Concomitant TP53 mutation in early-stage resected EGFR-mutated non-small cell lung cancer: a narrative approach in a genetically admixed Brazilian cohort.

TP53 mutations are frequent in non-small cell lung cancer (NSCLC) and have been associated with poor outcome. The prognostic and predictive relevance of EGFR/TP53 co-mutations in NSCLC is controversial. We analyzed lung tissue specimens from 70 patients with NSCLC using next-generation sequencing to determine EGFR and TP53 status and the association between these status with baseline patient and tumor characteristics, adjuvant treatments, relapse, and progression-free (PFS) and overall survival (OS) after surgical resection. We found the EGFR mutation in 32.9% of patients (20% classical mutations and 12.9% uncommon mutations). TP53 missense mutations occurred in 25.7% and TP53/EGFR co-mutations occurred in 43.5% of patients. Stage after surgical resection was significantly associated with OS (P=0.028). We identified an association between progression-free survival and poor outcome in patients with distant metastases (P=0.007). We found a marginally significant difference in OS between genders (P=0.057) and between mutant and wild type TP53 (P=0.079). In univariate analysis, distant metastases (P=0.027), pathological stage (IIIA-IIIB vs I-II; P=0.028), and TP53 status (borderline significance between wild type and mutant; P=0.079) influenced OS. In multivariable analysis, a significant model for high risk of death and poor OS (P=0.029) selected patients in stage IIIA-IIIB, with relapse and distant metastases, non-responsive to platin-based chemotherapy and erlotinib, with tumors harboring EGFR uncommon mutations, with TP53 mutant, and with EGFR/TP53 co-mutations. Our study suggested that TP53 mutation tends to confer poor survival and a potentially negative predictive effect associated with a non-response to platinum-based chemotherapy and erlotinib in early-stage resected EGFR-mutated NSCLC.

Clinical outcome of Brazilian patients with non-small cell lung cancer in early stage harboring rare mutations in epidermal growth factor receptor.

The common epidermal growth factor receptor (EGFR) mutations, such as the L858R point mutation in exon 21 and the in-frame deletional mutation in exon 19, have been definitively associated with response to EGFR-tyrosine kinase inhibitors (EGFR-TKI). However, the clinical outcome and response to treatment for many other rarer mutations are still unclear. In this study, we report the results of Brazilian patients in stage IB-IIIA non-small cell lung cancer (NSCLC) following complete resection with minimal residual disease and EGFR mutations treated with adjuvant chemotherapy and/or EGFR-TKIs. The frequency of EGFR mutations was investigated in 70 cases of early stage NSCLC. Mutations in exons 18 and 20, uncommon mutations in exons 19 and 21, as well as in exons 3, 7, 14, 16, 22, 27, and 28, and/or the presence of different mutations in a single tumor (complex mutations) are considered rare. EGFR mutations were detected in 23 tumors (32.9%). Fourteen cases carried rare mutations and were treated with platinum-based chemotherapy and two cases were treated with erlotinib. The clinical outcome is described case by case with references to the literature. Notably, we found two rare EGFR mutations and one of them with an unknown response to chemotherapy and/or EGFR-TKIs. We have provided complementary information concerning the clinical outcome and treatment of patients with early stage NSCLC for several rare EGFR mutations not previously or only rarely reported. Description of cases harboring rare mutations can support the decision-making process in this subset of patients.

Concomitant TP53 mutation in early-stage resected EGFR-mutated non-small cell lung cancer: a narrative approach in a genetically admixed Brazilian cohort

TP53 mutations are frequent in non-small cell lung cancer (NSCLC) and have been associated with poor outcome. The prognostic and predictive relevance of EGFR/TP53 co-mutations in NSCLC is controversial. We analyzed lung tissue specimens from 70 patients with NSCLC using next-generation sequencing to determine EGFR and TP53 status and the association between these status with baseline patient and tumor characteristics, adjuvant treatments, relapse, and progression-free (PFS) and overall survival (OS) after surgical resection. We found the EGFR mutation in 32.9% of patients (20% classical mutations and 12.9% uncommon mutations). TP53 missense mutations occurred in 25.7% and TP53/EGFR co-mutations occurred in 43.5% of patients. Stage after surgical resection was significantly associated with OS (P=0.028). We identified an association between progression-free survival and poor outcome in patients with distant metastases (P=0.007). We found a marginally significant difference in OS between genders (P=0.057) and between mutant and wild type TP53 (P=0.079). In univariate analysis, distant metastases (P=0.027), pathological stage (IIIA-IIIB vs I-II; P=0.028), and TP53 status (borderline significance between wild type and mutant; P=0.079) influenced OS. In multivariable analysis, a significant model for high risk of death and poor OS (P=0.029) selected patients in stage IIIA-IIIB, with relapse and distant metastases, non-responsive to platin-based chemotherapy and erlotinib, with tumors harboring EGFR uncommon mutations, with TP53 mutant, and with EGFR/TP53 co-mutations. Our study suggested that TP53 mutation tends to confer poor survival and a potentially negative predictive effect associated with a non-response to platinum-based chemotherapy and erlotinib in early-stage resected EGFR-mutated NSCLC.

Clinical outcome of Brazilian patients with non-small cell lung cancer in early stage harboring rare mutations in epidermal growth factor receptor

The common epidermal growth factor receptor (EGFR) mutations, such as the L858R point mutation in exon 21 and the in-frame deletional mutation in exon 19, have been definitively associated with response to EGFR-tyrosine kinase inhibitors (EGFR-TKI). However, the clinical outcome and response to treatment for many other rarer mutations are still unclear. In this study, we report the results of Brazilian patients in stage IB-IIIA non-small cell lung cancer (NSCLC) following complete resection with minimal residual disease and EGFR mutations treated with adjuvant chemotherapy and/or EGFR-TKIs. The frequency of EGFR mutations was investigated in 70 cases of early stage NSCLC. Mutations in exons 18 and 20, uncommon mutations in exons 19 and 21, as well as in exons 3, 7, 14, 16, 22, 27, and 28, and/or the presence of different mutations in a single tumor (complex mutations) are considered rare. EGFR mutations were detected in 23 tumors (32.9%). Fourteen cases carried rare mutations and were treated with platinum-based chemotherapy and two cases were treated with erlotinib. The clinical outcome is described case by case with references to the literature. Notably, we found two rare EGFR mutations and one of them with an unknown response to chemotherapy and/or EGFR-TKIs. We have provided complementary information concerning the clinical outcome and treatment of patients with early stage NSCLC for several rare EGFR mutations not previously or only rarely reported. Description of cases harboring rare mutations can support the decision-making process in this subset of patients.

Detection and Distribution of V1016Ikdr Mutation in the Voltage-Gated Sodium Channel Gene in Aedes aegypti (Diptera: Culicidae) Populations From Sergipe State, Northeast Brazil.

Aedes aegypti (L.) resistance to pyrethroids was recorded in Brazil few years after its introduction as the adulticide in the National Dengue Control Program campaigns. Altered susceptibility to pyrethroids had been reported in the state of Sergipe, northeast Brazil, through biological assays, even before its use against Ae. aegypti in the state. Metabolic and target-site resistance mechanisms were also revealed in samples from Aracaju, the capital of Sergipe. Herein, we investigated the presence and distribution of the kdr mutation V1016Ikdr in Ae. aegypti populations from different municipalities of the state. Aedes aegypti eggs were collected from seven municipalities located in areas showing different climatic types and infestation levels. Approximately 20 Ae. aegypti females from each municipality (total of 135 subjects) were individually submitted to allele-specific polymerase chain reaction (AS-PCR) for the 1016 site of the voltage-gated sodium channel (NaV). The V1016Ikdr mutation was found in subjects from all the municipalities under study with a high frequency of heterozygotes in several locations. Homozygous recessive subjects (resistant kdr genotype) were found only in one municipality. The results suggest a wide distribution of the V1016Ikdr mutation in the northeast Brazil, which indicates urgent need for monitoring the effectiveness of the pyrethroids currently used for vector control.

Diagnosis of Giardia infections by PCR-based methods in children of an endemic area

The present study was designed to estimate the prevalence of Giardia infection in preschool- and school-aged children living in an endemic area. Fecal samples from 573 children were processed by zinc sulfate centrifugal flotation, centrifugal sedimentation (using a commercial device for fecal concentration - TF-Test kit®) and polymerase chain reaction (PCR)-based methods. Of the stool samples assessed, 277 (48.3 percent) were positive for intestinal parasites and/or commensal protozoa. Centrifugal flotation presented the highest diagnostic sensitivity for Giardia infections. The kappa index revealed that both coproparasitological techniques closely agreed on the Giardia diagnosis (86 percent) versus satisfactory (72 percent) and poor (35 percent) concordances for commensal protozoan and helminth infections, respectively. Concerning Giardia molecular diagnosis, from the 71 microscopy-positive samples, specific amplification of gdh and tpi fragments was noted in 68 (95.7 percent) and 64 (90 percent) samples, respectively. Amplification of gdh and tpi genes was observed, respectively, in 95.7 percent and 90 percent of microscopy-positive Giardia samples. For 144 microscopy-negative samples, gdh and tpi gene amplification products were obtained from 8.3 percent and 35.9 percent samples, respectively. The agreement between these genes was about 40 percent. The centrifuge-flotation based method was the most suitable means of Giardia diagnosis assessed in the present study by combining accuracy and low cost.

Genotyping of Brazilian Giardia duodenalis human axenic isolates

Giardia duodenalis is a complex species that comprises at least seven distinct genetic groups (A to G), but only genotypes A and B are known to infect humans and a wide variety of other mammals. Regardless of biological, biochemical and antigenic analysis, several isolates maintained in vitro were not genetically typed yet. So, in the present study, five Brazilian axenic isolates obtained from asymptomatic and symptomatic patients were typed in order to determine the major genetic groups to which the isolates belonged. DNA was extracted from axenic trophozoites, fragments of glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes were amplified by PCR and the isolate genotyping was carried out using restriction fragment length polymorphism (RFLP) and DNA sequencing for both genes. The results revealed that all isolates were assigned to genotype A at both analyzed loci. Indeed, DNA sequence analysis classified the four isolates obtained from asymptomatic individuals into subtype AII, while the isolate obtained from the symptomatic patient was typed as subtype AI. Despite of the limited number of isolates assessed, the findings presented herein provide interesting insights on the occurrence of Giardia genotypes in Brazil and hold the perspective for future molecular and epidemiological investigations.

Characterization of the excretory/secretory products of Dermatobia hominis larvae, the human bot fly.

Proteolytic activity in excretory/secretory products (ESP) of first- (L1), second- (L2) and third-instar (L3) larvae of Dermatobia hominis was analyzed through gelatin-gel and colorimetric enzyme assays with the chromogenic substrates azocasein and BApNA. The functional characterization of proteases was based on inhibition assays including synthetic inhibitors. ESP were obtained from new-hatched larvae reared in the laboratory and from second- and third-instar larvae removed from naturally infested cattle. Gelatin-gel analysis evidenced few bands of proteolysis, predominantly of high apparent molecular masses, in ESP of L1, whereas in the gel of L2 and L3 ESP there was a wide range of proteolytic activity most of them not resolved in a single species. Azocasein assays revealed a progressive increase of protease activity from first- to third-instar larvae. Protease inhibitor assays revealed a predominance of metalloproteases in L1 ESP that could be related to a skin penetration process and to a diversion of host immune response. The predominance of serine proteases in L2 and L3 and the great tryptic activity presented by L3 ESP were attributed to an increasing trophic activity by the growing larvae, since the viability of adult flies strictly depends on larval abilities to assimilate nutrients from the host. Taking together, these results suggest that Dematobia larvae secrete/excrete different proteases that may be related to diverse functions during host penetration and infestation, which reinforces the relevance of the study of such proteolytic enzymes.

Morphological and molecular characterization of Strongyloides ophidiae (Nematoda, Strongyloididae).

The aim of the present study is to report morphological data from parasitic female, rhabditoid and filarioid larvae, free-living female worms and eggs of Strongyloides ophidiae (Nematoda, Strongyloididae). In addition, a molecular DNA analysis was carried out using a pool of eight S. ophidiae parasitic females. Samples were obtained from the small intestine of Oxyrhopus guibei (Serpentes, Colubridae) collected in the municipality of Lençóis Paulista, State of São Paulo, Brazil. DNA amplification by polymerase chain reaction (PCR) resulted in a 350 bp band for samples containing S. ophidiae and Strongyloides venezuelensis DNA. Strongyloides ophidiae nucleotide sequence analysis showed 98% similarity with Strongyloides procyonis and 97% with Strongyloides cebus, Strongyloides stercoralis, Strongyloides fuelleborni and Strongyloides sp. from snakes.

Characterization and transcriptional analysis of the promoter region of the Duffy blood group, chemokine receptor (DARC) gene in cattle.

The Duffy antigen is the only receptor for Plasmodium vivax, a hemoparasite of the phylum Apicomplexa and the cause of vivax malaria in humans. Resistance to this parasite in the majority of black African individuals and their descendents is due to a mutation in the gene promoter region, which blocks its transcription on erythrocytes. Regarding bovine babesiosis, it is known that taurine breeds are more susceptible to parasite infection than zebuine breeds. In order to verify whether the same human resistance occurs in bovine, the 5' flanking region of the DARC gene was isolated and characterized in Bos indicus and Bos taurus. Four single nucleotide polymorphisms were identified and genotyped (SNP1: EF_647729.1:g.91C>T; SNP2: EF_647729.1:g.405C>T; SNP3: EF_647729.1: g.433A>G and SNP4: EF_647729.1:g.588A>G), which showed significant frequency differences among 99 bovines of each species (n=198). Characterization of the isolated region revealed the presence of 6 putative haplotypes, 14 genotypes, which are formed by haplotypes, and numerous putative transcription factor binding sites. Only the thymine presence on SNPs 1 and 2, more common in B. indicus, was observed to alter some of the sites in this region. Despite this fact, analyses through real-time PCR on bovines that present the most common homozygote genotypes of each species, which contrast for all the polymorphism, revealed no difference on the DARC gene transcription. Thus, in principle, it was concluded that the polymorphisms identified would not be useful as molecular markers in an improvement program for resistance to babesiosis.

Taxonomic and phylogenetic relationships between neotropical species of ticks from genus Amblyomma (Acari: Ixodidae) inferred from second internal transcribed spacer sequences of rDNA.

The accurate specific identification of ticks is essential for the study, control and prevention of tick-borne diseases. Herein, we determined ribosomal nucleotide sequences of the second internal transcribed spacer (ITS2) of 15 Neotropical hard tick species of the genus Amblyomma Koch found in Brazil. Most of the studied ticks accidentally parasite humans and potentially act as vectors of zoonoses. Lengths of the ITS2 sequences ranged from 956 to 1,207 bp, whereas GC content varied from 62.4 to 66.9%. A matrix of ITS2 divergence was calculated with the ITS2 sequence data obtained showing divergence levels varying from 1.5 to 28.8%. The analysis indicated that this molecular marker can be useful for Amblyomma-specific identification. Phylogenetic inferences based on the ITS2 sequences were used to assess some issues in subgenus taxonomy.

Two digestive trypsins occur in three species of neotropical anophelines.

Trypsin activity increases in the midgut of Anopheles aquasalis, Anopheles albitarsis, and Anopheles darlingi after a bloodmeal. The activity returns to basal levels at the time the blood is completely digested. Affinity chromatography, reversed-phase high performance liquid chromatography (HPLC), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to sequentially purify the mosquito trypsins found in the midguts at 24 h after feeding. Aminoterminal sequencing of the purified trypsins showed the occurrence of two distinct trypsins in the midgut of each of the mosquitoes studied. The sequences obtained are similar to those of the trypsins of other hematophagous insects.

Functional morphology of adult female Culex quinquefasciatus midgut during blood digestion.

The adult female Culex quinquefasciatus midgut comprises a narrow anterior and a dilated posterior region, with epithelia composed of a monolayer of adjacent epithelial cells joined at the apical portion by septate junctions. Densely packed apical microvilli and an intricate basal labyrinth characterise each cell pole. Our morphological studies suggest that, during blood digestion, the anterior midgut region also participates in an initial absorptive stage which is probably related to the intake of water, salts and other small molecules. This activity peaked by 6h after bloodmeal feeding (ABF) and ended approximately 18 h ABF, when the peritrophic membrane was already formed. After this time, absorption only occurred in the posterior region, with morphologic and biochemical evidence of high synthetic activity related to the secretion of proteases. Chymotrypsin, elastase, aminopeptidase, and trypsin reached their maximum activity at around 36 h ABF. Digestion products were apparently absorbed and transported to the basal labyrinth, from where they should be released to the hemolymph. At 72 h ABF, proteolysis had already ended and protein levels had returned to those observed before blood meal. The epithelium of the posterior region, however, did not return to its initial morphology, appearing quite disorganised. Additionally, from 48 h ABF onwards some epithelial cells showed morphological signals of apoptosis.

Targeting Plasmodium ligands on mosquito salivary glands and midgut with a phage display peptide library.

Despite vast efforts and expenditures in the past few decades, malaria continues to kill millions of persons every year, and new approaches for disease control are urgently needed. To complete its life cycle in the mosquito, Plasmodium, the causative agent of malaria, has to traverse the epithelia of the midgut and salivary glands. Although strong circumstantial evidence indicates that parasite interactions with the two organs are specific, hardly any information is available about the interacting molecules. By use of a phage display library, we identified a 12-aa peptide--salivary gland and midgut peptide 1 (SM1)--that binds to the distal lobes of the salivary gland and to the luminal side of the midgut epithelium, but not to the midgut surface facing the hemolymph or to ovaries. The coincidence of the tissues with which parasites and the SM1 peptide interact suggested that the parasite and peptide recognize the same surface ligand. In support of this hypothesis, the SM1 peptide strongly inhibited Plasmodium invasion of salivary gland and midgut epithelia. These experiments suggest a new strategy for the genetic manipulation of mosquito vectorial capacity.

Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus.

Antifungal and antibacterial activities were detected in the hemolymph and gut contents of the cattle tick, Boophilus microplus. A peptide with antibacterial activity from the tick gut contents was purified to homogeneity by reversed-phase chromatography. The molecular mass of the purified peptide was 3,205.7 Da, measured by matrix-assisted laser desorption/ionization mass spectrometry. The amino acid sequence was obtained by Edman degradation and showed that the peptide was identical to a fragment of the bovine alpha-hemoglobin. A synthetic peptide based on the sequence obtained showed characterization data identical to those of the isolated material, confirming its structure. The synthetic peptide was active in micromolar concentrations against Gram-positive bacteria and fungi. These data led us to conclude that the antibacterial activity detected in tick gut contents is the result of enzymatic processing of a host protein, hemoglobin. This activity may be used by ticks as a defense against microorganisms.

Vitellogenin and yolk protein processing in Bothrops jararaca (Wied), a tropical venomous snake.

The plasma vitellogenin of Bothrops jararaca is composed of two subunits. The larger subunit (160 kDa) is phosphate rich and carbohydrate poor, while the smaller (110 kDa) is highly glycosylated and less phosphorylated. As in other vertebrates, the vitellogenin of B. jararaca is synthesized in the liver under estrogen control. The newly synthesized vitellogenin molecule is a 270 kDa polypeptide. This polypeptide originates the two subunits of the plasma vitellogenin by proteolytic cleavage. In the eggs of B. jararaca six main yolk polypeptides have been detected (113, 107, 104, 72, 27.2 and 20.7 kDa). Using phosphoprotein staining we have shown that the 72 kDa polypeptide is the larger phosvitin so far described in a vertebrate egg yolk. The 107 kDa yolk polypeptide also seems to be phosphorylated, but to a lesser extent than the phosvitin. The 104 kDa vitellin originates from the larger vitellogenin subunit while the 113 kDa vitellin originates from the smaller vitellogenin subunit. Based on these results we propose a general scheme for vitellogenin and vitellin processing in B. jararaca.

Musca domestica hemolymph ferritin.

We describe a method for the purification of ferritin from Musca domestica larval hemolymph. Musca ferritin occurs in hemolymph predominantly as a native protein with molecular weight equal to 550,000 and subunits of 26,000. The average iron content of purified ferritin was determined to be 3,000 +/ 600 iron atoms per molecule. The iron contents of ferritin was heterogeneous; both fully iron loaded molecules and apoferritin are probably present in the Musca hemolymph. The anti-ferritin serum raised in rabbit was able to recognize native ferritin but was not reactive with the protein subunits isolated by SDS-PAGE. The ferritin concentration in hemolymph attains a maximum of 0.28 mg/ml in the wandering stage larvae decreasing to 0.13 mg/ml at the middle of pupal stadium. The ferritin contents of midgut and fat bodies were also determined. Fat body ferritin content is greatly reduced when the feeding larva passes into wandering stage.

Processing of procathepsin from Musca domestica eggs.

The major source of amino acids for insect embryos are yolk proteins which accumulate in developing oocytes and are hydrolyzed during embryogenesis. Studies on Musca domestica embryogenesis indicated that a cathepsin B-like proteinase is responsible for yolk protein degradation (Ribolla et al., 1993). In this study, we report the purification of mature cathepsin and show that it is made up of a single 41 kDa polypeptide chain. The Musca domestica cathepsin NH2-terminal 11-residue sequence was determined (Ala-Pro-Lys-Tyr-Val-Asp-Tyr-Gly-Glu-Asn-Glu) and reveals homology with other cathepsins of the papain family. Experiments using serum anti-cathepsin show that the enzyme is stored in oocytes as a 55 kDa zymogen. The activation of the zymogen occurs in vitro only at low pH. In vitro activation in the presence of cysteine protease inhibitors is blocked at an intermediary polypeptide of 48 kDa. Kinetic studies of this activation process at pH 3.5 and 4.6 show that the zymogen is processed in a manner similar to that of pepsin (Foltmann, 1986) and papain (Vernet et al., 1991). We propose that Musca domestica cathepsin zymogen activation occurs in two steps. First, an intramolecular cleavage of the procathepsin polypeptide chain (55,000), induced by low pH gives rise to an intermediary polypeptide (48,000) which then undergoes autolysis to produce the mature enzyme (41,000).