Comprehensive serial biobanking in advanced NSCLC: feasibility, challenges and perspectives.

BACKGROUND: Availability of tumor material at baseline and disease progression is increasingly important for patient management in non-small-cell lung cancer (NSCLC), especially for the application of targeted therapies like tyrosine kinase inhibitors and for immune checkpoint inhibitor treatment. Here we report the experience of prospective biomaterial acquisition in advanced NSCLC from a pilot project. METHODS: Main objective was the longitudinal collection of high-quality, cryoconserved biopsies in addition to formalin-fixed paraffin-embedded (FFPE) biopsies required for routine diagnostics, along with blood samples and detailed clinical annotation using standardized questionnaires. RESULTS: Over five years, 205 patients were enrolled for the project, yielding 387 cryoconserved biopsies and 1,098 serum, plasma and buffy-coat samples. The feasibility of obtaining the cryoconserved biopsies in addition to the FFPE biopsies was 89% for newly diagnosed cases, but dropped down to 56% and 47% at first and second disease progression, respectively. While forceps biopsy was the preferred procedure for tissue acquisition, the highest tissue amounts were received using the cryobiopsy method. Biopsies had a median tumor cellularity of 34% and yielded in median 13.6 µg DNA and 12 µg RNA (median RIN =8). During the five-year project, a maximum of 38 follow-up blood samples per patient were assembled in up to four therapy lines. CONCLUSIONS: Despite the poor condition and limited prognosis of most NSCLC patients, this serial biomaterial acquisition including routine collection of cryoconserved biopsies is feasible to support individualized management. The standardized collection of high-quality material has enabled and enriched several translational research studies that can advance therapeutic options.

Mutation analysis of circulating plasma DNA to determine response to EGFR tyrosine kinase inhibitor therapy of lung adenocarcinoma patients.

Long-lasting success in lung cancer therapy using tyrosine kinase inhibitors (TKIs) is rare since the tumors develop resistance due to the occurrence of molecularly altered subclones. The aim of this study was to monitor tumors over time based on the quantity of mutant plasma DNA and to identify early indications for therapy response and tumor progression. Serial plasma samples from lung adenocarcinoma patients treated with TKIs were used to quantify EGFR and KRAS mutations in circulating DNA by digital PCR. Mutant DNA levels were compared with the courses of responses to treatment with TKIs, conventional chemotherapy, radiotherapy, or combinations thereof. Variations in plasma DNA mutation levels over time were found in 15 patients. We categorize three major courses: First, signs of therapy response are associated with a fast clearing of plasma DNA mutations within a few days. Second, periods of stable disease are accompanied by either absence of mutations or fluctuation at low levels. Finally, dramatic increase of mutational load is followed by rapid tumor progression and poor patient survival. In summary, the serial assessment of EGFR mutations in the plasma of NSCLC patients allows conclusions about controlled disease and tumor progression earlier than currently available methods.

Pseudokineococcus lusitanus gen. nov., sp. nov., and reclassification of Kineococcus marinus Lee 2006 as Pseudokineococcus marinus comb. nov.

A Gram-reaction-positive, motile, coccus-shaped actinobacterium, designated strain T2A-S27(T), was isolated from a roof tile in Oporto (Portugal) and studied using a polyphasic approach. The 16S rRNA gene sequence of the novel isolate showed high similarity to that of Kineococcus marinus KST3-3(T) (97.8 % sequence similarity). Strain T2A-S27(T) showed lower 16S rRNA gene sequence similarities with other members of the genus Kineococcus and members of the family Kineosporiaceae (<94 %). A phylogenetic tree, based on 16S rRNA gene sequences, showed that strain T2A-S27(T) formed a coherent clade with the type strain of K. marinus and Quadrisphaera granulorum. The isolate was characterized by the presence of meso-diaminopimelic acid in the cell-wall peptidoglycan, MK-9(H(2)) as the predominant menaquinone and a polar lipid profile consisting of diphosphatidylglycerol and phosphatidylglycerol. The fatty acid profile was dominated by anteiso-C(15 : 0). The DNA G+C content was 76.9 mol%. The low level of DNA-DNA relatedness to K. marinus (46-47 %) and the results of the chemotaxonomic and physiological studies clearly distinguished strain T2A-S27(T) from recognized species of the genus Kineococcus. On the basis of its phylogenetic position and phenotypic traits, strain T2A-S27(T) ( = LMG 24148(T)  = CECT 7306(T)  = DSM 23768(T)) represents a novel species of a new genus in the family Kineosporiaceae, for which the name Pseudokineococcus lusitanus gen. nov., sp. nov. is proposed. The misclassified species K. marinus is transferred to the new genus as Pseudokineococcus marinus comb. nov. The type strain of Pseudokineococcus marinus is KST3-3(T) ( = KCCM 42250(T)  = NRRL B-24439(T)).

Arousing sleeping genes: shifts in secondary metabolism of metal tolerant actinobacteria under conditions of heavy metal stress.

Numerous microbial habitats are strongly influenced by elevated levels of heavy metals. This type of habitat has developed either due to ore mining and metal processing or by pedogenesis above metal-rich base rocks. Most actinobacteria are soil-borne microbes with a remarkable capability for the synthesis of a broad variety of biologically active secondary metabolites. One major obstacle in identifying secondary metabolites, however, is the known phenomenon of sleeping gene clusters which are present, but silent under standard screening conditions. Here, we proceed to show that sleeping gene clusters can be awakened by the induction in heavy metal stress. Both, a chemical and a biological screening with extracts of supernatant and biomass of 10 strains derived from metal contaminated and non-contaminated environments was carried out to assay the influence of heavy metals on secondary metabolite patterns of metal tolerant actinobacteria. Metabolite patterns of cultures grown in complex and minimal media were compared to nickel (or cadmium) spiked parallels. Extracts of some strains grown in the presence of a metal salt displayed intense antibiosis against Escherichia coli, Mycobacterium smegmatis, Staphylococcus aureus and Candida albicans. Contrarily to the widely held opinion of metals as hindrance in secondary metabolism, metals thus can induce or enhance synthesis of possibly potent and medically relevant metabolites in metal tolerant strains. Hence, re-screening of existing strain libraries as well as identification of new strains from contaminated areas are valid strategies for the detection of new antibiotics in the future.

Direct detection of Kitasatospora species with a chaperone oligonucleotide microarray method lacking PCR amplification.

Identification of members of the genus Kitasatospora from soil samples has been introduced to evaluate occurrence of potential natural compound producers in different habitats. The microarray hybridization usually involves PCR amplification of the target DNA. Since PCR might lead to biased amplification, a diagnostic Kitasatospora microarray technique was improved by a protocol lacking PCR amplification prior to hybridization. The described advanced hybridization method used chaperone oligonucleotides for direct co-hybridization with genomic DNA on an oligonuclotide microarray with optical readout.

Pyrrole and indole alkaloids from an endophytic Fusarium incarnatum (HKI00504) isolated from the mangrove plant Aegiceras corniculatum.

Two new pyrrole alkaloids, N-[4-(2-formyl-5-hydroxymethyl-pyrrol-1-yl)-butyl]-acetamide (1) and N-[5-(2-formyl-5-hydroxymethyl-pyrrol-1-yl)-pentyl]-acetamide (2), and a new indole derivative (3aR,8aR)-3a-acetoxyl-1,2,3,3a,8,8a-hexahydropyrrolo-[2,3-b]indol (3) were isolated, together with ( - )-3a-hydroxyfuroindoline, (3aR,8aS)-1-acetyl-1,3,3a,8,8a-hexahydropyrrolo-[2,3-b]indol-3a-ol, and N-acetyltryptamine A, from an endophytic ascomycetous fungus, Fusarium incarnatum (HKI00504), which was isolated from the mangrove plant Aegiceras corniculatum. The structures of compounds 1-3 were determined on the basis of extensive spectroscopic data analyses.

Fodinicola feengrottensis gen. nov., sp. nov., an actinomycete isolated from a medieval mine.

A filamentous, Gram-positive actinobacterium was isolated from acidic rocks in a medieval alum slate mine and was investigated by means of a polyphasic taxonomic approach. A 16S rRNA gene sequence similarity study indicated that strain HKI 0501(T) forms an individual line of descent and is related to certain members of the suborder Frankineae, order Actinomycetales (<95 % sequence similarity). Distance-matrix and neighbour-joining analyses set the branching point of the novel isolate between two clades, one being represented by members of the genus Cryptosporangium (family 'Kineosporiaceae') and the other by members of the genera Frankia and Acidothermus (family Frankiaceae and family Acidothermaceae, respectively). The organism had meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan and xylose as the characteristic cell-wall sugar. The muramic acid in the peptidoglycan was found to be N-acetylated. The major menaquinones were MK-9(H(4)), MK-9(H(6)) and MK-9(H(8)) and the fatty acid profile was characterized by the predominance of iso-C(16 : 0), 10-methyl C(17 : 0), C(17 : 1) cis9 and 10-methyl iso-C(18 : 0). The polar lipids comprised diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and several unknown phospholipids and glycolipids. Mycolic acids were absent. The DNA G+C content was 65 mol%. The distinct phylogenetic position and the phenotypic markers that clearly separate the novel organism from all other members of the suborder Frankineae indicate that strain HKI 0501(T) represents a novel genus and species, for which the name Fodinicola feengrottensis gen. nov., sp. nov. is proposed. The type strain of Fodinicola feengrottensis is HKI 0501(T) (=DSM 19247(T) =JCM 14718(T)).

A-seco-oleane-type triterpenes from Phomopsis sp. (strain HKI0458) isolated from the mangrove plant Hibiscus tiliaceus.

From the fermentation broth of an unidentified Phomopsis sp. (strain HKI0458) isolated from the mangrove plant Hibiscus tiliaceus, four A-seco-oleane-type triterpenes, namely 3,4-seco-olean-11,13-dien-4,15alpha, 22beta,24-tetraol-3-oic acid (1), 3,4-seco-olean-11,13-dien-4,7beta,22beta,24-tetraol-3-oic acid (2), 3,4-seco- olean-13-en-4,7,15,22,24-pentaol-3-oic acid (3), and 3,4-seco-olean-13-en-4,15,22,24-tetraol-3-oic acid (4) were obtained. Their structures were elucidated by extensive spectroscopic (UV, IR, FABMS, and 2D NMR) data analyses.

Burkholderia rhizoxinica sp. nov. and Burkholderia endofungorum sp. nov., bacterial endosymbionts of the plant-pathogenic fungus Rhizopus microsporus.

Several strains of the fungus Rhizopus microsporus harbour endosymbiotic bacteria for the production of the causal agent of rice seedling blight, rhizoxin, and the toxic cyclopeptide rhizonin. R. microsporus and isolated endobacteria were selected for freeze-fracture electron microscopy, which allowed visualization of bacterial cells within the fungal cytosol by their two parallel-running envelope membranes and by the fine structure of the lipopolysaccharide layer of the outer membrane. Two representatives of bacterial endosymbionts were chosen for phylogenetic analyses on the basis of full 16S rRNA gene sequences, which revealed that the novel fungal endosymbionts formed a monophyletic group within the genus Burkholderia. Inter-sequence similarities ranged from 98.94 to 100%, and sequence similarities to members of the Burkholderia pseudomallei group, the closest neighbours, were 96.74-97.38%. In addition, the bacterial strains were distinguished from their phylogenetic neighbours by their fatty acid profiles and other biochemical characteristics. The phylogenetic studies based on 16S rRNA gene sequence data, together with conclusive DNA-DNA reassociation experiments, strongly support the proposal that these strains represent two novel species within the genus Burkholderia, for which the names Burkholderia rhizoxinica sp. nov. (type strain, HKI 454T=DSM 19002T=CIP 109453T) and Burkholderia endofungorum sp. nov. (type strain, HKI 456T=DSM 19003T=CIP 109454T) are proposed.

Kribbella aluminosa sp. nov., isolated from a medieval alum slate mine.

Three actinomycetes (strains HKI 0478(T), HKI 0479 and HKI 0480) isolated from the surfaces of rocks in the Feengrotten medieval alum slate mine (Thuringia, Germany) were examined in a polyphasic taxonomic study. The following morphological and chemotaxonomic features supported their classification as members of the genus Kribbella: the presence of ll-diaminopimelic acid in the cell-wall peptidoglycan; glucose together with minor amounts of mannose and ribose as the whole-cell sugars; polar lipids comprising phosphatidylcholine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and unknown phospho- and glycolipids; fatty acid profiles characterized by the predominance of anteiso-C(15 : 0), iso-C(16 : 0) and C(16 : 0) 9-methyl; and the presence of MK-9(H(4)) as the main menaquinone. The isolates had almost identical 16S rRNA gene sequences (99.9-100 %) and were most closely related to the type strains of Kribbella jejuensis (98.9 % sequence similarity), Kribbella swartbergensis and Kribbella solani (both 98.8 %). A wide range of genotypic and phenotypic markers as well as the low levels of DNA-DNA relatedness between strain HKI 0478(T) and the type strains of K. jejuensis (41.3 %), K. swartbergensis (18.6 %) and K. solani (14.2 %) distinguished the novel strains from their closest phylogenetic neighbours. On the basis of these results, strain HKI 0478(T) represents a novel member of the genus Kribbella, for which the name Kribbella aluminosa sp. nov. is proposed. The type strain is HKI 0478(T) (=DSM 18824(T) =JCM 14599(T)).

Amycolatopsis saalfeldensis sp. nov., a novel actinomycete isolated from a medieval alum slate mine.

Three actinomycetes isolated from the surfaces of rocks in a medieval slate mine were examined in a polyphasic taxonomic study. Chemotaxonomic and morphological characteristics of the isolates were typical of strains of the genus Amycolatopsis. The isolates had identical 16S rRNA gene sequences and formed a distinct phyletic line towards the periphery of the Amycolatopsis mediterranei clade, being most closely related to Amycolatopsis rifamycinica. The organisms shared a wide range of genotypic and phenotypic markers that distinguished them from their closest phylogenetic neighbours. On the basis of these results, a novel species, Amycolatopsis saalfeldensis sp. nov., is proposed. The type strain is HKI 0457(T) (=DSM 44993(T)=NRRL B-24474(T)).

Amycolatopsis nigrescens sp. nov., an actinomycete isolated from a Roman catacomb.

The taxonomic status of two actinomycetes isolated from the wall of a hypogean Roman catacomb was established based on a polyphasic investigation. The organisms were found to have chemical and morphological markers typical of members of the genus Amycolatopsis. They also shared a range of chemical, molecular and phenotypic markers which served to separate them from representatives of recognized Amycolatopsis species. The new isolates formed a branch in the Amycolatopsis 16S rRNA gene sequence tree with Amycolatopsis minnesotensis NRRL B-24435(T), but this association was not supported by a particularly high bootstrap value or by the product of the maximum-parsimony tree-making algorithm. The organisms were distinguished readily from closely related Amycolatopsis species based on a combination of phenotypic properties and from all Amycolatopsis strains by their characteristic menaquinone profiles, in which tetra-hydrogenated menaquinones with 11 isoprene units predominated. The combined genotypic and phenotypic data indicate that the isolates merit recognition as representing a novel species of the genus Amycolatopsis. The name proposed for this novel species is Amycolatopsis nigrescens sp. nov., with type strain CSC17Ta-90(T) (=HKI 0330(T)=DSM 44992(T)=NRRL B-24473(T)).

Practical thin layer chromatography techniques for diaminopimelic acid and whole cell sugar analyses in the classification of environmental actinomycetes.

For the determination of diaminopimelic acid (DAP) stereoisomers in whole cell hydrolysates as chemotaxonomic markers of actinomycetes, two new pyridine-free solvent systems for TLC on cellulose sheets have been introduced: methanol/0.05 m potassium hydrogenphthalate buffer pH 4 2:1 (v/v) and methanol/0.12 M dimethylaminopyridine (DMAP) in H(2)O pH 6 2:1 (v/v). The commercial (Sigma) DAP standard can be separated by repeated TLC into its three stereoisomers. Addition of a single stereoisomer to the samples supported the detection (presence or absence) of the relevant stereoisomer by HPLC. In the study on whole cell sugars, TLC both on cellulose and on silica gel revealed to be successful in the separation of the components in modified pyridine-free solvent systems. The procedures of DAP and sugar analyses are summarized in two flow-charts.

Myceligenerans crystallogenes sp. nov., isolated from Roman catacombs.

Three xylan-degrading actinobacterial strains were isolated from different sampling sites in the Roman catacombs of Domitilla and San Callisto. The organisms showed morphological and chemotaxonomic properties such as peptidoglycan type A4alpha, L-lys-L-thr-D-Glu; whole-cell sugars (glucose, mannose and galactose); octa-, hexa- and tetrahydrogenated menaquinones with nine isoprene units; phosphatidylglycerol and diphosphatidylglycerol as the major phospholipids; anteiso-C(15 : 0), iso-C(15 : 0) and iso-C(16 : 0) as the predominant fatty acids; and a DNA G+C content of 72 mol%. These features are consistent with affiliation of these isolates to the genus Myceligenerans. The three isolates shared a 16S rRNA gene similarity of 99.9 % and were most closely related to Myceligenerans xiligouense DSM 15700T (97.9 % sequence similarity). The low level of DNA-DNA relatedness (about 14 %) and the differences in phenotypic characteristics between the novel strains and M. xiligouense DSM 15700T justify the proposal of a novel species of the genus Myceligenerans, Myceligenerans crystallogenes sp. nov., with CD12E2-27T (= HKI 0369T = DSM 17134T = NCIMB 14061T = VTT E-032285T) as the type strain.

Design and evaluation of an oligonucleotide-microarray for the detection of different species of the genus Kitasatospora.

An oligonucleotide-microarray method was developed for the detection of Kitasatospora species in soil samples. The 16S-23S rDNA internal transcribed spacer (ITS) sequence of these antibiotics-producing actinomycetes was applied to design short oligonucleotide probes. Two different 26-mers were synthesized, specific to each species used. Additionally, four oligonucleotide probes were designed to evaluate the system. The oligonucleotides were spotted onto slides of the ArrayTube microarray system and examined with a new silver-labeling detection technique. Prior to hybridization analysis, the 16S-23S rDNA were amplified by polymerase chain reaction both from bacterial cells and environmental samples using two actinomycetes specific primers containing a 5' biotin labeling. The type strains of eight Kitasatospora species included in this study were K. phosalacinea DSM 43860, K. setae DSM 43861, K. cochleata DSM 41652, K. cystarginea DSM 41680, K. azatica DSM 41650, K. mediocidica DSM 43929, K. paracochleata DSM 41656, and K. griseola DSM 43859. The actinomycetes-specific primers were shown to amplify the entire 16S-23S rDNA ITS region from all tested strains. More importantly, the described technique allows the detection of Kitasatospora strains from soil samples by extracting metagenomic DNA followed by a PCR amplification step. This indicates that the oligonucleotide-microarray method developed in this study is a reliable tool for the detection of Kitasatospora species in environmental samples.

Structure determination of germacrane-type sesquiterpene alcohols from an endophyte Streptomyces griseus subsp.

Three germacrane-type sesquiterpene alcohols were isolated from an endophyte of mangrove plant Kandelia candel. Their structures were characterized as 1(10)E,5E-germacradiene-11-ol (1), 1(10)E,5E-germacradiene-3,11-diol (2), 1(10)E,5E-germacradiene-2,11-diol (3) based on the extensive NMR studies. Among them, 2 and 3 are identified as new compounds.

Agromyces subbeticus sp. nov., isolated from a cave in southern Spain.

An actinomycete, strain Z33(T), was isolated from a cyanobacterial biofilm in the Cave of Bats, near Zuheros (Cordoba, southern Spain). 16S rRNA gene sequence analysis showed that strain Z33(T) formed a distinct phyletic line within the genus Agromyces. This isolate could be readily distinguished from representatives of all recognized Agromyces species on the basis of a broad range of phenotypic characteristics and DNA-DNA relatedness data. Genotypic and phenotypic properties indicate that strain Z33(T) represents a novel species, for which the name Agromyces subbeticus sp. nov. is proposed. The type strain is Z33(T) (=HKI 0340(T)=DSM 16689(T)=NCIMB 14025(T)).

Isoptericola hypogeus sp. nov., isolated from the Roman catacomb of Domitilla.

In order to clarify the taxonomic position of an actinobacterium from the Roman catacomb of Domitilla, a combination of phenotypic characterization, phylogenetic analysis based on the 16S rRNA gene sequence and DNA-DNA relatedness studies was used. The results from the polyphasic taxonomic study of this organism showed that strain HKI 0342(T) (=DSM 16849(T)=NCIMB 14033(T)) should be considered as the type strain of a novel species of the genus Isoptericola, for which the name Isoptericola hypogeus sp. nov. is proposed.

Cervimycin A-D: a polyketide glycoside complex from a cave bacterium can defeat vancomycin resistance.

Cervimycins A-D are novel polyketide glycosides with significant activity against multi-drug-resistant staphylococci and vancomycin-resistant enterococci. They are produced by a strain of Streptomyces tendae, isolated from an ancient cave. The structures of the cervimycins were determined by performing extensive NMR and chemical degradation studies. All cervimycins have a common tetracyclic polyketide core that is substituted with unusual di- and tetrasaccharide chains, composed exclusively of trideoxysugars; however, they differ in the acetyl and carbamoyl ring substituent and in the highly unusual terminal methylmalonyl and dimethylmalonyl residues.

Agromyces italicus sp. nov., Agromyces humatus sp. nov. and Agromyces lapidis sp. nov., isolated from Roman catacombs.

A polyphasic study was carried out to clarify the taxonomic positions of three Gram-positive isolates from the Catacombs of Domitilla, Rome (Italy). 16S rRNA gene sequence comparisons placed these strains within the genus Agromyces. The morphological and chemotaxonomic characteristics of these isolates were consistent with the description of the genus Agromyces. The three isolates could be readily distinguished from one another and from representatives of all Agromyces species with validly published names by a broad range of phenotypic characteristics and DNA-DNA relatedness studies. Therefore, these isolates are proposed to represent three novel species of the genus Agromyces, Agromyces italicus sp. nov. (type strain CD1(T)=HKI 0325(T)=DSM 16388(T)=NCIMB 14011(T)), Agromyces humatus sp. nov. (type strain CD5(T)=HKI 0327(T)=DSM 16389(T)=NCIMB 14012(T)) and Agromyces lapidis sp. nov. (type strain CD55(T)=HKI 0324(T)=DSM 16390(T)=NCIMB 14013(T)).