Brigitte Wolf

Genetic analysis of multiple synchronous lesions of the colon adenoma–carcinoma sequence

The colorectal adenoma–carcinoma sequence represents a well-known paradigm for the sequential development of cancer driven by the accumulation of genomic defects. Although the colorectal adenoma–carcinoma sequence is well investigated, studies about tumours of different dignity co-existent in the same patient are seldom. In order to address the distribution of genetic alterations in different lesions of the same patient, we coincidently investigated carcinomas, adenomas and aberrant crypt foci in patients with sporadic colon cancer. By utilizing polymerase chain reaction, single-strand conformation polymorphism, heteroduplex-analysis, restriction fragment length polymorphism, protein truncation test and sequencing techniques we looked for mutations and microsatellite instability of APC, H- ras, K- ras, p53, DCC and the DNA repair genes hMLH1/hMSH2. In accordance with the suggested adenoma–carcinoma sequence of the colon, four patients reflected the progressive accumulation of genetic defects in synchronously appearing tumours during carcinogenesis. However, two patients with non-hereditary malignomas presented different genetic instabilities in different but synchronously appearing tumours suggesting non-clonal growth under almost identical conditions of the environment. Thus, sporadically manifesting multiple lesions of the colon were not necessarily driven by similar genetic mechanisms. Premalignant lesions may transform into malignant tumours starting from different types of genetic instability, which indicates independent and simultaneous tumorigenesis within the same organ.

LINE-1 induces hTERT and ensures telomere maintenance in tumour cell lines

A hallmark of cancer cells is an activated telomere maintenance mechanism, which allows prolonged survival of the malignant cells. In more than 80% of tumours, telomeres are elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Cancer cells are also characterized by expression of active LINE-1 elements (L1s, long interspersed nuclear elements-1). L1 elements are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Using L1-knockdown (KD), we show for the first time that L1 is critical for telomere maintenance in telomerase-positive tumour cells. The reduced length of telomeres in the L1-KD-treated cells correlated with an increased rate of telomere dysfunction foci, a reduced expression of shelterin proteins and an increased rate of anaphase bridges. The decreased telomere length was associated with a decreased telomerase activity and decreased telomerase mRNA level; the latter was increased upon L1 overexpression. L1-KD also led to a decrease in mRNA and protein expression of cMyc and KLF-4, two main transcription factors of telomerase and altered mRNA levels of other stem-cell-associated proteins such as CD44 and hMyb, as well as a corresponding reduced growth of spheroids. The KD of KLF-4 or cMyc decreased the level of L1-ORF1 mRNA, suggesting a specific reciprocal regulation with L1. Thus, our findings contribute to the understanding of L1 as a pathogenicity factor in cancer cells. As L1 is only expressed in pathophysiological conditions, L1 now appears to be target in the rational treatment of telomerase-positive cancer.

The biomarker TP53 divides patients with neoadjuvantly treated esophageal cancer into 2 subgroups with markedly different outcomes. A p53 Research Group study

BACKGROUND Fluorouracil and cisplatin have been used most frequently as neoadjuvant therapy for esophageal cancer. Both drugs are believed to act via a p53-dependent apoptosis pathway. The TP53 gene is frequently mutated in esophageal cancer. OBJECTIVE To test the value of TP53 as a biomarker prognosing outcome in patients with neoadjuvantly treated esophageal cancer. PATIENTS AND METHODS The investigation included 36 patients with primary operable esophageal cancer who were treated neoadjuvantly with cisplatin and fluorouracil. The TP53 genotype was assessed from paraffin-embedded diagnostic tumor biopsies using a standardized gene-specific TP53 sequencing protocol (mark53 kit; mark53 Ltd, Vienna, Austria). RESULTS Mutations in the TP53 gene were present in 50% of tumors. Two-year overall survival rates were 55.6% in patients with a normal TP53 marker status, compared with 16.7% in those with a mutant TP53 gene. In patients with normal TP53, neoadjuvant treatment resulted in significant advantages in terms of tumor-associated survival (P=.0049) and overall survival (P=.0304) compared with those with mutant TP53. The median tumor-associated survival was 34.2 months for patients with normal TP53, compared with 8.9 months for those with mutant TP53. The latter had a 3-fold higher risk of dying (hazard ratio, 3.01; 95% confidence interval, 1.359-6.86). CONCLUSIONS The biomarker TP53 divides esophageal cancer patients into 2 categories with markedly different outcomes: patients with a normal TP53 marker status may experience notable benefits from neoadjuvant chemotherapy with cisplatin/fluorouracil, whereas those with a mutant TP53 marker status appear to be at risk for lack of response.

Efficiency of the revised Bethesda guidelines (2003) for the detection of mutations in mismatch repair genes in Austrian HNPCC patients

The clinical diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) is based on the Amsterdam II criteria (ACII). The purpose of using the Bethesda guidelines (BG) is to select tumours for microsatellite analysis. Recently, the modified Amsterdam criteria (ACmod) and Bethesda guidelines (BGmod) were proposed to simplify definitions. We evaluated the efficiency of the ACmod and BGmod to identify patients with germ‐line mutations in MLH1 and MSH2 in 81 unrelated Austrian HNPCC families. Microsatellite (MS) analysis was performed in 55 tumours. The new criteria included more families than the old ones: BGmod, n = 81; BG, n = 72; ACmod, n = 52 and ACII, n = 35. The more stringent old criteria tended to show greater positive predictive value for association with a germ‐line mutation than the corresponding new criteria: BGmod, 23%; BG, 26%; ACmod, 31% and ACII, 37%. The larger number of patients analysed in the ACmod group resulted in greater sensitivity compared to the ACII. The increased workload for BGmod was not associated with greater sensitivity. Microsatellite instability (MSI) significantly enhanced specificity in all subgroups. We recommend the use of the ACmod criteria to select patients for primary sequence analysis, when microsatellite analysis is not possible. If the BG are used, we suggest that BG be given preference over BGmod, as the former signify a lesser workload.

TP53 Mutational Status and Prediction of Benefit from Adjuvant 5-Fluorouracil in Stage III Colon Cancer Patients.

We investigated the hypothesis that the varying treatment efficacy of adjuvant 5-fluorouracil (5FU) in stage III colon cancer is linked to the TP53 mutational status. ABCSG-90 was a prospective randomized trial in which effect of adjuvant 5FU was studied in stage III colon cancer patients. Tumor material of 70% of these patients (389/572) was available for analysis of the biomarker TP53 using a TP53-gene-specific Sanger sequencing protocol. Median follow-up was 88 months. TP53 mutation frequency was 33%. A significant interaction between TP53 status, outcomes and nodal category was found (P = 0.0095). In the N1 category, TP53 wildtype patients had significantly better overall survival than TP53 mutated (81.0% vs. 62.0% overall survival at 5 years; HR = 2.131; 95% CI: 1.344–3.378; P = 0.0010). In the N2 category, the TP53 status did not affect survival (P = 0.4992). In TP53 wildtype patients, the prognostic significance of N category was significantly enhanced (P = 0.0002). In TP53 mutated patients, survival curves of N1 and N2 patients overlapped and nodal category was no longer prognostic. The biomarker TP53 independently predicted effect of adjuvant 5FU in N1 colon cancer patients. TP53 was not predictive in N2 patients, in whom 5FU is known to have no effect.

Spectrum of germ-line MLH1 and MSH2 mutations in Austrian patients with hereditary nonpolyposis colorectal cancer

SummaryBACKGROUND: Germ-line mutations in mismatch repair genes are associated with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, which is characterized by susceptibility to cancer of the colon, endometrium, small bowel or urothelium at an unusually young age and with a high degree of penetration in all generations. MATERIAL AND METHODS: One hundred and nine individuals from 46 Austrian families who fulfilled the Amsterdam criteria (n = 29) or at least one of the Bethesda guidelines (n= 17) were analyzed for mutations in MLH1 and MSH2. Microsatellite instability was determined in the tumors of index persons and affected relatives. RESULTS AND CONCLUSION: High-grade instability was present in 60.6% of the tumor samples from index patients. Twenty-three germ-line DNA sequence variants in 24/46 families and four somatic mutations in three tumors were detected in MLH1 and MSH2. Fifteen mutations are novel. None of the newly identified germ-line variants was found in 100 alleles of healthy control individuals. We were able to characterize two intronic variants (MLH1 c.589-10T > A; MSH2 c.367-1G > A) with regard to their effect on mRNA. Both created new splice sites that replaced the regular ones. Germ-line mutations occurred in 44.8% of the families fulfilling the Amsterdam criteria and in 35.3% of the Bethesda patients. The detection of a pathogenic mutation was strongly correlated with microsatellite instability in the tumor DNA (p = 0.007). This study is the first comprehensive report of mutations in mismatch repair genes in Austrian patients with HNPCC.ZusammenfassungGRUNDLAGEN: Keimbahnmutationen in Mismatch-Reparatur-Genen stehen in ursächlichem Zusammenhang mit dem erblichen nicht-polypösen Kolonkarzinom-Syndrom. Diese Erkrankung zeichnet sich durch ein vermehrtes Auftreten von Karzinomen des Kolons, des Endometriums, des Dünndarmes oder des Urothels in einem außergewöhnlich jungen Alter aus. MATERIAL UND METHODEN: In dieser Studie untersuchten wir 109 Individuen aus 46 österreichischen Familien auf Mutationen in MLH1 und MSH2. Die Selektion der Patienten basierte auf den Amsterdam-Kriterien (n = 29) oder den Bethesda-Richtlinien (n = 17). In Tumorproben von Index-Patienten und erkrankten Verwandten wurde der Grad der Mikrosatelliteninstabilität ermittelt. ERGEBNISSE UND SCHLUSSBETRACHTUNG: Eine hochgradige Mikrosatelliteninstabilität konnte in 60,6% der Tumore von Index-Patienten nachgewiesen werden. In MLH1 und MSH2 wurden 23 DNA-Sequenzabweichungen in der Keimbahn von 24/46 Familien und 4 somatische Mutationen in 3 Tumoren festgestellt. Bei 15 Mutationen handelt es sich um neue, bislang noch nicht beschriebene genetische Veränderungen. Keine dieser neuen genetischen Varianten konnte in 100 Allelen gesunder Individuen nachgewiesen werden. Von 2, in Introns lokalisierten Varianten (MLH1 c.589-10T > A; MSH2 c.367-1G > A), konnten wir die Auswirkung auf die mRNA zeigen. In beiden Fällen entstanden neue Spleiß-Stellen, welche die Regulären ersetzten. Keimbahnmutationen traten in 44,8% der Amsterdam-Familien und in 35,3% der Bethesda-Patienten auf. Die Korrelation zwischen dem Nachweis einer pathogenen Keimbahnmutation und einer Mikrosatelliteninstabilität in der Tumor-DNA war hoch signifikant (p = 0,007). Diese Studie stellt den ersten umfassenden Bericht über Mutationen in Mismatch-Reparatur-Genen bei österreichischen Patienten mit erblichem nicht-polypösem Kolonkarzinom-Syndrom dar.

Strategy for prevention of cancers of the esophagus

The following, from the 12th OESO World Conference: Cancers of the Esophagus, includes commentaries on the animal reflux–inflammation models for Barretts esophagus and esophageal adenocarcinoma; genomic/epigenomic analyses; eflornithine‐based combinations; the molecular derangements that promote neoplastic transformation; the role of COX‐2 inhibitors, proton pump inhibitors, and phase II trials in Barretts adenocarcinoma; statins in chemoprevention and treatment of esophageal cancer; and biomarkers as potential targets in Barretts adenocarcinoma.

Potential of DNA methylation in rectal cancer as diagnostic and prognostic biomarkers

Background:Aberrant DNA methylation is more prominent in proximal compared with distal colorectal cancers. Although a number of methylation markers were identified for colon cancer, yet few are available for rectal cancer.Methods:DNA methylation differences were assessed by a targeted DNA microarray for 360 marker candidates between 22 fresh frozen rectal tumour samples and 8 controls and validated by microfluidic high-throughput and methylation-sensitive qPCR in fresh frozen and formalin-fixed paraffin-embedded (FFPE) samples, respectively. The CpG island methylator phenotype (CIMP) was assessed by MethyLight in FFPE material from 78 patients with pT2 and pT3 rectal adenocarcinoma.Results:We identified and confirmed two novel three-gene signatures in fresh frozen samples that can distinguish tumours from adjacent tissue as well as from blood with a high sensitivity and specificity of up to 1 and an AUC of 1. In addition, methylation of individual CIMP markers was associated with specific clinical parameters such as tumour stage, therapy or patients’ age. Methylation of CDKN2A was a negative prognostic factor for overall survival of patients.Conclusions:The newly defined methylation markers will be suitable for early disease detection and monitoring of rectal cancer.

TP53 germline mutation may affect response to anticancer treatments: analysis of an intensively treated Li–Fraumeni family

Li–Fraumeni syndrome (LFS) is a rare autosomal dominant inherited disorder associated with the occurrence of a wide spectrum of early-onset malignancies, the most prevalent being breast cancer and sarcoma. The presence of TP53 germline mutations in the majority of LFS patients suggests a genetic basis for the cancer predisposition. No special recommendations for the treatment of LFS patients have been made to date, except that of minimizing radiation. We hypothesized that TP53 germline mutations may be associated not only with cancer predisposition, but also with lack of response to chemo- and radiotherapy. Here, we present an Austrian LFS family whose members were intensively treated with chemo- and radiotherapy due to cancers that occurred at a predominantly young age, including eight breast cancers in six patients. Material from seven family members was screened for p53 mutation by Sanger sequencing and immunohistochemistry. A rare missense mutation in the tetramerization domain of exon 10 of the TP53 gene was found to segregate with malignant disease in this family. Lack of response to various chemotherapies and radiotherapy could be ascertained by histopathology of surgical specimens after neoadjuvant treatment, by cancer relapse occurring while receiving adjuvant systemic treatment and by the occurrence of second primaries in areas of adjuvant radiation. Our observations suggest that current standards of cancer treatment may not be valid for patients with LFS. In patients with TP53 germline mutation, cytotoxic treatment may bear not only the risk of tumor induction but also the risk of treatment failure.

Turning the tables on surgical oncology: the • Pancho trial unplugged

ZusammenfassungGRUNDLAGEN: Die neoadjuvante Therapie bringt bei Patienten mit Ösophaguskarzinom keine eindeutige Verbesserung des Gesamtüberlebens. Einen deutlichen Überlebensvorteil zeigen allerdings Patienten mit kompletter pathologischer Remission. METHODIK: Dieses Review fasst mehrere retrospektive klinische Studien zusammen, die p53 als potentiellen prädiktiven Marker für das Ansprechen auf Chemotherapie ausweisen. Um den Zusammenhang zwischen p53 und Chemotherapieansprechen auf hohem Evidenzniveau (Level I) zu untersuchen, wurde die • Pancho Studie initiiert. ERGEBNISSE: In der • Pancho Studie wird erstmals – prospektiv randomisiert – untersucht, ob eine Interaktion zwischen p53 und dem Ansprechen auf Chemotherapie besteht. In der vorliegenden Arbeit berichten wir über das • Pancho Studiendesign (Wechselwirkungsdesign), die Fallzahlberechnung, die Studienendpunkte und den Rekrutierungsstatus dieser Multizenter-Studie nach einem Jahr Laufzeit. SCHLUSSFOLGERUNGEN: Die • Pancho Studie testet erstmals in einem geeigneten Design, ob der p53 Genotyp ein valider Marker ist, um wirksame Chemotherapien zu selektieren und damit eine individuelle Krebstherapie zu ermöglichen.SummaryBACKGROUND: In esophageal cancer patients neoadjuvant therapy failed to demonstrate a clear benefit in overall survival. A significant advantage can be seen in patients with complete pathological response. METHODS: This review summarizes the results of retrospective clinical studies suggesting p53 as a predictive marker for chemotherapy response. To advance these findings to level of evidence I the • Pancho trial was initiated. RESULTS: The • Pancho trial represents the first prospective randomized trial testing the interaction between p53 and response to chemotherapy. The special design of the • Pancho trial (interaction design), the sample size considerations, the study endpoints and the 12 months accrual of this nationwide study are reported. CONCLUSIONS: The • Pancho trial evaluates for the first time whether the p53 genotype is qualified to select patients who will respond to certain chemotherapy and to guide cancer therapy.