Search

Background: Atypical EGFR mutations occur in 10%-30% of non-small-cell lung cancer (NSCLC) patients with EGFR mutations and their sensitivity to classical epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKI) is highly heterogeneous. Patients harboring one group of uncommon, recurrent EGFR mutations (G719X, S768I, L861Q) respond to EGFR-TKI. Exon 20 insertions are mostly insensitive to EGFR-TKI but display sensitivity to exon 20 inhibitors. Clinical outcome data of patients with very rare point and compound mutations upon systemic treatments are still sparse to date. Patients and methods: In this retrospective, multicenter study of the national Network Genomic Medicine (nNGM) in Germany, 856 NSCLC cases with atypical EGFR mutations including co-occurring mutations were reported from 12 centers. Clinical follow-up data after treatment with different EGFR-TKIs, chemotherapy and immune checkpoint inhibitors were available from 260 patients. Response to treatment was analyzed in three major groups: (i) uncommon mutations (G719X, S7681, L861Q and combinations), (ii) exon 20 insertions and (iii) very rare EGFR mutations (very rare single point mutations, compound mutations, exon 18 deletions, exon 19 insertions). Results: Our study comprises the largest thus far reported real-world cohort of very rare EGFR single point and compound mutations treated with different systemic treatments. We validated higher efficacy of EGFR-TKI in comparison to chemotherapy in group 1 (uncommon), while most exon 20 insertions (group 2) were not EGFR-TKI responsive. In addition, we found TKI sensitivity of very rare point mutations (group 3) and of complex EGFR mutations containing exon 19 deletions or L858R mutations independent of the combination partner. Notably, treatment responses in group 3 (very rare) were highly heterogeneous. Co-occurring TP53 mutations exerted a non-significant trend for a detrimental effect on outcome in EGFR-TKI-treated patients in groups 2 and 3 but not in group 1. Conclusions: Based on our findings, we propose a novel nNGM classification of atypical EGFR mutations.

BACKGROUND: Homologous recombination repair (HRR) enables fault-free repair of double-stranded DNA breaks. HRR deficiency is predicted to occur in around half of high-grade serous ovarian carcinomas. Ovarian cancers harbouring HRR deficiency typically exhibit sensitivity to poly-ADP ribose polymerase inhibitors (PARPi). Current guidelines recommend a range of approaches for genetic testing to identify predictors of sensitivity to PARPi in ovarian cancer and to identify genetic predisposition. DESIGN: To establish a European-wide consensus for genetic testing (including the genetic care pathway), decision making and clinical management of patients with recently diagnosed advanced ovarian cancer, and the validity of biomarkers to predict the effectiveness of PARPi in the first-line setting. The collaborative European experts' consensus group consisted of a steering committee (n = 14) and contributors (n = 84). A (modified) Delphi process was used to establish consensus statements based on a systematic literature search, conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. RESULTS: A consensus was reached on 34 statements amongst 98 caregivers (including oncologists, pathologists, clinical geneticists, genetic researchers, and patient advocates). The statements concentrated on (i) the value of testing for BRCA1/2 mutations and HRR deficiency testing, including when and whom to test; (ii) the importance of developing new and better HRR deficiency tests; (iii) the importance of germline non-BRCA HRR and mismatch repair gene mutations for predicting familial risk, but not for predicting sensitivity to PARPi, in the first-line setting; (iv) who should be able to inform patients about genetic testing, and what training and education should these caregivers receive. CONCLUSION: These consensus recommendations, from a multidisciplinary panel of experts from across Europe, provide clear guidance on the use of BRCA and HRR deficiency testing for recently diagnosed patients with advanced ovarian cancer. ispartof: ANNALS OF ONCOLOGY vol:33 issue:3 pages:276-287 ispartof: location:England status: published

DNA double-strand breaks may evoke cell death or cancer. Cells have developed two fundamentally different mechanisms of DNA double-strand repair: the accurate mechanism of homologous recombination repair and the error-prone nonhomologous end joining. The deficiency of homologous recombination repair (HRD) is a frequent feature of several solid tumor entities and is associated with sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitor therapy.Among other biomarkers, HRD testing provides an opportunity to guide PARP inhibitor therapy in solid tumors, but the basic principles are complex and the use and benefits of the different methodologies remain controversial. Knowledge of the underlying mechanisms at the molecular level will advance our understanding and will pave the way to introduce the testing of new biomarkers.An overview of the fundamental mechanisms of DNA repair is provided. Important terms like HRR, HRD, and BRCAness are defined, and analysis methods are described, especially with regard to their integration in molecular pathology routine diagnostics.Currently, at least testing of the BRCA mutation status and genomic instability using a composite HRD score should be implemented in laboratories to identify subgroups of patients who might benefit from PARP inhibitor therapies. A broad range of testing methods is available with pros and cons for introduction in the clinical setting. They have to be validated carefully to reliably inform treatment selection.Biomarkers to identify current homologous recombination deficiency status are needed to predict the benefit from PARP inhibitors and stratify their use in clinical management. Besides commercial assays, different tests might be used for the analysis of HRD. The application depends, among other things, on the local situation and has to be extensively validated.HINTERGRUND: DNA-Doppelstrangbrüche können Zelltod und Tumorentstehung verursachen. Die beiden unterschiedlichen Mechanismen der DNA-Doppelstrangreparatur sind die fehlerfreie homologe Rekombinationsreparatur und die fehleranfällige nichthomologe Endverknüpfung. Die Defizienz der homologen Rekombinationsreparatur (HRD) ist ein häufiges Merkmal in soliden Tumoren und ist assoziiert mit einer Sensitivität gegenüber einer Therapie mit Poly(ADP-Ribose)-Polymerasen(PARP)-Inhibitoren.Die Entscheidung für eine Therapie mit PARP-Inhibitoren kann durch die Bestimmung eines HRD-Scores gelenkt werden. Die Biologie der HRD ist komplex und Einsatz und Nutzen der verschiedenen Methoden werden kontrovers diskutiert. Die Kenntnis der zugrunde liegenden Mechanismen auf molekularer Ebene ist Voraussetzung für die Entwicklung und Integration neuer Biomarkertests.Der Artikel gibt einen Überblick über die wesentlichen Mechanismen der DNA-Reparatur. Begriffe wie HRR, HRD und BRCAness werden definiert und die zugehörigen Analysemethoden beschrieben, insbesondere in Hinblick auf ihren Einsatz in der molekularpathologischen Routinediagnostik.Um Patienten zu identifizieren, die von einer Therapie mit PARP-Inhibitoren profitieren, sollte die Bestimmung des BRCA-Mutationsstatus und der genomischen Instabilität flächendeckend etabliert werden. Ein breites Spektrum von Testverfahren zur Bestimmung eines zusammengesetzten HRD-Scores ist verfügbar. Bei einer Etablierung für die klinische Testung müssen diese Verfahren sorgfältig validiert werden.Biomarker zur Bestimmung des HRD-Status sind für die Vorhersage eines Therapieansprechens auf PARP-Inhibition unerlässlich. Neben kommerziellen Assays können im Labor verschiedene Verfahren genutzt werden. Ihr Einsatz ist unter anderem abhängig von lokalen Gegebenheiten und muss unter geeigneten Bedingungen validiert werden.

Background GNAQ and GNA11 mutant nonuveal melanoma represent a poorly characterized rare subgroup of melanoma with a gene mutation profile similar to uveal melanoma. Objectives To characterize these tumours in terms of clinical behaviour and genetic characteristics. Methods Patients with nonuveal GNAQ/11 mutated melanoma were identified from the prospective multicentre tumour tissue registry ADOREG, Tissue Registry in Melanoma (TRIM) and additional cooperating skin cancer centres. Extensive data on patient, tumour and treatment characteristics were collected retrospectively. Targeted sequencing was used to determine tumour mutational burden. Immunohistochemistry staining was performed for programmed death-ligand 1 and BRCA1-associated protein (BAP)1. Existing whole-exome cutaneous and uveal melanoma data were analysed for mutation type and burden. Results We identified 18 patients with metastatic GNAQ/11 mutant nonuveal melanoma. Tumours had a lower tumour mutational burden and fewer ultraviolet signature mutations than cutaneous melanomas. In addition to GNAQ and GNA11 mutations (nine each), six splicing factor 3b subunit 1 (SF3B1), three eukaryotic translation initiation factor 1A X-linked (EIF1AX) and four BAP1 mutations were detected. In contrast to uveal melanoma, GNAQ/11 mutant nonuveal melanomas frequently metastasized lymphatically and concurrent EIF1AX, SF3B1 and BAP1 mutations showed no apparent association with patient prognosis. Objective response to immunotherapy was poor with only one partial response observed in 10 treated patients (10%). Conclusions Our findings suggest that GNAQ/11 mutant nonuveal melanomas are a subtype of melanoma that is both clinically and genetically distinct from cutaneous and uveal melanoma. As they respond poorly to available treatment regimens, novel effective therapeutic approaches for affected patients are urgently needed. What is already known about this topic? The rare occurrence of GNAQ/11 mutations in nonuveal melanoma has been documented. GNAQ/11 mutant nonuveal melanomas also harbour genetic alterations in EIF1AX, SF3B1 and BAP1 that are of prognostic relevance in uveal melanoma. What does this study add? GNAQ/11 mutant nonuveal melanomas show metastatic spread reminiscent of cutaneous melanoma, but not uveal melanoma. GNAQ/11 mutant nonuveal melanomas have a low tumour mutational burden that is higher than uveal melanoma, but lower than cutaneous melanoma. What is the translational message? Primary GNAQ/11 mutant nonuveal melanomas are a subtype of melanoma that is clinically and genetically distinct from both cutaneous and uveal melanoma. As metastatic GNAQ/11 mutant nonuveal melanomas respond poorly to available systemic therapies, including immune checkpoint inhibition, novel therapeutic approaches for these tumours are urgently needed. Linked Comment: Rafei-Shamsabadi. Br J Dermatol 2020; 183:806-807.

Technical advancement and availability of high-throughput analysis has advanced molecular subtyping of most cancers. Thus, new possibilities for precision oncology have emerged.Therefore, we aimed to collect data regarding availability and use of next generation sequencing (NGS) for urothelial cancer within the uropathology working group of the German Society of Pathology.We collected data by questionnaires and additionally asked for sequencing results of bladder cancers in the participating institutions.A total of 13 university-affiliated institutes of pathology took part in the survey. All university institutes offer NGS-based molecular panel diagnostics and provide panels covering between 15 and 170 genes. Altogether, only 20 bladder cancers were sequenced in routine diagnostics and for 10 cancers potential targeted treatment options were available.So far, despite availability of NGS diagnostics at university institutes of pathology, only few bladder cancer samples have been sequenced. Based on current data from the molecular subtyping of bladder cancers, we recommend a step-by-step protocol with basic immunohistochemistry analysis and subsequent subtype-dependent analyses, e.g., alterations of the fibroblast growth factor receptors (FGFR) or comprehensive gene panel analyses.

Due to the increasing amount of data and sources of information, data evaluation is a crucial step in parallel sequencing.Illustration of pitfalls in evaluating the variant list of parallel sequencing and recommendations regarding software tools and databases.Description of filtering steps used, demonstration of criteria and recommendations for annotation by examples from everyday work, comparative analysis of databases with somatic variants, description of the installation of an individualized database.Variant filtering is a multistep process using information from different databases. The plausibility of variant calling should be verified using the Integrative Genomics Viewer and variants should be described according to the Human Genome Variation Society (HGVS) recommendations. Different databases, which all show advantages and disadvantages, are available for variant interpretation. An individualized database can be built up with the open-source tool cBioPortal.Different tools and databases might be used for the analysis of parallel sequencing data. The application depends on, amongst other things, the local situation and has to be extensively validated.

Summary Background Inhibitors of the mutated, constitutively activated BRAF protein have shown efficacy in the treatment of metastatic melanoma in clinical trials. Mutation analysis especially of the BRAF, NRAS and KIT genes is essential to identify patients suitable for targeted therapies and has been introduced into routine patient care. Objectives To correlate mutational status with clinical parameters including age, skin type, number of melanocytic naevi, primary tumour location, chronic sun damage and exposure to ultraviolet (UV) irradiation. The overall aim was to define subgroups with an increased or decreased likelihood of gene mutations. Additionally, the impact of activating BRAF mutations on clinical course was investigated. Methods In a single-centre, retrospective approach, mutation analysis was performed on patients with metastatic malignant melanoma. Clinical parameters were correlated with molecular findings. The total sun-burden score was assessed using a validated standardized questionnaire. Results The analysis included 141 patients with metastatic melanoma. Forty-four per cent of patients had activating BRAF mutations and were significantly younger than patients with wild-type BRAF or with NRAS mutations. KIT mutations were detected in only 3% of the patients. BRAF-mutated melanomas developed preferentially in intermittently sun-exposed areas of the body, and patients had significantly more melanocytic naevi. Once patients had progressed into stage IV disease, survival times were identical for those with BRAF-mutated and BRAF wild-type tumours. Conclusions Mutations of the BRAF gene are correlated with younger age, a higher number of melanocytic naevi and a tumour location in intermittently UV-exposed skin. Signs of chronic photodamage are not indicative of mutational status. Patients with metastatic melanoma with BRAF mutations showed a nonsignificant tendency to progress later to stage IV disease, but once metastases were present the prognosis was identical to that with BRAF wild-type tumours.

Due to their rarity and multiple subtypes, there is scant experience with sarcomas. Any effective targeted therapy depends on precise diagnosis of the tumor group using molecular markers and, increasingly, mutation testing. The necessary histopathological expertise and molecular diagnostic tools are usually only found at specialized centers. Using the Bonner GIST register as an example, the advantages of this kind of register from a diagnostic and therapeutic perspective will be discussed. Material submitted for gastrointestinal stromal tumors and other mesenchymal tumors, as well as the supervision of pathological referencing for national and international studies have made accurate diagnosis and appropriate therapy strategies ever more possible. The introduction of epidemiological as well as interdisciplinary sarcoma registers is a prerequisite for the improvement of sarcoma diagnostics and therapy.

The use of modern molecular techniques has gained importance in the diagnosis of sarcomas in recent years. Each of the analytical methods discussed here has its unique advantages and specific requirements. Cytogenetic screening methods which provide genome-wide information depend on the availability of fresh tissue. With the aid of fluorescence in situ hybridization and RT-polymerase chain reaction, specific events such as translocations in Ewing sarcoma, synovial sarcoma or alveolar rhabdomyosarcoma, as well as gene amplifications in well-differentiated and dedifferentiated liposarcoma or radiation-induced angiosarcoma and deletions in rhabdoid tumors or well-differentiated spindle cell liposarcoma can be detected in fresh and formalin fixed tissues. Molecular methods including Sanger sequencing, pyrosequencing and high resolution melting provide information about specific molecular aberrations on gene level. Here we review the most important molecular techniques currently used in sarcoma diagnosis, describe their relevance for differential diagnosis and point out specific examples.

Gastrointestinal stromal tumors (GIST) are the most frequent mesenchymal tumors of the gastrointestinal tract. Two thirds of them are located in the stomach, another 30% occur in the small bowel, while the remaining tumors occur in the rectum or more rarely in the oesophagus. GIST most commonly grow from the smooth muscular layer towards the serosal surface whereas development towards to the mucosal layer is less frequent. In the latter case ulceration may occur, leading to gastrointestinal bleeding as the main symptom. However, the majority of GIST of the stomach are asymptomatic, resulting in large tumors on initial diagnosis. Most gastric GIST are not visible on endoscopy but may be diagnosed by endosonography. Due to their location in the outer layers of the tubular gastrointestinal tract biopsy is often hindered of even impossible. GIST of the stomach differ from tumors in other locations with regard to their morphology, molecular pathology and prognosis. This present article provides an overview of these differences also with regard to possible therapeutic consequences.

Inflammatorische fibroide Polypen (IFP) wurden vor 60 Jahren von Vaněk erstmals als „submukoses Granulom mit eosinophiler Infiltration“ beschrieben. IFP stellen polypose Spindelzellproliferate in der Submukosa und Mukosa von Magen, Dunndarm und Kolon mit entzundlicher Infiltration dar. Die Lasion ist bislang als entzundlich-reaktiv angesehen worden. Neuere Daten zeigen, dass die lasionalen Spindelzellen den PDGFRA exprimieren, in der Mehrzahl der Falle sind aktivierende PDGFRA-Mutationen nachweisbar. IFP sind also echte benigne mesenchymale Tumoren des Gastrointestinaltrakts.

Gastrointestinale Stromatumoren (GIST) zeigen in etwa 50% der Falle einen aggressiven Phanotyp mit Rezidiven und Metastasen. Sie konnen immunhistochemisch sicher von anderen mesenchymalen Tumorentitaten unterschieden werden und tragen in bis zu 85% der Falle aktivierende Mutationen in den Rezeptortyrosinkinasen KIT oder PDGFRA. Die Aufklarung ihrer molekularen Pathogenese hatte einen therapeutischen Paradigmenwechsel zur Folge. Bei inoperablen oder metastasierten GIST stellt die Behandlung mit Tyrosinkinaseinhibitoren heute den Goldstandard dar mit einer Ansprechrate von bis zu 80% bei vergleichsweise geringen Nebenwirkungen. Die Kenntnis der KIT- bzw. PDGFRA-Mutation ist dabei sowohl prognostisch als auch therapeutisch von zentraler Bedeutung.

Hintergrund Inflammatorische fibroide Polypen (IFP) sind Proliferationen CD34-positiver Spindelzellen vor einem inflammatorischen Hintergrund in der Submukosa und Mukosa des Gastrointestinaltrakts. IFP kommen vor allem im Magen, aber auch im Dunndarm, Kolon und Osophagus vor. Atiologie und Pathogenese sind bislang unklar.

Gastrointestinale Stromatumoren (GISTs) sind die haufigsten mesenchymalen Tumoren des Magen-Darm-Trakts. Bezogen auf alle Neoplasien in diesem Organsystem machen sie etwa 1% aller Tumoren aus. Durch immunhistochemische Untersuchungen ist eine sichere Abgrenzung gegenuber anderen mesenchymalen Tumorentitaten wie z.B. myogenen und neurogenen Tumoren moglich. Seit die molekulare Pathogenese von GISTs durch den Nachweis aktivierender Mutationen in der Rezeptortyrosinkinase KIT vor 10 Jahren und wenige Jahre spater durch die Entdeckung ganz ahnlicher Mutationen im verwandten PDGFRα-Gen wesentlich besser verstanden wird, hat sich in rasantem Tempo ein therapeutischer Paradigmenwechsel vollzogen. Obwohl die chirurgische Entfernung des Tumors bei Operabilitat nach wie vor den ersten Schritt darstellt, bietet sich fur die fortgeschrittenen oder inoperablen Stadien des prinzipiell chemo- und radiotherapieresistenten Tumors mit dem Rezeptortyrosinkinaseinhibitor Imatinib (Glivec®, Novartis, Basel, Schweiz) eine sehr effiziente und erfolgreiche Therapieoption. Bis zu 80% der Patienten sprechen auf diese orale Behandlung an, die im Vergleich zu konventionellen zytoreduktiven Therapien deutlich besser vertragen wird. In einem Teil der primar inoperablen Tumoren kann durch eine praoperative Imatinibbehandlung die sekundare Operabilitat hergestellt werden. Schlieslich wird aktuell in Studien gepruft, ob bei primar R0-resezierten GISTs mit hohem Ruckfallrisiko eine postoperative adjuvante Imatinibtherapie die Rezidiv- bzw. Metastasenhaufigkeit senken kann. Die Ansprechrate auf die Behandlung wird wesentlich von der zugrundeliegenden molekularen Alteration im KIT- bzw. PDGFRα-Gen beeinflusst. Dem unterschiedlichen Ansprechen der verschiedenen Mutationstypen kann zumindest partiell durch unterschiedliche Dosierung von Imatinib begegnet werden. Zudem sind die verschiedenen Mutationstypen offenbar auch mit einem unterschiedlichen biologischen Verhalten assoziiert. Derzeit befinden sich neue Tyrosinkinaseinhibitoren und weitere, die intrazellularen Signaltransduktionskaskaden beeinflussende Therapeutika in Entwicklung, die auch bei imatinibresistenten GISTs wirksam sind. Die Kenntnis der zugrundeliegenden molekularen Alteration im KIT- oder PDGFRα-Gen gewinnt also sowohl prognostisch als auch therapeutisch zunehmend an Bedeutung. So muss die Mutationsanalyse zumindest fur GISTs mit hohem Risiko eines aggressiven klinischen Verhaltens heute empfohlen werden.

Molekularbiologische Untersuchungen in der Tumordiagnostik haben sich in den letzten Jahren zu einem wesentlichen Bestandteil der pathologischen Routinediagnostik entwickelt. Gangige Untersuchungsmethoden, die sich auch fur formalinfixiertes Gewebe eignen, sind die Polymerasekettenreaktion und die Fluoreszenz-in-situ-Hybridisierung. Die molekularen Analysen liefern zum einen Informationen, die die histologische und immunhistochemische Diagnostik erganzen, z. B. durch den Nachweis charakteristischer Genveranderungen bei bestimmten Tumorentitaten. Daruber hinaus geben sie aber auch die Moglichkeit, Aussagen zur Prognose oder Therapie von Tumorerkrankungen zu treffen. In der vorliegenden Arbeit werden die zurzeit etablierten Methoden beschrieben und neue Entwicklungen, v. a. aus dem Bereich der Therapiepradiktion, aufgezeigt.

Lymphomatoid contact dermatitis is a rare complication after tattooing, mostly occurring when red dye is used. More common is tattoo-induced chronic contact dermatitis. Pseudolymphoma-like contact-reactions are uncommon and the prognosis is to be considered unclear. Therefore excision is the most widely recommended treatment. We report the case of a 21-year-old patient, whose therapy-resistant contact dermatitis in the area of a red dyed tattoo proved to be a lymphocytic contact dermatitis.

Inhibitors of the mutated, constitutively activated BRAF protein have shown efficacy in the treatment of metastatic melanoma in clinical trials. Mutation analysis especially of the BRAF, NRAS and KIT genes is essential to identify patients suitable for targeted therapies and has been introduced into routine patient care.To correlate mutational status with clinical parameters including age, skin type, number of melanocytic naevi, primary tumour location, chronic sun damage and exposure to ultraviolet (UV) irradiation. The overall aim was to define subgroups with an increased or decreased likelihood of gene mutations. Additionally, the impact of activating BRAF mutations on clinical course was investigated.In a single-centre, retrospective approach, mutation analysis was performed on patients with metastatic malignant melanoma. Clinical parameters were correlated with molecular findings. The total sun-burden score was assessed using a validated standardized questionnaire.The analysis included 141 patients with metastatic melanoma. Forty-four per cent of patients had activating BRAF mutations and were significantly younger than patients with wild-type BRAF or with NRAS mutations. KIT mutations were detected in only 3% of the patients. BRAF-mutated melanomas developed preferentially in intermittently sun-exposed areas of the body, and patients had significantly more melanocytic naevi. Once patients had progressed into stage IV disease, survival times were identical for those with BRAF-mutated and BRAF wild-type tumours.Mutations of the BRAF gene are correlated with younger age, a higher number of melanocytic naevi and a tumour location in intermittently UV-exposed skin. Signs of chronic photodamage are not indicative of mutational status. Patients with metastatic melanoma with BRAF mutations showed a nonsignificant tendency to progress later to stage IV disease, but once metastases were present the prognosis was identical to that with BRAF wild-type tumours.

Due to their rarity and multiple subtypes, there is scant experience with sarcomas. Any effective targeted therapy depends on precise diagnosis of the tumor group using molecular markers and, increasingly, mutation testing. The necessary histopathological expertise and molecular diagnostic tools are usually only found at specialized centers. Using the Bonner GIST register as an example, the advantages of this kind of register from a diagnostic and therapeutic perspective will be discussed. Material submitted for gastrointestinal stromal tumors and other mesenchymal tumors, as well as the supervision of pathological referencing for national and international studies have made accurate diagnosis and appropriate therapy strategies ever more possible. The introduction of epidemiological as well as interdisciplinary sarcoma registers is a prerequisite for the improvement of sarcoma diagnostics and therapy.

The use of modern molecular techniques has gained importance in the diagnosis of sarcomas in recent years. Each of the analytical methods discussed here has its unique advantages and specific requirements. Cytogenetic screening methods which provide genome-wide information depend on the availability of fresh tissue. With the aid of fluorescence in situ hybridization and RT-polymerase chain reaction, specific events such as translocations in Ewing sarcoma, synovial sarcoma or alveolar rhabdomyosarcoma, as well as gene amplifications in well-differentiated and dedifferentiated liposarcoma or radiation-induced angiosarcoma and deletions in rhabdoid tumors or well-differentiated spindle cell liposarcoma can be detected in fresh and formalin fixed tissues. Molecular methods including Sanger sequencing, pyrosequencing and high resolution melting provide information about specific molecular aberrations on gene level. Here we review the most important molecular techniques currently used in sarcoma diagnosis, describe their relevance for differential diagnosis and point out specific examples.

Inflammatory fibroid polyps (IFP) were described by Vanek 60 years ago as "submucosal granuloma with eosinophilic infiltration". IFP represent polypous proliferations of spindle cells in the submucosa and mucosa of the stomach, small bowel and colon with inflammatory infiltration. The lesions have been regarded as inflammatory and reactive. Recent data show that the spindle cells express PDGFRA, and the majority of IFP harbour activating PDGFRA mutations. Therefore, IFP represent true benign mesenchymal tumors of the gastrointestinal tract.

Gastrointestinal stromal tumors (GIST) show an aggressive behavior with metastases and recurrences in up to 50% of cases. They can be clearly distinguished from other mesenchymal tumors by immunohistochemistry in the vast majority of cases. Of the tumors 85% carry somatic activating mutations in the receptor tyrosine kinases KIT or PDGFRA. The detection of these molecular events has changed the treatment of inoperable and metastatic GISTs dramatically as up to 80% of tumors respond well to tyrosine kinase inhibitors. This treatment has become the gold standard in the last few years with only few side effects. Knowledge of the underlying KIT or PDGFRA mutation is both relevant for the prognosis and treatment response.

Inflammatory fibroid polyps (IFP) are proliferations of CD34-positive spindle cells in the submucosa and mucosa of the gastrointestinal tract with an inflammatory infiltrate. IFP occur in the stomach, small bowel, colon and esophagus. To date, etiology and pathogenesis are unclear.A total of 29 IFP originating in the stomach, small bowel and colon were examined immunohistochemically, and mutational analyses of PDGFRA exons 10, 12, 14 and 18 were conducted.Activating mutations in PDGFRA exons 12 and 18 were found in 20 cases (69%). The mutational types are related to mutations known from gastrointestinal stromal tumors (GIST). D842V was the most frequent mutation. No activating mutations were found in exons 10 and 14.The majority of IFP reveal activating PDGFRA mutations. Our data indicate that IFP are true neoplasms (true benign tumors) and not reactive lesions. In terms of pathogenesis, the relationship between PDGFRA-mutant GISTs and IFP remains to be determined.

Receptor tyrosine kinases have been shown to be a challenging target for the treatment of hematologic diseases and solid tumors. One very effective tyrosine kinase inhibitor is imatinib mesylate inhibiting the KIT receptor tyrosine kinase in gastrointestinal stromal tumors (GISTs) which often carry activating mutations in the KIT gene. Our own observations show that the location of the underlying mutations influence the response to treatment with imatinib.In Bonn, nearly 1000 GISTs have been characterized molecularily before and/or under treatment with tyrosine kinase inhibitors. Tumor-DNA was extracted from paraffine-embedded material, amplified in all known hot spots of KIT (exons 9, 11, 13, 14, 17) and PDGFRcx (exons 12, 14, 18) and sequenced directly.The best response to treatment with imatinib is achieved in GISTs with an underlying KIT mutation in exon 11 encoding the juxtamembranous domain. Exon 9 mutated GISTs respond in only half of the cases. GISTs with mutations in the tyrosine kinase domain 1 or 2 are very rare (less than 1%) and are thought to be resistant. PDGFRalpha-mutated GISTs are resistant when carrying the most common point mutation D842V (exon 18) and may respond when deletions occur in exon 18. Low response rates are achieved in tumors without detectable mutation. Under treatment, secondary KIT mutations may occur leading to resistance to treatment.The molecular status of GISTs plays a central role for treatment response. Its evaluation will be mandatory in the future at least in tumors with intermediate or high risk criteria.

The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC) has been linked to activating mutations in the EGFR gene. So far these mutations have been extensively characterized in established cell lines. The aim of this study was to determine the effects of EGFR mutations on downstream signaling in human tumor specimens. We have looked for mutations of the EGFR gene in specimens of 67 patients with NSCLC and correlated these with EGFR phosphorylation and the activity of its three main downstream signaling cascades Akt, MAPK and Stat3 by immunohistochemistry. We show that the phosphorylation of tyrosine residues 922 and 1173, but not 1068, are primarily affected by the activating EGFR mutations. Akt activity was significantly higher in patients with EGFR mutations but we found no difference in Stat3 or MAPK phosphorylation. Our results suggest that EGFR mutations not only increase receptor activity, but also alter responses of downstream signaling cascades in human NSCLCs and that these finding differ from results obtained in cell lines.

Gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors in the gastrointestinal tract, metastasize in up to 50 % of cases and are resistant to conventional radio- and chemotherapy. They are characterized by the expression of the type III receptor tyrosine kinase KIT which is the most important diagnostic immunohistochemical feature. Genomically, the majority of GISTs carry heterozygous mutations in the KIT or the PDGF receptor alpha gene leading to an autophosphorylation of the respective receptor protein. The evaluation of the mutational status allows the subdivision of GISTs into different prognostic sub-groups. For example, GISTs carrying an activating mutation in PDGF receptor alpha are most often located in the stomach and seem to have a better prognosis than GISTs with a KIT mutation. Specific mutational subtypes of KIT mutations in exon 11 (esp. proximal deletions of codons tryptophane-557 and lysine-558) have a significantly higher metastatic risk than GISTs with KIT mutations located in the distal part of exon 11 (esp. insertions/duplications). GISTs in the small bowel most often carry KIT exon 9 mutations and have a worse prognosis than GISTs with exon 11 mutations. Mutational subtype in KIT or PDGF receptor alpha not only influences the biological behavior of GISTs but also their response to treatment with imatinib, a tyrosine kinase inhibitor also inhibiting ARG, PDGF receptor beta and BCR-ABL. KIT exon 11 mutated tumors show response rates of up to 80 % of cases whereas KIT exon 9 mutated GISTs respond in less then 50 %. GISTs without detectable KIT mutation in these both exons often are resistant to imatinib. The development of secondary resistance to imatinib in GIST patients occurs in up to 40% of cases and is partly due to secondary KIT mutations occuring additionally to the primary mutation. Actually, several studies evaluate the efficacy of alternative small molecules such as SU 11248, RAD001 and AMG706 inhibiting signal transduction pathways downstream of KIT and PDGF receptor alpha. In summary, mutational status in KIT or PDGF receptor alpha of GISTs is relevant for prognosis, for response to treatment and for further insights into mechanisms of treatment failure.

Most gastrointestinal stromal tumors (GISTs) carry gain-of-function mutations of the KIT gene encoding the receptor tyrosine kinase KIT. However, in a subset of GISTs no activating mutations are detectable in KIT. Recently, PDGFRalpha mutations have been identified as alternative oncogenic mechanism. We studied a panel of 100 GISTs for mutations in hot spot regions of KIT (exons 9, 11, 13 and 17) and PDGFRalpha (exons 12 and 18) and compared the results with pathomorphological and immunohistochemical data.DNA from formalin-fixed and paraffin-embedded tumor tissue was extracted after microdissection from serial sections. We performed single strand conformational polymorphism analysis and direct sequencing.We found PDGFRalpha mutations in 24 of 55 GISTs with wild-type sequence in exons 9, 11, 13 and 17 of KIT. All mutations were located in exon 18 of the PDGFRca gene which encodes the tyrosine kinase domain II. None of the 45 GISTs with detectable KIT mutation carried a mutation in the PDGFRalpha gene. Interestingly, all PDGFRalpha-mutated tumors were located in the stomach whereas GISTs with exon 9 and 13 KIT mutations occurred predominantly in the small bowel. Additionally, 21 of 24 GISTs carrying PDGFRalpha mutations displayed an epithelioid or mixed phenotype. In contrast, KIT-mutated GISTs exhibited almost always a spindled histology (38 of 45 cases).Our analysis provides evidence that GISTS represent distinctive entities with different genetic, biological and phenotypic features.

Experimental and clinical data support an infectious cause of atherosclerosis and thereby coronary artery disease. This study was intended to assess the prevalence and possible clinical associations of the presence of cytomegalovirus DNA within coronary samples from patients undergoing coronary artery bypass grafting.A coronary thrombendatherectomy was performed in 53 patients with advanced coronary artery disease. Two samples of each atheroma were used for further analysis and pathogen detection.In 30% of patients with advanced coronary artery disease cytomegalovirus DNA was detected in coronary samples as assessed by highly sensitive PCR methods. The occurrence of the virus within the vessels was characterized by an inhomogeneous distribution pattern.Due to an increased proportion of restenotic lesions and a higher degree of calcification in cytomegalovirus-positive lesions, a causative association between the virus presence and mechanisms of restenosis post angioplasty is further supported. Antiviral pharmacological interventions to prevent restenosis in high-risk patients, however, seem not to be justified by the data currently available.

Infiltrating ductal (DC) and lobular carcinoma (LC) of the breast represent the most frequently observed varieties of invasive breast cancer, characterized by differences in their histological and clinical properties. Although comparative genomic hybridization (CGH) of invasive breast carcinomas has revealed a complex and consistent pattern of DNA copy number changes, the data with regard to type specific aberrations are limited. A comprehensive study was therefore performed on 19 LCs and 29 DCs to ascertain type-specific differences of unbalanced DNA copy number changes by CGH. Statistical analysis revealed significantly higher frequencies for underrepresentation of chromosomes 16q (p0.01), 22 (p0.05), and 17q (p0.05), and a lower frequency for overrepresentation of chromosome 8q (p0.01) in LC. Similar frequencies of non-random chromosomal changes in LC and DC were obtained for gain of 1q (74%/59%) and loss of 19p (53%/52%), parts of 1p (42%/41%) and 11q (21%/24%). Less frequently, gains mainly involving parts of chromosomes 20q, 20p, 3q, and 5p and partial losses of chromosomes 17p and 13 were observed in both groups of tumours. Minimal regions of overlapping amplifications were mapped to 17q23 exclusively in DC (17%) and 11q13-q14 in both DC and LC (21% and 11%, respectively). High occurrences of DNA copy number decreases were detected at the distal part of chromosomes 1p, 19 and 22, but further analysis is required to confirm these imbalances. It is suggested that the observed differences are involved in the development of type-specific properties of DC and LC.

A variety of rheumatic syndromes have been associated with neoplasia. We report a case of occult metastatic breast cancer which presented with symptoms and signs of adult onset Still’s disease (AOSD). In July 2002, a 52 year old Caucasian woman presented with a 4 week history of high, spiking fevers (up to 40°C) that were accompanied by a transient macular rash, myalgia, and arthralgia affecting most joints. Initially, there had been a sore throat and painful lymphadenopathy in the left supraclavicular fossa. A lymph node was removed for histopathological examination and the lymphadenopathy resolved spontaneously. The past medical history was unremarkable other than a family history of breast cancer which had affected two second degree relatives. The physical and gynaecological examinations were …