Antiproliferative and antibiofilm potentials of endolichenic fungi associated with the lichen Nephroma laevigatum.

AIMS: The objective of this study was to explore the diversity of endolichenic fungi from Nephroma laevigatum and to investigate their antiproliferative and antibiofilm potential. METHODS AND RESULTS: Forty-six isolates were obtained and identified by DNA barcoding. They belonged to genera Nemania, Daldinia, Peziza and Coniochaeta. Six strains belonging to the most represented species were selected and tested for their antiproliferative and antibiofilm activities. Extracts were analysed by reversed-phase HPLC. Activities against fungal and bacterial biofilm were evaluated using tetrazolium salt (XTT) assay and crystal violet assay respectively. Antiproliferative responses of extracts were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis induction by two extracts was observed in two cell lines (HT-29 and PC-3) via morphological changes, pro-apoptotic and anti-apoptotic proteins analysis (Western blotting) and DNA fragmentation. Four extracts displayed activities against Candida albicans biofilm with IC50 values ranging from 25 to 200 µg ml-1 . All extracts were inactive against Staphylococcus aureus and Pseudomonas aeruginosa biofilms. The most active isolates against human colorectal (HT-29 and HCT116) and prostate (PC-3 and DU145) cancer cell lines were Nemania serpens (NL08) and Nemania aenea var. aureolatum (NL38) with IC50 values ranging from 13 to 39 µg ml-1 . These extracts induced an apoptotic process through activation of caspases 8 and 3, poly(ADP-ribose) polymerase cleavage and DNA fragmentation. CONCLUSIONS: Selected crude fungal extracts have antiproliferative and antibiofilm activities. Data suggest that this antipoliferative effect is due to apoptosis process. This is the first report showing the effects of endolichenic fungi from N. laevigatum. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the therapeutic potential of endolichenic fungi metabolites as sources for drug discovery programmes.

Acromegaly in Carney complex.

PURPOSE: Carney complex (CNC) is a rare autosomal dominant syndrome, characterized by mucocutaneous pigmentation, cardiac, cutaneous myxomas and endocrine overactivity. It is generally caused by inactivating mutations in the PRKAR1A (protein kinase cAMP-dependent type I regulatory subunit alpha) gene. Acromegaly is an infrequent manifestation of CNC, reportedly diagnosed in 10% of patients. METHODS: We here report the case of a patient who was concomitantly diagnosed with Carney complex, due to a new mutation in PRKAR1A ((NM_002734.3:c.80_83del, p.(Ile27Lysfs*101 in exon 2), and acromegaly. In parallel, we conducted an extensive review of published case reports of acromegaly in the setting of CNC. RESULTS: The 43-year-old patient was diagnosed with an acromegaly due to a GH-secreting pituitary microadenoma resistant to somatostatin analogs. He underwent transsphenoidal surgery in our tertiary referral center, which found a pure GH-secreting adenoma. In the literature, we identified 57 cases (24 men, 33 women) of acromegaly in CNC patients. The median age at diagnosis was 28.8 ± 12 year and there were 6 cases of gigantism. Acromegaly revealed CNC in only 4 patients. 24 patients had a microadenoma and two carried pituitary hyperplasia and/or multiple adenomas, suggesting that CNC may result in a higher proportion of microadenoma as compared to non-CNC acromegaly. CONCLUSIONS: Although it rarely reveals CNC, acromegaly is diagnosed at a younger age in this setting, with a higher proportion of microadenomas.

Enhanced synchronization of gamma activity between frontal lobes during REM sleep as a function of REM sleep deprivation in man.

UNLABELLED: Studies have shown that synchrony or temporal coupling of gamma activity is involved in processing and integrating information in the brain. Comparing rapid eye movement (REM) sleep to waking and non-REM (NREM) sleep, interhemispheric temporal coupling is higher, but lower between the frontal and posterior association areas of the same hemisphere. However, the homeostatic response of REM sleep temporal coupling after selective REM sleep deprivation (REMD) has not been studied. This study proposed exploring the effect of one night of selective REMD on the temporal coupling of cortical gamma activity during recovery REM sleep. Two groups of healthy subjects were subjected to either REMD by awakening them at each REM sleep onset, or to NREM sleep interruptions. Subjects slept four consecutive nights in the laboratory: first for adaptation, second as baseline, third for sleep manipulation, and fourth for recovery. Interhemispheric and intrahemispheric EEG correlations were analyzed during tonic REM (no eye movements) for the first three REM sleep episodes during baseline sleep, and recovery sleep after one night of selective REMD. Temporal coupling between frontal lobes showed a significant homeostatic rebound that increased during recovery REM sleep relative to baseline and controls. Results showed a rebound in temporal coupling between the two frontal lobes after REM sleep deprivation, indicating that the enhanced gamma temporal coupling that occurs normally during REM sleep has functional consequences. CONCLUSION: results suggest that synchronized activity during REM sleep may play an important role in integrating and reprocessing information.

T cell dysfunction in the diabetes-prone BB rat. A role for thymic migrants that are not T cell precursors.

Diabetes-prone BB (BB-DP) rats express several T cell dysfunctions which include poor proliferative and cytotoxic responses to alloantigen. The goal of this study was to determine the origin of these T cell dysfunctions. When BB-DP rats were thymectomized, T cell depleted, and transplanted with neonatal thymus tissue from diabetes-resistant and otherwise normal DA/BB F1 rats, the early restoration of T cell function proceeded normally on a cell-for-cell basis; i.e., peripheral T cells functioned like those from the thymus donor. Because the thymus in these experiments was subjected to gamma irradiation before transplantation and there was no evidence of F1 chimerism in the transplanted BB-DP rats, it appeared that the BB-DP T cell precursors could mature into normally functioning T cells if the maturation process occurred in a normal thymus. If the F1 thymus tissue was treated with dGua before transplantation, the T cells of these animals functioned poorly like those from untreated BB-DP rats. dGua poisons bone marrow-derived cells, including gamma radiation-resistant cells of the macrophage/dendritic cell lineages, while sparing the thymic epithelium. Therefore, the reversal of the T cell dysfunction depends on the presence in the F1 thymus of gamma radiation-resistant, dGua-sensitive F1 cells. Conversely, thymectomized and T cell-depleted F1 rats expressed T cell dysfunction when transplanted with gamma-irradiated BB thymus grafts. T cell responses were normal in animals transplanted with dGua-treated BB thymus grafts. With increasing time after thymus transplantation, T cells from all animals gradually expressed the functional phenotype of the bone marrow donor. Taken together these results suggest that BB-DP bone marrow-derived cells that are not T cell precursors influence the maturation environment in the thymus of otherwise normal BB-DP T cell precursors.

Spontaneous fusion in vivo between normal host and tumor cells: possible contribution to tumor progression and metastasis studied with a lectin-resistant mutant tumor.

Previous studies demonstrated that growth in DBA/2 mice of MDW4, a wheat germ agglutinin-resistant (WGAr) mutant of the highly metastatic MDAY-D2 DBA/2 mouse tumor, led to the emergence of WGA-sensitive (WGAs) revertants having higher ploidy levels at the site of inoculation as well as at distant visceral metastases. The results implied that MDW4 was nonmetastatic but progressed to become metastatic in vivo only after a cellular change took place which was accompanied by extinction of the WGAr phenotype and acquisition of a higher number of chromosomes. Results presented here provide strong and direct evidence for the underlying mechanism being spontaneous cell fusion in vivo between the MDW4 (WGAr) tumor cells and normal host cells, at least some of which are of bone marrow origin. Thus, growth of the H-2d MDW4 tumor cells in (C3H X DBA/2)F1 (H-2k X H-2d) or (C57BL/6 X DBA/2)F1 (H-2b X H-2d) mice led to the appearance of WGAs revertants bearing the H-2k or H-2b major histocompatibility complex antigens associated with the C3H or C57BL/6 parental strains, respectively. Similarly, WGAs revertants of MDW4 were found to express H-2k antigens after growth in CBA/HT6T6 (H-2k) leads to DBA/2 bone marrow radiation chimeras. Attempts to mimic the in vivo hybridization process were successful in that in vitro somatic cell fusion between an ouabain-resistant (OuaR), 6-thioguanine-resistant (Thgr) derivative of the MDW4 mutant and either normal bone marrow or spleen cells resulted in loss of the WGAr phenotype in the hybrids (thus showing its recessive character) and increased malignant properties in vivo. An analysis of spontaneous frequencies of re-expression of various drug resistance genetic markers in several hybrid metastatic cells was also consistent with chromosome segregation of the sensitive alleles. The results show that tumor progression and the emergence of metastatic cell variants could arise as a consequence of tumor X host cell fusion followed by chromosome segregation. We also discuss the possibility that this type of event may normally be a very rare one during the growth of tumors, the frequency of which can be artificially amplified by the use of certain classes of lectin-resistant mutants carrying particular cell surface alterations.

Coincidental acquisition of growth autonomy and metastatic potential during the malignant transformation of factor-dependent CCL39 lung fibroblasts.

After sc implantation into BALB/c nude mice, factor-dependent and premalignant Chinese hamster lung fibroblasts (CCL39) developed into tumors that occasionally disseminated and produced pulmonary metastases. Unlike CCL39 cells that required several growth factors (insulin, alpha-thrombin, epidermal growth factor) to proliferate in culture, metastatic tumor cells divided autonomously in serum-free medium. The re-implantation of independently isolated primary tumors revealed that they comprised factor-independent clones present at a variable frequency, as well as malignant clones that disseminated to the lungs after sc or iv inoculation. The resulting metastases invariably contained cells that were able to divide in serum-free medium and that presumably represented the progeny of autonomous variants populating the primary tumors. Among ten CCL39 variants selected in vitro for reduced growth factor requirements, two were metastatic upon sc and iv inoculation. These cell lines were the only ones that replicated rapidly in the absence of growth factors. Unlike myc transfectants, CCL39 fibroblasts that were transformed with an activated Harvey ras oncogene or that were infected with polyomavirus were both metastatic and autonomous. Taken together, these observations are consistent with the notion that the metastatic potential of CCL39 tumor cells coincides with their ability to obviate growth factor requirements and thus to divide in an autonomous fashion.

Differential tumorigenicity of growth factor-dependent versus -independent CCL39 lung fibroblast lines in nude mice.

Several cell lines derived from CCL39 Chinese hamster fibroblasts were characterized so that comparison could be made of their tumorigenic potential and their ability to proliferate in vitro in response to growth factor stimulation. Tumor formation was assessed by implanting single-cell suspensions into the flanks (sc), peritoneal cavity (ip), or tail vein (iv) of BALB/c athymic nude mice, whose ages ranged from 8-10 days (neonatal), to 5-7 weeks (adult), to more than 5 months (old). Serum, insulin (INS), epidermal growth factor (EGF), and alpha-thrombin (THR) were tested for promoting short-term cell proliferation in serum-free medium. CCL39 fibroblasts arrestable in the Go phase required INS and EGF or THR to divide optimally. They formed sc tumors after a long latent period and only in the young and adult mice. Among such tumors, those composed of growth factor-independent variants became lethal ip and iv and grew sc earlier and rapidly in all age groups. These properties were shared by polyomavirus-transformed CCL39 fibroblasts and 1 mutant that divided without growth factors. Mutants that escaped only partially the growth factor requirements had been implanted ip but not iv. They grew at the sc site in newborn and adult mice, with kinetics comparable to that of CCL39 cells. Their expansion in old animals was variable, transient or slow. Neither CCL39 nor 39T10 tumor cells were susceptible to cytotoxic reactions mediated by activated macrophages or natural killer cells. These observations indicate that distinct anatomical regions in nude mice and factors related to their age contribute to present CCL39 heterotransplants with environments that are more or less permissive to their growth. These observations also suggest that cells that achieve growth factor autonomy are more malignant than cells in which growth factor controls of division are less stringent but not abrogated.

Genotypic and phenotypic evolution of a murine tumor during its progression in vivo toward metastasis.

To follow the cellular progeny of the multiple-drug-marked benign murine tumor cell line MDW4 during its progression in vivo toward metastatic spread in DBA/2 mice, the following parameters were analyzed: retention of the drug-resistant markers ouabain resistance (OuaR) and thioguanine resistance (ThgR), lectin-resistance pattern (WGAR), and the karyotype of cell populations (and clones derived from these cells) removed at intervals from the solid tumor growing at the site of inoculation, as well as distant metastatic nodules. It was determined that the initially homogeneous inoculum composed of OuaR, ThgR, and WGAR hypotetraploid cells (mode: 68 +/- 2 chromosomes) was gradually overgrown and replaced by a new population of cells that were either OuaR or ouabain-sensitive but that became thioguanine-and lectin-sensitive and hyperploid (mode: 95 +/- 5). Regardless of the composition of the individual drug marker combinations, only cells with high chromosome contents were found to be able to disseminate to distant visceral organs and to rapidly produce metastases upon sc or iv reinjection. The presence of the same number of metacentric chromosomes in metastatic cells as in MDW4 and the coextinction of two recessive drug-resistant markers (WGAR and ThgR) suggested that cells endowed with invasive-metastatic potential represent the product of spontaneous somatic hybridization between the original nonmetastatic MDW4 cells and normal host cells of unknown origin. Such a fusion was followed by more or less extensive chromosome segregation that accounts for the karyotype mosaicism and the occasional drug marker heterogeneity identified in cell populations of metastatic nodules.

Genetic evidence for progressive selection and overgrowth of primary tumors by metastatic cell subpopulations.

We have exploited random insertions of transfected DNA as unique clonotypic markers to follow cell lineages during primary and metastatic tumor growth of a mouse mammary adenocarcinoma, SP1. Southern analysis was undertaken of primary solid tumors and metastases obtained after injection of a pooled population of individual SP1 transfectants, or reconstituted mixtures of genetically marked metastatic and unmarked nonmetastatic cells. Here we provide evidence for the reproducible selection and eventual overgrowth of primary tumors by genotypically distinct metastatic clones, thereby illustrating that late-state, advanced primary tumors can evolve to become biologically similar, or even identical, to distant metastases. The selective growth advantage of metastatic cells within primary tumors was shown to occur despite the fact that tumors generated by both metastatic and nonmetastatic SP1 cell populations grew at comparable growth rates when injected and analyzed separately. The extent of the local growth advantage manifested by individual metastatic clones varied considerably, from 5- to 50-fold. Clonal overgrowth was also observed whether the tumor cells were injected ectopically, or orthotopically (i.e., into the mammary fat). This type of experimental approach should provide new insights into the dynamics of tumor progression and metastasis, the lineage relationship of primary tumors to metastases, the influence of clonal interactions on tumor behavior, and the physiological changes which are causative of malignant disease.

Metastatic conversion of factor-dependent lung fibroblasts (CCL39) requires over-expression of oncogenes which induce high rate of autonomous replication.

Several classes of oncogenes were tested for their ability to confer cellular growth autonomy and the metastatic phenotype on CCL39 lung fibroblasts. v-sis as well as highly but not weakly expressed activated ras, v-fps and myc genes were susceptible to relieve CCL39 cells from their dependence on exogenous growth factors. However, based on growth rate estimations, ras and fps cells divided 2 to 3 times more rapidly than myc and sis cells in serum-free medium. All ras and fps cells produced pulmonary metastases in 60-100% of young nude mice, following subcutaneous or intravenous injection. Acquisition of factor-independent growth during in vivo passage was demonstrated in two instances. Animals developed either no or sporadic metastases after implantation of transfected cells expressing v-sis, normal Ha-ras, myc or no foreign oncogene. The results are consistent with the notion that the rate at which tumor cells can proliferate independently from growth factor stimulation is a good predictor of their metastatic potential. Oncogenes such as activated ras and fps appear more efficient than myc and sis to induce the metastatic conversion of preneoplastic CCL39 cells and to abrogate Go-arrest controls of division.

v-fps protein-tyrosine kinase coordinately enhances the malignancy and growth factor responsiveness of pre-neoplastic lung fibroblasts.

The v-fps oncoprotein was expressed in a pre-neoplastic, growth factor-dependent Chinese hamster lung fibroblast line (CCL39) to study its effect on growth controls and on the induction of malignancy. Two transfectants were characterized which expressed low (39FPS-8) or high (51FPS-6) levels of P130gag-f ps protein-tyrosine kinase activity. 39FPS-8 cells still arrested in quiescence when deprived of growth factors, but developed an increased sensitivity to the mitogenic actions of epidermal growth factor (20-fold) and alpha-thrombin (50-fold), although not to insulin. In contrast, 51FPS-6 cells completely escaped growth controls, divided in serum-free medium, and were insensitive to further growth factor stimulation. Both transfectants produced rapidly growing tumors in nude mice that formed pulmonary metastases from a subcutaneous site, unlike the parental cells which are non-metastatic. 51FPS-6 cells were comparatively more efficient than 39FPS-8 cells in colonizing the lungs after intravenous inoculation. The v-fps tyrosine kinase therefore induces a partial to complete relaxation of growth factor-mediated controls on the CCL39 cell cycle, with the extent of factor independence reflecting the amount of P130gag-f ps synthesized. This reduction in growth factor requirements correlates with the capacity of v-fps to confer the attributes of metastatic tumors upon preneoplastic CCL39 fibroblasts. We speculate that increased sensitivity to growth factor stimulation represents a common mechanism by which tumor cells acquire metastatic properties.

Progressive relaxation of Go-arrest controls and altered responsiveness to insulin, EGF and thrombin in CCL39 lung fibroblasts over-expressing myc and ras oncogenes.

Factor-dependent hamster lung fibroblasts (CCL39) were transfected with plasmid vectors expressing normal Ha-ras, T24 Ha-ras, V-Ki-ras or myc oncogenes placed under the transcriptional control of potent viral or inducible (metallothionein-I) promoters. The rate at which clonal isolates proliferated in monolayer cultures in serum-free medium or in response to progression (insulin, EGF) and competence growth factors (alpha-thrombin) was found to vary according to the level of oncogene expression. Low levels of T24 ras gene did not abrogate Go-arrest controls but rendered CCL39 cells responsive to both insulin and EGF. Moderate levels (3- to 10-fold) of T24 and Ki-ras genes induced autonomous growth in serum-free medium and attenuated the cell responsiveness to all three factors. Growth of highest ras expressors was enhanced by low (less than 100 microM) concentrations of suramin, and was partially or not inhibited at higher concentrations. In contrast, EGF but not insulin, was able to recruit quiescent cells expressing moderate (less than or equal to 5-fold) myc levels. Higher levels conferred growth autonomy as well as hypersensitivity to insulin, EGF and thrombin. Suramin abolished the self-replication of such myc cells, thus suggesting an autocrine mechanism of proliferation. Hence, both myc and ras genes provided CCL39 cells with distinct growth competence functions which enabled these cells to respond overall more efficiently to functionally distinct classes of mitogens, or to sustain a completely autonomous replication, depending on the abundance of their respective products. Negative autoregulatory mechanisms may additionally be set into motion in this system when activated ras oncogenes are excessively expressed.

A non-equilibrium thermodynamics analysis of active transport within the framework of the chemiosotic theory.

The proton circuit devised by Mitchell in the chemiosmotic theory was subjected to analysis using the formalism of irreversible thermodynamics. The phenomenological coefficients and the degree of coupling relating co-permeant flows were derived from anion/H+, substrate/H+, cation/H+ and anion/anion biporter models. Linearity and equality of the cross-coefficients in Onsager relations were always satisfied. Macroscopic flows leading to charges splitting, such as oxido-reduction, hydro-dehydratation and transhydrogenase, are driven by a composite thermodynamic force which includes the proton-motive component. Multiple coupling occurs in the circuit when it is assumed that the net inward flux of protons becomes zero, i.e. when the circulation of protons reaches a stationary state. Under these conditions, oxidative phosphorylation, ATPase- or respiration-linked transhydrogenase and uptake of anion or cation against their electrochemical gradient may be predicted, in agreement with known experimental evidence.

Effects of selective REM sleep deprivation on prefrontal gamma activity and executive functions.

Given that the dorsolateral prefrontal cortex is involved in executive functions and is deactivated and decoupled from posterior associative regions during REM sleep, that Gamma temporal coupling involved in information processing is enhanced during REM sleep, and that adult humans spend about 90 min of every 24h in REM sleep, it might be expected that REM sleep deprivation would modify Gamma temporal coupling and have a deteriorating effect on executive functions. We analyzed EEG Gamma activity and temporal coupling during implementation of a rule-guided task before and after REM sleep deprivation and its effect on verbal fluency, flexible thinking and selective attention. After two nights in the laboratory for adaptation, on the third night subjects (n=18) were randomly assigned to either selective REM sleep deprivation effectuated by awakening them at each REM sleep onset or, the same number of NREM sleep awakenings as a control for unspecific effects of sleep interruptions. Implementation of abstract rules to guide behavior required greater activation and synchronization of Gamma activity in the frontopolar regions after REM sleep reduction from 20.6% at baseline to just 3.93% of total sleep time. However, contrary to our hypothesis, both groups showed an overall improvement in executive task performance and no effect on their capacity to sustain selective attention. These results suggest that after one night of selective REM sleep deprivation executive functions can be compensated by increasing frontal activation and they still require the participation of supervisory control by frontopolar regions.

Twenty-nine day study of stability for six different parenteral nutrition mixtures.

: BACKGROUND: The aim of the study was to assess the particle size stability of six parenteral nutrition regimens, fitted to various pathologies, and used by the University Hospital of Limoges. The mixtures contained glucose (30 or 50%), amino acids (Hyperamine(R)25), and either long-chain triglycerides (20% Intralipide(R)) or a combination of medium and long-chain triglycerides (20% Médialipide(R)). The regimens were not supplemented. RESULTS: The visual examinations, particle size analysis and physico-chemical tests, carried out during a long storage period, did not reveal any significant evolution of the lipid emulsions. All the tested formulae were stable for 28 days at 4 degrees C plus 24 h at room temperature. CONCLUSIONS: It was concluded that the choice of lipid emulsions depends, for these formulae, on the metabolic and clinical needs of the treated patients.

[Use of a medium and long chain triglyceride mixture for parenteral nutrition in a university hospital: results of an internal audit]. / Utilisation d'un mélange de triglycérides à chaînes longues et chaînes moyennes pour la nutrition parentérale en hôpital universitaire. Résultats d'un audit interne.

OBJECTIVES: The aim of this study was to determine whether a medium and long chain triglyceride mixture for parenteral nutrition is used in accordance with indications and contraindications in hospital practice. METHODS: Patient data recorded in 30 consecutive patients included illness, nutritional status, laboratory findings before nutrition as well as indications and contraindications for parenteral nutrition. RESULTS: When expressed in g.kg-1.day-1 maximal recommended doses of the mixture were exceeded in 32% but there was no excess when expressed in g.kg-1.hr-1. Serious hepatic insufficiency was present in 11% of the patients, 38% had hypertriglyceridemia and one had serious coagulopathy. There were 3 contraindications for the mixture. CONCLUSION: Indications for using this emulsion were respected, but there were contraindications in 45% of the cases. These contraindications are however questionable as is the daily dosage. Because the mixture seems better for use in many cases of parenteral nutrition, it would appear best to discuss the prescription case by case.

[Structure, ultrastructure and microanalysis of the enamel of teeth near maxillary clefts]. / Structure, ultrastructure et microanalyse de l'émail des dents voisines des fentes maxillaires.

Teeth located in the vicinity of cleft palate were studied using correlated light microscopy, microradiography, SEM, TEM and microanalysis. Structural, ultrastructural and chemical abnormalities were found in enamel. The enamel surface presented micro and gross hypoplasia, associated with hypomineralization. Microradiography showed the antenatal enamel to be more mineralized than postnatal enamel. Microanalysis showed the Ca/P ratio measured at the enamel surface to be higher in the teeth associated with cleft palate, compared with controls. The Ca/P value was due to a Ca increase and a P decrease in the enamel surface. The Ca increase was correlated with a decrease in Mg, supporting the hypothesis of a Mg/Ca substitution in the apatitic structure.

Diagnostic sensitivity of three tumour markers in non-small cell lung cancer: a pilot study.

BACKGROUND: Three tumour markers (CEA, CYFRA 21.1 and CA125) were evaluated for diagnostic sensitivity in newly diagnosed, untreated non-small cell lung cancer. METHODS: In the 24 patients studied, the tumours were classified histologically as 15 squamous cell carcinomas and 9 adenocarcinomas. In 19 cases, the disease was confined to the lung (M0); 5 cases presented with metastatic disease at the time of diagnosis (M1). RESULTS: CA125 displayed the best overall sensitivity (62%) and also when only localised disease was evaluated (63%). CA125 was the most sensitive marker for adenocarcinomas (89%), with values differing significantly with histological type (p < 0.005). CYFRA 21.1 was most sensitive in squamous cell carcinomas (53%); this was the only marker which was elevated in all cases involving metastatic disease, and exhibited a significant correlation with stage (p<0.02). CEA presented the poorest overall sensitivity (42%). The overall sensitivity of the three-tumour marker association was 79% and the best combination of two markers was CYFRA 21.1 + CA125 (75%). CONCLUSIONS: This pilot study allows recommendation of the associated use of these two markers as first choice of diagnostic aid in non-small cell lung cancer. Further measurements, including specificity studies in benign lung diseases, should be performed to confirm these results.