Proteomic analysis of brain metastatic lung adenocarcinoma reveals intertumoral heterogeneity and specific alterations associated with the timing of brain metastases.

BACKGROUND: Brain metastases are associated with considerable negative effects on patients' outcome in lung adenocarcinoma (LADC). Here, we investigated the proteomic landscape of primary LADCs and their corresponding brain metastases. MATERIALS AND METHODS: Proteomic profiling was conducted on 20 surgically resected primary and brain metastatic LADC samples via label-free shotgun proteomics. After sample processing, peptides were analyzed using an Ultimate 3000 pump coupled to a QExactive HF-X mass spectrometer. Raw data were searched using PD 2.4. Further data analyses were carried out using Perseus, RStudio and GraphPad Prism. Proteomic data were correlated with clinical and histopathological parameters and the timing of brain metastases. Mass spectrometry-based proteomic data are available via ProteomeXchange with identifier PXD027259. RESULTS: Out of the 6821 proteins identified and quantified, 1496 proteins were differentially expressed between primary LADCs and corresponding brain metastases. Pathways associated with the immune system, cell-cell/matrix interactions and migration were predominantly activated in the primary tumors, whereas pathways related to metabolism, translation or vesicle formation were overrepresented in the metastatic tumors. When comparing fast- versus slow-progressing patients, we found 454 and 298 differentially expressed proteins in the primary tumors and brain metastases, respectively. Metabolic reprogramming and ribosomal activity were prominently up-regulated in the fast-progressing patients (versus slow-progressing individuals), whereas expression of cell-cell interaction- and immune system-related pathways was reduced in these patients and in those with multiple brain metastases. CONCLUSIONS: This is the first comprehensive proteomic analysis of paired primary tumors and brain metastases of LADC patients. Our data suggest a malfunction of cellular attachment and an increase in ribosomal activity in LADC tissue, promoting brain metastasis. The current study provides insights into the biology of LADC brain metastases and, moreover, might contribute to the development of personalized follow-up strategies in LADC.

A standardised diagnostic approach to pituitary neuroendocrine tumours (PitNETs): a European Pituitary Pathology Group (EPPG) proposal.

The 2017 World Health Organization (WHO) classification proposes to type and subtype primary adenohypophyseal tumours according to their cell lineages with the aim to establish more uniform tumour groups. The definition of atypical adenoma was removed in favour of high-risk adenoma, and the assessment of proliferative activity and invasion was recommended to diagnose aggressive tumours. Recently, the International Pituitary Pathology Club proposed to replace adenoma with the term of pituitary neuroendocrine tumour (PitNET) to better reflect the similarities between adenohypophyseal and neuroendocrine tumours of other organs. The European Pituitary Pathology Group (EPPG) endorses this terminology and develops practical recommendations for standardised reports of PitNETs that are addressed to histo- and neuropathologists. This brief report presents the results of EPPG's consensus for the reporting of PitNETs and proposes a diagnostic algorithm.

Next-generation sequencing identifies novel mitochondrial variants in pituitary adenomas.

PURPOSE: Disrupted mitochondrial functions and genetic variants of mitochondrial DNA (mtDNA) have been observed in different human neoplasms. Next-generation sequencing (NGS) can be used to detect even low heteroplasmy-level mtDNA variants. We aimed to investigate the mitochondrial genome in pituitary adenomas by NGS. METHODS: We analysed 11 growth hormone producing and 33 non-functioning [22 gonadotroph and 11 hormone immunonegative] pituitary adenomas using VariantPro™ Mitochondrion Panel on Illumina MiSeq instrument. Revised Cambridge Reference Sequence (rCRS) of the mtDNA was used as reference. Heteroplasmy was determined using a 3% cutoff. RESULTS: 496 variants were identified in pituitary adenomas with overall low level of heteroplasmy (7.22%). On average, 35 variants were detected per sample. Samples harbouring the highest number of variants had the highest Ki-67 indices independently of histological subtypes. We identified eight variants (A11251G, T4216C, T16126C, C15452A, T14798C, A188G, G185A, and T16093C) with different prevalences among different histological groups. T16189C was found in 40% of non-recurrent adenomas, while it was not present in the recurrent ones. T14798C and T4216C were confirmed by Sanger sequencing in all 44 samples. 100% concordance was found between NGS and Sanger method. CONCLUSIONS: NGS is a reliable method for investigating mitochondrial genome and heteroplasmy in pituitary adenomas. Out of the 496 detected variants, 414 have not been previously reported in pituitary adenoma. The high number of mtDNA variants may contribute to adenoma genesis, and some variants (i.e., T16189C) might associate with benign behaviour.

Breast cancer brain metastases show increased levels of genomic aberration-based homologous recombination deficiency scores relative to their corresponding primary tumors.

Background: Based on its mechanism of action, PARP inhibitor therapy is expected to benefit mainly tumor cases with homologous recombination deficiency (HRD). Therefore, identification of tumor types with increased HRD is important for the optimal use of this class of therapeutic agents. HRD levels can be estimated using various mutational signatures from next generation sequencing data and we used this approach to determine whether breast cancer brain metastases show altered levels of HRD scores relative to their corresponding primary tumor. Patients and methods: We used a previously published next generation sequencing dataset of 21 matched primary breast cancer/brain metastasis pairs to derive the various mutational signatures/HRD scores strongly associated with HRD. We also carried out the myChoice HRD analysis on an independent cohort of 17 breast cancer patients with matched primary/brain metastasis pairs. Results: All of the mutational signatures indicative of HRD showed a significant increase in the brain metastases relative to their matched primary tumor in the previously published whole exome sequencing dataset. In the independent validation cohort, the myChoice HRD assay showed an increased level in 87.5% of the brain metastases relative to the primary tumor, with 56% of brain metastases being HRD positive according to the myChoice criteria. Conclusions: The consistent observation that brain metastases of breast cancer tend to have higher HRD measures may raise the possibility that brain metastases may be more sensitive to PARP inhibitor treatment. This observation warrants further investigation to assess whether this increase is common to other metastatic sites as well, and whether clinical trials should adjust their strategy in the application of HRD measures for the prioritization of patients for PARP inhibitor therapy.

Simulating the selfing and migration of Luehea divaricata populations in the Pampa biome to investigate the conservation potential of their genetic resources.

Computer simulations are an important tool for developing conservation strategies for forest species. This study used simulations to investigate the genetic, ecological, and reproductive patterns that contribute to the genetic structure of the tree Luehea divaricata Mart. & Zucc. in five forest fragments in the Brazilian Pampa biome. Using the EASYPOP model, we determined the selfing and migration rates that would match the corresponding genetic structure of microsatellite marker data (based on observed and expected heterozygosity parameters). The simulated reproductive mode was mixed, with a high rate of outcrossing (rate = 0.7). This was consistent with a selfing-incompatible system in this species, which reduced, but did not prevent, selfing. The simulated migration rate was 0.02, which implied that the forest fragments were isolated by distance, and that the inbreeding coefficients were high. Based on Nei's gene diversity analysis, 94% of the genetic variability was distributed within the forest fragments, and only 6% of the genetic diversity was caused by differences between them. Furthermore, the minimum viable population and minimum viable area genetic conservation parameters (which determine conservation potential in the short and long term) suggested that only the Inhatinhum forest fragment had the short-term potential to maintain its genetic diversity. However, in the long term, none of the forest fragments proved to be sustainable, indicating that the populations will require intervention to prevent a decline in genetic variability. The creation of ecological corridors could be a useful solution to connect forest fragments and enhance gene flow between them.

Morphological and molecular characterization of Cladosporium cladosporioides species complex causing pecan tree leaf spot.

The objective of this study was to characterize species of the Cladosporium cladosporioides complex isolated from pecan trees (Carya illinoinensis) with symptoms of leaf spot, based on morphological and molecular approaches. Morphological attributes were assessed using monosporic cultures on potato dextrose agar medium, which were examined for mycelial growth, sporulation, color, and conidia and ramoconidia size. Molecular characterization comprised isolation of DNA and subsequent amplification of the translation elongation factor 1α (TEF-1α) region. Three species of the C. cladosporioides complex were identified: C. cladosporioides, Cladosporium pseudocladosporioides, and Cladosporium subuliforme. Sporulation was the most important characteristic differentiating species of this genus. However, morphological features must be considered together with molecular analysis, as certain characters are indistinguishable between species. TEF-1αcan be effectively used to identify and group isolates belonging to the C. cladosporioides complex. The present study provides an important example of a methodology to ascertain similarity between isolates of this complex causing leaf spot in pecan trees, which should facilitate future pathogenicity studies.

Study of the genetic diversity and structure of a natural population of Nectandra megapotamica (Spreng.) Mez. using RAPD markers.

Nectandra megapotamica (Spreng.) Mez. is a tree species that naturally occurs in the Atlantic Forest, Brazil. The aim of this study was to evaluate the genetic diversity and structure of a natural population of 12 N. megapotamica individuals using random amplified polymorphic DNA markers. Eleven primers were used in this study, producing 81 bands, of which 98.99% were polymorphic. Analysis using STRUCTURE defined three different clusters (K = 3), results that were consistent with those of principal coordinates analysis. Both Nei's genetic diversity (h = 0.33) and Shannon's diversity index (I = 0.49) were relatively high. Analysis of molecular variance indicated that 24.89% of the genetic variability was among clusters, while the remaining 75.11% was within clusters. The Mantel test showed a weak correlation between genetic and geographic distances (r = 0.25, P = 0.105). Overall, the results revealed high levels of genetic diversity within clusters and high genetic differentiation among clusters without any spatial pattern of genetic variability. In addition, gene flow was independent of the geographical distribution and was compatible with the hierarchical island model.

Morphological and molecular characterization of Fusarium spp pathogenic to pecan tree in Brazil.

The occurrence of Fusarium spp associated with pecan tree (Carya illinoinensis) diseases in Brazil has been observed in recent laboratory analyses in Rio Grande do Sul State. Thus, in this study, we i) obtained Fusarium isolates from plants with disease symptoms; ii) tested the pathogenicity of these Fusarium isolates to pecan; iii) characterized and grouped Fusarium isolates that were pathogenic to the pecan tree based on morphological characteristics; iv) identified Fusarium spp to the species complex level through TEF-1α sequencing; and v) compared the identification methods used in the study. Fifteen isolates collected from the inflorescences, roots, and seeds of symptomatic plants (leaf necrosis or root rot) were used for pathogenicity tests. Morphological characterization was conducted using only pathogenic isolates, for a total of 11 isolates, based on the mycelial growth rate, sporulation, colony pigmentation, and conidial length and width variables. Pathogenic isolates were grouped based on morphological characteristics, and molecular characterization was performed by sequencing TEF-1α genes. Pathogenic isolates belonging to the Fusarium chlamydosporum species complex, Fusarium graminearum species complex, Fusarium proliferatum, and Fusarium oxysporum were identified based on the TEF-1α region. Morphological characteristics were used to effectively differentiate isolates and group the isolates according to genetic similarity, particularly conidial width, which emerged as a key morphological descriptor in this study.

First Report of "Cylindrocarpon" pauciseptatum Associated with Black Foot Disease of Grapevine in Brazil.

Since 1999, the decline of American grapevines (Vitis labrusca L.) has been common in Rio Grande do Sul, Brazil (1). In August 2012, V. labrusca with black foot symptoms were collected in vineyards in the Serra Gaúcha Region. Symptomatic plants had low vigor, vascular lesions, delayed budding, and decline and death of vines. Symptomatic roots had necrotic lesions and reduced biomass. Fungal isolations were made from necrotic root and crown fragments (own-rooted cultivar) on potato dextrose agar (PDA) medium amended with 0.5 g L-1 streptomycin sulfate. Putative colonies of "Cylindrocarpon" pauciseptatum Schroers & Crous were obtained from single macroconidia isolations. Two isolates were used to confirm the identity of isolated colonies: Cy12UFSM and Cy13UFSM. After incubation in the dark for 10 days at 20°C, the isolated mycelial colonies, which were cottony white to felty in texture, became dark orange to brown. Both isolates produced chlamydospores in chains at 40 days. Chlamydospores of Cy12UFSM and Cy13UFSM were 9 to 12 µm and 5 to 11.5 µm in diameter. Sporodochia formation on carnation leaf agar (CLA) medium was observed after 30 days. To encourage development of conidia, the isolates were grown on spezieller nährstoffarmer agar (SNA) medium for five weeks at 20°C with addition of two pieces of 1 cm2 filter paper. Microconidia of Cy12UFSM were 4 to 8.5 × 3.5 to 5 µm and those of Cy13UFSM were 3.5 to 7.5 × 3 to 5 µm. Macroconida were predominantly 3-septate (Cy12UFSM was 36 to 45 × 7.5 to 9 µm and Cy13UFSM was 30 to 38 × 7.5 to 8 µm), but 1-, 2- septate macroconidia were observed. The sizes of the three spore types and colony morphology for our isolates were similar to those described by Schroers et al. (3) for "C." pauciseptatum. To further confirm the identity of Cy12UFSM and Cy13UFSM, multi-gene DNA sequence analysis (rDNA-ITS, ß-tubulin, and histone H3) was conducted using primer pairs ITS1 and ITS4 (4), Bt2a and Bt2b, and H3-1a and H3-1b (2), which amplify the ITS1-5.8S rRNA-ITS2 genes, part of the ß-tubulin gene, and the histone H3 gene, respectively. Sequences of these three regions had 99, 99, and 97% similarity with references sequences of "C." pauciseptatum (isolate Cy238; accessions ITS [JF735307]; ß-tubulin [JF735435], and histone H3 [JF735582], respectively). To evaluate pathogenicity, 4-month-old rooted cuttings of V. labrusca cv. Bordô were inoculated with two isolates by immersing them in a conidial suspension (106 conidia ml-1) for 60 min. Ten single-vine replicates were used for each isolate, and 10 water-inoculated vines were included as controls. Thirty days after inoculation, vines were re-inoculated with 40 ml of a 106 conidia ml-1 suspension to ensure root infection. After 4 months, the inoculated plants had reduced root mass relative to controls (39.18% for Cy12UFSM and 18.27% for Cy13UFSM). Inoculated plants also had root and crown necrosis, vascular lesions, shoot decline, and vine mortality (60 and 80% mortality for Cy12UFSM and Cy13UFSM, respectively). All water-inoculated control plants remained symptomless. The fungi Cy12UFSM and Cy13UFSM were re-isolated from infected woody tissues, confirming Koch's postulates. To our knowledge, this is the first report of "C." pauciseptatum associated with black foot disease of grapevine in Brazil, which may potentially impact the sustainability of grapevine nurseries and vineyard productivity. References: (1) L. R. Garrido et al. Fitopatol. Brasil. 29:548, 2004. (2) N. L. Glass et al. Appl. Environ. Microbiol. 61:1323, 1995. (3) H. J. Schoers et al. Mycol. Res. 112:82, 2008. (4) T. J. White et al. Amplification Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Multiplicação in vitro de segmentos apicais caulinares de segurelha (Satureja hortensis L. ) / In vitro multiplication of apical shoot segments of Summer Savory (Satureja hortensis L. )

O presente estudo teve por objetivo avaliar o efeito de BAP, na presença e ausência de ANA, sobre a multiplicação in vitro de segmentos apicais caulinares de Satureja hortensis. Os explantes foram isolados de plântulas germinadas in vitro e cultivados em meio nutritivo MS. O delineamento experimental utilizado foi o inteiramente casualizado, em esquema fatorial 2x5, correspondendo às concentrações de ANA (0 e 1 µM) e de BAP (0; 5; 10; 15 e 20 µM), com seis repetições, cada uma composta por três explantes. Para a porcentagem de explantes com brotações houve efeito significativo para o fator BAP, aumentando à medida que cresce a concentração da citocinina. A variável "número de brotos por explante" apresentou interação entre os fatores, havendo a maior formação de brotos na presença de ANA na faixa de 10 - 15 µM de BAP. Na presença da auxina, o maior valor ocorreu com 15 µM de BAP tendendo a diminuir independente da presença de ANA. O enraizamento dos segmentos apicais foi maior na presença de ANA e ausência de BAP, diminuindo com o aumento da citocinina. O número de folhas foi influenciado pela maior concentração de BAP sendo que a 15 µM foi observado o maior número, tendendo à queda com aumento da concentração. A concentração de 15 µM de BAP, independente de ANA, proporcionou os melhores resultados na multiplicação da espécie produzindo aumento de brotações e número de folhas, à exceção do enraizamento que foi influenciado pela auxina.

This study aimed to evaluate the effect of BAP in the presence and absence of NAA, on the in vitro multiplication of shoot apical segments of Satureja hortensis. The explants were isolated from seedlings germinated in vitro and cultured in nutrient medium MS. The experimental design was completely randomized in a 2x5 factorial arrangement, corresponding to the concentrations of NAA (0 and 1 µM) and BAP (0, 5, 10, 15 and 20 µM), with six replicates, each consisting of three explants. For the percentage of explants with shoots, there was no significant effect for the factor BAP, increasing as the concentration of BAP grows. The variable number of shoots per explant showed interaction between factors, with the highest shoot formation in the presence of NAA and BAP in the range of 10 -15 µM. In the presence of auxin, the highest value occurred with 15 µM BAP, tending to decline regardless of the presence of NAA. The rooting of the apical segments was higher in the presence of NAA and absence of BAP, decreasing with increasing cytokinin. Leaf number was influenced by the higher concentration of BAP, and the amount of 15 µMhadthe largest number, tending to decrease with increasing concentration. The concentration of 15 µM BAP, regardless of NAA, provided better results in the multiplication of the species, producing increased number of shoots and leaves, except for rooting, whichwas influenced by auxin.

First Report of Fusarium sambucinum Associated on Pinus elliottii Seeds in Brazil.

An elevated incidence of the fungal genus Fusarium was ascertained during a health quality analysis of a batch of Pinus elliottii Englm. seeds obtained from the Florestas Institute for Agricultural and Forest Research (Fundação Estadual de Pesquisa Agropecuária [FEPAGRO] Florestas) in Santa Maria (29° 39' 55″ S and 53° 54' 45″ W), state of Rio Grande do Sul, Brazil. This genus comprised about 75% of all fungal genera observed in a blotter test. The fungus was then isolated and purified to perform pathogenicity tests. Healthy seeds of P. elliottii were inoculated by contact with fungal mycelium for 48 h (3). Forty-two days after inoculation, a reduction was observed in the germination potential of the seeds; however, those seeds that germinated developed normally until, as seedlings, they suffered damping-off. Fusarium was isolated from the affected vegetal material by transferring mycelium tips to potato dextrose agar (PDA) medium in petri dishes in order to morphologically identify the species. After 72 h, a tan mycelial pad 5.5 cm in diameter had formed. After transfer to carnation leaf agar (CLA), pale orange sporodochia that formed macroconidia could be observed. The macronidia were relatively short and narrow (40.2 × 4.7 µm), each containing a mean of 5 septa; the apical cell was pointed, while the basal one was foot-shaped (2,4). The chlamydospores formed in clusters, while the conidiogenous cells could be seen on top of monophialides. Primer pairs ITS1 and ITS4, EF1-T and EF1-567R, and ßtub-F and ßtub were employed to amplify the three regions ITS1.8S ITS2, elongation factor - 1α (TEF 1-α), and ß-tubulin, respectively. The sequences of these three regions showed 97, 95, and 99% of similarity with Fusarium sambucinum Fückel, respectively. The pathogen was reinoculated on P. elliottii seeds in order to complete Koch's postulates. The pathogenicity test was repeated with the same conditions described before and the results were confirmed. No occurrence of damping-off was observed in the control seedlings. The inoculated seedlings showed, besides damping-off, a visible reduction in root system expansion as well as reductions in fresh and dry tissue weight. F. sambucinum has already been reported on P. radiata D. Don in New Zealand, causing root rot and dieback (1); however, in Brazil, the present study is, to the best of our knowledge, the first to report the association of this pathogen with P. elliottii. References: (1) M. A. Dick and K. Dobbie. N. Z. Plant Prot. 55:58, 2002. (2) W. Gerlach and H. Nirenberg. The Genus Fusarium - A Pictorial Atlas. Biologische Bundesanstalt für Land - und. Forstwirtschaft, Berlin, 1982. (3) M. Lazarotto et al. Summa Phytopathol. 36:134, 2010. (4) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual, 1st ed. Wiley-Blackwell, Hoboken, NJ, 2006.

Rare provisional entity: primary cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma in a young woman.

Primary cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma is a rare and provisional entity, characterised by cutaneous involvement and aggressive clinical behaviour. The case is here presented of a young woman with concurrent cutaneous and systemic involvement. Despite multi-agent chemotherapy, only partial remission could be achieved, and the patient died from therapy-resistant respiratory and circulatory failure. This case report is intended to add to the data collected on this rare entity, with only about 20 cases as yet described.

Activity of the notch-signalling pathway in circulating human chronic lymphocytic leukaemia cells.

Dysregulation of the Notch-pathway has been implicated in the pathogenesis of chronic lymphocytic leukaemia (B-CLL). We characterized the mRNA expression of Notch pathway elements in circulating normal B- and B-CLL cells, and compared expression profiles with clinical and prognostic data. Similar expression profiles were found in normal B-cells and B-CLL cells, however, most B-CLL samples showed lower Hairy/Enhancer of Split-1 expression than normal B-cells, which suggests that the pathway is not over-activated in B-CLL. The expression of Notch-pathway genes did not correlate with other prognostic factors of B-CLL. The importance of Notch-signalling in CLL cells in lymphatic tissue microenvironments remains to be determined.

Richter's and prolymphocytic transformation of chronic lymphocytic leukemia are associated with high mRNA expression of activation-induced cytidine deaminase and aberrant somatic hypermutation.

Chronic lymphocytic leukemia (CLL) is an indolent B-cell non-Hodgkin's lymphoma that may transform into higher-grade lymphoma. The transformation involves an increased number of prolymphocytic cells, termed prolymphocytic transformation (PLT) or the development of diffuse large B-cell lymphoma (DLBL), also referred to as Richter's transformation (RT). To analyze whether activation-induced cytidine deaminase (AID), which is essential for somatic hypermutation (SHM) of normal B-cells, and malfunction of SHM termed aberrant somatic hypermutation (ASHM) are associated with higher-grade transformation of CLL, AID mRNA expression and the mutation pattern of c-MYC, PAX-5 and RhoH genes were analyzed in eight cases of CLL without transformation and in 21 cases that showed RT or PLT. Chronic lymphocytic leukemia cases, which showed no transformation or eventually transformed into higher-grade lymphoma, showed low levels of AID mRNA expression and low frequency of mutations of c-MYC, PAX-5 and RhoH genes. In both RT and PLT, high-levels of AID mRNA expression and high-frequency mutations of c-MYC, PAX-5 and RhoH genes were detected. These results indicate that AID expression and ASHM are associated with higher-grade transformation of CLL and provide further evidences that AID expression and ASHM may be activated during the clonal history of B-cell lymphomas.

Clonal selection in the bone marrow involvement of follicular lymphoma.

To characterize the pathways of bone marrow (BM) involvement of follicular lymphoma (FL), we performed morphological and immunophenotypical analysis of tumor cells from lymph nodes (LNs) and corresponding BMs in 21 patients with FL. In three cases, genealogical trees were constructed based on the immunoglobulin variable region heavy chain (IgV(H)) gene sequences of tumor clones from LNs and BMs. Results showed that FLs within the BMs display identical or lower cytological grades than in the LNs. In the majority of cases, different proportions of tumor cells expressed bcl-2, CD10 and Ki67 in LNs and BMs. Tumor cells in the BM showed ongoing somatic hypermutation of the IgV(H) genes; the distribution of these mutations was highly consistent with antigen selection. The topology of the genealogical trees revealed that different subclones populate the LN and BM and BM infiltration may occur at different points of the clonal evolution of FL. Early descendants of the original tumor clone and derivatives of diversified tumor clones may invade the BM. These results suggest that the BM involvement of FL is associated with intensive clonal selection of tumor cells, and the BM provides a microenvironment similar to the germinal centers of LNs, where tumor cells retain their biological nature.