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Aims The advent of specific ALK-targeting drugs has radically changed the outcome of patients with ALK translocated non-small-cell lung cancer (NSCLC). However, emerging resistance to treatment with ALK inhibitors in these patients remains a major concern. In previous studies, we analysed two ALK+ patient cohorts (TP53 wild-type/TP53 mutated) in terms of copy number alterations. All patients belonging to the TP53 wild-type group had mainly genetically stable genomes, with one exception showing chromosomal instability and amplifications of several gene loci, including TERT. Here, we aimed to determine the prevalence of TERT amplifications in these ALK+ lung cancer patients by analysing an independent cohort of 109 ALK translocated cases. We further analysed the copy numbers of numerous cancer-relevant genes and other genetic aberrations. Methods and results The prevalence of TERT amplifications was determined by means of FISH analyses. Copy numbers of 87 cancer-relevant genes were determined by NanoString nCounter® technology, FoundationOne® and lung-specific NGS panels in some of these TERT-amplified samples, and clinical data on patients with TERT-amplified tumours were collected. Our data revealed that five (4.6%) of all 109 analysed ALK+ patients harboured amplification of TERT and that these patients had genetically unstable genomes. Conclusions Our preliminary study shows that ALK+ adenocarcinomas should be evaluated in the context of their genomic background in order to more clearly understand and predict patients' individual course of disease.

Although non-small-cell lung cancer is a leading cause of cancer-related deaths, the molecular characterization and classification of its genetic alterations has drastically changed treatment options and overall survival within the last few decades. In particular, tyrosine kinase inhibitors targeting specific molecular alterations, among other MET, have greatly improved the prognosis of non-small-cell lung cancer patients. Here, we compare the genomic background of a subset of non-small-cell lung cancer cases harboring either a MET high-level amplification (n = 24) or a MET exon 14 skipping mutation (n = 26), using next-generatison sequencing, fluorescence in situ hybridization, immunohistochemistry, and Nanostring nCounter® technology. We demonstrate that the MET-amplified cohort shows a higher genetic instability, compared with the mutant cohort (p

The anaplastic lymphoma kinase (ALK) rearrangement defines a distinct molecular subtype of non-small cell lung cancer (NSCLC). Despite the excellent initial efficacy of ALK inhibitors in patients with ALK+ lung cancer, resistance occurs almost inevitably. To date, there is no reliable biomarker allowing the identification of patients at higher risk of relapse. Here, we analysed a subset of 53 ALK+ tumours with and without TP53 mutation and ALK+ NSCLC cell lines by NanoString nCounter technology. We found that the co-occurrence of early TP53 mutations in ALK+ NSCLC can lead to chromosomal instability: 24% of TP53-mutated patients showed amplifications of known cancer genes such as MYC (14%), CCND1 (10%), TERT (5%), BIRC2 (5%), ORAOV1 (5%), and YAP1 (5%). MYC-overexpressing ALK+ TP53-mutated cells had a proliferative advantage compared to wild-type cells. ChIP-Seq data revealed MYC-binding sites within the promoter region of EML4, and MYC overexpression in ALK+ TP53-mutated cells resulted in an upregulation of EML4-ALK, indicating a potential MYC-dependent resistance mechanism in patients with increased MYC copy number. Our study reveals that ALK+ NSCLC represents a more heterogeneous subgroup of tumours than initially thought, and that TP53 mutations in that particular cancer type define a subset of tumours that harbour chromosomal instability, leading to the co-occurrence of pathogenic aberrations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Atypical fibroxanthomas (AFX) and pleomorphic dermal sarcomas (PDS) are frequent cutaneous sarcomas typically arising on sun-exposed skin in elderly patients. In contrast to AFX, which generally do not recur after complete excision, PDS locally recur in up to 50% and metastasize in up to 20%. We recently detected characteristic UV-induced TP53 mutations as potential driver mutation in almost all PDS investigated as well as activating PIK3CA and RAS gene mutations in around one third of our tumors representing targets for personalized treatments in patients with unresectable or metastasized PDS. In the present study, we identified amplifications and deletions in a small part of the PDS (6 of 27 cases) but not in AFX suggesting that copy number variations (CNV) might not be an initial event in tumor development but rather important during tumor progression. In addition to BRAF, KNSTRN, IDH1 and PDGFRA amplification, CNV analyses revealed deletions in the CDKN2A, KIT and PDGFRA genes. In cases where an appropriate FISH assay was established, the CNV results could be verified by FISH analysis. Amplification of BRAF, KIT or PDGFRA and/or losses of CDKN2A might represent bad prognostic markers, although larger studies are needed to clarify their association with prognosis or progression in PDS.

The anaplastic lymphoma kinase (ALK) rearrangement defines a distinct molecular subtype of non‐small cell lung cancer (NSCLC). Despite the excellent initial efficacy of ALK inhibitors in patients with ALK+ lung cancer, resistance occurs almost inevitably. To date, there is no reliable biomarker allowing the identification of patients at higher risk of relapse. Here, we analysed a subset of 53 ALK+ tumours with and without TP53 mutation and ALK+ NSCLC cell lines by NanoString nCounter technology. We found that the co‐occurrence of early TP53 mutations in ALK+ NSCLC can lead to chromosomal instability: 24% of TP53‐mutated patients showed amplifications of known cancer genes such as MYC (14%), CCND1 (10%), TERT (5%), BIRC2 (5%), ORAOV1 (5%), and YAP1 (5%). MYC‐overexpressing ALK+ TP53‐mutated cells had a proliferative advantage compared to wild‐type cells. ChIP‐Seq data revealed MYC‐binding sites within the promoter region of EML4, and MYC overexpression in ALK+ TP53‐mutated cells resulted in an upregulation of EML4–ALK, indicating a potential MYC‐dependent resistance mechanism in patients with increased MYC copy number. Our study reveals that ALK+ NSCLC represents a more heterogeneous subgroup of tumours than initially thought, and that TP53 mutations in that particular cancer type define a subset of tumours that harbour chromosomal instability, leading to the co‐occurrence of pathogenic aberrations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Purpose: MET is a potential therapeutic target in lung cancer and both MET tyrosine kinase inhibitors and monoclonal antibodies have entered clinical trials. MET signaling can be activated by various mechanisms, including gene amplification. In this study, we aimed to investigate MET amplification status in adeno- and squamous cell carcinomas of the lung. We propose clearly defined amplification scores and provide epidemiologic data on MET amplification in lung cancer. Experimental Design: We evaluated the prevalence of increased MET gene copy numbers in 693 treatment-naïve cancers by FISH, defined clear cutoff criteria, and correlated FISH results to MET IHC. Results: Two thirds (67%) of lung cancers do not have gains in MET gene copy numbers, whereas 3% show a clear-cut high-level amplification (MET/centromer7 ratio ≥2.0 or average gene copy number per nucleus ≥6.0 or ≥10% of tumor cells containing ≥15 MET copies). The remaining cases can be subdivided into intermediate- (6%) and low-level gains (24%). Importantly, MET amplifications occur at equal frequencies in squamous and adenocarcinomas without or with EGFR or KRAS mutations. Conclusion: MET amplification is not a mutually exclusive genetic event in therapy-naïve non–small cell lung cancer. Our data suggest that it might be useful to determine MET amplification (i) before EGFR inhibitor treatment to identify possible primary resistance to anti-EGFR treatment, and (ii) to select cases that harbor KRAS mutations additionally to MET amplification and, thus, may not benefit from MET inhibition. Furthermore, our study provides comprehensive epidemiologic data for upcoming trials with various MET inhibitors. Clin Cancer Res; 21(4); 907–15. ©2014 AACR.

Reliable detection of anaplastic lymphoma kinase (ALK) rearrangements is a prerequisite for personalized treatment of lung cancer patients, as ALK rearrangements represent a predictive biomarker for the therapy with specific tyrosine kinase inhibitors. Currently, fluorescent in situ hybridization (FISH) is considered to be the standard method for assessing formalin-fixed and paraffin-embedded tissue for ALK inversions and translocations. However, FISH requires a specialized equipment, the signals fade rapidly and it is difficult to detect overall morphology and tumor heterogeneity. Chromogenic in situ hybridization (CISH) has been successfully introduced as an alternative test for the detection of several genetic aberrations. This study validates a newly developed ALK CISH assay by comparing FISH and CISH signal patterns in lung cancer samples with and without ALK rearrangements. One hundred adenocarcinomas of the lung were included in this study, among them 17 with known ALK rearrangement. FISH and CISH were carried out and evaluated according to the manufacturers' recommendations. For both assays, tumors were considered positive if ≥15% of tumor cells showed either isolated 3' signals or break-apart patterns or a combination of both. A subset of tumors was exemplarily examined by using a novel EML4 (echinoderm microtubule-associated protein-like 4) CISH probe. Red, green and fusion CISH signals were clearcut and different signal patterns were easily recognized. The percentage of aberrant tumor cells was statistically highly correlated (P0.001) between FISH and CISH. On the basis of 86 samples that were evaluable by ALK CISH, we found a 100% sensitivity and 100% specificity of this assay. Furthermore, EML4 rearrangements could be recognized by CISH. CISH is a highly reliable, sensitive and specific method for the detection of ALK gene rearrangements in pulmonary adenocarcinomas. Our results suggest that CISH might serve as a suitable alternative to FISH, which is the current gold standard.

KIT exon 9 mutations in gastrointestinal stromal tumors (GISTs) are highly relevant and have direct therapeutic implications. In this context, we established and validated a fast and sensitive high-resolution melting assay. Analyzing 126 primary and 18 metastatic KIT exon 9-mutated cases from our registry, we demonstrate that the mutational spectrum of exon 9 is broader than previously thought and describe 3 novel mutations. Including these cases and the common p.A502_Y503dup mutation, we provide a comprehensive list of all known KIT exon 9 mutations according to the Human Genome Variation Society nomenclature. Two of the newly described mutations were associated with an aggressive phenotype and tumor progression while being treated with 400 mg imatinib, indicating that also GIST with rare exon 9 mutations could be treated with increased imatinib dosage. On the basis of >1500 GISTs from our registry, we have determined the frequency of KIT exon 9 mutations to be 9.2% among all GISTs and 22.5% among small-bowel cases. We describe for the first time that nearly 20% of exon 9-mutated GIST occur in the stomach or rectum. Furthermore, we provide first evidence that exon 9-mutated GISTs metastasize significantly more often to the peritoneum than to the liver. Performing extensive statistical analyses on data from our registry and from the literature, we demonstrate that KIT exon 9 mutations are neither associated with intermediate-risk/high-risk status nor overrepresented among metastatic lesions. Thus, we conclude that exon 9 mutations per se do not have prognostic relevance.

About 10-15% of gastrointestinal stromal tumors (GISTs) carry wild-type sequences in all hot spots of KIT and platelet-derived growth factor receptor alpha (PDGFRA) (wt-GISTs). These tumors are currently defined by having no mutations in exons 9, 11, 13, and 17 of the KIT gene and exons 12, 14, and 18 of the PDGFRA gene. Until now, the analysis of further exons is not recommended. However, we have previously published a report on a KIT exon 8 germline mutation, which was associated with familial GIST and mastocytosis. We therefore investigated whether KIT exon 8 mutations might also occur in sporadic GIST. We screened a cohort of 145 wt-GISTs from a total of 1351 cases from our registry for somatic mutations in KIT exon 8. Two primary GISTs with an identical exon 8 mutation (p.D419del) were detected, representing 1.4% of all the cases analyzed. Based on all GISTs from our registry, the overall frequency of KIT exon 8 mutations was 0.15%. The first tumor originating in the small bowel of a 53-year-old male patient had mostly a biphasic spindled-epithelioid pattern with a high proliferative activity (14 mitoses/50 HPF) combined with a second low proliferative spindle cell pattern (4/50 HPF). The patient developed multiple peritoneal metastases 29 months later. The second case represented a jejunal GIST in a 67-year old woman who is relapse-free under adjuvant imatinib treatment. We conclude that about 1-2% of GISTs being classified as 'wild type' so far might, in fact, carry KIT mutations in exon 8. Moreover, this mutational subtype was shown to be activating and imatinib sensitive in vitro. We therefore propose that screening for KIT exon 8 mutations should become a routine in the diagnostic work-up of GIST and that patients with an exon 8 mutation and a significant risk for tumor progression should be treated with imatinib.

Aims and methods: Desmoid-type fibromatosis (desmoid) is a fibroblastic tumour that shows locally aggressive growth. Mesenteric desmoid is a rare lesion that shares morphological and biological features with fibromatoses occurring in the abdominal wall or in extraabdominal sites, but differs in terms of gross appearance and clinical presentation. We report on a series of 56 cases of mesenteric desmoids from our consultation files and compare them with cases of non-mesenteric desmoids and retroperitoneal fibrosis. Results: Primary diagnosis of desmoid-type fibromatosis was correct in 42%, and gastrointestinal stromal tumour was a common misdiagnosis. Nuclear expression of β-catenin was detected in 91.6% of all desmoids. Mutational analysis of exon 3 of the β-catenin gene (CTNNB1) revealed that mesenteric desmoids carried mutations significantly more often (51/56, 91.1%) than non-mesenteric tumours (20/28; 71.4%; P = 0.027). p.T41A occurred significantly more frequently in mesenteric fibromatoses (80.4%) than in abdominal wall and extra-abdominal fibromatoses (46.4%; P = 0.002). Two novel mutations (p.S45C and p.D32G) were found. In retroperitoneal fibrosis, mutations and nuclear β-catenin expression were absent. β-Catenin-negative desmoids either carried a CTNNB1 mutation or were associated with Gardner syndrome. Conclusions: Our study provides evidence that some clinical and genetic features of mesenteric desmoids differ from those of non-mesenteric fibromatosis, and corroborates the usefulness of mutational analysis, especially in diagnosing β-catenin-negative mesenteric desmoids.

Huss S, Wardelmann E, Goltz D, Binot E, Hartmann W, Merkelbach-Bruse S, Buttner R & Schildhaus H-U (2012) Histopathology 61, 59–68 Activating PDGFRA mutations in inflammatory fibroid polyps occur in exons 12, 14 and 18 and are associated with tumour localization Aims: Inflammatory fibroid polyps (IFP) are mesenchymal tumours of the gastrointestinal tract. This study was performed to broaden the base of evidence of the pathogenic role of PDGFR mutations in IFP with particular regard to clinicopathological data and mutational patterns among IFP subtypes. Methods and results: Molecular analysis of 38 tumours revealed activating mutations in three different exons of PDGFRA in 25 IFP. For the first time we report two cases with PDGFRA-exon 14 mutations (p.N659K; p.[N659K(+)T665A]). The results of our study and cases reported earlier indicate clearly that there is a localization-specific pattern: exon 12 mutations predominate in the small intestine, while exon 18 mutations occur frequently in the stomach (P

Soft tissue sarcomas are a heterogeneous group of tumors with many different subtypes. In 2014 an estimated 12,020 newly diagnosed cases and 4,740 soft tissue sarcoma related deaths can be expected in the United States. Many soft tissue sarcomas are associated with poor prognosis and therapeutic options are often limited. The evolution of precision medicine has not yet fully reached the clinical treatment of sarcomas since therapeutically tractable genetic changes have not been comprehensively studied so far. We analyzed a total of 484 adult-type malignant mesenchymal tumors by MET fluorescence in situ hybridization and MET and hepatocyte growth factor immunohistochemistry. Eleven different entities were included, among them the most common and clinically relevant subtypes and tumors with specific translocations or complex genetic changes. MET protein expression was observed in 2.6% of the cases, all of which were either undifferentiated pleomorphic sarcomas or angiosarcomas, showing positivity rates of 14% and 17%, respectively. 6% of the tumors showed hepatocyte growth factor overexpression, mainly seen in undifferentiated pleomorphic sarcomas and angiosarcomas, but also in clear cell sarcomas, malignant peripheral nerve sheath tumors, leiomyosarcomas and gastrointestinal stromal tumors. MET and hepatocyte growth factor overexpression were significantly correlated and may suggest an autocrine activation in these tumors. MET FISH amplification and copy number gain were present in 4% of the tumors (15/413). Two samples, both undifferentiated pleomorphic sarcomas, fulfilled the criteria for high level amplification of MET, one undifferentiated pleomorphic sarcoma reached an intermediate level copy number gain, and 12 samples of different subtypes were categorized as low level copy number gains for MET. Our findings indicate that angiosarcomas and undifferentiated pleomorphic sarcomas rather than other frequent adult-type sarcomas should be enrolled in screening programs for clinical trials with MET inhibitors. The screening methods should include both in situ hybridization and immunohistochemistry. peerReviewed

Summary The suitability of the recently described red fluorescent protein dsRED from reef corals for use as a reporter in plant molecular biology was investigated. Based on the clone pDSRED (Clontech), plant expression vectors were constructed for constitutive dsRED expression in the cytosol, the endoplasmic reticulum and the vacuole. Fluorescence microscopy of tobacco BY2 suspension culture cells transiently expressing the plant vectors generated proved that cytosolic expression of the dsRED gives rise to readily detectable levels of red fluorescence, whereas expression in the ER was poor. Vacuolar dsRED expression did not result in any significant fluorescence. dsRED transgenic tobacco SR1 plants were generated to test the sensitivity of dsRED as a reporter in an autofluorescent background, and to identify the possible impact of the introduced fluorescent protein on morphogenesis, plant development and fertility. During the transformation and regeneration phase plants did not show any abnormalities, indicating that dsRED is not interfering with plant development and morphogenesis. Regenerated plants were analysed by PCR, Western blot and fluorescence microscopy for the presence and expression of the transferred genes. The filter sets chosen for fluorescence microscopy proved to be able to block the red chlorophyll fluorescence completely, allowing specific dsRED detection. Best expression levels were obtained with dsRED targeted to the cytosol or chloroplasts. ER-targeted expression of dsRED also gave rise to readily detectable fluorescence levels, whereas vacuolar expression yielded no fluorescence. dsRED transgenic plant lines expressing the protein in the cytosol, ER or chloroplast proved to be fertile. Seed set and germination were normal, except that the seeds and seedlings maintained the red fluorescence phenotype.

The mutational status of KIT and PDGFRA is highly relevant for prognosis and therapy prediction in gastrointestinal stromal tumors (GIST). PDGFRA exon 18 mutations have direct therapeutic implications since it is crucial to distinguish mutations associated with sensitivity to tyrosine kinase inhibitors from those causing primary resistance, eg, the most common exon 18 mutation p.D842V. In response to a growing demand for reliable, faster and more sensitive methods we established and validated a high-resolution melting (HRM) assay for PDGFRA exon 18. A total of 159 GIST samples were comparatively analyzed by HRM and direct Sanger sequencing. We demonstrate that HRM provides highly reliable mutational results with higher sensitivity and shorter time to diagnosis compared to Sanger sequencing. We determined the sensitivity threshold of HRM at 6% of mutated alleles. PDGFRA exon 18 wild-type status and the most common p.D842V resistance mutation (together representing >90% of the cases) can be detected specifically by HRM. Other rare mutations can be pre-screened by HRM and afterwards determined precisely by DNA sequencing. In this way we detected four novel mutations in PDGFRA exon 18, two of which were associated with an aggressive clinical course. Including these new mutations, we provide a comprehensive overview of all 60 currently known subtypes of PDGFRA exon 18 mutations in GIST. Seven of them (accounting for about 75% of all exon 18-mutated GISTs) are reported to be resistant to imatinib. However, there are at least 10 other mutations which are regarded as sensitive to tyrosine kinase inhibitors.

Male breast cancer (MBC) is a rare disease. To date, therapy is mainly based on studies and clinical experiences with breast cancer in women. Only little is known about molecular typing of MBC, particularly with regard to potential biological predictors for adjuvant therapy. In female breast cancer tumors with chromosome 17 centromere (CEP17) duplication, HER2 and/or Topoisomerase II alpha (Topo II-α) gene alterations have been suggested to be associated with poor prognosis and increased sensitivity to anthracycline-containing regimens. In a well characterized cohort of 96 primary invasive MBC, we studied CEP17, HER2 and Topo II-α alterations by fluorescence in-situ hybridization (FISH), and expression of hormone receptors (HR), HER2 and Ki67 by immunohistochemistry to define molecular subtypes. Tumor characteristics and follow-up data were available and correlated with molecular findings. HER2 amplification and Topo II-α amplification/deletion were exceptionally rare in MBC (6.3% and 3.1%, respectively). CEP17 polysomy were found in 9.4% of tumors. HER2, Topo II-α and CEP17 gene alterations were not correlated to patients outcome. 96.9% of our cases were HR positive. Triple negative tumors were found in only 3.1% of the cases. In nodal negative tumors luminal A subtypes were significantly associated with better overall survival. Our results provide evidence for a predominant male breast cancer phenotype, characterized by HR expression and a lack of HER2/Topo II-α alterations and CEP17 duplicates. Therefore, the impact of anthracycline sensitivity linked to HER2/Topo II-α alterations as found in female breast cancer has low clinical significance for this specific male breast cancer phenotype.

We characterized genome alterations in 1255 clinically annotated lung tumors of all histological subgroups to identify genetically defined and clinically relevant subtypes. More than 55% of all cases had at least one oncogenic genome alteration potentially amenable to specific therapeutic intervention, including several personalized treatment approaches that are already in clinical evaluation. Marked differences in the pattern of genomic alterations existed between and within histological subtypes, thus challenging the original histomorphological diagnosis. Immunohistochemical studies confirmed many of these reassigned subtypes. The reassignment eliminated almost all cases of large cell carcinomas, some of which had therapeutically relevant alterations. Prospective testing of our genomics-based diagnostic algorithm in 5145 lung cancer patients enabled a genome-based diagnosis in 3863 (75%) patients, confirmed the feasibility of rational reassignments of large cell lung cancer, and led to improvement in overall survival in patients with EGFR-mutant or ALK-rearranged cancers. Thus, our findings provide support for broad implementation of genome-based diagnosis of lung cancer.

Desmoid-type fibromatosis (desmoid) is a fibroblastic tumour that shows locally aggressive growth. Mesenteric desmoid is a rare lesion that shares morphological and biological features with fibromatoses occurring in the abdominal wall or in extraabdominal sites, but differs in terms of gross appearance and clinical presentation. We report on a series of 56 cases of mesenteric desmoids from our consultation files and compare them with cases of non-mesenteric desmoids and retroperitoneal fibrosis. Primary diagnosis of desmoid-type fibromatosis was correct in 42%, and gastrointestinal stromal tumour was a common misdiagnosis. Nuclear expression of β-catenin was detected in 91.6% of all desmoids. Mutational analysis of exon 3 of the β-catenin gene (CTNNB1) revealed that mesenteric desmoids carried mutations significantly more often (51/56, 91.1%) than non-mesenteric tumours (20/28; 71.4%; P = 0.027). p.T41A occurred significantly more frequently in mesenteric fibromatoses (80.4%) than in abdominal wall and extra-abdominal fibromatoses (46.4%; P = 0.002). Two novel mutations (p.S45C and p.D32G) were found. In retroperitoneal fibrosis, mutations and nuclear β-catenin expression were absent. β-Catenin-negative desmoids either carried a CTNNB1 mutation or were associated with Gardner syndrome.Our study provides evidence that some clinical and genetic features of mesenteric desmoids differ from those of non-mesenteric fibromatosis, and corroborates the usefulness of mutational analysis, especially in diagnosing β-catenin-negative mesenteric desmoids.

We recently reported fibroblast growth factor receptor-type 1 (FGFR1) amplification to be associated with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. This makes FGFR1 a novel target for directed therapy in these tumors. To reproducibly identify patients for clinical studies, we developed a standardized reading and evaluation strategy for FGFR1 fluorescence in-situ hybridization (FISH) and propose evaluation criteria, describe different patterns of low- and high-level amplifications and report on the prevalence of FGFR1 amplifications in pulmonary carcinomas. A total of 420 lung cancer patients including 307 squamous carcinomas, 100 adenocarcinomas of the lung and 13 carcinomas of other types were analyzed for FGFR1 amplification using a dual color FISH. We found heterogeneous and different patterns of gene copy numbers. FGFR1 amplifications were observed in 20% of pulmonary squamous carcinomas but not in adenocarcinomas. High-level amplification (as defined by an FGFR1/centromer 8 (CEN8) ratio ≥2.0, or average number of FGFR1 signals per tumor cell nucleus ≥6, or the percentage of tumor cells containing ≥15 FGFR1 signals or large clusters ≥10%) was detected at a frequency of 16% and low-level amplification (as defined by ≥5 FGFR1 signals in ≥50% of tumor cells) at a frequency of 4%. We conclude that FGFR1 amplification is one of the most frequent therapeutically tractable genetic lesions in pulmonary carcinomas. Standardized reporting of FGFR1 amplification in squamous carcinomas of the lung will become increasingly important to correlate therapeutic responses with FGFR1 inhibitors in clinical studies. Thus, our reading and evaluation strategy might serve as a basis for identifying patients for ongoing and upcoming clinical trials.

Inflammatory fibroid polyps (IFP) are mesenchymal tumours of the gastrointestinal tract. This study was performed to broaden the base of evidence of the pathogenic role of PDGFR mutations in IFP with particular regard to clinicopathological data and mutational patterns among IFP subtypes.Molecular analysis of 38 tumours revealed activating mutations in three different exons of PDGFRA in 25 IFP. For the first time we report two cases with PDGFRA-exon 14 mutations (p.N659K; p.[N659K(+)T665A]). The results of our study and cases reported earlier indicate clearly that there is a localization-specific pattern: exon 12 mutations predominate in the small intestine, while exon 18 mutations occur frequently in the stomach (P0.001). Codons 567-571 of PDGFRA represent an IFP specific mutational hot spot and are affected most frequently by deletions. Furthermore, in our series IFP of the stomach share common features. In contrast to intestinal IFP, gastric tumours occur at higher age, show heavy inflammation and tend to be smaller. IFP located in the small intestine are frequently associated with intussusception.We conclude that there is a 'small bowel' and a 'gastric' phenotype of IFPs which are associated with exon 12 and exon 18 PDGFRA mutations, respectively.

1589 Background: The incidence of SPLC in Germany was reported to be 1.2 per 100,000 inhabitants in 2007 and the prevalence of second primary cancers reported by the US International Cancer Institute's Surveillance, Epidemiology and End Results Program has more than doubled over the last 25 years. Recently there has been tremendous progress in the ability to detect driver mutations in lung cancer and large studies presented the frequencies of driver mutations in unselected patient populations. We conducted an association study of known genetic driver events and clinical characteristics with the occurence of SPLC. Methods: Within the Network Genomic Medicine Lung Cancer, a local molecular screening network encompassing hospitals and office-based oncologists in the catchment area of the Center for Integrated Oncology Köln Bonn, we retrospectively reviewed the medical records of 375 molecularly annotated NSCLC patients for their SPLC status and clinical characteristics (age at diagnosis, histology, sex, smoking status). Molecular analysis for lung adenocarcinoma included the EGFR, ALK, BRAF, KRAS and PIK3CA status and for squamous cell lung cancer the FGFR1 and PIK3CA status. Results: 84 of 375 (22,4%) patients were SPLCs. The median age of SPLC diagnosis was 70 yrs. (Range 53 - 85 yrs.) and differed significantly from the rest of the population with a median age of 65 yrs. (Range 28 - 86 yrs.). The frequency of SPLCs was not significantly different between males and females (p = 0,259). In univariate logistic regression analysis active and former smoking, positive PIK3CA mutation and FGFR1 amplification status as well as age were significantly associated with the occurrence of SPLC. In a multivariate logistic regression analysis including the variables mentioned above only PIK3CA mutations (OR 9,48 95% CI 1,83 - 49,1) (p = 0,007) and age (OR 1,06 95% CI 1,03 - 1,1) (p = 0,015) could be confirmed to be independent prognosticators for the occurrence of SPLCs. Conclusions: The occurrence of SPLC was associated with age and PIK3CA mutations in our study population. Further comprehensive investigations on this topic are needed to confirm our findings and to further understand the association between mutation status and SPLCs.

Background: Lung cancer is one of the leading health care issues worldwide. Easy, reliable and standardized tests for predictive diagnoses and targeted therapies are needed. A small subset of pulmonary adenocarcinomas (AC) harbor therapeutically relevant EML4-ALK translocations, however FISH-based diagnosis is sometimes compromised by the different types of chromosome 2p rearrangements and tumor cell aneuploidy. The incidence of squamous cell carcinomas (SC) of the lung increases dramatically but targeted therapeutic options are not well established for this subgroup of NSCLC. Recently, Fibroblast Growth Factor Receptor 1 (FGFR1) amplification in SC was described to be associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients. Material and Methods: A total of 400 NSCLC samples (routinely processed FFPE material of biopsies and surgical specimens) were included in this study. For detection of EML4-ALK translocations a novel triple color FISH assay was used: two probes labeled orange and green flank the breakpoint region of ALK in telomeric and centromeric direction, and a third probe, labeled in blue, spans the entire EML4 gene. The results of the hybridisation patterns were compared with that of the conventional two color ALK break apart probe. For testing the entire spectrum of ALK translocations also ALK lymphomas and inflammatory myofibroblastic tumors were investigated. Normal tissues and breast carcinomas served as negative controls. A dual color FGFR1/CEN8 probe was used to evaluate the prevalence of FGFR1 amplification in SC and to develop an evaluation strategy for FGFR1 FISH assays. Results & Conclusions: Using the triple color EML4-ALK probe specific signal patterns were found for different types of ALK rearrangements. An inversion of chromosome 2p results in (1) split (separated) orange and green signals, (2) a split (doubled) blue signal and (3) a colocalisation (fusion) of the separated orange and green signals with blue signals. Additional specific signals patterns were seen in cases with inversions/deletions and interstitial deletions alone. Even ALK translocations without involvement of EML4 were clearly detectable. Aberrant split/fusion signals were not observed in normal tissues and aneuploid negative controls. The novel triple color split/fusion approach decreases the number of questionable or borderline cases at high sensitivity and specificity and allows the diagnosis of specific types of ALK translocations. Thus we present a novel triple color FISH that leads to an improved detection rate of EML4-ALK translocated patients. Investigating a subset of 254 SC with a two colour FISH assay we found FGFR1 amplifications in 21.3% of SCC but not in AC. We developed an evaluation strategy and observed different FGFR1 amplification patterns. Low amplification levels (as defined by ≥5 FGFR1 signals in ≥50% of tumor cells) were rare with a fequency of 6.3% while high level amplifications (as defined by a FGFR1/CEN8 ratio ≥2.0, or average number of FGFR1 signals per tumor cell nucleus ≥6, or the percentage of tumor cells containing ≥15 FGFR1 signals or large clusters ≥10%) occurred in 15% of all SC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A200.