Histological and molecular plasticity of ALK-positive non-small-cell lung cancer under targeted therapy: a case report.

With medical progress in cancer therapy, tyrosine kinase inhibitors (TKIs) became a standard of care for many cancer types. But the broad range of possible targeted therapies was accompanied by a plethora of potential resistance mechanisms, of which many have still to be identified. Here, we present the case of a patient with an EML4-ALK translocated non-small-cell lung cancer treated with four different TKIs. Her tumor developed not only a well-known ALK-TKI resistance mutation but also underwent a histological transformation from adenocarcinoma to squamous cell carcinoma. To confirm a shared monoclonal origin of the phenotypically different tumors, a phylogenetic reconstruction was conducted: This revealed a cluster of mutations including NFE2L2, KMT2D, and MLH1, which are possible triggering events for the transformation.

Assigning evidence to actionability: An introduction to variant interpretation in precision cancer medicine.

Modern concepts in precision cancer medicine are based on increasingly complex genomic analyses and require standardized criteria for the functional evaluation and reporting of detected genomic alterations in order to assess their clinical relevance. In this article, we propose and address the necessary steps in systematic variant evaluation consisting of bioinformatic analysis, functional annotation and clinical interpretation, focusing on the latter two aspects. We discuss the role and clinical application of current variant classification systems and point out their scope and limitations. Finally, we highlight the significance of the molecular tumor board as a platform for clinical decision-making based on genomic analyses.

Combination of Crizotinib and Osimertinib in T790M+ EGFR-Mutant Non-Small Cell Lung Cancer with Emerging MET Amplification Post-Osimertinib Progression in a 10-Year Survivor: A Case Report.

Tyrosine kinase inhibitors (TKIs) represent the standard treatment for patients with non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations. The duration of the response is, however, limited in time owing to the development of resistance mechanisms to both first- and second-generation agents such as MET oncogene amplification. This report describes the successful results obtained with the combination of the third-generation TKI osimertinib with the multitargeted TKI and MET inhibitor crizotinib in a patient with EGFR-mutant NSCLC with emerging MET amplification with a tolerable toxicity profile.

KRAS/GNAS-testing by highly sensitive deep targeted next generation sequencing improves the endoscopic ultrasound-guided workup of suspected mucinous neoplasms of the pancreas.

Pancreatic cysts or dilated pancreatic ducts are often found by cross-sectional imaging, but only mucinous lesions can become malignant. Therefore, distinction between mucinous and non-mucinous lesions is crucial for adequate patient management. We performed a prospective study including targeted next generation sequencing (NGS) of cell-free DNA in the diagnostic endoscopic ultrasound (EUS)-guided workup. Pancreatic cyst(s) or main duct fluid obtained by EUS-guided FNA was analysed by carcinoembryonic antigen (CEA), cytology and deep targeted NGS of 14 known gastrointestinal cancer genes (AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53, APC) with a limit of detection down to variant allele frequency of 0.01%. Results were correlated to histopathology and clinical follow-up. One hundred and thirteen patients with pancreatic cyst(s) and/or a dilated pancreatic main duct (≥5 mm) were screened. Sixty-six patients had to be excluded, mainly due to inoperability or small cyst size (≤10 mm). Forty-seven patients were enrolled for further analysis. A final diagnosis was available in 27 cases including 8 negative controls. In 43/47 (91.5%) of patients a KRAS- and/or GNAS-mutation was diagnosed by NGS. 27.0% of the KRAS-mutated and 10.0% of the GNAS-mutated lesions harbored multiple mutations. KRAS/GNAS-testing by NGS, cytology, and CEA had a sensitivity and specificity of 94.7/100%, 38.1/100%, and 42.1/75.0%, respectively. KRAS/GNAS-testing was significantly superior to CEA (P = .0209) and cytology (P = .0016). In conclusion, KRAS/GNAS-testing by deep targeted NGS is a suitable method to distinguish mucinous from non-mucinous pancreatic lesions, suggesting its usage as a single diagnostic test. Results must be confirmed in a larger cohort.

Targeting rare and non-canonical driver variants in NSCLC - An uncharted clinical field.

OBJECTIVES: Implementation of tyrosine kinase inhibitors (TKI) and other targeted therapies was a main advance in thoracic oncology with survival gains ranging from several months to years for non-small-cell lung cancer (NSCLC) patients. High-throughput comprehensive molecular profiling is of key importance to identify patients that can potentially benefit from these novel treatments. MATERIAL AND METHODS: Next-generation sequencing (NGS) was performed on 4500 consecutive formalin-fixed, paraffin-embedded specimens of advanced NSCLC (n = 4172 patients) after automated extraction of DNA and RNA for parallel detection of mutations and gene fusions, respectively. RESULTS AND CONCLUSION: Besides the 24.9 % (n = 1040) of cases eligible for approved targeted therapies based on the presence of canonical alterations in EGFR exons 18-21, BRAF, ROS1, ALK, NTRK, and RET, an additional n = 1260 patients (30.2 %) displayed rare or non-canonical mutations in EGFR (n = 748), BRAF (n = 135), ERBB2 (n = 30), KIT (n = 32), PIK3CA (n = 221), and CTNNB1 (n = 94), for which targeted therapies could also be potentially effective. A systematic literature search in conjunction with in silico evaluation identified n = 232 (5.5 %) patients, for which a trial of targeted treatment would be warranted according to available evidence (NCT level 1, i.e. published data showing efficacy in the same tumor entity). In conclusion, a sizeable fraction of NSCLC patients harbors rare or non-canonical alterations that may be associated with clinical benefit from currently available targeted drugs. Systematic identification and individualized management of these cases can expand applicability of precision oncology in NSCLC and extend clinical gain from established molecular targets. These results can also inform clinical trials.

RNA-Based Detection of Gene Fusions in Formalin-Fixed and Paraffin-Embedded Solid Cancer Samples.

Oncogenic gene fusions are important drivers in many cancer types, including carcinomas, with diagnostic and therapeutic implications. Hence, sensitive and rapid methods for parallel profiling in formalin-fixed and paraffin-embedded (FFPE) specimens are needed. In this study we analyzed gene fusions in a cohort of 517 cases where standard treatment options were exhausted. To this end the Archer® DX Solid tumor panel (AMP; 285 cases) and the Oncomine Comprehensive Assay v3 (OCA; 232 cases) were employed. Findings were validated by Sanger sequencing, fluorescence in situ hybridization (FISH) or immunohistochemistry. Both assays demonstrated minimal dropout rates (AMP: 2.4%; n = 7/292, OCA: 2.1%; n = 5/237) with turnaround times of 6-9 working days (median, OCA and AMP, respectively). Hands-on-time for library preparation was 6 h (AMP) and 2 h (OCA). We detected n = 40 fusion-positive cases (7.7%) with TMPRSS2::ERG in prostate cancer being most prevalent (n = 9/40; 22.5%), followed by other gene fusions identified in cancers of unknown primary (n = 6/40; 15.0%), adenoid cystic carcinoma (n = 7/40; 17.5%), and pancreatic cancer (n = 7/40; 17.5%). Our results demonstrate that targeted RNA-sequencing of FFPE samples is feasible, and a well-suited approach for the detection of gene fusions in a routine clinical setting.

Spatial and Temporal Heterogeneity of Panel-Based Tumor Mutational Burden in Pulmonary Adenocarcinoma: Separating Biology From Technical Artifacts.

BACKGROUND: Tumor mutational burden (TMB) is an emerging biomarker used to identify patients who are more likely to benefit from immuno-oncology therapy. Aside from various unsettled technical aspects, biological variables such as tumor cell content and intratumor heterogeneity may play an important role in determining TMB. METHODS: TMB estimates were determined applying the TruSight Oncology 500 targeted sequencing panel. Spatial and temporal heterogeneity was analyzed by multiregion sequencing (two to six samples) of 24 pulmonary adenocarcinomas and by sequencing a set of matched primary tumors, locoregional lymph node metastases, and distant metastases in five patients. RESULTS: On average, a coding region of 1.28 Mbp was covered with a mean read depth of 609x. Manual validation of the mutation-calls confirmed a good performance, but revealed noticeable misclassification during germline filtering. Different regions within a tumor showed considerable spatial TMB variance in 30% (7 of 24) of the cases (maximum difference, 14.13 mut/Mbp). Lymph node-derived TMB was significantly lower (p = 0.016). In 13 cases, distinct mutational profiles were exclusive to different regions of a tumor, leading to higher values for simulated aggregated TMB. Combined, intratumor heterogeneity and the aggregated TMB could result in divergent TMB designation in 17% of the analyzed patients. TMB variation between primary tumor and distant metastases existed but was not profound. CONCLUSIONS: Our data show that, in addition to technical aspects such as germline filtering, the tumor content and spatially divergent mutational profiles within a tumor are relevant factors influencing TMB estimation, revealing limitations of single-sample-based TMB estimations in a clinical context.

Combined targeted DNA and RNA sequencing of advanced NSCLC in routine molecular diagnostics: Analysis of the first 3,000 Heidelberg cases.

Tyrosine kinase inhibitors currently confer the greatest survival gain for nonsmall cell lung cancer (NSCLC) patients with actionable genetic alterations. Simultaneously, the increasing number of targets and compounds poses the challenge of reliable, broad and timely molecular assays for the identification of patients likely to benefit from novel treatments. Here, we demonstrate the feasibility and clinical utility of comprehensive, NGS-based genetic profiling for routine workup of advanced NSCLC based on the first 3,000 patients analyzed in our department. Following automated extraction of DNA and RNA from formalin-fixed, paraffin-embedded tissue samples, parallel sequencing of DNA and RNA for detection of mutations and gene fusions, respectively, was performed using PCR-based enrichment with an ion semiconductor sequencing platform. Overall, 807 patients (27%) were eligible for currently approved, EGFR-/BRAF-/ALK- and ROS1-directed therapies, while 218 additional cases (7%) with MET, ERBB2 (HER2) and RET alterations could potentially benefit from experimental targeted compounds. In addition, routine capturing of comutations, e.g. TP53 (55%), KEAP1 (11%) and STK11 (11%), as well as the precise typing of fusion partners and involved exons in case of actionable translocations including ALK and ROS1, are prognostic and predictive tools currently gaining importance for further refinement of therapeutic and surveillance strategies. The reliability, low dropout rates (<5%), minimal tissue requirements, fast turnaround times (6 days on average) and lower costs of the diagnostic approach presented here compared to sequential single-gene testing, highlight its practicability in order to support individualized decisions in routine patient care, enrollment in molecularly stratified clinical trials, as well as translational research.

Next generation sequencing of the cellular and liquid fraction of pancreatic cyst fluid supports discrimination of IPMN from pseudocysts and reveals cases with multiple mutated driver clones: First findings from the prospective ZYSTEUS biomarker study.

Approximately half of all pancreatic cysts are neoplastic, mainly comprising intraductal papillary mucinous neoplasms (IPMN), which can progress to invasive carcinoma. Current Fukuoka guidelines have limited sensitivity and specificity in predicting progression of asymptomatic pancreatic cysts. We present first results of the prospective ZYSTEUS biomarker study investigating (i) whether detection of driver mutations in IPMN by liquid biopsy is technically feasible, (ii) which compartment of IPMN is most suitable for analysis, and (iii) implications for clinical diagnostics. Twenty-two patients with clinical inclusion criteria were enrolled in ZYSTEUS. Fifteen cases underwent endoscopic ultrasound (EUS)-guided fine-needle aspiration and cytological diagnostics. Cellular and liquid fraction of the cysts of each case were separated and subjected to deep targeted next generation sequencing (NGS). Clinical parameters, imaging findings (EUS and MRI), and follow-up data were collected continuously. All IPMN cases (n = 12) showed at least one mutation in either KRAS (n = 11) or GNAS (n = 4). Three cases showed both KRAS and GNAS mutations. Six cases harbored multiple KRAS/GNAS mutations. In the three cases with pseudocysts, no KRAS or GNAS mutations were detected. DNA yields were higher and showed higher mutation diversity in the cellular fraction. In conclusion, mutation detection in pancreatic cyst fluid is technically feasible with more robust results in the cellular than in the liquid fraction. Current results suggest that, together with imaging, targeted sequencing supports discrimination of IPMN from pseudocysts. The prospective design of ZYSTEUS will provide insight into diagnostic value of NGS in preoperative risk stratification. Our data provide evidence for an oligoclonal nature of IPMN.

Synonymous EGFR variant p.Q787Q is neither prognostic nor predictive in patients with lung adenocarcinoma.

Patients with non-small cell lung cancer (NSCLC) harboring activating mutations in the Epidermal Growth Factor Receptor (EGFR) benefit from targeted therapies. A synonymous polymorphism (rs1050171, p.Q787Q) was shown to be associated with improved overall survival (OS) in colorectal cancer patients. As data in NSCLC are limited, we retrospectively analyzed associations of p.Q787Q with clinicopathological parameters including clinical response and outcome in patients with lung adenocarcinoma (ADC) who received tyrosine kinase inhibitor (TKI) therapy. Of 642 ADC patients whose tumors were profiled by next generation sequencing, 102 (15.9%) carried EGFR mutations targetable by TKIs (30.4% male patients, median age 65.1 y, 19.6% smokers with 12.8 median pack years). Seventy-nine patients (77.5%) received TKI therapy either as a first- or second-line therapy. Of the 102 EGFR-mutant tumors, 72 (70.6%) exhibited the p.Q787Q polymorphism and another 12 (11.8%) cases with p.Q787Q harbored an additional TKI insensitive mutation (p.T790M). The polymorphism was neither associated with classic clinicopathological parameters nor with overall survival (21.1 months vs. 20.1 months; P-value = 0.91) or clinical response (P-value = 0.122). The patients with p.T790M had worse survival compared to EGFR activating mutation carriers with and without p.Q787Q when analyzed as a separate group (27.5 months, P-value = 0.02). In conclusion, p.Q787Q is neither a suitable prognostic nor predictive biomarker for ADC patients receiving anti-EGFR therapy in first- or second-line of therapy. © 2016 Wiley Periodicals, Inc.

Copy number changes of clinically actionable genes in melanoma, non-small cell lung cancer and colorectal cancer-A survey across 822 routine diagnostic cases.

Targeted deep massive parallel sequencing has been implemented in routine molecular diagnostics for high-throughput genetic profiling of formalin-fixed paraffin-embedded (FFPE) cancer samples. This approach is widely used to interrogate simple somatic mutations but experience with the analysis of copy number variations (CNV) is limited. Here, we retrospectively analyzed CNV in 822 cancer cases (135 melanoma, 468 non-small cell lung cancers (NSCLC), 219 colorectal cancers (CRC)). We observed a decreasing frequency of CNV in clinically actionable genes from melanoma to NSCLC to CRC. The overall cohort displayed 168 (20%) amplifications in 17 druggable targets. The majority of BRAF mutant melanomas (54%) showed co-occurring CNV in other genes, mainly affecting CDKN2A. Subsets showed clustered deletions in ABL1, NOTCH1, RET or STK11, GNA11, and JAK3. Most NRAS mutant melanomas (49%) harbored CNVs in other genes with CDKN2A and FGFR3 being most frequently affected. Five BRAF/NRASwt tumors had co-amplifications of KDR, KIT, PDGFRA and another six mutated KIT. Among all NSCLC, we identified 14 EGFRamp (with ten EGFRmut) and eight KRASamp (with seven KRASmut). KRASmut tumors displayed frequent amplifications of MYC (n = 10) and MDM2 (n = 5). Fifteen KRAS/EGFR/BRAFwt tumors had MET mutations/amplifications. In CRC, amplified IGF2 was most prevalent (n = 13) followed by MYC (n = 9). Two cases showed amplified KRAS wildtype alleles. Two of the KRASmut cases harbored amplifications of NRAS and three KRASwt cases amplification of EGFR. In conclusion, we demonstrate that our approach i) facilitates detection of CNV, ii) enables detection of known CNV patterns, and iii) uncovers new CNV of clinically actionable genes in FFPE tissue samples across cancers. © 2016 Wiley Periodicals, Inc.

Genotyping of colorectal cancer for cancer precision medicine: Results from the IPH Center for Molecular Pathology.

Cancer precision medicine has opened up new avenues for the treatment of colorectal cancer (CRC). To fully realize its potential, high-throughput sequencing platforms that allow genotyping beyond KRAS need to be implemented and require performance assessment. We comprehensively analyzed first-year data of 202 consecutive formalin-fixed paraffin embedded (FFPE) CRC samples for which prospective genotyping at our institution was requested. Deep targeted genotyping was done using a semiconductor-based sequencing platform and a self-designed panel of 30 CRC-related genes. Additionally, microsatellite status (MS) was determined. Ninety-seven percent of tumor samples were suitable for sequencing and in 88% MS could be assessed. The minimal drop-out rates of 6 and 25 cases, respectively were due to too low amounts or heavy degradation of DNA. Of 557 nonsynonymous mutations, 90 (16%) have not been described in COSMIC at the time of data query. Forty-three cases (22%) had double- or triple mutations affecting a single gene. Sixty-four percent had genetic alterations influencing oncological therapy. Eight percent of patients (MSI phenotype: 6%; mutated POLE: 2%) were potentially eligible for treatment with immune checkpoint inhibitors. Of 56% of KRASwt CRC that potentially qualified for anti-EGFR treatment, 30% presented with mutations in BRAF/NRAS. Mutated PIK3CA was detected in 21%. In conclusion, we here present real-life routine diagnostics data that not only demonstrate the robustness and feasibility of deep targeted sequencing and MS-analysis of FFPE CRC samples but also contribute to the understanding of CRC genetics. Most importantly, in more than half of the patients our approach enabled the selection of the best treatment currently available. © 2016 Wiley Periodicals, Inc.

TERT promoter hotspot mutations are recurrent in myxoid liposarcomas but rare in other soft tissue sarcoma entities.

BACKGROUND: Recently, recurrent point mutations in the telomerase reverse transcriptase (TERT) promoter region have been found in many human cancers, leading to a new transcription factor binding site, increased induction of TERT and subsequently to telomere maintenance. We determined the prevalence of TERT promoter mutations in soft tissue sarcomas of 341 patients comprising 16 entities and in 16 sarcoma cell lines covering 7 different soft tissue sarcoma types. METHODS: The sarcoma tissue samples were collected from the archives of the Institute of Pathology, University of Heidelberg and were composed of 39 myxoid liposarcomas (MLS), 61 dedifferentiated liposarcomas, 15 pleomorphic liposarcomas, 27 leiomyosarcomas, 25 synovial sarcomas (SS), 35 malignant peripheral nerve sheath tumors (MPNST), 40 undifferentiated pleomorphic sarcomas, 17 myxofibrosarcomas, 9 low grade fibromyxoid sarcomas, 10 cases of dermatofibrosarcoma protuberans, 31 solitary fibrous tumors (SFT), 8 extraskeletal myxoid chondrosarcomas, 9 angiosarcomas, 6 alveolar soft part sarcomas, 5 clear cell sarcomas and 4 epithelioid sarcomas. Sarcoma cell lines were obtained from the raising laboratories. A 193 bp fragment of the TERT promoter region covering the hot-spot mutations C228T and C250T was amplified, and direct sequencing of the PCR products was performed. RESULTS: TERT promoter mutations were detected in 36/341 sarcomas. They were highly recurrent in MLS (29/39; 74%) and were in the present MLS series not associated with the phenotype (myxoid vs. round cell variant), tumor grade, tumor site and patients' median age or gender. In the remaining cases, TERT promoter mutations were found only in 7/302 sarcoma samples and confined to SFTs (4/31; 13%), MPNSTs (2/35; 6%), and SSs (1/25; 4%). Within the collection of sarcoma cell lines examined, TERT promoter mutations were detected in two MLS and in one of three MPNST cell lines. CONCLUSIONS: TERT promoter mutations are frequent in MLSs including their round cell variants, representing the most prevalent mutation identified in this sarcoma entity to date, and in a minor fraction of SFTs, MPNSTs and SSs. The majority of sarcomas are devoid of TERT promoter hotspot mutations. These data suggest that telomere maintenance through increased expression of telomerase plays an important role in the pathogenesis especially of MLS.

EGFR, KRAS, BRAF and ALK gene alterations in lung adenocarcinomas: patient outcome, interplay with morphology and immunophenotype.

Numerous studies have been published on single aspects of pulmonary adenocarcinoma (ADC). To comprehensively link clinically relevant ADC characteristics, we evaluated established morphological, diagnostic and predictive biomarkers in 425 resected ADCs. Morphology was reclassified. Cytokeratin-7, thyroid transcription factor (TTF)1, napsin A, thymidylate synthase and excision repair cross-complementing rodent repair deficiency complementation group-1 expression, anaplastic lymphoma kinase rearrangements as well as epidermal growth factor receptor (EGFR), V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue (KRAS) and v-Raf murine sarcoma viral oncogene homologue B1 (BRAF) mutations were analysed. All characteristics were correlated with clinical and survival parameters. Morphological ADC subtypes were significantly associated with smoking history and distinct patterns of diagnostic biomarkers. KRAS mutations were prevalent in male smokers, while EGFR mutations were associated with female sex, nonsmoking and lepidic as well as micropapillary growth patterns. TTF1 expression (hazard ratio (HR) for overall survival 0.61, p=0.021) and BRAF mutations (HR for disease-free survival 2.0, p=0.046) were found to be morphology- and stage-independent predictors of survival in multivariate analysis. Adjuvant radio-/chemotherapy, in some instances, strongly impacted on the prognostic effect of both diagnostic and predictive biomarkers. Our data draw a comprehensive picture of the prevalence and interplay of established histological and molecular ADC characteristics. These data will help to develop time- and cost-effective diagnostic and treatment algorithms for ADC.

Clinical and molecular characteristics of HNSCC patients with brain metastases: a retrospective study.

Among the metastasis patterns of head and neck squamous cell carcinoma (HNSCC), intracranial spread is a rare but dreaded event. To date only very few cases have been reported and clinical and molecular data are sparse. We screened our archives for HNSCC patients from 1992 to 2005 who were diagnosed with brain metastases (BM). For retrospective analysis, all clinico-pathological data including disease-free survival (DFS), local progression-free survival (LPFS), and overall survival (OS) were compiled. Additionally, we assessed the mutational status of the TP53 gene and the prevalence of HPV serotypes by PCR and Sanger sequencing. Immunohistochemistry was applied to detect p16INK4A expression levels as surrogate marker for HPV infection. The prevalence rate of BM in our cohort comprising 193 patients with advanced HNSCC was 5.7%. Of 11 patients with BM, 3 were female and 9 were male. Seven of the primary tumors were of oropharyngeal origin (OPSCC). LPFS of the cohort was 11.8 months, DFS was 12.1 months and OS was 36.0 months. After the diagnosis of BM, survival was 10.5 months. Five tumors showed a mutation in the TP53 gene, while five of the seven OPSCC tumors had a positive HPV status displaying infection with serotype 16 in all cases. Compared with patients who harbored TP53wt/HPV-positive tumors, patients with TP53 mutations showed a poor prognosis. Compared with the whole cohort, the interval between diagnosis of the primary and the detection of BM was prolonged in the HPV-infected OPSCC subgroup (26.4 vs. 45.6 months). The prognosis of HNSCC patients with BM is poor. In our cohort, most tumors were OPSCC with the majority being HPV positive. Our study points toward a putatively unusual metastatic behavior of HPV-positive OPSCC.

Knowledge and valuation of Andean agroforestry species: the role of sex, age, and migration among members of a rural community in Bolivia.

BACKGROUND: Agroforestry is a sustainable land use method with a long tradition in the Bolivian Andes. A better understanding of people's knowledge and valuation of woody species can help to adjust actor-oriented agroforestry systems. In this case study, carried out in a peasant community of the Bolivian Andes, we aimed at calculating the cultural importance of selected agroforestry species, and at analysing the intracultural variation in the cultural importance and knowledge of plants according to peasants' sex, age, and migration. METHODS: Data collection was based on semi-structured interviews and freelisting exercises. Two ethnobotanical indices (Composite Salience, Cultural Importance) were used for calculating the cultural importance of plants. Intracultural variation in the cultural importance and knowledge of plants was detected by using linear and generalised linear (mixed) models. RESULTS AND DISCUSSION: The culturally most important woody species were mainly trees and exotic species (e.g.Schinus molle, Prosopis laevigata, Eucalyptus globulus). We found that knowledge and valuation of plants increased with age but that they were lower for migrants; sex, by contrast, played a minor role. The age effects possibly result from decreasing ecological apparency of valuable native species, and their substitution by exotic marketable trees,loss of traditional plant uses or the use of other materials (e.g. plastic) instead of wood. Decreasing dedication to traditional farming may have led to successive abandonment of traditional tool uses, and the overall transformation of woody plant use is possibly related to diminishing medicinal knowledge. CONCLUSIONS: Age and migration affect how people value woody species and what they know about their uses.For this reason, we recommend paying particular attention to the potential of native species, which could open promising perspectives especially for the young migrating peasant generation and draw their interest in agroforestry. These native species should be ecologically sound and selected on their potential to provide subsistence and promising commercial uses. In addition to offering socio-economic and environmental services,agroforestry initiatives using native trees and shrubs can play a crucial role in recovering elements of the lost ancient landscape that still forms part of local people's collective identity.

Optimized algorithm for Sanger sequencing-based EGFR mutation analyses in NSCLC biopsies.

Pulmonary adenocarcinoma patients harboring EGFR mutations can benefit from tyrosine kinase inhibitor therapy. Reliable molecular analyses and precise pathological reporting of the EGFR mutational status are factors essential for patient treatment and outcome. More than 70 % of all EGFR mutation analyses are performed on non-small cell lung cancer (NSCLC) biopsies. However, biopsies may not be sufficient for mutation analysis due to low tumor content and admixture with non-neoplastic cells. To define the minimal concentration of tumor cells required for reliable EGFR mutational diagnostics by Sanger sequencing and to develop an algorithm for routine diagnostics on biopsy material, we determined total numbers of tumor and non-tumor cells, calculated the tumor cell concentration and serially diluted DNA from EGFR-mutated NSCLC by adding DNA of non-tumor cells from the same section. A counted tumor cell concentration of 30 %, which refers to a histologically estimated concentration of 40 %, is necessary for reliable detection of all mutations. Based on these data, we developed an algorithm for evidence-based EGFR mutation analysis by Sanger sequencing in biopsy specimens, which was subsequently applied to 461 diagnostic cases. Optimized diagnostic testing results in 80 % reliable EGFR mutation analyses of biopsy specimens, while in 20 % of cases re-biopsies had to be recommended.

Potential of vetiver (vetiveria zizanioides (L.) Nash) for phytoremediation of petroleum hydrocarbon-contaminated soils in Venezuela.

Venezuela is one of the largest oil producers in the world. For the rehabilitation of oil-contaminated sites, phytoremediation represents a promising technology whereby plants are used to enhance biodegradation processes in soil. A greenhouse study was conducted to determine the tolerance of vetiver (Vetiveria zizanioides (L.) Nash) to a Venezuelan heavy crude oil in soil. Additionally, the plant's potential for stimulating the biodegradation processes of petroleum hydrocarbons was tested under the application of two fertilizer levels. In the presence of contaminants, biomass and plant height were significantly reduced. As for fertilization, the lower fertilizer level led to higher biomass production. The specific root surface area was reduced under the effects of petroleum. However, vetiver was found to tolerate crude-oil contamination in a concentration of 5% (w/w). Concerning total oil and grease content in soil, no significant decrease under the influence of vetiver was detected when compared to the unplanted control. Thus, there was no evidence of vetiver enhancing the biodegradation of crude oil in soil under the conditions of this trial. However, uses of vetiver grass in relation to petroleum-contaminated soils are promising for amelioration of slightly polluted sites, to allow other species to get established and for erosion control.

DNA microarray analysis of pancreatic malignancies.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. To improve the prognosis, novel molecular markers and targets for earlier diagnosis and adjuvant and/or neoadjuvant treatment are needed. Recent advances in human genome research and high-throughput molecular technologies make it possible to cope with the molecular complexity of malignant tumors. With DNA array technology, mRNA expression levels of thousand of genes can be measured simultaneously in a single assay. As several studies using microarrays in PDAC have already been published, this review attempts to compare the published data and therefore to validate the results. In addition, the applied techniques are discussed in the context of pancreatic malignancies.

TSC1 and TSC2 mutations in tuberous sclerosis, the associated phenotypes and a model to explain observed TSC1/ TSC2 frequency ratios.

UNLABELLED: Tuberous sclerosis (TSC) is a multisystem disease with manifestations in the central nervous system, skin, kidneys, heart, and other visceral organs. The development of TSC is associated with alterations within a gene on chromosome 9q34 ( TSC1) and a gene on chromosome 16p13 ( TSC2). Most de-novo patients show a mutation in TSC2, whereas only 50% of all familial cases can be related to TSC2 mutations. In the present study, 68 unrelated patients with confirmed clinical manifestations of TSC were tested for mutations in the TSC1 and TSC2 genes. In total, we studied 59 sporadic cases and 9 familial cases, including one large family with TSC2 linkage. Two pathogenic mutations were found in TSC1. The TSC2 gene analysis revealed 29 mutations, including 3 large deletions and 26 small mutations, 15 of them truncating. CONCLUSION: the TSC1-TSC2 mutation ratio in our group of patients differs significantly from the 1:1 ratio previously predicted on the basis of linkage studies. There is an obvious paradox between the observed frequency of TSC1 mutations in familial cases and sporadic cases. An interestingly mild phenotype, observed in one of our TSC1 mutation carriers, led to the elaboration of a model that provides a plausible explanation for this paradox. We propose the presence of a very mildly affected patient group with TSC1-related disease who are not regularly detected by clinical diagnosis.