Evangelos Manolis

Expression of p16INK4A and alterations of the 9p21‐23 chromosome region in non‐small‐cell lung carcinomas: Relationship with tumor growth parameters and ploidy status

The 9p21‐23 chromosome region harbors a number of known and putative tumor‐suppressor genes (TSGs). The best characterized gene in this area is p16INK4A (CDKN2A). Alterations of its product have been observed in various malignancies, including non‐small‐cell lung carcinomas (NSCLCs). We earlier investigated the mechanisms underlying p16INK4A inactivation. In the present study, we examined, in a series of 87 NSCLCs, its relationship with the kinetic parameters [proliferation index (PI) and apoptotic index (Al)] and the ploidy status of the tumors. In addition, we extended our previous LOH analysis of the 9p21‐23 region by examining flanking areas of p16INK4A. Aberrant p16 expression was observed in 41.4% of the carcinomas. A significant association was found with increased PI (p = 0.037), but not with apoptosis. Aneuploid tumors were more frequently correlated with abnormal p16 staining (p = 0.05). A high frequency of allelic imbalance (Alm) was noticed at the D9S161 (51.3%) and D9S157 (64.5%) loci, which lie approximately 4cM centromeric and 7cM telomeric, respectively, to CDKN2A. Abnormal p16INK4A expression was strongly correlated with Alm at D9S161 (p = 0.004). Allelic losses at D9S157 occurred more frequently in early stages (p = 0.018) and were significantly associated with deletions at D9S161 (p = 0.035). We conclude that, in a sub‐set of NSCLCs, (i) abnormal p16 expression contributes to tumor growth mainly by increasing the proliferative activity in the initial stages of carcinogenesis; (ii) the association with aneuploidy merely reflects the impact of aberrant p16 on proliferative activity; and (iii) other putative TSGs possibly reside within the 9p21‐23 region that possibly co‐operate in certain cases with CDKN2A in the development of NSCLCs. Int. J. Cancer (Pred. Oncol.) 89:133–141, 2000.

Hepatic stimulator substance administration ameliorates liver regeneration in an animal model of fulminant hepatic failure and encephalopathy.

Abstract: Aims/Background: Hepatic stimulator substance (HSS) is a liver‐specific growth factor implicated in hepatocellular proliferation and hepatoprotection in models of acute liver injury. In the present study, we examined the effect of exogenous HSS administration on liver proliferating capacity and survival outcome in an experimental animal model of fulminant hepatic failure (FHF) and encephalopathy, induced by repeated injections of thioacetamide (TAA) in rats.

Sensitive differential detection of genetically related mycobacterial pathogens in archival material.

A polymerase chain reaction (PCR) assay targeted to the immunogenic protein MPB64 gene was used to detect members of the Mycobacterium tuberculosis complex, and an outward-primed PCR (OPPCR) designed on the IS6110 element allowed differentiation between Mycobacterium bovis and Mycobacterium tuberculosis. Additionally, the amplification of IS1110 and 16S ribosomal RNA sequences combined with a dot blotting assay were able to differentially detect Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium paratuberculosis. The validity of the experimental procedure was tested on reference material and formalin-fixed paraffin-embedded samples from patients with tuberculosis, sarcoidosis, or Crohn disease. We demonstrated mycobacterial DNA in 59 of 75 cases with histologic lesions typical of tuberculosis; we detected M tuberculosis and M paratuberculosis in 6 of 25 sarcoidosis cases and in 7 of 20 Crohn disease specimens, respectively. The proposed diagnostic procedure is directly applicable to archival material and allows differentiation of genetically related mycobacterial pathogens in more detail than other molecular methods. It provides a tool for the diagnostic study of tuberculosis, sarcoidosis, and Crohn disease.

Hepatic stimulator substance activity in animal model of fulminant hepatic failure and encephalopathy

Hepatic stimulator substance (HSS) is a known liver-specific but species-nonspecific growth factor. In the present study we examined the activity of the endogenously produced HSS in an established experimental model of fulminant hepatic failure (FHF) and encephalopathy, induced by repeated injections of thioacetamide (TAA). FHF was induced by three consecutive intraperitoneal injections of TAA (400 mg/kg body weight) in rats, at time intervals of 24 hr. The animals were killed at 0, 6, 12, or 18 hr following the last injection of TAA. The rate of tritiated thymidine incorporation into hepatic DNA, the enzymatic activity of liver thymidine kinase (EC 2.7.1.21), and the assessment of mitotic index in hepatocytes were used to estimate liver regeneration. HSS extract obtained from the livers of TAA-treated rats, sacrificed at the above-mentioned time points was tested for its activity. Increased HSS activity was noted in all TAA-treated animals, presenting a peak at 12 hr following the third TAA dose, suggesting active participation of this growth factor in hepatocyte replication in this animal model of FHF and encephalopathy. It may also be suggested that up-regulation of HSS activity could be used in future as a therapeutic approach in FHF.

Comparison of the Enzyme-Linked Immunosorbant Assay III, Recombinant Immunoblot Third Generation Assay, and Polymerase Chain Reaction Method in the Detection of Hepatitis C Virus Infection in Haemodialysis Patients

Hepatitis C virus (HCV) serotyping assays have evolved from simple antibody screening tests to complex RNA‐based qualitative and quantitative methods. The objective of this study was to compare the HCV screening results from 161 patients in long‐term maintenance haemodialysis (HD) as assessed by the recently developed Enzyme Linked Immunosorbant Assay III (ELISA III), confirmed by the Recombinant Immunoblot 3rd generation assay (RIBA 3rd) and determined by the qualitative HCV reverse transcription polymerase chain reaction (RT‐PCR) method. One hundred sixty‐one HD patients were tested for the presence of anti‐HCV antibodies by the ELISA III and confirmed by the RIBA 3rd. HCV RNA was determined by an HCV RT‐PCR method. All reported results that were designated as discrepant, anti‐HCV (+) and/or HCV RNA (+) were further investigated by means of a quantitative HCV RT‐PCR assay. Reported results obtained from ELISA III and qualitative RT‐PCR assays were HCV positive for 16/161 patients (9,93%) and these were designated as anti‐HCV (+)/HCV RNA (+). Subsequently, these 16 anti‐HCV positive/161 HD patients were confirmed by the RIBA 3rd. Three individuals anti‐HCV (–)/RIBA (+)/ HCV RNA (–)], the viral load that was reported from the quantitative RT‐PCR was less than the assay detection level (< 2,000 viral copies/ml). In view of previous observations, our findings suggest that ELISA III remains still a highly reliable and valuable assay. However, despite the cost, the combination of both ELISA III and qualitative RT‐PCR allows a definitive classification on HCV diagnosis. J. Clin. Lab. Anal. 13:122–125, 1999.

Emergence of vancomycin-intermediate Staphylococcus aureus and S. sciuri, Greece.

To the Editor: Staphylococcal isolates with reduced susceptibility to glycopeptides, such as vancomycin and teicoplanin, are a serious public health problem because staphylococci frequently show multidrug resistance, and glycopeptides are the only remaining effective drugs. Since the early reports of glycopeptide-resistant staphylococci, teicoplanin resistance has become more common than vancomycin resistance, particularly among coagulase-negative staphylococcal species (1–3). In cases of staphylococci with reduced susceptibility to vancomycin (vancomycin-intermediate staphylococci), an increasing number of strains showing heteroresistance are reported (strains that contain subpopulations of cells at frequencies >10-6 for which the vancomycin MICs are 8 µg/mL to 16 µg/mL); homogeneous resistance still appears to be rare (2,4–7). In northern Greece, resistance to teicoplanin has recently been documented in S. haemolyticus strains isolated from clinical infections (8). We report the first bloodstream infections in Greece associated with S. aureus and S. sciuri strains that have homogeneous intermediate-resistance to vancomycin (MIC = 8 µg/mL). In our department, all clinically significant staphylococcal isolates are screened for reduced susceptibility to vancomycin and teicoplanin by an agar incorporation method (9), which has been routinely performed since January 1999. An inoculum of 104 CFU/spot from a log-phase broth culture was spread on Mueller-Hinton agar plates containing appropriate antibiotic concentrations. The strains were incubated for a full 24 hours before the MICs were read. When a reduced susceptibility to vancomycin was observed (MIC 8 to 16 µg/mL), the test was repeated for confirmation of the result and the strains were also tested by National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution (9) and E-test (AB Biodisk, Solna, Sweden) with BHI agar (Oxoid, Ltd., Basingstoke, Hampshire, UK) and an inoculum density adjusted to 0.5 McFarland value. S. aureus ATCC 29213, which had MICs for vancomycin of 1 µg/mL and for teicoplanin of 0.5 µg/mL, was used as a control for the estimation of the MICs. Two vancomycin-intermediate staphylococcal isolates (one S. aureus and one S. sciuri) were recovered in our hospital during December 2000 and April 2001, respectively. The organisms were identified with the Vitek system (bioMerieux Vitek, La Balme les Grottes, France). Slide-coagulase test and Staph ID 32 API system (API system, bioMerieux) confirmed identification. Susceptibility to 18 antimicrobial agents was evaluated with the Vitek system according to the recommendations of the manufacturer, and carriage of the mecA gene was confirmed with a polymerase chain reaction (PCR) that amplifies a 449-bp product. The first strain (S. aureus) was recovered from a 52-year-old man who was hospitalized after a severe traffic accident. The patient had multiple injuries, including an external laryngeal trauma, pelvic ring disruption, and various fractures of the extremities. He underwent immediate tracheotomy, and a neurosurgical operation was performed to evacuate an extracerebral hematoma. Ceftazidime, clindamycin, ciprofloxacin, metronidazole, teicoplanin, and vancomycin were periodically administered as prophylaxis. An oxacillin-resistant S. aureus isolate was recovered from two blood cultures 4 weeks after the patient’s admission. The strain was also resistant to tobramycin, macrolides, tetracyclines, rifampicin, and fusidic acid, and had intermediate resistance to vancomycin (MIC 8 µg/mL) and teicoplanin (MIC 16 µ/mL) by all tested methods (agar dilution, broth microdilution, and E-test). The strain was susceptible to chloramphenicol, co-trimoxazole, fosfomycin, gentamicin, kanamycin, nitrofurantoin, and ofloxacin. The removal of an intravenous catheter and treatment with gentamicin and vancomycin eradicated the infection. The second strain (S. sciuri) was recovered from a 35-year-old man who was an intravenous drug user. He was admitted with renal failure, electrolyte disturbances, and acute respiratory distress, which necessitated intubation and mechanical ventilation. The patient became febrile, and multiple courses of antibiotics (amikacin, cefepime, ciprofloxacin, metronidazole, and vancomycin, alone or in combinations) were administered before the S. sciuri strain was isolated. Seven weeks after his admission, an oxacillin-resistant S. scuiri strain that had cross-resistance to aminoglycosides, macrolides, quinolones, rifampicin, and tetracycline was found in subsequent blood cultures. The MIC of the strain for vancomycin was 8 μg/mL and for teicoplanin 16 µg/mL by the agar dilution method, and the result was confirmed by the E-test and the broth microdilution method. The strain was susceptible only to cotrimoxazole, fosfomycin, and nitrofurantoin. The patient improved clinically and was subsequently discharged on cotrimoxazole and vancomycin therapy. In both cases, the MICs of vancomycin remained stable after repeated subcultures in a drug-free medium. PCR amplification showed that both staphylococcal strains carried the mecA gene. However, the vanA, vanB, and vanC genes were not amplified in any strain. Vancomycin-intermediate staphylococci have been sporadically reported from clinical infections after prolonged exposure to vancomycin or preexisting infection with methicillin-resistant staphylococci (5,7). In our hospital, high rates of methicillin-resistant staphylococci are detected, and vancomycin has been the only treatment uniformly effective against staphylococcal infections. However, this is the first report of infection caused by vancomycin-intermediate S. aureus in Greece. In addition, S. sciuri, a species considered taxonomically the most primitive among staphylococci and found primarily in rodents and primitive mammals, has not been implicated previously in human infections caused by vancomycin-intermediate strains in our region or elsewhere. Although various studies have described staphylococci with reduced susceptibility to vancomycin, the existence of isolates that have the homogeneous vancomycin-intermediate phenotype is rather limited (4–7). In this report, the vancomycin MIC for both staphylococcal isolates was repeatedly 8 µg/mL, and a confluent growth was observed after 24 hours on Mueller Hinton agar containing vancomycin at a concentration of 4 µg/mL. Discrete colonies were detected only in plates containing 6 μg/mL of vancomycin but not in plates containing 8 µ/mL of the drug even when a prolonged incubation of 48 hours and an inoculum of 106 CFU/spot were used. The Vitek system recorded correctly both isolates as having intermediate level of resistance to glycopeptides; this result was particularly important given the wide use of this commercial system in many hospital laboratories. However, as was reported for previous glycopeptide-intermediate staphylococci (2,5), both isolates appeared to be susceptible to vancomycin when tested by the disk diffusion method, with zones of 15 mm and 16 mm for the S. aureus and the S. sciuri isolates, respectively. Vancomycin-resistant staphylococci had not been detected in our hospital until December 2000. Therefore, the emergence of vancomycin-resistant staphylococci is a recent development, suggesting a potential for wider dissemination. Since the NCCLS agar dilution method we used is not sensitive for the detection of heterogeneous resistance phenotypes (7), screening for vancomycin-resistant subpopulations (mainly those with MIC for vancomycin of 4 µg/mL) in the vancomycin-susceptible isolates is important. Whether the mechanisms responsible for homogeneous intermediate resistance to vancomycin in our staphylococci are similar to those described in isolates from Japan and elsewhere still remains to be answered.

High frequency of concomitant nm23-H1 and E-cadherin transcriptional inactivation in primary non-inheriting colorectal carcinomas

This study evaluated the added significance of nm23-H1 to that of E-cadherin in determining metastatic proclivity in primary sporadic colorectal carcinomas (SCRCs). A clinical cohort of 52 SCRCs was examined for the significance of nm23-H1 and E-cadherin mRNA levels and E-cadherin protein expression levels into the progression of colorectal tumor invasion, determined by their relevance compared with conventional biological markers. A more than twofold decreased expression of nm23-H1 mRNA was reported in 28/52 (54%) of the carcinomas and was positively associated with the presence of nodal metastases and Astler-Coller stages B1 and B2 in 29% and 35% of the SCRCs, respectively. Reduced expression of E-cadherin mRNA was reported in 38.5% of the carcinomas and was similarly associated with stages Astler-Coller B1 and B2 in 27% of the SCRCs. Decreased E-cadherin immunohistochemical expression (grades II and III) was observed in 67% of the samples. E-cadherin mRNA and protein expression were significantly related to each other. The nm23-H1 (+)/E-cadherin (+) coexpression profile was observed in 31% and was significantly related to the absence of lymph node metastases in 31% and stages Astler-Coller B1 and B2 in 29% of the carcinomas examined. Furthermore, the nm23-H1 (−)/E-cadherin (+) coexpression profile was coupled to decreased E-cadherin immunohistochemical protein detection (grade II) in 21% of the cases examined. These findings suggest that impairment of nm23-H1 expression is an early event into the progression of colorectal metastasis that precedes E-cadherin transcriptional silencing in the majority of SCRCs examined. Nm23-H1 may therefore play an important role in suppressing the early steps of metastasis in sporadic cases of colorectal carcinomas.

c-mos immunoreactivity is an indicator of good prognosis in lung cancer

Reports concerning the expression of cytoplasmic components of the mitogen‐activating protein kinase (MAPK) pathway in lung cancer are limited. One of the molecules participating in this pathway is the product of the c‐mos proto‐oncogene. In vitro investigations, in somatic cells, have shown that c‐mos expression has opposing effects on cell cycle progression suggesting that it may represent an important determinant of aberrant cell function. In this study we analysed, by immunohistochemical means, its status in a series of lung carcinomas and correlated the findings with clinicopathological parameters and survival of the patients.

Transcriptional impairment of β-catenin/E-cadherin complex is not associated with β-catenin mutations in colorectal carcinomas

We report the absence of β-catenin mutations in 63 sporadic colorectal carcinomas (SCRCs) with demonstrated decreased β-catenin and E-cadherin mRNA expression and E-cadherin protein expression in a subset of carcinomas examined, suggesting that β-catenin mutations are an extremely rare phenomenon in SCRCs and are not responsible for the transcriptional impairment of the β-catenin/E-cadherin adhesion complex observed in these tumours.

Allelic imbalance at the 5q14 locus is associated with decreased apoptotic rate in non-small cell lung carcinomas (NSCLCs). Possible synergistic effect with p53 gene alterations on apoptosis.

Deletions in the 5q14 region have been described in a variety of neoplasms, such as testicular germ cell tumors, ovarian, gastric and lung cancer. The high frequency of allelic losses observed in this region implies the presence of putative tumor suppressor gene(s) (TSGs). In the present study, we investigated in a series of 56 non-small cell lung carcinomas (NSCLCs) the allelic imbalance (Alm) within the 5q14 region, employing the D5S644 marker, and its relationship with p53 abnormalities, the kinetic parameters [proliferation index (PI) and apoptotic index (AI)] and the ploidy status of the carcinomas. AI at D5S644 was found at a frequency of 51.2%. The rather high percentage of Alm in stage I tumors suggests an early involvement in NSCLC development. LOH at 5q14 was associated with decreased AR in lung tumors insinuating the presence of a putative TSG(s) (P=0.008). Simultaneous alterations of both p53 and D5S644 locus were the most frequent pattern observed (37.5%). Cases demonstrating this profile also exhibited a marked decrease in AI (P=0.001). These findings imply a synergistic mechanism of co-operation between different TSGs. However, proliferation activity was dependent only on p53 status, leading to the assumption that the putative TSG(s) present at 5q14 may probably be involved in normal apoptotic procedures. Further studies are needed to identify the candidate gene(s).