Changes in the Location of Cutaneous Melanoma Over the Past 30 Years. A Retrospective Observational Study. / Cambios en la localización del melanoma cutáneo en los últimos 30 años. Estudio observacional retrospectivo.

BACKGROUND: The location of cutaneous melanoma is associated with photoexposure. OBJECTIVES: To retrospectively analyze changes in the location of cutaneous melanoma over the past 30 years. PATIENTS AND METHODS: All patients treated at our hospital for cutaneous melanoma from 1988 through 2017 were prospectively collected. Data obtained in cases diagnosed from 1988 through June 2006 were compared to those diagnosed from July 2006 through 2017. RESULTS: A total of 1,937 patients (876 men and 1061 women; median age, 57 years; interquartile range 27) were diagnosed with primary cutaneous melanoma. The location of melanoma was head and neck (470 cases), trunk (745 cases), upper limbs (239 cases), and lower limbs (483 cases). From July 2006 through 2017 we detected an increase in the incidence of head and neck melanomas (19.9% vs 28.6%, p <0.001). A drop in the incidence of melanomas located in the lower extremities was also seen in women (39.8% vs 30.4%, p <0.001), and in the trunk men (57.5% vs 47.3%, p=0.003). In the multivariate analyses, only the decrease in melanomas located in lower extremities in women remained significant. CONCLUSION: The increased incidence of head and neck melanomas in both sexes and the decrease in trunk melanomas in men can be attributed to the aging of our population. The reduction in the incidence of melanomas in the lower extremities in women could be associated with changes in photoexposure patterns. Analyzing the factors possibly associated with these changes would contribute to better understanding the pathogenesis of cutaneous melanoma for prevention purposes.

Impact of switching to raltegravir and/or adding losartan in lymphoid tissue fibrosis and inflammation in people living with HIV. A randomized clinical trial.

BACKGROUND: Persistent inflammation and immune activation are associated with lymph node fibrosis and end-organ diseases in treatment-suppressed people living with HIV (PLWH). We investigated the effect of switching to raltegravir and/or adding losartan on lymphoid tissue fibrosis and on the inflammatory/immune-activation mediators in treated HIV patients. METHODS: Chronic HIV-infected patients treated with two nucleoside reverse transcriptase inhibitors (2NRTI) and one non-NRTI (NNRTI) or protease inhibitor (PI) during at least 48 weeks were randomized to four groups (n = 48): 2NRTI + efavirenz (EFV), 2NRTI + EFV + losartan, 2NRTI + raltegravir and 2NRTI + raltegravir + losartan for 48 weeks. Tonsillar biopsy and peripheral blood markers of CD4 and CD8 T-lymphocyte activation and senescence, monocyte activation and soluble markers of inflammation were determined at baseline and at week 48 and compared between groups. RESULTS: No changes in lymphoid tissue architecture were observed. Adding losartan had no impact on lymphocyte subsets. Conversely, patients who switched to raltegravir showed a higher decrease in all activated [CD4+CD38+HLA-DR+, -0.3 vs. 0.48 (P = 0.033); CD8+CD38+ HLA-DR+, -1.6 vs. 1.3 (P = 0.02)] and senescent [CD4+CD28-CD57+, -0.3 vs. 0.26 (P = 0.04); CD8+CD28-CD57+, -6.1 vs. 3.8 (P = 0.002)] T lymphocytes. In addition, the median CD4/CD8 ratio increased by 0.35 in patients in the raltegravir group vs. 0.03 in the other arms (P = 0.002). Differences between groups in monocyte subpopulations or soluble inflammation markers were not observed. CONCLUSIONS: Losartan had no effect on lymphoid fibrosis or immune activation/inflammation. Conversely, switching to a regimen with raltegravir significantly decreased activated and senescent T-lymphocyte subpopulations and increased CD4/CD8 ratio in successfully treated PLWH.

Symbiotic performance and induction of systemic resistance against Cercospora sojina in soybean plants co-inoculated with Bacillus sp. CHEP5 and Bradyrhizobium japonicum E109.

Soybean is an economically very important crop throughout the word and particularly in Argentina. Soybean yield may be affected by many factors such as the lack of some essential nutrients or pathogens attack. In this work we demonstrated that the co-inoculation of the native biocontrol bacterium Bacillus sp. CHEP5 which induces resistance against Cercospora sojina in soybean and the nitrogen fixing strain Bradyrhizobium japonicum E109, was more effective in reducing frog leaf spot severity than the inoculation of the biocontrol agent alone. Probably, this is related with the increase in the ability to form biofilm when both bacteria are growing together. Furthermore, Bacillus sp. CHEP5 inoculation did not affect Bradyrhizobium japonicum E109 symbiotic behavior and flavonoids composition of root exudates in pathogen challenged plants. These results suggest that co-inoculation of plants with rhizobia and biocontrol agents could be a strategy to improve soybean production in a sustainable system.

An oxidative burst and its attenuation by bacterial peroxidase activity is required for optimal establishment of the Arachis hypogaea-Bradyrhizobium sp. symbiosis.

AIMS: The main purpose of this study was to determine whether the Arachis hypogaea L. root oxidative burst, produced at early stages of its symbiotic interaction with Bradyrhizobium sp. SEMIA 6144, and the bacterial antioxidant system are required for the successful development of this interaction. METHODS AND RESULTS: Pharmacological approaches were used to reduce both plant oxidative burst and bacterial peroxidase enzyme activity. In plants whose H2 O2 levels were decreased, a low nodule number, a reduction in the proportion of red nodules (%) and an increase in the bacteroid density were found. The symbiotic phenotype of plants inoculated with a Bradyrhizobium sp. SEMIA 6144 culture showing decreased peroxidase activity was also affected, since the biomass production, nodule number and percentage of red nodules in these plants were lower than in plants inoculated with Bradyrhizobium sp. control cultures. CONCLUSIONS: We demonstrated for the first time that the oxidative burst triggered at the early events of the symbiotic interaction in peanut, is a prerequisite for the efficient development of root nodules, and that the antioxidant system of bradyrhizobial peanut symbionts, particularly the activity of peroxidases, is counteracting this oxidative burst for the successful establishment of the symbiosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results provide new insights into the mechanisms involved in the development of the symbiotic interaction established in A. hypogaea L. a legume infected in an intercellular way.

Role of reactive oxygen species generation and Nod factors during the early symbiotic interaction between bradyrhizobia and peanut, a legume infected by crack entry.

AIMS: We evaluated whether reactive oxygen species (ROS) production and the plant antioxidant system are involved in the symbiotic interaction between bradyrhizobia and legumes infected by crack entry, without intracellular infection threads (IT) formation, such as Arachis hypogaea L. (peanut). The role of bradyrhizobial Nod factors (NF) in modulating the plants' oxidative burst was also analysed. METHODS AND RESULTS: Histochemical and quantitative procedures were used to detect ROS levels in inoculated and in NF-treated peanut roots. Increase in root H2O2 production was determined at 10 min postinoculation with Bradyrhizobium sp. SEMIA 6144 or after NF addition. ROS production was modulated by NF. From 15 to 30 min postinoculation, the compatibility of Bradyrhizobium sp.-peanut interaction depends mostly on the H2O2 detoxification via catalase. CONCLUSIONS: We demonstrated for the first time that the early events of the symbiotic interaction in legumes invaded by crack entry trigger an increase in ROS production (represented exclusively by a higher H2O2 content) in which NADPH-oxidase seems not to be involved. NF modulate this response by enhancing the plant antioxidant machinery, contributing to the creation of adequate conditions for symbiosis development. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data provide new insights into the mechanism involves in the symbiotic interaction that establish legumes infected by crack entry and suggest that ROS response shows differences compared with legumes invaded by IT formation.

The biocontrol agent Bacillus sp. CHEP5 primes the defense response against Cercospora sojina.

Glycine max (soybean) production can be dramatically affected by frogeye leaf spot (FLS) caused by Cercospora sojina Hara. The inoculation of biocontrol agents may be an alternative strategy for C. sojina control. The native biocontrol bacterium Bacillus sp. CHEP5 reduced the severity of FLS in soybean by inducing systemic resistance. We suggest that the defense response was primed since the expression of the defense related gene GmAOS was enhanced in induced plants treated with both methyl jasmonate and C. sojina. Furthermore, as GmAOS is related to jasmonic acid biosynthesis, we assume that this phytohormone is involved in induced systemic resistance signaling defense pathway in soybean against C. sojina.

Patterns of visceral metastasis in cutaneous melanoma: a descriptive study.

BACKGROUND AND OBJECTIVES: Some types of cancer tend to spread to certain organs. In the case of melanoma, uveal melanoma spreads almost exclusively to the liver, while cutaneous melanoma spreads to the liver and other organs. Although important advances have been made in our understanding of the molecular mechanisms underlying melanoma, few recent studies have focused on the patterns of visceral metastasis in cutaneous melanoma. The aim of this study was to retrospectively investigate whether clinicopathologic variants of cutaneous melanoma and primary tumor site might be associated with pattern and time of onset of metastasis to visceral sites, including the central nervous system (CNS). MATERIALS AND METHODS: We included patients diagnosed with cutaneous melanoma between 1988 and 2009 with at least 2 years' follow-up. RESULTS: Of the 1083 patients studied, 92 developed visceral metastasis. The CNS was affected in 21 cases, the lungs in 24, the liver in 17, the digestive tract in 7, and multiple organs simultaneously in 23. Metastasis to the lungs, the liver, and the digestive tract occurred within 5 years in most cases, while metastasis to the CNS and multiple organs occurred later (>5 years in 38% and 43% of cases, respectively). CONCLUSIONS: Unlike uveal melanoma, cutaneous melanoma spreads to different organs without any particular predilection. We observed no significant associations between the site of visceral metastasis and either clinicopathologic variant or location of the primary tumor. Metastasis occurred within 5 years of diagnosis in most cases, but it can occur after 10 years.

Discrimination of glaucomatous from non-glaucomatous optic neuropathy with swept-source optical coherence tomography.

OBJECTIVE: To assess the ability of optic nerve head (ONH) parameters, peripapillary retinal nerve fiber layer (pRNFL), and macular ganglion cell layer (GCL) thickness measurements with swept-source optical coherence tomography (SS-OCT), to discriminate between glaucomatous and non-glaucomatous optic neuropathy (GON and NGON). METHODS: This retrospective cross-sectional study involved 189 eyes of 189 patients, 133 with GON and 56 with NGON. The NGON group included ischemic optic neuropathy, previous optic neuritis, and compressive, toxic-nutritional, and traumatic optic neuropathy. Bivariate analyses of SS-OCT pRNFL and GCL thickness and ONH parameters were performed. Multivariable logistic regression analysis was employed to obtain predictor variables from OCT values, and the area under the receiver operating characteristic curve (AUROC) was calculated to differentiate between NGON and GON. RESULTS: Bivariate analyses showed that the overall and inferior quadrant of the pNRFL was thinner in the GON group (P=0.044 and P<0.01), while patients with NGON had thinner temporal quadrants (P=0.044). Significant differences between the GON and NGON groups were identified in almost all the ONH topographic parameters. Patients with NGON had thinner superior GCL (P=0.015), but there were no significant differences in GCL overall and inferior thickness. Multivariate logistic regression analysis demonstrated that vertical cup-to-disc ratio (CDR), cup volume, and superior GCL provided independent predictive value for differentiating GON from NGON. The predictive model of these variables along with disc area and age achieved an AUROC=0.944 (95% CI 0.898-0.991). CONCLUSIONS: SS-OCT is useful in discriminating GON from NGON. Vertical CDR, cup volume, and superior GCL thickness show the highest predictive value.

Ocular axial length influence on peripapillary retinal nerve fiber layer thickness measurement with optical coherence tomography.

BACKGROUND: Optical coherence tomography (OCT) allows the measurement of the peripapillary optic nerve fiber layer (RNFL) thickness. The effect of ocular axial length (AL) on RNFL thickness measurement may be relevant in the interpretation of OCT results in diagnosing optic nerve diseases. PURPOSE: To assess the influence of ocular AL on RNFL thickness and on optic disc topographic parameters (optic disc area, rim area and cup volume) measured by OCT, in healthy individuals. METHODS: A sample of 109 healthy eyes classified into 3 groups according to AL (A: AL<22mm; B: AL 22-24.5mm; C: AL>24.5mm) was studied. RNFL thickness and optic disc topographic parameters were measured using Swept-Source OCT Triton (Topcon) and were compared between groups using a variance analysis. Correlation between the AL and the study variables was performed using a Pearson's correlation coefficient test. RESULTS: The RNFL thickness was lower in eyes with higher AL in the superior (r=-0.41; p<0.001), inferior (r=0.58; p<0.001) and nasal (r=-0.43; p<0.001) quadrants, in the mean value of the RNFL (r=-0.49; p<0.001), optic disc area (r=-0.40; p<0.001) and rim area (r=-0.25; p=0.01). CONCLUSIONS: AL is negatively correlated with RNFL thickness and optic disc topographic parameters measured by Swept-Source OCT Triton (Topcon).

[Descriptive analysis of cutaneous recurrence patterns in patients with melanoma]. / Análisis descriptivo de los patrones de recidiva cutánea en los pacientes con melanoma.

BACKGROUND AND OBJECTIVES: Few studies have addressed cutaneous recurrence of melanoma. The aim of this retrospective study was to analyze the characteristics and prognostic significance of the different patterns of cutaneous recurrence. MATERIAL AND METHODS: Patients diagnosed with melanoma between 1988 and 2008 at Hospital de Bellvitge, Barcelona, Spain and for whom data were available for at least 2 years of follow-up were included in the study. Local recurrence was defined as melanoma invasion of the skin adjacent to the scar left by excision of the primary tumor, regional metastasis or recurrence as metastasis restricted to the area drained by a regional lymph node station, and distant cutaneous metastasis as metastasis occurring outside this area. The relationship between cutaneous recurrence pattern and age, sex, primary tumor site, tumor subtype, Breslow depth, and ulceration was assessed. RESULTS: Eighty-five out of 1,080 patients (7.87%) had cutaneous recurrence. In 71 of those patients (83.53%; 27 men and 44 women; mean age, 60.68 years), this was the first indication of melanoma recurrence. Thirty-two patients had local recurrence, 32 regional metastasis, and 7 distant metastasis. Significant differences were observed in survival time from diagnosis of the primary tumor (P=.044) and from diagnosis of cutaneous recurrence (P<.001) according to the type of recurrence. CONCLUSIONS: Our results suggest that the pattern of cutaneous recurrence is prognostically significant and related to the site of the primary tumor given that the majority of local and regional recurrences occurred in primary tumors located on the lower limbs and head.

Role of ethylene in effective establishment of the peanut-bradyrhizobia symbiotic interaction.

Ethylene has been implicated in nitrogen fixing symbioses in legumes, where rhizobial invasion occurs via infection threads (IT). In the symbiosis between peanut (Arachis hypogaea L.) and bradyrhizobia, the bacteria penetrate the root cortex intercellularly and IT are not formed. Little attention has been paid to the function of ethylene in the establishment of this symbiosis. The aim of this article is to evaluate whether ethylene plays a role in the development of this symbiotic interaction and the participation of Nod Factors (NF) in the regulation of ethylene signalling. Manipulation of ethylene in peanut was accomplished by application of 1-aminocyclopropane-1-carboxylic acid (ACC), which mimics applied ethylene, or AgNO3, which blocks ethylene responses. To elucidate the participation of NF in the regulation of ethylene signalling, we inoculated plants with a mutant isogenic rhizobial strain unable to produce NF and evaluated the effect of AgNO3 on gene expression of NF and ethylene responsive signalling pathways. Data revealed that ethylene perception is required for the formation of nitrogen-fixing nodules, while addition of ACC does not affect peanut symbiotic performance. This phenotypic evidence is in agreement with transcriptomic data from genes involved in symbiotic and ethylene signalling pathways. NF seem to modulate the expression of ethylene signalling genes. Unlike legumes infected through IT formation, ACC addition to peanut does not adversely affect nodulation, but ethylene perception is required for establishment of this symbiosis. Evidence for the contribution of NF to the modulation of ethylene-inducible defence gene expression is provided.

Holmes-Adie syndrome as an early manifestation of systemic lupus erythematosus. / Síndrome de Holmes-Adie como primera manifestación de lupus eritematoso sistémico.

Lupus is an autoimmune disease with multiple manifestations and multiorgan damage. Neuro-ophthalmic disorders are the less common ophthalmological manifestations of lupus. Adie's tonic pupil is mostly idiopathic and may rarely be caused by autoimmune disorders. The combination of abnormal pupil size and a decrease or loss of deep tendon reflexes is usually called Holmes-Adie syndrome. A case is reported of Holmes-Adie syndrome as an early manifestation of systemic lupus erythematosus.

Interaction among Arachis hypogaea L. (peanut) and beneficial soil microorganisms: how much is it known?

The leguminous crop Arachis hypogaea L. (peanut) is originally from South America and then was disseminated to tropical and subtropical regions. The dissemination of the crop resulted in peanut plants establishing a symbiotic nitrogen-fixing relationship with a wide diversity of indigenous soil bacteria. We present in this review, advances on the molecular basis for the crack-entry infection process involved in the peanut-rhizobia interaction, the diversity of rhizobial and fungal antagonistic bacteria associated with peanut plants, the effect of abiotic and biotic stresses on this interaction and the response of peanut to inoculation.

Endoglin modulates cellular responses to TGF-beta 1.

Endoglin is a homodimeric membrane glycoprotein which can bind the beta 1 and beta 3 isoforms of transforming growth factor-beta (TGF-beta). We reported previously that endoglin is upregulated during monocyte differentiation. We have now observed that TGF-beta itself can stimulate the expression of endoglin in cultured human monocytes and in the U-937 monocytic line. To study the functional role of endoglin, stable transfectants of U-937 cells were generated which overexpress L- or S- endoglin isoforms, differing in their cytoplasmic domain. Inhibition of cellular proliferation and downregulation of c-myc mRNA which are normally induced by TGF-beta 1 in U-937 cells were totally abrogated in L-endoglin transfectants and much reduced in the S-endoglin transfectants. Inhibition of proliferation by TGF-beta 2 was not altered in the transfectants, in agreement with the isoform specificity of endoglin. Additional responses of U-937 cells to TGF-beta 1, including stimulation of fibronectin synthesis, cellular adhesion, platelet/endothelial cell adhesion molecule 1 (PECAM-1) phosphorylation, and homotypic aggregation were also inhibited in the endoglin transfectants. However, modulation of integrin and PECAM-1 levels and stimulation of mRNA levels for TGF-beta 1 and its receptors R-I, R-II, and betaglycan occurred normally in the endoglin transfectants. No changes in total ligand binding were observed in L-endoglin transfectants relative to mock, while a 1.5-fold increase was seen in S-endoglin transfectants. The degradation rate of the ligand was the same in all transfectants. Elucidating the mechanism by which endoglin modulates several cellular responses to TGF-beta 1 without interfering with ligand binding or degradation should increase our understanding of the complex pathways which mediate the effects of this factor.

Snail silencing effectively suppresses tumour growth and invasiveness.

The transcription factor Snail has been recently proposed as an important mediator of tumour invasion because of its role in downregulation of E-cadherin and induction of epithelial-mesenchymal transitions (EMT). This behaviour has led to the consideration of Snail as a potential therapeutic target to block tumour progression. In this report, we provide evidence for this hypothesis. We show that silencing of Snail by stable RNA interference in MDCK-Snail cells induces a complete mesenchymal to epithelial transition (MET), associated to the upregulation of E-cadherin, downregulation of mesenchymal markers and inhibition of invasion. More importantly, stable interference of endogenous Snail in two independent carcinoma cell lines leads to a dramatic reduction of in vivo tumour growth, accompanied by increased tumour differentiation and a significant decrease in the expression of MMP-9 and angiogenic markers and invasiveness. These results indicate that use of RNA interference can be an effective tool for blocking Snail function, opening the way for its application in new antiinvasive therapies.

Incorporation of a laminin-derived peptide (SIKVAV) on polymer-modified adenovirus permits tumor-specific targeting via alpha6-integrins.

Effective gene therapy for disseminated metastatic cancer is currently impossible because of poor delivery of vector to target sites. Modification of viral vectors to target advanced cancer has long been a challenge. In this study, we aimed to redirect adenovirus tropism to infect prostate cancer cells via alpha6beta1 integrins, whose expression is upregulated during prostate cancer progression. To ablate normal mechanisms of infection and provide a framework for attachment of targeting ligands, viruses were non-genetically modified with pHPMA-ONp polymer. Addition of polymer-coated virus to prostate cells showed significantly reduced transgene expression compared with unmodified virus. To restore infectivity, an alpha6-integrin binding peptide (-SIKVAV-) derived from laminin was incorporated onto the surface of the polymer-coated viruses. Photon correlation spectroscopic analysis revealed a small increase in the mean diameter of the particles following retargeting. Addition of -SIKVAV- peptide restored virus infectivity of PC-3 cells in a ligand concentration-dependent manner that was significantly improved following removal of unincorporated polymer and peptide. Competition assays using cells preincubated with Ad5 fiber protein or free -SIKVAV- peptide confirmed that entry of retargeted viruses was mediated via the incorporated ligand. Application of retargeted viruses to a panel of human cell lines revealed varying levels of transduction efficiency. Flow cytometric analysis of cells using anti-alpha6 integrin and anti-beta1 integrin antibodies demonstrated that for prostate cells, greater transduction efficiency correlated with higher levels of expression of both integrin subunits. Furthermore with the exception of LNCaP cells, increased alpha6beta1 integrin expression correlated with advanced disease. Intravenous administration of retargeted viruses to tumor-bearing mice resulted in slower plasma clearance and greatly reduced liver tropism, and hence toxicity compared with unmodified virus, while maintaining reporter gene expression in the tumor. The data suggest that YESIKVAVS-retargeted viruses have potential for systemic delivery for the treatment of metastatic disease.

The NADPH oxidase NOX4 represses epithelial to amoeboid transition and efficient tumour dissemination.

Epithelial to mesenchymal transition is a common event during tumour dissemination. However, direct epithelial to amoeboid transition has not been characterized to date. Here we provide evidence that cells from hepatocellular carcinoma (HCC), a highly metastatic cancer, undergo epithelial to amoeboid transition in physiological environments, such as organoids or three-dimensional complex matrices. Furthermore, the NADPH oxidase NOX4 inhibits this transition and therefore suppresses efficient amoeboid bleb-based invasion. Moreover, NOX4 expression is associated with E-cadherin levels and inversely correlated with invasive features. NOX4 is necessary to maintain parenchymal structures, increase cell-cell and cell-to-matrix adhesion, and impair actomyosin contractility and amoeboid invasion. Importantly, NOX4 gene deletions are frequent in HCC patients, correlating with higher tumour grade. Contrary to that observed in mesenchymal cell types, here NOX4 suppresses Rho and Cdc42 GTPase expression and downstream actomyosin contractility. In HCC patients, NOX4 expression inversely correlates with RhoC and Cdc42 levels. Moreover, low expression of NOX4 combined with high expression of either RhoC or Cdc42 is associated with worse prognosis. Therefore, loss of NOX4 increases actomyosin levels and favours an epithelial to amoeboid transition contributing to tumour aggressiveness.

Adenovirus E1A orchestrates the urokinase-plasminogen activator system and upregulates PAI-2 expression, supporting a tumor suppressor effect.

Invasiveness and metastatic potential are the two most important properties defining malignancy. The adeno-virus E1A (Ad-E1A) gene has a dual effect as a proliferative gene and as a tumor-suppressor gene, decreasing tumor growth and the metastatic potential of malignant cells. In order to study genes related with the antimetastatic effect of Ad-E1A in human cells, we performed a microarray analysis using OncoChiptrade mark. In three independent experiments, NIH3T3, IMR90 and MDA MB 435 cells were infected with pLPC retroviruses carrying the adenovirus 12S E1A gene or the GFP gene. We analyzed cDNA expression by using the CNIO OncoChipTM, a cDNA microarray containing a total of 6386 genes represented by 7237 clones. uPA, uPAr, tPA, PAI-1 and PAI-2 were also studied at RNA and protein levels. Microarrays of cDNA expression, RT-PCR and Western blot performed in IMR90 E1A-expressing cells showed downregulation of uPA, uPAr, tPA, PAI-1 and upregulation of PAI-2. These results were confirmed in NIH3T3 and MDA MB 435 breast carcinoma cells, with PAI-2 upregulation by RT-PCR and Western blot. In addition, zymographic analysis demonstrated that E1A expression greatly reduced the gelatinase activity of the pro-MMP2 and -MMP9 proteins. We propose that adenovirus E1A may orchestrate the expression of most members of the urokinase-plasminogen activation system, downregulating potentially invasive genes and upregulating PAI-2, which is associated with a better prognosis in human tumors.