Wolfgang Dietmaier

Multiple Mutation Analyses in Single Tumor Cells with Improved Whole Genome Amplification

Combining whole genome amplification (WGA) methods with novel laser-based microdissection techniques has made it possible to exploit recent progress in molecular knowledge of cancer development and progression. However, WGA of one or a few cells has not yet been optimized and systematically evaluated for samples routinely processed in tumor pathology. We therefore studied the value of established WGA protocols in comparison to an improved PEP (I-PEP) PCR method in defined numbers of flow-sorted and microdissected tumor cells obtained both from frozen as well as formalin-fixed and paraffin-embedded tissue sections. In addition, the feasibility of I-PEP-PCR for mutation analysis was tested using clusters of 50-100 unfixed tumor cells obtained by touch preparation of ten breast carcinomas by conventional sequencing of exon 7 and 8 of the p53 gene. Finally, immunocytochemically stained microdissected single disseminated tumor cells from bone marrow aspirates were investigated with respect to mutations in codon 12 of Ki-ras by restriction fragment length polymorphism (RFLP)-PCR after I-PEP-PCR. The modified I-PEP-PCR protocol was superior to the original PEP-PCR and DOP-PCR protocols concerning amplification of DNA from one cell (efficiency rate I-PEP-PCR 40% versus PEP-PCR 15% and DOP-PCR 30%) and five cells (efficiency rate I-PEP-PCR 100% versus PEP-PCR 33% and DOP-PCR 20%). Preamplification by I-PEP allowed 100% sequence accuracy in > 4000 sequenced base pairs and Ki-ras mutation detection in isolated single disseminated tumor cells. For reliable microsatellite analysis of I-PEP-preamplified DNA, at least 10 unfixed cells from fluorescence-activated cell sorting, 10 cells from frozen tissue, or at least 30 cells from formalin-fixed and paraffin-embedded tissue sections were required. Thus, I-PEP-PCR allowed multiple reliable microsatellite analyses suited for microsatellite instability and losses of heterozygosity and mutation analysis even at the single cell level, rendering this technique a powerful new tool for molecular analyses in diagnostic and experimental tumor pathology.

Minute Gastric Sclerosing Stromal Tumors (GIST Tumorlets) Are Common in Adults and Frequently Show c-KIT Mutations

Multifocal hyperplasia of interstitial cells of Cajal (ICC hyperplasia) is a precursor of hereditary gastrointestinal stromal tumors (GISTs) in patients with germline mutations of c-KIT or PDGFRA, but precursor lesions of sporadic GISTs have not been defined yet. Small hyalinizing stromal tumors of the proximal stomach (referred to in this study as GIST tumorlets) were collected prospectively from 98 consecutive autopsies and additional cases were retrieved from surgical pathology files (total n=57). GIST tumorlets were grossly detectable in 22.5% consecutive autopsies performed in individuals older than 50 years. All lesions were located in the cardia, fundus, or proximal body, and ranged in size from 1 to 10 mm (4 mm). Similar lesions were not detected in the antrum, duodenum, and the remainder of the bowel. Histologically, the spindle cell subtype comprised all cases, with hyalinization and calcification in 57% of cases. The spindle cells were immunohistochemically positive for vimentin, CD117, and CD34. Twenty-four cases yielded sufficient DNA for subsequent molecular analysis, which showed c-KIT mutations in 11 cases (46%) and PDGFRA mutations in 1 case (4%). Sporadic GIST tumorlets of the proximal stomach are common in the general population over the age of 50 years and frequently show somatic c-KIT mutations. GIST tumorlets probably represent the grossly recognizable counterpart of sporadic ICC hyperplasia caused by somatic c-KIT or PDGFRA mutations. Early hyalinization and calcification seems to confer limited growth potential, and complete regression of such lesions is common. GIST tumorlets likely represent preclinical (preneoplastic) lesions that need additional stimuli to evolve into clinical GISTs, raising the possibility of a hyperplasia-neoplasia sequence in the development of sporadic GISTs.

Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation.

The adoptive transfer of CD4+CD25+ natural regulatory T cells (Treg) is a promising strategy for the treatment of autoimmune diseases and the prevention of alloresponses after transplantation. Clinical trials exploring this strategy require efficient in vitro expansion of this rare cell population. Protocols developed thus far rely on high‐grade purification of Treg prior to culture initiation, a process still hampered by the lack of Treg cell‐specific surface markers. Depletion of CD127+ cells was shown to separate activated conventional T cells from natural Treg cell populations allowing the isolation of highly enriched FOXP3+ cells with all functional and molecular characteristics of natural Treg. Here, we demonstrate that upon in vitro expansion, CpG methylation in a conserved region within the FOXP3 gene locus increased in CD4+CD25+CD127low Treg, correlating with loss of FOXP3 expression and emergence of pro‐inflammatory cytokines. Further analysis identified CD45RA−FOXP3+ memory‐type Treg as the main source of converting cells, whereas CD45RA+FOXP3+ Treg from the same donors showed no conversion within 3 wk of in vitro expansion. Thus, Treg cell lineage differentiation does not seem to represent a final fate decision, as natural Treg can lose their cell‐type‐specific characteristics after repetitive TCR stimulation.

Evidence for oligoclonality and tumor spread by intraluminal seeding in multifocal urothelial carcinomas of the upper and lower urinary tract

Multifocality and recurrence of urothelial carcinoma may result from either the field effect of carcinogens leading to oligoclonal tumors or monoclonal tumor spread. Previous molecular studies, favoring the monoclonality hypothesis, are mostly limited to the urinary bladder. We investigated genetic alterations in a total of 94 synchronous or metachronous multifocal tumors from 19 patients with at least one tumor both in the upper and lower urinary tract. Loss of heterozygosity (LOH) was determined using eight markers on chromosome 9 and one marker on 17p13 (p53). Microsatellite instability was investigated at six loci and protein expression of MSH2 and MLH1 was evaluated by immunohistochemistry. In addition, exons 5–9 of the p53 gene were sequenced. Deletions at chromosome 9 were found in 73% of tumors and at 17p13 in 18% of tumors. There was no significant difference in the frequency of LOH in the upper and lower urinary tract. Deletions at 9p21 were significantly correlated with invasive tumor growth. The pattern of deletion revealed monoclonality of all tumors in nine patients. In five patients there were at least two tumor clones with different genetic alterations. In four of these patients the different clones occured in the bladder and subsequently in the ureter and renal pelvis. All four patients with p53 mutations revealed identical mutations in all tumors. Thus, multifocal urothelial carcinomas are frequently monoclonal, whereas others show oligoclonality, providing molecular evidence for field cancerization. Intraluminal tumor cell seeding appears to be an important mechanism of multifocal occurence and recurrence of urothelial carcinomas.

Pegylated liposomal doxorubicin-efficacy in patients with recurrent high-grade glioma

Doxorubicin exhibits high efficacy in malignant glioma cell cultures. Nonetheless, as a standard formulation, doxorubicin has not been used clinically, due to poor penetration of the blood‐brain barrier. Furthermore, doxorubicin is known to induce tumor resistance genes. To address both of these issues, the authors investigated the use of pegylated liposomal doxorubicin (Caelyx™; Essex Pharma, Munich, Germany) alone (Trial 1) and in combination with tamoxifen (Trial 2) in two sequentially performed nonrandomized prospective Phase II trials involving patients with recurrent high‐grade glioma.

CD133 Expression and Cancer Stem Cells Predict Prognosis in High-grade Oligodendroglial Tumors

High‐grade oligodendroglial tumors, that is, anaplastic oligodendroglial tumors and glioblastomas with oligodendroglial component, differ significantly in terms of prognosis and response to chemotherapy. Differentiation might be difficult because the histological differences are vague and reliable markers are not established. We correlated the presence of putative cancer stem cells (CSC) in high‐grade oligodendroglial tumors (WHO grades III and IV) with clinical outcome. Tumors with favorable prognosis neither contained CSC nor did they show CD133 expression. Tumor cells resembled lineage‐restricted progenitor cells with limited proliferative capacity and differentiation profile. In contrast, CD133 expression and stem cell‐like tumor cells characterized tumors with poor prognosis. They showed neurosphere‐like growth, differentiated into cells of all neural lineages, and were tumorigenic in nude mice. In our series, CSC and expression of CD133 predicted the clinical course of disease better than the histological grading. To confirm these results, we retrospectively analyzed 36 high‐grade oligodendroglial tumors. Again, CD133 expression indicated shorter survival and predicted clinical outcome more reliable than the histological assessment.

Genotype-Phenotype Comparison of German MLH1 and MSH2 Mutation Carriers Clinically Affected With Lynch Syndrome: A Report by the German HNPCC Consortium

PURPOSE Lynch syndrome is linked to germline mutations in mismatch repair genes. We analyzed the genotype-phenotype correlations in the largest cohort so far reported. PATIENTS AND METHODS Following standard algorithms, we identified 281 of 574 unrelated families with deleterious germline mutations in MLH1 (n = 124) or MSH2 (n = 157). A total of 988 patients with 1,381 cancers were included in this analysis. RESULTS We identified 181 and 259 individuals with proven or obligatory and 254 and 294 with assumed MLH1 and MSH2 mutations, respectively. Age at diagnosis was younger both in regard to first cancer (40 v 43 years; P < .009) and to first colorectal cancer (CRC; 41 v 44 years; P = .004) in MLH1 (n = 435) versus MSH2 (n = 553) mutation carriers. In both groups, rectal cancers were remarkably frequent, and the time span between first and second CRC was smaller if the first primary occurred left sided. Gastric cancer was the third most frequent malignancy occurring without a similarly affected relative in most cases. All prostate cancers occurred in MSH2 mutation carriers. CONCLUSION The proportion of rectal cancers and shorter time span to metachronous cancers indicates the need for a defined treatment strategy for primary rectal cancers in hereditary nonpolyposis colorectal cancer patients. Male MLH1 mutation carriers require earlier colonoscopy beginning at age 20 years. We propose regular gastric surveillance starting at age 35 years, regardless of the familial occurrence of this cancer. The association of prostate cancer with MSH2 mutations should be taken into consideration both for clinical and genetic counseling practice.

Risks of Less Common Cancers in Proven Mutation Carriers With Lynch Syndrome

PURPOSE Patients with Lynch syndrome are at high risk for colon and endometrial cancer, but also at an elevated risk for other less common cancers. The purpose of this retrospective cohort study was to provide risk estimates for these less common cancers in proven carriers of pathogenic mutations in the mismatch repair (MMR) genes MLH1, MSH2, and MSH6. PATIENTS AND METHODS Data were pooled from the German and Dutch national Lynch syndrome registries. Seven different cancer types were analyzed: stomach, small bowel, urinary bladder, other urothelial, breast, ovarian, and prostate cancer. Age-, sex- and MMR gene-specific cumulative risks (CRs) were calculated using the Kaplan-Meier method. Sex-specific incidence rates were compared with general population incidence rates by calculating standardized incidence ratios (SIRs). Multivariate Cox regression analysis was used to estimate the impact of sex and mutated gene on cancer risk. RESULTS The cohort comprised 2,118 MMR gene mutation carriers (MLH1, n = 806; MSH2, n = 1,004; MSH6, n = 308). All cancers were significantly more frequent than in the general population. The highest risks were found for male small bowel cancer (SIR, 251; 95% CI, 177 to 346; CR at 70 years, 12.0; 95% CI, 5.7 to 18.2). Breast cancer showed an SIR of 1.9 (95% CI, 1.4 to 2.4) and a CR of 14.4 (95% CI, 9.5 to 19.3). MSH2 mutation carriers had a considerably higher risk of developing urothelial cancer than MLH1 or MSH6 carriers. CONCLUSION The sex- and gene-specific differences of less common cancer risks should be taken into account in cancer surveillance and prevention programs for patients with Lynch syndrome.

Smoking and cancer-related gene expression in bronchial epithelium and non-small-cell lung cancers.

Tobacco smoking is the leading cause of lung cancer worldwide. Gene expression in surgically resected and microdissected samples of non‐small‐cell lung cancers (18 squamous cell carcinomas and nine adenocarcinomas), matched normal bronchial epithelium, and peripheral lung tissue from both smokers (n = 22) and non‐smokers (n = 5) was studied using the Affymetrix U133A array. A subset of 15 differentially regulated genes was validated by real‐time PCR or immunohistochemistry. Hierarchical cluster analysis clearly distinguished between benign and malignant tissue and between squamous cell carcinomas and adenocarcinomas. The bronchial epithelium and adenocarcinomas could be divided into the two subgroups of smokers and non‐smokers. By comparison of the gene expression profiles in the bronchial epithelium of non‐smokers, smokers, and matched cancer tissues, it was possible to identify a signature of 23 differentially expressed genes, which might reflect early cigarette smoke‐induced and cancer‐relevant molecular lesions in the central bronchial epithelium of smokers. Ten of these genes are involved in xenobiotic metabolism and redox stress (eg AKR1B10, AKR1C1, and MT1K). One gene is a tumour suppressor gene (HLF); two genes act as oncogenes (FGFR3 and LMO3); two genes are involved in matrix degradation (MMP12 and PTHLH); three genes are related to cell differentiation (SPRR1B, RTN1, and MUC7); and five genes have not been well characterized to date. By comparison of the tobacco‐exposed peripheral alveolar lung tissue of smokers with non‐smokers and with adenocarcinomas from smokers, it was possible to identify a signature of 27 other differentially expressed genes. These genes are involved in the metabolism of xenobiotics (eg GPX2 and FMO3) and may represent cigarette smoke‐induced, cancer‐related molecular targets that may be utilized to identify smokers with increased risk for lung cancer. Copyright

Frequent genetic alterations in flat urothelial hyperplasias and concomitant papillary bladder cancer as detected by CGH, LOH, and FISH analyses.

Flat urothelial hyperplasia, defined as markedly thickened urothelium without cytological atypia, is regarded in the new WHO classification as a urothelial lesion without malignant potential. Frequent deletions of chromosome 9 detected by fluorescence in situ hybridization (FISH) have been previously reported in flat urothelial hyperplasias found in patients with papillary bladder cancer. Using comparative genomic hybridization (CGH) and microsatellite analysis, these hyperplasias and concomitant papillary tumours of the same patients were screened for other genetic alterations to validate and extend the previous findings. Eleven flat hyperplasias detected by 5‐ALA‐induced fluorescence endoscopy and ten papillary urothelial carcinomas (pTaG1–G2) from ten patients were investigated. After microdissection, the DNA of the lesions was pre‐amplified using whole genome amplification (I‐PEP‐PCR). Loss of heterozygosity (LOH) analyses were performed with five microsatellite markers at chromosomes 9p, 9q, and 17p. CGH was performed using standard protocols. In 6 of 11 hyperplasias and 7 of 10 papillary tumours, deletions at chromosome 9 were simultaneously shown by FISH, LOH, and CGH analyses. There was a good correlation between FISH, LOH, and CGH analyses, with identical results in 6 of 10 patients. In addition to deletions at chromosome 9, further genetic alterations were detected by CGH in 9 of 10 investigated hyperplasias, including changes frequently found in invasive papillary bladder cancer (loss of chromosomes 2q, 4, 8p, and 11p; gain of chromosome 17; and amplification at 11q12q13). There was considerable genetic heterogeneity between hyperplasias and papillary tumours, but a clonal relationship was suggested by LOH and/or CGH analyses in 5 of 10 cases. These data support the hypothesis that flat urothelial hyperplasias can display many genetic alterations commonly found in bladder cancer and could therefore be an early neoplastic lesion in the multistep development of invasive urothelial carcinoma. Copyright