population. Evaluation of CTCs as prognostic marker should compromise EpCAM (+) and (−) subpopulations.

Diagnostic Value of Quantitative Hepatic Copper Determination in Patients With Wilson’s Disease

BACKGROUND & AIMS A 5-fold increase of hepatic copper concentration is considered as the best available test for diagnosis of hepatic Wilsons disease (WD). However, the sensitivity and specificity of this test have never been fully investigated. METHODS Copper content was measured by flame atomic absorption spectroscopy in 114 liver biopsies obtained at diagnosis of WD, in 219 patients with noncholestatic liver diseases (including 144 with chronic hepatitis C and 44 with nonalcoholic fatty liver disease), and in 26 without evidence of liver disease. RESULTS Liver copper content was >250 microg/g in 95 WD patients (83.3%), between 50 and 250 microg/g in 15, and below 50 microg/g in 4. It did not correlate with age (r(2) = .003), the grade of fibrosis, or the presence of stainable copper. Liver copper content was >250 or between 50 and 250 microg/g in 3 (1.4%) and 20 (9.1%) of 219 patients with noncholestatic liver diseases, respectively. By lowering the cutoff from 250 to 75 microg/g, the sensitivity of liver copper content to diagnose WD increased from 83.3% (95% confidence interval, 75.2%-89.6%) to 96.5% (91.3%-99.1%), but the specificity decreased from 98.6% (96.0%-99.7%) to 95.4% (91.8%-97.8%). CONCLUSIONS There is no gold standard for the diagnosis of WD. Liver copper content is a useful parameter, but a value below 250 microg/g does not exclude WD. Diagnosis requires the combination of a variety of clinical and biochemical tests.

Mammalian target of rapamycin pathway activity in hepatocellular carcinomas of patients undergoing liver transplantation.

Background. Because mammalian target of rapamycin (mTOR) inhibitors combine anticancer and immunosuppressive properties we investigated: 1) the activation status and prognostic significance of the mTOR pathway in hepatocellular carcinoma (HCC) tissues of patients undergoing orthotopic liver transplantation (OLT) for HCC, and 2) the single and combinatorial efficacy of RAD001 in HCC cells. Methods. PTEN, p-AKT, p-mTOR, p-p70S6K, and p-4EBP-1 were analyzed by immunohistochemistry in explanted HCCs of 166 patients undergoing OLT. Efficacy of RAD001 as mono- and combination therapy with doxorubicin was tested in Hep3B and SNU398 cells. Results. The mTOR pathway is activated in about 40% of patients undergoing OLT for HCC but no direct correlation between up- and downstream proteins was observed. We found no influence of mTOR pathway protein expression on disease free survival (DFS) or overall survival (OS). There was a marked single agent and chemo-sensitizing effect of RAD001 against HCC cells in vitro. Conclusion. The mTOR pathway is active in 40% of patients with HCC undergoing OLT, but has no influence of DFS or OS. No direct correlation was observed between up- and downstream proteins limiting the use of upstream proteins to predict mTOR activity. Prospective clinical trials are needed to test whether the activation status of the mTOR pathway in HCCs predicts the antitumor effect of rapamycin derivative in the posttransplantation course.

Treg‐Therapy Allows Mixed Chimerism and Transplantation Tolerance Without Cytoreductive Conditioning

Establishment of mixed chimerism through transplantation of allogeneic donor bone marrow (BM) into sufficiently conditioned recipients is an effective experimental approach for the induction of transplantation tolerance. Clinical translation, however, is impeded by the lack of feasible protocols devoid of cytoreductive conditioning (i.e. irradiation and cytotoxic drugs/mAbs). The therapeutic application of regulatory T cells (Tregs) prolongs allograft survival in experimental models, but appears insufficient to induce robust tolerance on its own. We thus investigated whether mixed chimerism and tolerance could be realized without the need for cytoreductive treatment by combining Treg therapy with BM transplantation (BMT). Polyclonal recipient Tregs were cotransplanted with a moderate dose of fully mismatched allogeneic donor BM into recipients conditioned solely with short‐course costimulation blockade and rapamycin. This combination treatment led to long‐term multilineage chimerism and donor‐specific skin graft tolerance. Chimeras also developed humoral and in vitro tolerance. Both deletional and nondeletional mechanisms contributed to maintenance of tolerance. All tested populations of polyclonal Tregs (FoxP3‐transduced Tregs, natural Tregs and TGF‐β induced Tregs) were effective in this setting. Thus, Treg therapy achieves mixed chimerism and tolerance without cytoreductive recipient treatment, thereby eliminating a major toxic element impeding clinical translation of this approach.

Prevalence and clinical importance of hypertransaminasaemia in coeliac disease.

OBJECTIVE To assess the prevalence and potential pathogenetic factors of hypertransaminasaemia in patients with coeliac disease prior to initiation of a gluten-free diet (GFD) and to assess the course of transaminases on a GFD. PATIENTS A retrospective study was made of 178 patients with coeliac disease (130 women, 48 men; median age 36 years; range 17-84 years) at the gastroenterological department of a university hospital. METHODS Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured prior to initiation of a GFD and at 3, 6 and 12 months of GFD. Intestinal permeability, a test for functional integrity of the small bowel, was investigated before starting a GFD in 116 patients by an oral test using lactulose and mannitol. RESULTS In 72 patients (40.4%) AST and/or ALT were increased prior to initiation of a GFD. Within 1 year on a GFD ALT and AST normalized except in eight cases (4.6%). The intestinal permeability index (% lactulose/% mannitol in 5 h urine) was higher in patients with elevated (median 0.34; range 0.03-1.43) than in patients with normal transaminases (0.11; 0.02-1.28) (P < 0.0001) and correlated with AST (tau = 0.34; P < 0.0001) and ALT (tau = 0.32; P < 0.0001). In five cases with hypertransaminasaemia a liver biopsy was performed prior to initiation of a GFD. Two patients had mild to moderate hepatitis with septal fibrosis. The other three had minimal lymphocytic infiltrates of the portal tracts. Inflammatory alterations of the bile ducts were not found. CONCLUSION Hypertransaminasaemia before GFD is frequent in coeliac patients, correlates with intestinal permeability and normalizes on a GFD in most patients. In cases of persistently elevated liver function tests of unknown origin underlying coeliac disease should be considered.

The mRNA of L-type calcium channel elevated in colon cancer : Protein distribution in normal and cancerous colon

Previous reports indicate that the mRNA for the cardiac isoform of the voltage-gated L-type calcium channel (alpha(1C)) is elevated in colon cancer. The aim of these experiments was to verify that the mRNA for alpha(1C) was significantly increased in tumors of two separate populations of patients when compared to normal adjacent mucosa. The second aim was to measure the distribution of alpha(1C) using immunocytochemistry in normal human colon and in colon cancer and to determine what might regulate the channel expression. Biopsies were taken from patients with various stages of colon cancer and nearby normal mucosa were used as control. RNA was prepared and mRNA level measured by semiquantitative reverse transcriptase-polymerase chain reaction. The mRNA of the calcium channel was compared with other markers including beta-actin. The mRNA for alpha(1C) was increased significantly in colon cancers compared to nearby adjacent mucosa. Using confocal microscopy alpha(1C) was localized mainly at the apical membrane in the surface epithelium of normal human colon with less distribution on the lateral and basal membranes. The channel was localized on the lateral and basal membranes in crypt cells. Calcium channel localization appeared to be nearer nuclei in colon cancer samples, in part because of the smaller size of the cells. Likewise, cultured Caco-2 and T84 cells showed a membrane distribution. Western blotting indicated that alpha(1C) protein was increased in nonconfluent cultures of colonic carcinoma cells compared to confluent cells and immunocytochemistry confirms that there is more calcium channel protein in cells that are nonconfluent. We conclude that the increase in mRNA of alpha(1) subunit of the cardiac isoform of the L-type calcium channel may be a useful marker of colon cancer compared to other markers because the increase is large and this increase can be documented on small samples using a simple semiquantitative reverse transcriptase-polymerase chain reaction. We found that alpha(1C) protein is increased when colonic cells are nonconfluent or dividing which may account for the increase in cancer.

Cells obtained from colorectal microadenomas mirror early premalignant growth patterns in vitro

LT97, a permanent cell line consisting of epithelial cells with an early premalignant genotype was established from small colorectal polyps. LT97 cells have lost both alleles of the APC tumour suppressor gene. In addition, they carry a mutated Ki-ras oncogene, while TP53 is normal. LT97 growth characteristics are thus representative of early adenomas. They had to be passaged as multicellular aggregates indicating a dependency of survival on cell-cell contact and in accordance with their premalignant genotype were not capable of growth in soft agar. LT97 cells did express both the EGF-receptor and small amounts of TGF(alpha) establishing an autocrine growth or survival pathway. However, in spite of autocrine TGF(alpha) production, growth was strongly dependent on exogenous growth factors--mainly EGF, insulin and HGF. Inhibition of the EGF-receptor kinase induced apoptosis at an IC(50) concentration of 4 micromolar indicating that TGF(alpha) activated survival pathways in the early adenoma cell.