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K Zatloukal

Publications and source records attributed to K Zatloukal.

At least 19 recordsLinked to original sources
PubMed2006

Are the Mallory bodies and intracellular hyaline bodies in neoplastic and non-neoplastic hepatocytes related?

Mallory bodies (MBs) and intracellular hyaline bodies (IHBs) are cytoplasmic hepatocellular inclusions that consist of aggregated proteins. MBs are characteristically associated with alcoholic and non-alcoholic steatohepatitis, but may also be found in chronic cholestatic and metabolic (eg copper intoxication) diseases and hepatocellular neoplasms, particularly hepatocellular carcinomas. IHBs have hitherto only been described in hepatocellular carcinoma cells. In the present study hepatocellular carcinomas (HCCs) and a case of idiopathic copper toxicosis were evaluated with respect to the presence and mutual relationship of MBs and IHBs. IHBs alone were present in 8.6%, MBs alone in 16.1% and both types of inclusion in 7.5% of HCCs. It is shown that IHBs may also occur in non-neoplastic hepatocytes in association with idiopathic copper toxicosis, together with MBs. In HCCs and idiopathic copper toxicosis, MBs and IHBs may be present within the same cell. Moreover, hybrid inclusions holding an intermediate position between MBs and IHBs regarding light microscopy, ultrastructure and composition exist. MBs and IHBs contain p62, a stress-inducible adapter protein, as the major constituent. In MBs p62 is associated with keratins, whereas classical IHBs lack keratins. Light microscopic, electron microscopic and immunohistochemical data suggest a close pathogenetic relationship between MBs and IHBs. Both types of inclusion are the result of over-expression and accumulation of the stress protein p62. If p62 is induced alone, or at least prevails, IHBs may arise by aggregation. However, if abnormal keratins are present in addition to p62, p62 associates and co-aggregates with keratins, finally leading to classical MBs.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Prognostic relevance of tumour-associated macrophages and von Willebrand factor-positive microvessels in colorectal cancer.

Tumour-associated macrophages (TAM) are involved in tumour angiogenesis and anti-tumour immune response. In colorectal cancer (CRC), an association of high microvascular density (MVD) and unfavourable prognosis has been reported by some investigators. However, heterogeneous patient groups were studied. We, therefore, analysed the correlation between TAM and MVD and the prognostic relevance of MVD, TAM and T lymphocyte infiltration for long-term survival in a homogeneous group of 70 patients with moderately differentiated cancers of the International Union Against Cancer (UICC) stages II and III, who did not receive chemotherapy. MVD was evaluated using immunohistochemistry with antibodies against CD34 and von Willebrand factor (vWF). TAM and T lymphocytes were visualised with antibodies against CD68 and CD3, respectively. Statistical analysis did not reveal a significant correlation between TAM and T lymphocyte numbers and MVD. Multivariate analysis of immunohistochemical data from all CRC patients and the subgroup of patients with UICC stage-II CRC identified TAM- and vWF-positive microvessel numbers as prognostically relevant markers. Low numbers of TAM- and high numbers of vWF-positive microvessels were associated with an unfavourable prognosis. In conclusion, TAM- and vWF-positive microvessel numbers may serve as independent prognostic markers for patients with UICC stage-II and -III CRC and may help to identify patients with an unfavourable prognosis.

Aged↗
PubMed2003

Nonviral gene transfer into fetal mouse livers (a comparison between the cationic polymer PEI and naked DNA).

We investigated the efficacy and safety of the cationic polymer polyethylenimine (PEI) as a potential tool for intrauterine gene delivery into livers of fetal mice in the last trimester of pregnancy (E17.5). Using luciferase as a reporter gene, transferrin-conjugated and ligand-free PEI/DNA complexes (containing 3 microg DNA) with varying PEI-nitrogen/DNA-phosphate (N/P) ratios and different PEI forms, branched (800, 25 kDa) and linear (22 kDa), were compared with naked DNA. Transgene expression was measured 48 h after administration of PEI/DNA complexes or naked DNA. Highest luciferase activity (9.8 x 10(3) relative light units (RLU)/mg of tissue protein) was observed with ligand-free PEI22/DNA mixtures at N/P 6.0. In addition, this formulation was associated with very low toxicity as compared to the other PEI/DNA-injected groups. Using beta-galactosidase as a reporter gene, transfection of single, but also small, clusters of cells was demonstrated throughout the liver. Injection of 3 microg naked DNA resulted in an 11-fold lower transgene expression value (0.9 x 10(3) RLU/mg of tissue protein) as compared to PEI22/DNA complexes. However, the administration of higher concentrated naked DNA (9 microg) into fetal livers yielded expression levels of 3.2 x 10(4) RLU/mg of tissue protein, a more than three-fold increase compared to PEI22/DNA complexes. Furthermore, the gene transfer efficacy of concentrated naked DNA was approximately 40 times higher in fetuses than in adults (0.8 x 10(3) RLU/mg of tissue protein), indicating that fetal tissue is especially amenable to the uptake and expression of naked DNA.

3T3 Cells↗
PubMed2001

Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene.

The Raf kinases play a key role in relaying signals elicited by mitogens or oncogenes. Here, we report that c-raf-1(-/-) embryos are growth retarded and die at midgestation with anomalies in the placenta and in the fetal liver. Although hepatoblast proliferation does not appear to be impaired, c-raf-1(-/-) fetal livers are hypocellular and contain numerous apoptotic cells. Similarly, the poor proliferation of Raf-1(-/-) fibroblasts and hematopoietic cells cultivated in vitro is due to an increase in the apoptotic index of these cultures rather than to a cell cycle defect. Furthermore, Raf-1- deficient fibroblasts are more sensitive than wild- type cells to specific apoptotic stimuli, such as actinomycin D or Fas activation, but not to tumor necrosis factor-alpha. MEK/ERK activation is normal in Raf-1-deficient cells and embryos, and is probably mediated by B-RAF. These results indicate that the essential function of Raf-1 is to counteract apoptosis rather than to promote proliferation, and that effectors distinct from the MEK/ERK cascade must mediate the anti-apoptotic function of Raf-1.

Animals↗
PubMed2001

Gene expression patterns associated with the metastatic phenotype in rodent and human tumors.

Using subtractive technology, we have generated metastasis-associated gene expression profiles for rat mammary and pancreatic adenocarcinomas. Several genes whose expression is thought to be related to tumor progression such as c-Met, urokinase-type plasminogen activator receptor, ezrin, HMG-1, oncomodulin, cathepsin, and caveolin were thereby isolated. Half of the metastasis-associated clones showed no significant homology to genes with known function. Notably, several of the metastasis-associated clones were also expressed in metastatic lines but not in nonmetastatic lines of other tumor models. Furthermore, in situ hybridization using selected clones documents the relevance of these results for human cancer because strong expression in tumor cells including metastases was detected in human colorectal cancer samples and, to a lesser extent, in mammary cancer samples. These data support the concept that tumors express a "metastatic program" of genes.

Animals↗
PubMed2001

Identification of tumor antigens in renal cell carcinoma by serological proteome analysis.

We have investigated the suitability of proteomics for identification of tumor-associated antigens. First, we compared the proteomes of nontumorous kidney and renal cell carcinoma (RCC) by two-dimensional gel electrophoresis (2-DE) and silver staining. Protein patterns were markedly different (approximately 800 spots in RCCs versus approximately 1400 spots in kidney). 2-DE immunoblotting revealed five RCC-specific spots, reproducibly reactive with RCC-patient but not healthy donor control sera. Two of these antigens were isolated by preparative 2-DE, and identified by Edman sequencing of tryptic peptides. The first antigen, smooth muscle protein 22-alpha (SM22-alpha), is an actin-binding protein of unknown function predominantly expressed in smooth muscle cells. In situ hybridization revealed that SM22-alpha is not expressed in the malignant cells but in mesenchymal cells of the tumor stroma. The second antigen represents carbonic anhydrase I (CAI), an isoform usually not expressed in kidney. Interestingly, a different isoform (CAXII) has previously been identified by serological expression cloning as an antigen overexpressed in some RCCs. In additional assays, antibodies to recombinant CAI or SM22-alpha were detected in sera from 3/11 or 5/11 RCC patients, respectively, whereas sera from 13 healthy individuals did not react. In conclusion, serological proteome analysis may be a new tool for the identification of tumor-associated antigens.

Amino Acid Sequence↗
PubMed2001

[The increasing importance of tumor and tissue banks in the light of genomic and proteomic research].

Recent technological advances in genome and proteome research offer new perspectives for diagnosis and therapy. The DNA chip technology as well as high-resolution two-dimensional gel electrophoresis in combination with mass spectrometry is able to provide comprehensive information on gene and protein expression patterns, which allow insights into the dynamic and functional aspects of diseases. The application of these techniques depends on the availability of unfixed fresh or cryopreserved tissue with short ischaemia time. For this reason tissue banks are of increasing importance. The pathologist with his expertise and responsibility for histopathological diagnosis, plays a central role in the collection of the human tissues, in accordance with medical, legal and ethical standards, not only for diagnostic purposes, but also for research. The scientific value of a tissue bank is markedly increased if tissue samples are accompanied by detailed patient data as well as blood samples. Informed consent given by the patient is an essential requirement for the use of human tissue banks in biomedical research. The informed consent should not be restricted to scientific investigations but also include the potential commercial use of the data generated.

Animals↗
PubMed2001

[Alcoholic and nonalcoholic steatohepatitis. Histopathologic and pathogenetic considerations].

Alcoholic (ASH) and nonalcoholic (NASH) steatohepatitis show an almost identical morphology. Since the clinical picture is not characteristic, liver biopsy is still the diagnostic gold standard. ASH and NASH are morphologically characterized by a combination of steatosis, hepatocellular injury (ballooning degeneration, apoptosis, necrosis), perivenular and pericellular fibrosis, and inflammation (mostly neutrophils). A definitive differentiation of ASH and NASH is only possible by exclusion of alcohol abuse. Although NASH comprises a syndrome with a multifactorial etiology, adipositas seems to be the most constant associated causal factor. The pathogenesis of both diseases is still unclear. Clinical evidence and experimental studies suggest an important toxic role of reactive oxygen species (oxidative stress). According to our experience, ballooning of hepatocytes is a constant morphologic feature of ASH and NASH and already present in the early stages of disease. Ballooned cells often (but not always) contain Mallory bodies (alcoholic hyalin), which are irregular cytoplasmic inclusions consisting of keratins and nonkeratin components, including ubiquitin. Ballooning is associated with a disturbance and finally almost disappearance of the keratin-intermediate filament cytoskeleton. In our studies on the pathogenesis of ASH and NASH, we concentrated on these cytoskeletal alterations and Mallory body formation. It could be shown that in the early stages overexpression and hyperphosphorylation of keratins take place. Moreover, the 1:1 ratio of keratin type I (keratin 18) and type II (keratin 8) necessary for the assembly of intermediate filaments is disturbed and the equilibrium shifted toward keratin 8. Thus, the pool of soluble keratin 8 increases. The resulting keratin monomers are sensitive to misfolding and either degraded or aggregated as inclusion bodies. If the proteolytic capacity is impaired (e.g., by inhibition of the proteasomal system) in the chronically stressed cell aggregation prevails,finally leading to Mallory body formation. Convincing evidence exists on the basis of clinical and experimental studies that keratins exert a nonskeletal protective function in simple epithelia (e.g., liver cells). Disturbance of the keratin system may thus significantly contribute to cell damage.

Biopsy↗
PubMed2001

Sequence of events in the assembly of Mallory body components in mouse liver: clues to the pathogenesis and significance of Mallory body formation.

BACKGROUND/AIMS: Chronic intoxication of mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or griseofulvin (GF) results in appearance of Mallory bodies (MBs) and alterations of the keratin cytoskeleton, which are reversible upon drug withdrawal but recur after readministration within 2-3 days. METHODS: DDC- or GF-treated and recovered mice were reintoxicated with the original drugs but also colchicine and lumicolchicine. Cytoskeletal alterations of hepatocytes and MB formation were monitored by immunofluorescence microscopy using keratin, MB-specific antibodies, antibodies to phosphoepitopes and to HSP70. Keratin 8/18 mRNA expression and protein levels were determined by competitive reverse transcription-polymerase chain reaction, in situ-hybridization and western blotting. RESULTS: Duration of pretreatment was important for the efficiency of MB triggering. Rapid increase of keratin 8/18 mRNA and proteins was found in all reintoxicated mice concomitant with MB formation, whereby keratin 8 prevailed over keratin 18. Keratins and a protein with heat shock characteristics (M(M) 120-1 antigen) were the earliest detectable MB components, whereas ubiquitination and phosphorylation followed later. CONCLUSIONS: Overproduction of keratins is a major but not the only step responsible for MB formation. Additional components (e.g. M(M) 120-1 antigen) and excess of keratin 8 over keratin 18 are essential.

Animals↗
PubMed2001

Effects of ursodeoxycholic and cholic acid feeding on hepatocellular transporter expression in mouse liver.

BACKGROUND AND AIMS: Cholestasis is associated with retention of potentially toxic bile acids and alterations in hepatocellular transporter expression. Conversely, nontoxic ursodeoxycholic acid (UDCA) stimulates bile secretion and counteracts cholestasis. This study aimed to determine the effects of UDCA and cholic acid (CA) on the expression of hepatocellular transporters for bile acids (Ntcp, Bsep), organic anions (Oatp1, Mrp2), organic cations (Mdr1a/b), and phospholipids (Mdr2) in mouse liver. METHODS: Bile flow/composition was analyzed in UDCA- or CA-fed mice. Transporter expression was studied by reverse-transcription polymerase chain reaction, Western blotting, and immunofluorescence microscopy. RESULTS: UDCA had no effect on basolateral Ntcp and down-regulated Oatp1, whereas canalicular Bsep and Mrp2 were up-regulated. CA down-regulated basolateral Ntcp and Oatp1, whereas canalicular Bsep, Mrp2, and Mdr1a/b were up-regulated. Neither UDCA nor CA affected Mdr2 expression. Both UDCA and CA stimulated biliary bile acid and glutathione excretion, although only CA increased phospholipid and cholesterol excretion. CONCLUSIONS: Down-regulation of basolateral and up-regulation of canalicular transporters in response to CA may represent a defense mechanism, in an attempt to prevent hepatocellular accumulation of potentially toxic bile acids. The therapeutic effects of UDCA may be caused in part by stimulation of canalicular transporter expression in the absence of hepatocellular toxicity.

Administration, Oral↗
PubMed2001

Hepatobiliary transporter expression in percutaneous liver biopsies of patients with cholestatic liver diseases.

Reduced hepatobiliary transporter expression could explain impaired hepatic uptake and excretion of bile salts and other biliary constituents resulting in cholestasis and jaundice. Because little is known about alterations of hepatobiliary transport systems in human cholestatic liver diseases, it was the aim of this study to investigate such potential changes. Hepatic mRNA levels in hepatobiliary transport systems for bile salts (NTCP, BSEP), organic anions (OATP2, MRP2, MRP3), organic cations (MDR1), phospholipids (MDR3), and aminophospholipids (FIC1) were determined in 37 human liver biopsies and control livers by competitive reverse-transcription polymerase chain reaction (RT-PCR). Transporter tissue distribution was investigated by immunofluorescence microscopy. In patients with inflammation-induced icteric cholestasis (mainly cholestatic alcoholic hepatitis), mRNA levels of NTCP, OATP2, and BSEP were reduced by 41% (P <.001), 49% (P <.005), and 34% (P <.05) compared with controls, respectively. In addition, NTCP and BSEP immunostaining was reduced. MRP2 mRNA levels remained unchanged, but canalicular immunolabeling for MRP2 was also decreased. mRNA expression of MRP3, MDR1, MDR3, and FIC1 remained unchanged. In contrast to the alterations of transporter expression in inflammation-induced icteric cholestasis, transporter expression did not change in anicteric cholestasis caused by primary biliary cirrhosis (PBC) stages I and II. In conclusion, reduced expression of hepatobiliary transport systems for bile salts and other organic anions may contribute to inflammation-induced cholestasis in humans. Reduction of transporter gene expression can occur at the mRNA level as observed for NTCP, OATP2, and BSEP. However, reduced MRP2 immunostaining in the presence of conserved MRP2 mRNA levels suggests an additional role for posttranscriptional/posttranslational mechanisms.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2000

The tumor-associated shift in immunoglobulin G1/G2 is expressed at the messenger RNA level of peripheral blood B lymphocytes in patients with gynecologic malignancies.

BACKGROUND: Previously, it could be demonstrated that human patients with malignant diseases of various tissues exhibited characteristic and highly significant changes in the serum patterns of immunoglobulin (Ig)G subclasses, consisting of a decrease in IgG1 and an increase in IgG2 relative to total IgG. The aim of the current study was to determine whether this phenomenon was detectable at the level of IgG-producing B lymphocytes. METHODS: Using a competitive reverse transcriptase polymerase chain reaction specific to IgG1 and IgG2, the gene expression of these 2 IgG subclasses in peripheral B cells from 10 patients with carcinomas of various sites within the female reproductive tract and 10 healthy controls was quantitatively determined, in parallel with the concentrations of the respective serum proteins. RESULTS: Absolute levels of IgG subclass messenger ribonucleic acid (mRNA) showed a slight but not significant decrease in IgG1 and an increase in IgG2 in patients with gynecologic malignancies. However, the ratio of IgG1 to IgG2 expression showed a highly significant (P < 0.001) decrease in tumor patients compared with healthy controls, and corresponded to the change in the ratio of IgG1 to IgG2 serum proteins. CONCLUSIONS: These data suggest that the shifts in the serum patterns of IgG1 and IgG2 observed in patients with gynecologic malignancies are due to irregular biosynthesis of these IgG subclasses at the B-cell level.

B-Lymphocytes↗
PubMed2000

Hepatocyte cytokeratins are hyperphosphorylated at multiple sites in human alcoholic hepatitis and in a mallory body mouse model.

Alcoholic hepatitis (AH) is associated with cytokeratin 8 and 18 (CK8/18) accumulation as cytoplasmic inclusion bodies, termed Mallory bodies (MBs). Studies with MB mouse models and cultured hepatocytes suggested that CK8/18 hyperphosphorylation might be involved in MB formation. However, no data exist on phosphorylation of CK8/18 in human AH. In this study, antibodies that selectively recognize phosphorylated epitopes of CK8 or CK18 were used to analyze CK8/18 phosphorylation states in normal human and murine livers, human AH biopsies, and livers of 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC)-intoxicated mice, the last serving as model for MB induction. Hepatocyte cytokeratins become hyperphosphorylated at multiple sites in AH and in DDC-intoxicated mice. Hyperphosphorylation of CK8/18 occurred rapidly, after 1 day of DDC intoxication and preceded architectural changes of the cytoskeleton. In long-term DDC-intoxicated mice as well as in human AH, MBs preferentially contain hyperphosphorylated CK8/18 as compared with the cytoplasmic cytokeratin intermediate filament network suggesting that CK8/18 hyperphosphorylation may play a contributing role in MB pathogenesis. Furthermore, the site-specific phosphorylation of cytokeratin in different stages of MB induction provides indirect evidence for the involvement of a variety of protein kinases known to be activated in stress responses, mitosis, and apoptosis.

Animals↗
PubMed2000

Cytokeratin 8 protects from hepatotoxicity, and its ratio to cytokeratin 18 determines the ability of hepatocytes to form Mallory bodies.

In alcoholic hepatitis, a severe form of alcohol-induced toxic liver injury, as well as in experimental intoxication of mice with the porphyrinogenic drugs griseofulvin and 3,5-diethoxycarbonyl-1, 4-dihydrocollidine, hepatocytes form cytoplasmic protein aggregates (Mallory bodies; MBs) containing cytokeratins (CKs) and non-CK components. Here we report that mice lacking the CK8 gene and hence CK intermediate filaments in hepatocytes, but still expressing the type I partner, ie, the CK18 gene, do not form MBs but suffer from extensive porphyria and progressive toxic liver damage, leading to the death of a considerable number of animals (7 of 12 during 12 weeks of intoxication). Our observations show that 1) in the absence of CK8 as well as in the situation of a relative excess of CK18 over CK8 no MBs are formed; 2) the loss of CK8 is not compensated by other type II CKs; and 3) porphyria and toxic liver damage are drastically enhanced in the absence of CK8. Our results point to a protective role of CKs in certain types of toxic liver injury and suggest that MBs by themselves are not harmful to hepatocytes but may be considered as a product of a novel defense mechanism in hepatocytes.

Animals↗
PubMed2000

The affinity of MCF7 breast cancer cells to hyaluronan substrates of different molecular weight and concentrations in an in vitro model.

The affinity of MCF7 breast cancer cells to hyaluronan (HA) was investigated in an in vitro model. The cells form a tightly adhering monolayer on native HA with a concentration of 5 mg/ml. On native HA at higher concentrations the cells reduce their adhesion to the substrate in favor of increased intercellular bonds, resulting in a cluster-like aggregate that tends to detach from the substrate. Aggregate formation is accomplished after 12 h incubation. The phenomenon is independent of the CD44 receptor. Degradation of native HA by hyaluronidase abolishes aggregate formation even at high HA concentrations in favor of formation of a firmly adhering monolayer. This model may help to understand tumor spread on HA tissue structures and may explain therapy successes with hyaluronidase in tumor patients.

Adenocarcinoma↗
PubMed1999

Functions of c-Jun in liver and heart development.

Mice lacking the AP-1 transcription factor c-Jun die around embryonic day E13.0 but little is known about the cell types affected as well as the cause of embryonic lethality. Here we show that a fraction of mutant E13.0 fetal livers exhibits extensive apoptosis of both hematopoietic cells and hepatoblasts, whereas the expression of 15 mRNAs, including those of albumin, keratin 18, hepatocyte nuclear factor 1, beta-globin, and erythropoietin, some of which are putative AP-1 target genes, is not affected. Apoptosis of hematopoietic cells in mutant livers is most likely not due to a cell-autonomous defect, since c-jun-/- fetal liver cells are able to reconstitute all hematopoietic compartments of lethally irradiated recipient mice. A developmental analysis of chimeras showed contribution of c-jun-/- ES cell derivatives to fetal, but not to adult livers, suggesting a role of c-Jun in hepatocyte turnover. This is in agreement with the reduced mitotic and increased apoptotic rates found in primary liver cell cultures derived from c-jun-/- fetuses. Furthermore, a novel function for c-Jun was found in heart development. The heart outflow tract of c-jun-/- fetuses show malformations that resemble the human disease of a truncus arteriosus persistens. Therefore, the lethality of c-jun mutant fetuses is most likely due to pleiotropic defects reflecting the diversity of functions of c-Jun in development, such as a role in neural crest cell function, in the maintenance of hepatic hematopoiesis and in the regulation of apoptosis.

Animals↗
PubMed1999

Immunotherapy of metastatic malignant melanoma by a vaccine consisting of autologous interleukin 2-transfected cancer cells: outcome of a phase I study.

We performed a phase I trial to evaluate the safety and tolerability of repeated skin injections of IL-2-transfected autologous melanoma cells into patients with advanced disease. Cell suspensions, propagated from excised metastases, were IL-2 gene transfected by adenovirus-enhanced transferrinfection and X-irradiated prior to injection. Vaccine production was successful in 54% of the patients. Fifteen patients (37%) received two to eight skin vaccinations of either 3 x 10(6) (intradermal) or 1 x 10(7) (half intradermal, half subcutaneous) transfected melanoma cells per vaccination (secreting 140-17,060 biological response modifier program units of IL-2/10(6) cells/24 hr). Analyses of safety and efficacy were carried out in 15 and 14 patients, respectively. Overall, the vaccine was well tolerated. All patients displayed modest local reactions (erythema, induration, and pruritus) and some experienced flu-like symptoms. Apart from newly appearing (4 of 14) and increasing (5 of 14) anti-adenovirus and newly detectable anti-nuclear antibody titers (1 of 15), recipients developed de novo or exhibited increased melanoma cell-specific delayed-type hypersensitivity (DTH) reactions (8 of 15) and vitiligo (3 of 15) and showed signs of tumor regression (3 of 15). This supports the idea of a vaccine-induced or -amplified anti-cancer immune response. None of the patients exhibited complete or partial regressions, but five of them experienced periods of disease stabilization. Three of these individuals received more than the four planned vaccinations and their mean survival time was 15.7 +/- 3.5 months as compared to 7.8 +/- 4.6 months for the entire patient cohort. These data indicate that IL-2-producing, autologous cancer cells can be safely administered to stage IV melanoma patients and could conceivably be of benefit to patients with less advanced disease.

Adult↗