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Based on new trial data regarding immune checkpoint inhibitors (ICIs), the detection of high-grade microsatellite instability (MSI-H) or underlying deficient mismatch repair protein (dMMR) is now becoming increasingly important for predicting treatment response. For the first time, a PD‑1 ICI (pembrolizumab) has been approved by the European Medicines Agency (EMA) for first-line treatment of advanced (stage IV) dMMR/MSI‑H colorectal cancer (CRC). Further indications, such as dMMR/MSI‑H endometrial carcinoma (EC), have already succeeded (Dostarlimab, 2nd line treatment) and others are expected to follow before the end of 2021. The question of optimal testing in routine diagnostics should therefore be re-evaluated. Based on a consideration of the strengths and weaknesses of the widely available methods (immunohistochemistry and PCR), a test algorithm is proposed that allows quality assured, reliable, and cost-effective dMMR/MSI‑H testing. For CRC and EC, testing is therefore already possible at the primary diagnosis stage, in line with international recommendations (NICE, NCCN). The clinician is therefore enabled from the outset to consider not only the predictive but also the prognostic and predispositional implications of such a test when counseling patients and formulating treatment recommendations. As a basis for quality assurance, participation in interlaboratory comparisons and continuous documentation of results (e.g., QuIP Monitor) are strongly recommended.

Based on new trial data regarding immune checkpoint inhibitors (ICIs), the detection of high-grade microsatellite instability (MSI-H) or underlying deficient mismatch repair protein (dMMR) is now becoming increasingly important for predicting treatment response. For the first time, a PD‑1 ICI (pembrolizumab) has been approved by the European Medicines Agency (EMA) for first-line treatment of advanced (stage IV) dMMR/MSI‑H colorectal cancer (CRC). Further indications, such as dMMR/MSI‑H endometrial carcinoma (EC), have already succeeded (Dostarlimab, 2nd line treatment) and others are expected to follow before the end of 2021. The question of optimal testing in routine diagnostics should therefore be re-evaluated. Based on a consideration of the strengths and weaknesses of the widely available methods (immunohistochemistry and PCR), a test algorithm is proposed that allows quality assured, reliable, and cost-effective dMMR/MSI‑H testing. For CRC and EC, testing is therefore already possible at the primary diagnosis stage, in line with international recommendations (NICE, NCCN). The clinician is therefore enabled from the outset to consider not only the predictive but also the prognostic and predispositional implications of such a test when counseling patients and formulating treatment recommendations. As a basis for quality assurance, participation in interlaboratory comparisons and continuous documentation of results (e.g., QuIP Monitor) are strongly recommended.

Molecular investigations are crucial for further developments in precision medicine. RNA sequencing, alone or in combination with further omic-analyses, resulted in new therapeutic strategies. In this context, biobanks represent infrastructures to store tissue samples and body fluids in combination with clinical data to promote research for new predictive and prognostic biomarkers as well as therapeutic candidate molecules. Until today, the optimal storage conditions are a matter of debate especially with view to the storage temperature. In this unique approach we compared parallel samples from the same tumour, one half stored at − 80 °C and one half in the vapor phase of liquid nitrogen, with almost identical pre-analytical conditions. We demonstrated that RNA isolated from breast cancer samples revealed significantly higher RINe-values after 10 years of storage in the vapor phase of liquid nitrogen compared to storage at − 80 °C. In contrast, no significant difference was found regarding the DIN-values after DNA isolation. Morphological changes of the nucleus and cytoplasm, especially in the samples stored at − 80 °C, gave insights to degenerative effects, most possibly due to the storage protocol and its respective peculiarities. In addition, our results indicate that exact point-to point documentation beginning at the sample preparation is mandatory.

Background: Lung cancer has emerged as a global public health problem and is the most common cause of cancer deaths by absolute cases globally. Besides tobacco, smoke infectious diseases such as human papillomavirus (HPV) might be involved in the pathogenesis of lung cancer. However, data are inconsistent due to differences in study design and HPV detection methods. Aim: A systematic meta‐analysis was performed to examine the presence of HPV‐infection with lung cancer. Methods and Results: All studies in all languages were considered for the search concepts “lung cancer” and “HPV” if data specific to HPV prevalence in lung cancer tissue were given. This included Journal articles as well as abstracts and conference reports. As detection method, only HPV PCR results from fresh frozen and paraffin‐embedded tissue were included. Five bibliographic databases and three registers of clinical trials including MEDLINE, Embase, Cochrane Library, and ClinicalTrials.gov were searched through February 2020. A total 4298 publications were identified, and 78 publications were selected, resulting in 9385 included lung cancer patients. A meta‐analysis of 15 case‐control studies with n = 2504 patients showed a weighted overall prevalence difference of 22% (95% CI: 12%‐33%; P

After introduction of the Bethesda microsatellite test panel demonstration of microsatellite instability (MSI) and/or loss of mismatch repair proteins (MMRD) was primarily used as a marker for cancer predisposition of Lynch syndrome (LS, previous: HNPCC). Nowadays MSI/MMRD has become an important biomarker to predict therapy response to checkpoint immunotherapies. MSI can be determined either by polymerase chain reaction (PCR)-based technologies with or without specification of fragment sizes or next generation sequencing (NGS) methods. Depending on the individual tumor entities, these test methods are used differently. Currently, MSI/MMRD is a tumor biomarker which covers a broad spectrum of indications in tumor pathology, especially in colorectal, endometrial and gastric cancer. In advanced carcinomas, MSI is an established predictor of therapy response to checkpoint-directed immunotherapies.

Simple Summary Due to the approval of immune checkpoint inhibitor therapy for microsatellite instability-high or mismatch repair-deficient advanced solid tumors, testing of both biomarkers has gained interest in recent years. Available testing systems were established in the context of Lynch Syndrome for colorectal cancer, thus differences between microsatellite profiles across cancer types may lead to false data interpretation using validated tests for another tumor entity. The present study deals with challenges during microsatellite instability testing in endometrial cancer (EC) and provides a comprehensive comparative study of four different PCR-based approaches which could help to improve microsatellite instability (MSI) testing in future screenings. A validation strategy has been developed for the Idylla system, which can guide the method transfer to other tumor entities, and a screening procedure for EC has been proposed. By direct comparison, this study was able to highlight advantages and limitations of each system in an extensive manner. Abstract Microsatellite instability (MSI), a common alteration in endometrial cancers (EC) is known as a biomarker for immune checkpoint therapy response alongside screening for Lynch Syndrome (LS). However, former studies described challenging MSI profiles in EC hindering analysis by using MSI testing methods intensively validated for colorectal cancer (CRC) only. In order to reduce false negatives, this study examined four different PCR-based approaches for MSI testing using 25 EC samples already tested for mismatch repair deficiency (dMMR). In a follow up validation set of 75 EC samples previously tested both for MMR and MSI, the efficiency of a seven-marker system corresponding to the Idylla system was further analyzed. Both Bethesda and Promega marker panels require trained operators to overcome interpretation complexities caused by either hardly visible additional peaks of one and two nucleotides, or small shifts in microsatellite repeat length. Using parallel sequencing adjustment of bioinformatics is needed. Applying the Idylla MSI assay, an evaluation of input material is more crucial for reliable results and is indispensable. Following MMR deficiency testing as a first-line screening procedure, additional testing with a PCR-based method is necessary if inconclusive staining of immunohistochemistry (IHC) must be clarified.

Introduction: Tumor mutational burden (TMB) is a quan-titative assessment of the number of somatic mutations within a tumor genome. Immunotherapy benefit has been associated with TMB assessed by whole-exome sequencing (wesTMB) and gene panel sequencing (psTMB). The initiatives of Quality in Pathology (QuIP) and Friends of Cancer Research have jointly addressed the need for harmonization among TMB testing options in tissues. This QuIP study identifies critical sources of variation in psTMB assessment. Methods: A total of 20 samples from three tumor types (lung adenocarcinoma, head and neck squamous cell car-cinoma, and colon adenocarcinoma) with available WES data were analyzed for psTMB using six panels across 15 testing centers. Interlaboratory and interplatform variation, including agreement on variant calling and TMB classifica-tion, were investigated. Bridging factors to transform psTMB to wesTMB values were empirically derived. The impact of germline filtering was evaluated. Results: Sixteen samples had low interlaboratory and interpanel psTMB variation, with 87.7% of pairwise com-parisons revealing a Spearman & rsquo;s r greater than 0.6. A wesTMB cut point of 199 missense mutations projected to psTMB cut points between 7.8 and 12.6 mutations per megabase pair; the corresponding psTMB and wesTMB classifications agreed in 74.9% of cases. For three-tier classification with cut points of 100 and 300 mutations, agreement was observed in 76.7%, weak misclassification in 21.8%, and strong misclassification in 1.5% of cases. Confounders of psTMB estimation included fixation arti-facts, DNA input, sequencing depth, genome coverage, and variant allele frequency cut points. Conclusions: This study provides real-world evidence that all evaluated panels can be used to estimate TMB in a routine diagnostic setting and identifies important param-eters for reliable tissue TMB assessment that require careful control. As complex or composite biomarkers beyond TMB are likely playing an increasing role in therapy prediction, the efforts by QuIP and Friends of Cancer Research also delineate a general framework and blueprint for the eval-uation of such assays. (C) 2020 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Das adenosquamose Karzinom des Kolons stellt einen sehr seltenen Subtyp des Kolonkarzinoms dar, fur den es bislang aufgrund zu geringer Fallzahlen keine speziell etablierte Therapie gibt. Aufgrund seiner Seltenheit ist ebenfalls wenig uber sein biologisches Verhalten und seinen molekularen Hintergrund bekannt, auch wenn Fallberichte eine schlechtere Prognose im Vergleich zu den Adenokarzinomen des Kolons ohne speziellen Subtyp beschreiben. Der hier dargestellte Fall berichtet uber die erste erfolgreiche Immun-Checkpoint-Blockade-Therapie eines metastasierten, sporadisch hochgradig mikrosatelliteninstabilen, rechtsseitigen, adenosquamosen Kolonkarzinoms eines 40-jahrigen Mannes.

Aberrant promoter methylation of tumor relevant genes frequently occurs in early steps of carcinogenesis and during tumor progression. Epigenetic alterations could be used as potential biomarkers for early detection and for prediction of prognosis and therapy response in lung cancer. The present study quantitatively analyzed the methylation status of known and potential gatekeeper and tumor suppressor genes [O-6-methylguanine-DNA methyltransferase (MGMT), Ras association domain family member 1A (RASSF1A), Ras protein activator like 1 (RASAL1), programmed cell death 4 (PDCD4), metastasis suppressor 1 (MTSS1) and tumor suppressor candidate 3 (TUSC3)] in 42 lung cancers and in corresponding non-malignant bronchus and lung tissue using bisulfite-conversion independent methylation-quantification of endonuclease-resistant DNA (MethyQESD). Methylation status was associated with clinical and pathological parameters. No methylation was found in the promoter regions of PDCD4 and MTSS1 of either compartment. MGMT, RASSF1A and RASAL1 showed sporadic (up to 26.2%) promoter methylation. The promoter of TUSC3, however, was frequently methylated in the tumor (59.5%), benign bronchus (67.9%) and alveolar lung (31.0%) tissues from each tumor patient. The methylation status of TUSC3 was significantly associated with smaller tumor size (P=0.008) and a longer overall survival (P=0.013). Pooled blood DNA of healthy individuals did not show any methylation of either gene. Therefore, methylation of TUSC3 shows prognostic and pathobiological relevance in lung cancer. Furthermore, quantitative detection of TUSC3 promoter methylation appears to be a promising tool for early detection and prediction of prognosis in lung cancer. However, additional studies are required to confirm this finding.

Carcinomas of the ampulla of Vater are rare and assumed to generally arise from preexisting adenomas (adenoma-carcinoma sequence). Histologically, distinct subtypes can be distinguished that were shown to differ significantly in terms of clinical outcome. Since pathologists usually receive bioptic tissue samples of ampullary tumors obtained during endoscopy, accurate classification of carcinoma subtypes can sometimes be difficult on morphological criteria alone. We therefore performed immunohistochemistry using a panel of established marker proteins (CK7, CK20, p21, p27, ESA, bax, and ephrin-B2) on 175 carcinoma, 111 adenoma, and 152 normal mucosa specimens of the ampulla of Vater and identified distinct immunoprofiles for every carcinoma subtype. Fluorescence in situ hybridization analyses of therapeutic target genes (c-myc, EGFR1, CCND1, HER2) found CCND1 to represent the most frequently amplified gene in our series (7.5%).

Background: The prognosis of metastatic non-small cell lung cancer (NSCLC) is still poor. Activating epithelial growth factor receptor (EGFR) mutations are important genetic alterations with dramatic therapeutical implications. Up to now, in contrast to Asian populations only limited data on the prevalence of those mutations are available from patients with Caucasian and especially European ethnicity. Methods: In this multicentre study, 1201 unselected NSCLC patients from Southern Germany were tested in the daily clinical routine for EGFR mutation status. Results: Activating EGFR mutations were found in 9.8% of all tumours. Mutations in exons 18, 19 and 21 accounted for 4.2%, 61.9% and 33.1% of all mutations, respectively. Non-smokers had a significantly higher rate of EGFR mutations than smokers or ex-smokers (24.4% vs 4.2% P

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.

Object In craniopharyngiomas, cystic growth causes pressure on vital structures of the adjacent brain, leading to significant morbidity. However, the molecular pathogenesis of this cyst formation remains unknown. Carbonic anhydrase IX (CA IX) is a tumor-associated, hypoxia-inducible enzyme, which can cause fluid production and development of cysts. The authors investigated CA IX expression in craniopharyngiomas and its correlation with the extent of cyst formation. In addition, the major pathways of CA IX regulation, hypoxia and p53 mutation, were analyzed. Methods Expression of CA IX was analyzed in 20 craniopharyngioma patients by means of in situ hybridization and immunohistochemistry. Preoperative imaging was used to quantify cyst volume. To analyze putative hypoxic induction of CA IX, immunohistochemical staining for HIF-1α and VEGF was performed. Since p53 negatively regulates CA IX expression, we also analyzed the tumors for p53 mutation by direct sequencing. Results Significant CA IX was found in 85% of the 20 cases. The extent of CA IX expression was significantly correlated with cyst volume. HIF-1α expression was largely absent in all tissue samples, whereas moderate VEGF expression was present in a subset of cases but without correlation to cyst volume. No p53 mutation was found in any of the analyzed tumors. Conclusions Carbonic anhydrase IX, which is virtually absent in normal brain, is significantly upregulated in craniopharyngiomas and shows a significant association with cyst size. The mechanisms of regulation remain unknown, since neither hypoxia nor p53 appears to play a role. These results indicate that inhibition of CA IX may be a potential target for the adjuvant treatment in patients with cystic craniopharyngiomas.

OBJECTIVE: Chronic pancreatitis (CP) is a predisposing disease for pancreatic carcinoma (PC), however, precise molecular mechanisms of cancer development in the background of CP are ill defined. METHODS: A total of 443 laser-microdissected pancreatic intraepithelial neoplasias (PanINs), acinar-ductal metaplasia (ADM), and normal ducts from 21 patients with CP were analyzed for loss of heterozygosity (LOH) and immunohistochemical protein expression of p53, p16, and DPC4. Pancreatic intraepithelial neoplasias were analyzed for mutations in p53, p16, and Ki-ras genes by ABI sequencing. Aneuploidy was determined by fluorescence in situ hybridization with probes for chromosomes 3, 7, 8, and 17. RESULTS: Loss of heterozygosity rate in PanIN-1 and ADM was between 1.7% (p53) and 5.8% (p16). In PanIN-3, p53 protein overexpression and loss of expression for p16 and DPC4 protein were seen. Heterozygous mutations of p53 and p16 without LOH were found in PanIN-1A and ADM, whereas homozygous mutations were found in PanIN-3. Aneuploidy increased from PanIN-1A to PanIN-3. Ki-ras mutations were discovered first in PanIN-1. CONCLUSIONS: Heterozygous mutations of p53- and p16 genes together with chromosomal instability occur early in CP and are clonally expanded, but final inactivation mostly by LOH happens later in pancreatic carcinogenesis. Determination of aneuploidy in pancreatic juice may be of value for early detection and risk assessment in patients with long-standing CP.

Microsatellite instability (MSI) is a hallmark of hereditary non-polyposis colorectal cancer (HNPCC), but also occurs in about 12%-15% of sporadic colorectal cancer (CRC) where it is a consequence of an epigenetic inactiva- tion of MLH1. High frequency MSI (MSI-H; i.e. at least two of five specified microsatellite markers show instability) was shown in a large me- ta-analysis and in recent trials to be a positive prognostic marker for overall survival in CRC. MSI-H or mismatch repair deficiency (MMRD) was also shown to be a marker for ineffectiveness of adjuvant 5-fluorouracil (5- FU) based chemotherapy in CRC. At present, there are no guidelines defining the need for microsatellite analysis before chemothera- py. However, studies published to date pro- vide data suggesting that MSI-H CRC patients should not receive adjuvant chemotherapy, with the exception of patients with other fac- tors or poor prognosis (e.g. T4 tumors, G3/G4 status, blood or lymphatic vessel invasion). MSI or MMRD testing can contribute to a more individualized therapy of CRC and should be performed prior to planned 5-FU monotherapy or adjuvant chemotherapy in a non-metastatic setting.

Promoter hypermethylation occurs in various tumors and leads to silencing of tumor-relevant genes. Thus, promoter methylation analysis (MA) has been established as an important tool in cancer research and diagnostics. Here we present MethyQESD (methylation-quantification of endonuclease-resistant DNA) as a fast, easy, precise and reliable method for quantitative MA without the need of bisulfite-treatment or fluorescent probes. Though MethyQESD principally works with any gene promoter we established MethyQESD for the mismatch repair gene MLH1 and tested its utility to differentiate between sporadic microsatellite unstable (MSI-H) colorectal cancer and hereditary nonpolyposis colorectal cancer (HNPCC) by quantitative MLH1 MA. We investigated formalin-fixed and paraffin-embedded tissue samples from a previously published, well-characterized tumor collective comprising 25 HNPCC, 14 sporadic MSI-H CRC and 16 sporadic microsatellite stable (MSS) CRC. We found a high accuracy of MethyQESD by spiking experiments with dilution series of methylated (SW48 cancer cell line) and unmethylated (blood) DNA (Pearson's r=0.9997 (proximal MLH1 promoter region), r=0.9976 (distal MLH1 promoter region)). MethyQESD and conventional quantitative MA using of 96 formalin-fixed and paraffin-embedded CRC showed a high degree of concordance of both methods (Pearson's r=0.885). HNPCC tumors showed either null MLH1 methylation or a significantly lower degree of MLH1 methylation than sporadic MSI-H CRC (P

APC, a tumor suppressor gene in the Wnt pathway, stabilizes beta-catenin and controls cell growth. Mutation of APC or beta-catenin leads to nuclear accumulation of beta-catenin and transcription of cyclin D1/cyclin A. Pulmonary artery sarcoma (PAS) were studied by morphologic, immunohistochemical, and molecular genetic methods of the Wnt pathway. Eighteen cases were included: mean age 52 years, primary intraluminal location with typical clinical presentation. PAS were classified as epithelioid (n = 4) or malignant fibrous histiocytoma (MFH; spindled/pleomorphic, n = 4), myxofibrosarcoma (n = 8), and one each hemangiopericytoma-like or malignant inflammatory myofibroblastic tumor-like. The tumor cells demonstrated vimentin, focal actins, and rare focal desmin positivity. All but one were grade 2 or 3 by FNCLCC grading. Alteration in chromosome 5q21 (APC) was found in 4/14 PAS by LOH, mostly epithelioid-type; an MFH-type case demonstrated microsatellite instability (MSI) and nuclear beta-catenin. Cyclin D1 was expressed in seven tumors, all myxofibrosarcoma-type. No mutations were detected in APC or beta-catenin. In summary, PAS are predominantly intermediate grade myxofibrosarcoma in middle-aged males, and fatal in two-thirds of patients. Despite myofibroblastic phenotype, APC/beta-catenin pathway changes are rare. Cyclin D1, only expressed in the myxofibrosarcoma-type, is likely transcribed via factors other than beta-catenin.

Primary and metastatic so-called malignant fibrous histiocytoma/undifferentiated high-grade pleomorphic sarcoma (MFH) is rare in the gastrointestinal (GI) tract with approximately 50 primary and five metastatic cases reported so far. We evaluated two primary gastric and three metastatic intestinal high-grade pleomorphic sarcomas with features of storiform-pleomorphic MFH. Gastric tumours occurred in a 79-year-old man and a 68-year-old woman. One patient died post-operatively, and the other was disease-free at 6 months. Three patients presented with GI metastasis 24, 60 and 0 months after diagnosis of MFH of the heart (n = 1) and the thigh (n = 2). Metastases were located in the small (n = 1) and large bowel (n = 2) and were characteristically pedunculated and polypoid with oedematous haemorrhagic stroma. Concurrent metastases (brain, lung, bone) were present in all three cases. Tumours expressed alpha-smooth muscle actin (four of five), platelet-derived growth factor receptor (PDGFR) alpha (three of three) and PDGFRbeta (two of three) but were negative for CD117, CD34 and other lineage-specific markers. Ultrastructural examination revealed myo/fibroblastic features. Both gastric MFH were wild type for KIT and PDGFRalpha. In conclusion, primary and metastatic MFH of the GI tract commonly express PDGFRalpha and show a myo/fibroblastic phenotype. They should be distinguished from a variety of primary and metastatic pleomorphic neoplasms, in particular high-grade sarcomatous GI stromal tumours (GIST), pleomorphic leiomyosarcoma, sarcomatoid carcinoma and other mimics.

Low-grade myofibroblastic sarcoma (LGMS) represents a rare soft tissue neoplasm with a predilection for the head and neck. Intra-abdominal LGMS are rare with only four unequivocal examples reported so far. Two further cases in females in their 60s and 70s are analysed here.Immunohistochemical stains were applied on fresh-cut sections using the avidin-biotin complex method and the following antibodies: vimentin, alpha-SMA, desmin, h-caldesmon, S-100, CD117, CD34, fibronectin, HMB45, Pan-keratin, Ki-67, beta-catenin, MDM2, PDGFRalpha, PDGFRbeta and ALK-1. Genomic DNA was isolated from microdissected formalin-fixed paraffin-embedded tumour tissue and examined for KIT and PDGFRA mutations by PCR and direct sequencing of KIT and PDGFRA. Ultrastructural studies were also performed.The tumours arose in the mesentery and the pelvic peritoneum. Both revealed features intermediate between conventional fibrosarcoma and leiomyosarcoma with fascicles of spindled, stellated or plump cells possessing fusiform indented vesicular nuclei and pale eosinophilic cytoplasm. Mitotic activity ranged from 1 to 15 per 10 HPFs. The tumour cells strongly expressed vimentin, variably alpha-smooth muscle actin and fibronectin, but were negative for CD117, S-100, desmin, h-caldesmon, beta-catenin, ALK-1, MDM2, PDGFRalpha and PDGFRbeta. One tumour showed a weak expression of CD34. Molecular analysis revealed a wild-type KIT, exons 9, 11 and 13, and PDGFRA, exons 12 and 18. The patients developed multiple peritoneal recurrences at 5, 13 and 25 months, and 10, 19, 25 and 32 months, and were alive at 25 and 32 months, respectively. Distant metastases were not detected.Abdominopelvic LGMS follows a more aggressive clinical course characterised by a higher propensity for local recurrence, contrasting their more superficially located counterparts. LGMS may mimic a variety of benign and low-grade malignant neoplasms and might be under-recognised.

Summary Deregulation of protease expression and activity is known to play an important role in tumour progression of malignant melanoma. The serpin maspin, a tumour suppressor in breast and prostate cancer was described as an inhibitor of cell migration and inducer of cell adhesion between the basement membrane and extracellular matrix resulting in inhibition of tumour metastasis. In contrast, overexpression of maspin is correlated with poor prognosis in other cancers. However, little is known about expression, regulation and function of maspin in malignant melanoma. In this study, we found loss of maspin expression in malignant melanoma cells compared with normal human epidermal melanocytes, which was analysed by quantitative real-time PCR, Western blot analysis, immunohistochemistry and microarray. For functional studies, melanoma cell clones stably transfected with a maspin expression vector were tested for changes in proliferation, migration and invasion. Although we could not see differences in proliferation and migration, we detected strongly reduced invasive capacity in the melanoma cell clones in which maspin is re-expressed compared with control. Reduced invasive potential was also detected in three different melanoma cell lines transiently transfected with a maspin expression vector. Furthermore, exogenously added maspin alone was sufficient to reduce invasion in MelIm significantly, indicating that maspin directly inhibits invasion on the cell surface. In summary, we believe that maspin is a tumour suppressor in malignant melanoma.

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.

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.

In hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, more than 90% of the carcinomas show microsatellite instability (MSI) due to a loss of mismatch repair (MMR) function. Although adenomas are very common in HNPCC and demonstrate an accelerated adenoma-carcinoma sequence, data about the prevalence and development of MSI in these early neoplastic lesions are lacking. To determine whether MSI and loss of MMR-protein expression are already present in early stages of tumorigenesis and could therefore be used as a screening tool to identify HNPCC patients before they develop an invasive carcinoma, we analyzed 71 adenomas of 36 HNPCC patients during a 5-year follow-up study. These 36 patients were part of a cohort of 122 HNPCC patients who were investigated at the Institute of Pathology, Klinikum Kassel, as part of the multicentric German HNPCC Consortium, which currently serves more than 2,880 registered families. The diagnosis of HNPCC was based either on the detection of a pathogenic germline mutation in the MSH2, MLH1, or MSH6 genes or in cases where a pathogenic mutation was not found; diagnosis of HNPCC was made, because all patients fulfilled the Amsterdam or Bethesda criteria and revealed a high degree of MSI (MSI-H) as well as loss of one of the MMR proteins by IHC in the cancer tissue. We found that most adenomas (58/71) were MSI-H and had loss of MMR-protein expression. Of the 71 adenomas, 3 were MSI-H with expression of all MMR proteins, and 3 out of 71 displayed loss of a MMR protein with the microsatellites being classified as microsatellite stable (MSS). However, 7 of the 31 adenomas that were located more than 5 cm away from the carcinoma revealed an MSS status (n=6) or low in MSI (n=1) and expressed all MMR proteins. In summary, a significant percentage of HNPCC-associated adenomas (7/31, 22.6%) developing at a distance of more than 5 cm from the corresponding carcinoma did not show the MSI-H MMR-deficient phenotype and expressed all MMR genes. To our knowledge, this is the first study that shows that in most HNPCC patients, the mutator pathway is already detectable in adenomas, but MMR-proficient adenomas can also be found. Therefore, screening for MMR deficiency should not be applied routinely in adenomas with the goal to identify HNPCC patients.

Nephrogenic metaplasia or nephrogenic adenoma of the urinary tract may present a diagnostic challenge in surgical pathology practice. Previous case reports suggest the possibility of nephrogenic metaplasia progressing to clear cell adenocarcinoma, but a malignant potential of nephrogenic metaplasia is generally not acknowledged. A case of a 70-year-old female patient with multiple recurrences of nephrogenic metaplasia of the urinary bladder and subsequent development of clear cell adenocarcinoma is described. Immunohistochemical studies help to differentiate the 2 entities. Results of molecular studies, particularly comparative genomic hybridization analysis, suggest clonal evolution of nephrogenic metaplasia to clear cell adenocarcinoma in this case.

Pleural effusions may result from various inflammatory, hemodynamic, or neoplastic conditions. A common diagnostic problem lies in distinguishing malignant from benign pleural effusions using routine cytological evaluation. We studied pleural fluid samples obtained from 14 patients with histologically confirmed malignancy and from 6 patients with benign pleural effusions using 12 microsatellite markers from 8 different chromosomal regions. Supernatants and cellular sediments of all 20 pleural fluid samples were analyzed. Routine cytological examination was 100% specific for malignancy but was only 57% sensitive. Microsatellite analyses of pleural fluid supernatants showed genetic alterations in tumor patients only. However, 50% of pleural effusions that were considered negative for malignancy by routine cytological analysis showed either loss of heterozygosity or microsatellite instability. The sensitivity of pleural fluid examination rose to 79% when routine cytological assessment was supplemented by molecular studies. Our data suggest that microsatellite analysis increases the sensitivity of cytological pleural fluid examination in assessing potential malignancy and that combining cytological and molecular methods may improve yield and certainty in diagnostically challenging cases.

Summary Background RNASEL is thought to be a susceptibility gene for hereditary prostate cancer and encodes the endoribonuclease RNase L, which has a role in apoptosis and is a candidate tumour-suppressor protein. A common sequence variation in RNASEL , Arg462Gln, has been associated with hereditary and sporadic prostate cancer, and the Gln variant has about three-fold reduced RNase activity in vitro. In view of the association between the age of onset of hereditary non-polyposis colorectal cancer and functionally different variants of P53 , which play a key part in the apoptotic pathway, we aimed to assess whether the Arg462Gln variation of RNASEL affects the age of onset of hereditary non-polyposis colorectal cancer. Methods We screened 251 patients with hereditary non-polyposis colorectal cancer who were unrelated, had pathogenic germline mutations in MSH2 (n=141) or MLH1 (n=110), and had colorectal carcinoma as the first tumour, for variation at codon 462 of RNASEL and compared them with 439 healthy controls. Findings The median age of onset was 40 years (range 17–75) for patients with an Arg/Arg genotype at codon 462, 37 years (13–69) for patients with an Arg/Gln genotype, and 34 years (20–49) for those with a Gln/Gln genotype (p=0·0198). Only the RNASEL genotype had a significant effect on age of onset (p=0·0062) in an additive mode of inheritance. Pair-wise comparisons between genotype groups showed that the two homozygous groups (ie, Arg/Arg vs Gln/Gln) differed significantly in age of disease onset (mean age difference 4·8 years [SD 1·7], p=0·0044). Interpretation A sequence variation in the prostate-cancer-susceptibility gene RNASEL has a role in a different, unassociated malignant disease. Genotypes at RNASEL codon 462 are associated with age of onset of hereditary non-polyposis colorectal cancer in a dose-dependent way, and might have a role in preventive strategies for this disease.

Methylation of promoter regions and frameshift mutations in microsatellites of the coding sequence (CDS) of genes are frequently associated with loss of expression in microsatellite instable (MSI) colorectal carcinoma. In a panel of 40 MSI and 24 microsatellite stable (MSS) colorectal tumours as well as six cultured colorectal carcinoma cell lines hypermethylation of the TIMP3-promoter was found in 28% of MSI and 25% of MSS tumours, respectively. Additionally, three MSI tumours and one cell line displayed instability of a C7-repeat located in the CDS of the TIMP-3 gene. TIMP-3 fulfils all important criteria for being a target gene in the mutator pathway. Thus, TIMP-3 might be a factor of general importance for colorectal carcinogenesis.

Although microsatellite instability (MSI) testing is a useful tool for molecular screening of hereditary nonpolyposis colorectal cancer (HNPCC) carcinomas, conflicting results have been obtained in colorectal adenomas. This might result from different techniques of tissue sampling and MSI analysis. Alternatively, some HNPCC-associated adenomas may follow a molecular route that differs from the MSI pathway. In the present study we examined the MSI status of 18 adenomas from 17 HNPCC patients by comparing manual adenoma dissection under gross visual control with laser microdissection of single adenoma crypts. After manual gross dissection, 50% (9 of 18) and 11.1% (2 of 18) of the adenomas displayed high-level (MSI-H) and low-level (MSI-L) MSI, respectively. The same set of adenomas split into 83.3% (15 of 18) MSI-H and 5.6% (1 of 18) MSI-L after laser microdissection. The expression pattern of mismatch repair (MMR) proteins showed a higher concordance rate with the MSI status in laser-dissected (94%) than gross-dissected (47%) adenomas. Whereas two adenomas remained microsatellite stable (MSS) and MMR proficient even after laser-assisted dissection, two MSI-H cases showed either rare instabilities at coding microsatellites or intratumoral heterogeneity of MSI with and without MSH2 expression. This suggests that in some adenomas development of MMR dysfunction occurs stepwise with MSI, arising before complete loss of MMR gene expression, whereas other HNPCC-associated adenomas might develop independently of MMR deficiency.

Maspin, a member of the serpin family, has been reported to suppress metastasis and angiogenesis in breast and prostate cancers. Overexpression of maspin was associated with adverse prognostic features in several other tumours. In this study, expression of maspin was analysed in 41 colorectal carcinomas with high-frequency microsatellite instability (MSI-H) and 159 microsatellite stable colorectal cancers (MSS/MSI-L) by immunohistochemistry (IHC) and partly by relative quantitative real-time RT-PCR and western blot analyses. Significant upregulation of maspin expression was found in MSI-H tumours compared to both MSS/MSI-L tumours and matched benign colonic mucosa. Increased maspin expression was also found in three MSI-H colon cancer cell lines, but not in three MSS colon cancer cell lines by RT-PCR and western blot analyses. Regulation of maspin expression depended on promoter methylation as tissue specimens and cell lines expressing maspin showed unmethylated maspin promoters, whereas promoter hypermethylation was found in specimens with loss of maspin expression. Intense nuclear maspin immunostaining was seen specifically in MSI-H tumours (p = 0.013), de-differentiated tumours (p = 0.006), and at the invasion front. These findings provide new insights into the role of maspin in colorectal cancer progression and may be useful for diagnosis and treatment strategies.

Clinical criteria, microsatellite analysis (MSA) and immunohistochemistry (IHC) are important diagnostic tools for identification of hereditary nonpolyposis colorectal cancer (HNPCC) patients who are likely to carry pathogenic germline mutations in mismatch repair genes. Based on MSA and IHC results and subsequent mutation analyses of 1,119 unrelated index patients meeting the Amsterdam II criteria or the classical Bethesda guidelines, we analyzed the value of these tools to predict MLH1 and MSH2 mutations with the aim of establishing optimal strategies for their most efficient sequential use. The overall prevalence of pathogenic germline mutations in our cohort was 20.6% (95% CI = 18.3-23.0%) and 61.8% (95% CI = 56.8-66.6%), respectively, after MSA/IHC-based preselection. IHC was highly predictive (99.1%) and specific (99.6%) with regard to MSA. However, 14 out of 230 mutations (6%) escaped detection by IHC. Thus, IHC cannot be recommended to substitute MSA fully. Nonetheless, IHC is important to indicate the gene that is likely to be affected. To combine both methods efficiently, we propose a novel screening strategy that provides 2 alternative ways of sequential IHC and MSA application, either using IHC or MSA in the first place. A logistic regression model based on the age of the index patient at first tumor diagnosis and the number of fulfilled HNPCC criteria is used to allocate individual patients to that alternative pathway that is expected to be least expensive. A cost analysis reveals that about 25% of the costs can be saved using this strategy.

Methylthioadenosine phosphorylase (MTAP) is known as a ubiquitously expressed house keeping gene important in biochemical salvage processes. The MTAP gene is localized on the human chromosomal region 9p21, a region often deleted in cancer. Recently, several groups including our own have shown that MTAP serves as a tumour suppressor gene. The aim of this study was to analyse the role of MTAP in colon carcinoma and normal colon epithelium and the regulation of gene expression. To examine MTAP RNA and protein expression, we screened six colon carcinoma cell lines and human primary colon epithelial cells by RT-PCR and immunoblotting. MTAP expression was confirmed in vivo by immunohistochemical staining of normal colon tissue compared to adenoma and colon carcinoma. Interestingly, we found strong MTAP mRNA and protein expression by colon carcinoma cell lines but no expression by colonic epithelial cells. To analyse the regulation of MTAP expression, promoter studies were performed and revealed control of MTAP expression by LEF/TCF/beta-catenin. Furthermore, we demonstrated a significant correlation between MTAP protein expression and tumour progression as the intensity of MTAP protein staining increased from normal tissue to carcinoma. In addition, the recently postulated association between MTAP activity and interferon (IFN) sensitivity was confirmed in colon epithelial cells showing only little response to IFN-gamma, in contrast to the carcinoma cell lines. In summary, these data indicate for the first time that MTAP is not expressed in normal human colonic epithelium but is strongly upregulated in colon carcinoma. This finding may be of clinical significance concerning the homeostasis of normal colon epithelium and potential treatment of colon carcinoma.

Microsatellite instability at mono- and dinucleotide repeats is the hallmark of the hereditary non-polyposis cancer syndrome (HNPCC) and is related to deficient DNA mismatch repair. In contrast, a distinct form of microsatellite instability at selective tetranucleotide repeats (EMAST or elevated microsatellite alterations at selected tetranucleotides) was described in several non-HNPCC cancer types. EMAST is probably unrelated to mismatch repair defects. We investigated the frequency of microsatellite instability at mononucleotide, dinucleotide and tetranucleotide repeats in a series of 75 ovarian carcinomas (53 serous and 22 non-serous). Microsatellite analysis was carried out using 5 mono- and dinucleotide markers from the National Cancer Institute Consensus Panel and 6 tetranucleotide markers, which have been reported as frequently unstable in the literature. High frequency of microsatellite instability (MSI-H) at mono- and dinucleotide repeats was observed in 9% and a low frequency (MSI-L) in 21% of serous carcinomas. MSI-H was detected in 4% and MSI-L in 18% of non-serous carcinomas. Nine percent of serous carcinomas showed instability at multiple and 9% at single tetranucleotide loci. All non-serous carcinomas were stable at tetranucleotide loci. In summary, EMAST (e.g., tumors with tetranucleotide instability without concomitant MSI-H) was observed in 13% of ovarian serous carcinomas. All EMAST positive tumors were of advanced stage. We conclude that EMAST occurs as a distinct form of microsatellite instability in ovarian cancer. EMAST seems to be particularly frequent in advanced serous carcinomas. Its clinical significance needs to be investigated.

Gene expression profiling of matched colorectal carcinomas and metastases could reveal key molecular events involved in tumour progression and metastasis. Expression profiles have been created from 25 colorectal carcinomas (CRCs, pT1-4), corresponding normal colonic mucosa, and 14 liver metastases using cDNA arrays containing 1176 cancer-related genes (Clontech). Hierarchical clustering clearly distinguished carcinomas from non-cancerous tissues, separated tumours into high-stage (pT4 and extensive lymph node or distant metastases) and low-stage (< or =pT3) groups, and correlated with the histopathological classification in 87% (33/38 cases). Most primary tumours and matched liver metastases clustered on terminal branches of the dendrogram. Statistical analysis (Mann-Whitney U-test) revealed 40 tumour-specific genes (29 up-regulated, 11 down-regulated) which allowed identification of malignant tissue samples by cluster analysis. A specific expression signature in matching metastases was not found, but a set of 23 classifier genes with statistically significant expression patterns in high- and low-stage tumours was identified. These genes may represent important targets in colorectal carcinogenesis and might provide useful clinicopathological tools in the management of colorectal cancer.

BACKGROUND 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. METHODS Twenty patients were included in each trial. Progression-free survival at 6 months (PFS-6) and toxicity were the primary endpoints. Expression of the tumor resistance proteins multidrug resistance protein 1 (MDR-1) and multiple resistance protein (MRP) was evaluated by immunohistochemical methods and by sestamibi–single-photon emission computed tomography (SPECT). RESULTS The overall response rate (including cases of disease stabilization) was 40% in both Trial 1 and Trial 2. PFS-6 was 15%, and the median time to disease progression was 17 weeks. It is noteworthy that 40% of patients with Grade III tumors had long-term responses, which lasted for up to 3 years. There was no significant difference between Trial 1 and Trial 2 in terms of efficacy. Both regimens were well tolerated, with the main side effect being palmoplantar erythrodysesthesia. The authors found no correlation between clinical response and expression of tumor resistance genes or between clinical response and SPECT data. CONCLUSIONS Pegylated liposomal doxorubicin administered alone or in combination with tamoxifen is safe and moderately effective in patients with recurrent high-grade glioma. None of the putative predictors for response that were evaluated proved to be significant in this setting. Cancer 2004. © 2004 American Cancer Society.

Aims: To evaluate the usefulness of molecular markers in predicting histopathological and clinical response to preoperative high dose chemotherapy (HDCT) and survival of patients with advanced gastric cancer. Methods: In a phase II trial, 25 patients with metastatic gastric cancer received preoperative tandem HDCT consisting of etoposide, cisplatin, and mitomycin, followed by autologous bone marrow transplantation to achieve surgical resectability. Samples before and after treatment, from normal and tumour tissue, were characterised histopathologically, and both p53 and BAX expression was analysed by immunohistochemistry. Pretreatment formalin fixed, paraffin wax embedded samples from normal and tumour tissue were microdissected, and the extracted DNA was preamplified using improved primer extension preamplification polymerase chain reaction. Detection of microsatellite instability (MSI) or loss of heterozygosity (LOH) was performed using markers for p53, BAX, BAT25, BAT26, D2S123, D17S250, and APC. Exons 5–9 of the p53 gene were sequenced directly on ABI 373. Results: Four parameters were significantly associated with response to chemotherapy and prolonged overall survival: positive p53 immunostaining, positive p53 mutation status before chemotherapy, strong histological regression induced by preoperative HDCT, and surgical treatment. Patients’s sex or age, tumour location or stage, lymph node status, Lauren classification, MSI, or LOH did not influence duration of survival significantly in this high risk population. Conclusion: Positive p53 immunostaining and p53 mutation status in pretreatment tumour biopsies might be useful molecular predictors of response and prognosis in patients with advanced gastric cancer treated by preoperative HDCT.

Urothelial carcinoma of the renal pelvis and ureter may develop as a manifestation of hereditary nonpolyposis colorectal cancer syndrome (HNPCC), a disorder characterized by mutation or inactivation of a number of DNA mismatch repair genes and detectable as microsatellite instability (MSI). Some urothelial carcinomas display areas of endophytic, or inverted, growth. In this study, urothelial cancers of the upper urinary tract (n = 132) from patients treated at 2 tertiary care centers were studied to identify an association between growth pattern and MSI. Thirty-five neoplasms were microsatellite unstable (26.5%), and MSI was more frequent in papillary lesions than in sessile urothelial cancers (P = .033). The amount of inverted growth was estimated as a percentage of the total tumor. The interobserver and intraobserver concordance in recognizing inverted growth was good, and 65.7% of microsatellite-unstable tumors exhibited at least 20% of an inverted growth component, compared with only 17.5% of microsatellite-stable tumors (P < .0001). In this series, inverted growth predicted MSI with a sensitivity and specificity of .82. Inverted growth in urothelial carcinomas of the upper urinary tract may serve as a marker lesion for MSI and may help identify patients who should be offered testing for HNPCC.

Urothelial carcinoma of the renal pelvis and ureter may develop as a manifestation of the hereditary nonpolyposis colorectal cancer syndrome that is characterized by mutations in a number of DNA mismatch repair genes and detectable as microsatellite instability. In this study, we examined microsatellite instability and the clinicopathologic features of urothelial carcinoma of the renal pelvis (n = 61) and ureter (n = 53) from 114 consecutive patients surgically treated from 1985-1992. Clinical data were obtained through chart review. Matched normal and tumor DNA was extracted from paraffin-embedded tissue, and a panel of six microsatellite loci was analyzed. The male-female ratio was 2.8:1 with a median age of 70 years (range, 28 to 92 y). Microsatellite analysis was successful in 67 tumors, and 21 (31.3%) patients had tumors that exhibited microsatellite instability. Patients with microsatellite-unstable tumors were significantly more likely to have additional nonurologic cancers (P =.015) including colorectal carcinoma (P =.001) compared with patients with tumors that did not exhibit microsatellite instability. In addition, patients with microsatellite-unstable tumors showed more colorectal cancers in their family (P =.026) and were more likely to have higher grade urothelial carcinoma of the upper tract (P =.028). Grade and stage, but not microsatellite status, were the strongest predictors of cancer-specific survival. This study found the highest frequency of microsatellite instability in upper urothelial tract carcinomas reported to date and highlights upper tract urothelial carcinoma as a marker of the hereditary nonpolyposis colorectal cancer syndrome in some patients. These findings reinforce the importance of obtaining cancer histories in patients with upper tract urothelial carcinoma to subsequently identify individuals with the hereditary nonpolyposis colorectal cancer syndrome and at-risk relatives for surveillance and management programs.

Epidermal growth factor receptor (EGFR) is highly expressed in squamous cell carcinoma (SCC). The response of patients with lung cancer to EGFR inhibitors is significantly associated with the presence of EGFR mutations. Although these drugs have already been used for the treatment of advanced cutaneous SCC, the knowledge about EGFR mutations in this cancer is limited to one previous study in the US population. We analysed the presence of EGFR and concomitant HRAS mutations in a German cohort of 31 patients with cutaneous SCC by direct sequencing of EGFR and SNaPshot analysis of concomitant RAS mutations. We found a low prevalence of EGFR mutations (1/31; 3%) and HRAS mutations (1/31; 3%). The detected P741L EGFR mutation was proven to be somatic. Our results indicate that both EGFR and HRAS mutations are rare events in the carcinogenesis of cutaneous SCC, and therefore, only a small subgroup of patients will benefit from the screening for EGFR mutations in the run-up to targeted therapies with EGFR inhibitors.

Today, molecular diagnostic tests are widely used in clinical medicine with polymerase chain reaction (PCR)-based techniques being of particular interest. In tissue specimens, however, false-positive and false-negative results can be obtained if pathomorphological and processing aspects are not considered. We therefore studied the impact of tissue sampling in three widely used diagnostic tests: (1) assessment of clonality in B-cell non-Hodgkin's lymphoma, (2) analysis of microsatellite instability (MSI) in colorectal neoplasia, and (3) demonstration of mycobacterium tuberculosis. Tissue sections of routinely formalin-fixed and paraffin-embedded diagnostic specimens were taken, and the significance of sampling was systematically investigated using laser microdissection or processing of the entire section. PCR analyses were done according to standard protocols. False-positive pseudo-monoclonality was obtained in small gastrointestinal biopsies and in laser microdissected lymph follicles of non-neoplastic tonsils. False negativity (pseudo-stability) could be demonstrated in a case with hereditary rectal adenoma if whole tissue sections were taken without microdissection of the most dysplastic subareas. Demonstration of mycobacteria failed in tissue sections of a formalin-fixed lymph node that was positive after complete digestion or in fresh frozen material of the same patient. In diagnostic molecular pathology, sampling error is an important source of false-positive and false-negative results, particularly if disease- and tissue-specific morphological features, such as sample size, type of fixation, and intralesional heterogeneity, are ignored.

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 occurred 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 occurrence and recurrence of urothelial carcinomas.

Multifocality and recurrence are clinically important features of urothelial carcinomas of the urinary bladder. Recent molecular genetic studies have suggested that multifocal urothelial carcinomas are monoclonally derived from an identical transformed progenitor cell. However, most of these studies investigated advanced and poorly differentiated tumors. The study presented focuses on early papillary tumors, including 52 superficial well-differentiated multifocal and recurrent bladder carcinomas from 10 patients. Microdissection separating urothelium from stromal cells was considered essential to obtain pure tumor cell populations. Genetic analysis was carried out by applying two different methods. Dual color fluorescence in situ hybridization (FISH) with centromeric probes for chromosomes 9 and 17 and gene-specific probes for chromosome loci 9q22, 9p21, and 17p13 was carried out in parallel to loss of heterozygosity (LOH) analyses applying 5 microsatellite markers on these chromosomes. Overall, deletions on chromosome 9p were found in 47 tumors (90%), at chromosome 9q in 36 tumors (69%) and at chromosome 17p in 3 tumors (6%). There was a very high correlation of the results between FISH and LOH analysis. Ten early superficial papillary tumors showed deletion of chromosome 9p without deletion of 9q, suggesting 9p deletions as a very early event in the development of papillary urothelial carcinoma. Although in four patients, all investigated tumors showed identical genetic alterations and one patient showed no genetic alterations at the loci investigated, in five patients, two or more clones with different deletions were found. In four of these patients, the results are compatible with clonal divergence and selection of different cell subpopulations derived from a common progenitor cell. However, in one patient different alleles in two markers at chromosome 9 were deleted, favoring an independent evolution of two recurring tumor cell clones. In summary, we could show that there is considerable genetic heterogeneity in early multifocal and recurring urothelial carcinoma and demonstrated the occurrence of two independent clones in at least one patient as an indicator of possible initial oligoclonality of bladder cancer.

Introduction: Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy. Although the clinical course is usually rather benign, a subset of tumors is more aggressive. The ret/PTC oncogene was found only in PTC, with varying frequencies of up to 30%. Recently, two new variants of ret/PTC could be identified in post-Chernobyl PTCs, which raised the possibility that the prevalence of ret/PTC in non-radiation-induced PTCs might be higher than previously described. Normal thyroid cells do not express wild-type ret, but there is evidence that ret activation from any cause, including wild-type ret, occurs in more than a half of papillary tumors. Methods: We used reverse transcription polymerase chain reaction and sequencing to examine wild-type ret and all five forms of ret/PTC known today in 99 PTCs from Hannover, Dusseldorf, Halle and Regensburg. Our method could also detect other variants within the known breakpoint regions. The presence of the ret tyrosine-kinase domain was examined by immunohistochemistry. Results: Seven PTC1-positive tumors and one PTC3-positive tumor (8%), but none with the new variants or other variants of PTC1, 2 or 3 could be detected. Of 43 tumors examined, 20 showed expression of wild-type ret mRNA and staining of ret protein located predominantly to the cell membrane. Conclusion: Variants of ret/PTC do not substantially contribute to non-radiation-related ret/PTC-positive tumors, and the prevalence of ret/PTC in Germany is low in contrast to the high rate of wild-type ret expression. Thus, expression of wild-type ret should be examined for pathogenic, prognostic and possible therapeutic implications.

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 3%) 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.

Seborrheic keratosis (SK) represents a frequent epidermal skin tumor. Although lacking a malignant potential, these tumors reveal multiple oncogenic mutations. A previous study identified activating mutations in 89% of SK, particularly in FGFR3 and PIK3CA genes. The aim of this study was to identify further oncogenic mutations in human SK. Therefore, we screened for mutations in EGFR, FGFR2, PIK3R1, HRAS, KRAS, and NRAS genes using both Sanger sequencing of selected exons and a multiplex SNaPshot assay in 58 SK of 14 patients. We identified a somatic EGFR p.L858R mutation in 1 SK. Furthermore, the HRAS mutations p.G13R (2/58 SK) and p.Q61L (2/58 SK) were found. These mutations have not been described in human SK yet. In addition, 1 SK revealed the KRAS p.G12V mutation, which has already been reported in SK. No mutations were detected in FGFR2, PIK3R1, and NRAS genes. The results of this study suggest that activating mutations of EGFR, HRAS, and KRAS contribute to the pathogenesis of human SK, although at a lower frequency than FGFR3 and PIK3CA mutations. FGFR2, PIK3R1, and NRAS mutations obviously do not have a significant role in the development of SK.

DUSP4 (MKP-2), a member of the mitogen-activated protein kinase phosphatase (MKP) family and potential tumor suppressor, negatively regulates the MAPKs (mitogen-activated protein kinases) ERK, p38 and JNK. MAPKs play a crucial role in cancer development and progression. Previously, using microarray analyses we found a conspicuously frequent overexpression of DUSP4 in colorectal cancer (CRC) with high frequent microsatellite instability (MSI-H) compared to microsatellite stable (MSS) CRC. Here we studied DUSP4 expression on mRNA level in 38 CRC (19 MSI-H and 19 MSS) compared to matched normal tissue as well as in CRC cell lines by RT-qPCR. DUSP4 was overexpressed in all 19 MSI-H tumors and in 14 MSS tumors. Median expression levels in MSI-H tumors were significantly higher than in MSS-tumors (p < 0.001). Consistently, MSI-H CRC cell lines showed 6.8-fold higher DUSP4 mRNA levels than MSS cell lines. DUSP4 expression was not regulated by promoter methylation since no methylation was found by quantitative methylation analysis of DUSP4 promoter in CRC cell lines neither in tumor samples. Furthermore, no DUSP4 mutation was found on genomic DNA level in four CRC cell lines. DUSP4 overexpression in CRC cell lines through DUSP4 transfection caused upregulated expression of MAPK targets CDC25A, CCND1, EGR1, FOS, MYC and CDKN1A in HCT116 as well as downregulation of mismatch repair gene MSH2 in SW480. Furthermore, DUSP4 overexpression led to increased proliferation in CRC cell lines. Our findings suggest that DUSP4 acts as an important regulator of cell growth within the MAPK pathway and causes enhanced cell growth in MSI-H CRC.

Background Activating RAS mutations in the germline cause rare developmental disorders such as Costello syndrome. Somatic RAS mutations are found in approximately 30% of human cancers. Keratinocytic epidermal nevi (KEN) represent benign congenital skin lesions arranged along Blaschko's lines. A subgroup of KEN is caused by hotspot oncogenic FGFR3 and PIK3CA mutations in mosaicism, but the majority lack these mutations. Methods This study screened 72 KEN for activating mutations in RAS genes and other oncogenes. Results Activating RAS mutations were identified in 28/72 (39%) of KEN. HRAS was the most commonly affected oncogene (86%), with the HRAS p.G13R substitution representing a new hotspot mutation. Conclusion These results indicate that activating RAS somatic mutations leading to mosaicism result in benign KEN of the skin. Given the prevalence of KEN, mosaic HRAS mutations appear to be more common in patients than germline ones. These findings identify KEN as a mosaic RASopathy and lend further support to the notion that genetic mosaicism is an important contributor to disease.

Background: Carcinomas of the Vaterian system are rare and presumably arise from pre-existing adenomas. According to the cancer stem cell (CSC) hypothesis, only a small subset of tumor cells has the ability to initiate and develop tumor growth. In colorectal cancer, CD44, CD133, CD166 and EpCAM have been proposed to represent CSC marker proteins and their expression has been shown to correlate with patient survival. Aims: To evaluate a potential role of these CSC proteins in tumors of the ampulla of Vater, we investigated their expression in 175 carcinoma, 111 adenoma and 152 normal mucosa specimens arranged in a Tissue Microarray format. Materials and methods: Membranous immunoreactivity for each protein marker was scored semi-quantitatively by evaluating the number of positive tumor cells over the total number of tumor cells. Median protein expression levels were used as cut-off scores to define protein marker positivity. Clinical data including survival time were obtained by retrospective analysis of medical records, tumor registries or direct contact. Results: The expression of all evaluated marker proteins differed significantly between normal mucosa, adenoma and carcinoma samples. In all markers, we found a tendency towards more constant expression from normal to neoplastic tissue. EpCAM expression was significantly correlated with better patient survival. The increased expression of CD44s, CD166 and CD133 from normal mucosa samples to adenoma and carcinoma was linked to tumor progression. However, there was no statistically significant correlation with survival. Conclusion: Our findings indicate, that in ampullary carcinomas, loss of expression of EpCAM may be linked to a more aggressive tumor phenotype.

The aim of the present prospective study was to assess the diagnostic benefit of UroVysion (VysisAbbott Laboratories, Downers Grove, IL) in the follow-up of patients with a history of high-grade non–muscle-invasive urothelial carcinoma of the bladder (NMIBC). An unselected cohort of 25 patients with a history of high-grade NMIBC was prospectively followed up by office-based cystoscopy, cytology, and UroVysion in 210 events. The sensitivity and specificity for standard combined cystoscopy and cytology were 78% and 83%, respectively. UroVysion yielded a considerably higher detection rate with 94% and 93%, respectively. In 89% of the follow-up events of patients with a history of previous carcinoma in situ (CIS) and negative cystoscopy but a positive UroVysion finding, CIS recurrence was noticed within 5 months. UroVysion is a worthwhile approach in patients with previous CIS, a high risk for the development of CIS, or previous unequivocal cytology suggestive of CIS, especially during or shortly after instillation therapy. According to the 2008 guidelines of the European Association of Urology for non–muscle-invasive urothelial carcinoma of the bladder (NMIBC), the risk of recurrence and progression as determined by clinical and histopathologic parameters should be reflected in follow-up strategies and intervals of cystoscopy and cytology. 1,2 While this approach is an improvement of rather rigid schemes regarded as the “gold standard” for 30 years, it does not pose a true advance. No real progress in follow-up approaches has been established. 3