Frequent allelic losses on chromosomes 2q, 18q, and 22q in advanced non-small cell lung carcinoma.

Although it is widely accepted that tumor suppressor genes play an important role in the genesis and progression of human cancer, little is known about genetic events that accumulate during multistage lung carcinogenesis. Thus, to determine a subset of tumor suppressor genes that are involved in the genesis and progression of non-small cell lung carcinoma (NSCLC), 22 brain metastases and 23 stage I primary lung tumors were examined for allelic losses at 40 loci on 10 chromosomes including the loci of 5 tumor suppressor genes, APC, WT1, RB, p53, and DCC. The incidence of allelic losses on chromosomes 3p, 13q, and 17p was high (> 60%) in both primary tumors and brain metastases. In brain metastases, a high incidence of allelic losses (> 60%) was also observed at loci on chromosomes 2q, 18q, and 22q, and the incidence of allelic losses on these chromosomes in brain metastases was significantly higher than that in primary tumors (P < 0.05). In two cases of brain metastases with corresponding primary lung tumors, sequential accumulation of allelic losses during progression of primary lung tumors was observed on several chromosomes including chromosomes 2q and 18q. These results indicate that, besides loss of heterozygosity for chromosomes 3p, 13q, and 17p, loss of heterozygosity for chromosomes 2q, 18q, and 22q also occurs frequently in advanced NSCLCS. Thus, it is possible that loss of heterozygosity on chromosomes 2q, 18q, and 22q occurs late in the progression of NSCLC and/or causes phenotypic alterations of NSCLC cells into more aggressive ones.

Mutations in the p16INK4/MTS1/CDKN2, p15INK4B/MTS2, and p18 genes in primary and metastatic lung cancer.

We examined the genomic status of cyclin-dependent kinase-4 and -6 inhibitors, p16INK4,p15INK4B, and p18, in 40 primary lung cancers and 31 metastatic lung cancers. Alterations of the p16INK4 gene were detected in 6 (2 insertions and 4 homozygous deletions) of 22 metastatic non-small cell lung cancers (NSCLCs; 27%), but none were detected in 25 primary NSCLCs, 15 primary small cell lung cancers (SCLCs), or 9 metastatic SCLCs, indicating that mutation in the p16INK4 gene is a late event in NSCLC carcinogenesis. Although three intragenic mutations of the p15INK4B gene were detected in 25 primary NSCLCs (12%) and five homozygous deletions of the p15INK4B gene were detected in 22 NSCLCs (23%), no genetic alterations of the p15INK4B gene were found in primary and metastatic SCLCs. The p18 gene was wild type in these 71 lung cancers, except 1 metastatic NSCLC which showed loss of heterozygosity. We also examined alterations of these three genes and expression of p16INK4 in 21 human lung cancer cell lines. Alterations of the p16INK4 and p15INK4B genes were detected in 71% of the NSCLC cell lines (n = 14) and 50% of the NSCLC cell lines (n = 14), respectively, but there were none in the 7 SCLC cell lines studied. No p18 mutations were detected in these 21 cell lines. These results indicate that both p16INK4 and p15INK4B gene mutations are associated with tumor progression of a subset of NSCLC, but not of SCLC, and that p15INK4B mutations might also be an early event in the molecular pathogenesis of a subset of NSCLC.

Reconstitution of the RB gene suppresses the growth of small-cell lung carcinoma cells carrying multiple genetic alterations.

Multiple genetic alterations, including inactivation of the RB gene, occur commonly in small-cell lung carcinoma (SCLC). To assess a functional role of RB inactivation in the development of SCLC, an RB expression plasmid was introduced by stable transfection into SCLC cell lines, Lu-135 and N417, in which the RB gene was inactivated. Lu-135 and N417 cells transfected with the wild-type RB gene formed G418-resistant colonies twofold less efficiently than those with a mutated RB gene or with the control vector. Intact exogenous wild-type RB genes were detected only in approximately 20% of G418-resistant clones; three of 14 in Lu-135 and three of 16 in N417, respectively. Transcripts from the transfected RB gene were also detected in two of these three clones from Lu-135 and two of three from N417 but the amount of RB mRNA and protein was less than one fifth of that in normal fibroblast cells WI-38. Furthermore, clones with exogenous wild-type RB expression showed either reduced growth rates in culture or suppressed tumorigenicity in nude mice. These findings suggest that functional correction of the RB gene is sufficient to suppress the growth of SCLC cells, even though several other genetic alterations in the cells remain uncorrected.

Allelotype of neuroblastoma.

Although relatively high incidence of loss of heterozygosity (LOH) on chromosomes 1p, 11q, and 14q have been reported in neuroblastoma, it is still unclear whether or not LOH occurs specifically on these chromosome arms in neuroblastoma since only a few chromosomal arms have been examined for LOH in previous studies. Therefore, we screened 81 cases of tumors for LOH on all 22 autosomes and chromosome X using 35 restriction fragment length polymorphism markers and eight microsatellite markers. High incidence of LOH (> 20%) was observed on six chromosome arms; 1p (26%), 2q (30%), 9p (36%), 11q (24%), 14q (22%), and 18q (31%). Frequencies of LOH on other chromosome arms were less than 13%. Patients with 9p LOH in the tumors showed statistically significant association with advanced stage of the disease and poor prognosis (P = 0.037 and P = 0.003, respectively) independently from N-myc amplification, while LOH on other chromosomes did not show association with stage, prognosis, and N-myc amplification. Thus, besides LOH on chromosomes 1p, 11q, and 14q, LOH on chromosomes 2q, 9p, and 18q also occurs relatively frequently in neuroblastoma, indicating the involvement of multiple tumor suppressor genes in the development of neuroblastoma. It is possible that there is a novel tumor suppressor gene on chromosome 9p which is involved in the progression of neuroblastoma.

Germ-line p53 mutation is uncommon in patients with triple primary cancers.

We examined five patients with multiple primary cancers who had a history of three different types of primary cancers for germ-line p53 mutations. The germ-line p53 mutation was detected in a patient who conformed to the Li-Fraumeni syndrome, but not in the other four patients. The diagnosis of these four patients did not fall in the category of Li-Fraumeni syndrome. This result indicates that germ-line p53 mutations are uncommon even in patients with triple primary cancers.

Microsatellite instability in gastric cancer prone families.

We examined for germ-line p53 mutations and microsatellite instability in three gastric cancer patients who had family histories of gastric cancer aggregation. Although no germ-line p53 mutation was detected in these three cases, the replication error (RER) phenotype was observed in two of them. One base deletion in the sequence of ten repeating adenines of the type II transforming growth factor-beta receptor gene was detected in one of these two cases. Furthermore, there were young patients of 50 years and downward in their families. Therefore, it is possible that inherited disorders in mismatch repair systems contribute to high susceptibility to gastric cancers in these families.

[A fulminating case of Edwardsiella tarda septicemia with necrotizing fasciitis].

A 67-year-old Japanese male, suffering from liver cirrhosis with hepatoma, was admitted to the Yokohama National Hospital because of ascites retention. On physical examination, his abdomen was massively distended with ascites and his lower extremities were edematous. Laboratory findings on admission revealed hypoalbuminemia, moderate icterus, pancytopenia and hepatitis C virus antibody positivity. After admission, abdominal distention and edema were improved with the use of diuretics. On the 15th day of hospitalization, the patient noted diarrhea and bowel movements that occurred 10 times a day. On the following day, his body temperature rose to over 39 degrees C. On the morning of the 17th day, he complained of severe pain in the right lower extremity. Swelling and erythema over his right lower leg were evident. The skin lesion spread rapidly over the knee and became necrotic. His right leg became increasingly swollen with the development of edema and hemorrhagic bullae. About 4 hrs after the emergence of the skin lesion, his blood pressure fell to less than 60 mmHg. Laboratory findings suggested disseminated intravascular coagulation and multiple organ failure due to serious bacterial infection. In spite of vigorous treatment including administration of antibiotics, dopamine, gabexate mesilate and plasma, he did not recover from the state of shock and died about 14 hrs after the appearance of leg pain. Bacterial culture of the blood and contents of the bullae grew a gram negative rod identified as Edwardsiella tarda (E. tarda). Histological findings showed necrotizing fasciitis. E. tarda has recently become recognized as a pathogenic bacteria, particularly in patients with an underlying illness. This is the first reported case of E. tarda septicemia with necrotizing fasciitis.

[Two cases of methicillin-resistant Staphylococcus aureus (MRSA) sepsis following craniotomy].

We report here two cases of MRSA sepsis following craniotomy. In case 1, a petroclival meningioma was subtotally removed and lumbar drainage was inserted postoperatively to prevent cerebrospinal fluid leakage. Ventriculo-peritoneal shunt was performed after meningitis was treated with vancomycin and panipenem/betamipron. Two weeks after the procedure, the patient revealed continuous spiking fevers related to MRSA sepsis, which did not improve with vancomycin and arbekacin administration. The focus of infection was found by scintigraphy and CT by 67Ga to be spondylo-diskitis at the level of L2-L3. The lesion was removed and bone from the iliac crest grafted. In case 2, seven days after surgery for multiple meningioma, the patient exhibited spiking fevers and swelling in the left leg. The central venous catheter was removed from the left femoral vein and MRSA was found from blood culture. The patient was treated with arbekacin (200 mg/day). Venous thrombosis diagnosed by CT was treated with heparin. Symptoms related to the infection and laboratory data did not improve because the concentration of arbekacin in the blood did not reach an effective level. The symptoms markedly improved when the dose of arbekacin was doubled (400 mg/day).

Donor cell-derived leukemia after cord blood transplantation and a review of the literature: differences between cord blood and BM as the transplant source.

Donor cell-derived leukemia (DCL) is a rare complication of SCT. Here, we present a case of DCL following cord blood transplantation (CBT) and review the clinical features of previously reported DCL. To our knowledge, this is the first report comparing clinical characteristics of DCL from the standpoint of the transplant source, with umbilical cord blood and BM. AML and myelodysplastic syndrome (MDS) were recognized more frequently in DCL after CBT, whereas the incidence of AML and ALL was similar after BMT. The median duration between the occurrence of DCL following CBT and BMT was 14.5 and 36 months, respectively. DCL occurred in a significantly shorter period after CBT than after BMT. Abnormal karyotypes involving chromosome 7 were observed in 52.4% of CBT recipients and 17.3% of BMT recipients; this was a statistically significant difference. Particularly, the frequency of monosomy 7 was significantly higher in DCL after CBT than after BMT. The types of abnormal karyotypes in DCL following BMT were similar to those characteristically observed in adult de novo AML and MDS. DCL patients generally have a poor prognosis in both groups. SCT is the best treatment for curing DCL. DCL appears to have different clinical features according to the transplant source.

Combined effects of vancomycin and imipenem against methicillin-resistant Staphylococcus aureus (MRSA) in vitro and in vivo.

Infectious disease caused by methicillin-resistant Staphylococcus aureus (MRSA) often develops in compromised hosts in whom a single administration of vancomycin is usually not effective. We assessed the combined antimicrobial effects of vancomycin and imipenem against MRSA growth in vitro as well as in vivo using a neutropenic mouse thigh infection model. Synergic and additive effects of the two drugs were observed for 34 and two, respectively, of the 36 clinical isolates of MRSA, as determined by the chequerboard method. For MRSA strain N, postantibiotic effect (PAE) values obtained in vitro were 1.9-2.6 h for vancomycin and 2.6-3.5 h for imipenem, while higher values of 2.7-4.4 h were obtained for the combination of vancomycin and imipenem. In vivo, a single administration of vancomycin at 1 or 2 mg/kg or of imipenem/cilastatin at 5 mg/kg produced growth curves similar to those in controls, with a suppressive time of 0 h. Imipenem/cilastatin at 10 mg/kg demonstrated a suppressive time of 3.1 h. Combinations of vancomycin 1 mg/kg plus imipenem/cilastatin 5 mg/kg, vancomycin 1 mg/kg plus imipenem/cilastatin 10 mg/k and vancomycin 2 mg/kg plus imipenem/cilastatin 10 mg/kg demonstrated suppressive times of 2.9, 3.9 and 5.2 h, respectively, indicating that combined administration was effective. Simultaneous administration of the two drugs was more effective than sequential administration. Thus, combined administration of vancomycin and imipenem/cilastatin proved effective for MRSA in vivo. The combined effects seemed to be synergic, since a single administration of either drug did not show anti-MRSA effects at the doses used in the combined regimen.

Microsatellite instability in primary and metastatic colorectal cancers.

Microsatellite instability characterizes a sub-set of sporadic colorectal cancers (CRCs) as well as CRCs from patients with hereditary non-polyposis colorectal cancer (HNPCC). In order to clarify when the cells acquire a replication-error phenotype (RER) during colorectal-tumor progression, we examined the incidence of RER in 80 primary tumors and 36 liver metastases at 8 microsatellite loci; 1 mono-, 5 di-, 1 tetra- and 1 pentanucleotide. RER were detected in 20.1% (17/80) of primary tumors, including 5 tumors showing RER at 2 or more loci (RER2), while the incidence of RER in liver metastases (22.2%, 8/36) was almost the same as that in primary tumors, and there was only one RER2 case in metastases. There were 3 cases in which both primary tumors and liver metastases had the same type of RER at the same locus, and there were 2 cases that showed RER in primary tumors but not in liver metastases. In contrast, there was no case in which RER was detected in a metastasis but not in the corresponding primary tumor. The RER phenotype did not show correlation with any clinicopathological parameters of cancer-cell aggressiveness, such as clinical staging, histological grade and survival. These results indicate that a sub-set of CRCs acquire the RER phenotype in the relatively early stages of colorectal carcinogenesis, and that the RER phenotype is not associated with aggressiveness of CRCs.

Microsatellite instability in primary and metastatic lung carcinomas.

Fifty-seven primary lung carcinomas and 35 metastatic lung carcinomas were analyzed for microsatellite instability at II different chromosomal loci. Although no instability was detected in 37 small cell lung carcinomas (SCLC), it was frequently detected in non-small cell lung carcinomas (NSCLC) (16/55, 29%). In NSCLC, the incidence of replication errors (RERs) in metastatic tumors (12/22, 55%) was significantly higher than that in primary tumors (4/33, 12%) (P = 0.0021). Among 10 pairs of primary tumors and corresponding metastases, there were 4 cases which manifested the identical RER phenotypes in both primary and metastatic tumors. In two cases, RER phenotypes were detected in metastatic but not in primary tumors. Never was an RER phenotype found only in a primary tumor but not in the metastases. RERs were detected more frequently in stage III or IV tumors (3/8, 38%) than stage I or II tumors (1/25, 4%) (P = 0.0359). Tumor cells with allelic losses on chromosome arm 3p or 18q tended to have RER phenotypes (P = 0.0432 and P = 0.0187, respectively). The data suggest that microsatellite instability is common in NSCLC but not in SCLC, and that genomic instability appears late in tumor progression and plays an important role in the acquisition of more malignant phenotypes in NSCLC.

Induction of apoptosis but not G1 arrest by expression of the wild-type p53 gene in small cell lung carcinoma.

Multiple genetic alterations, including inactivation of the p53 and RB genes and loss of heterozygosity on chromosome 3p, occur commonly in small cell lung carcinoma (SCLC). To assess the biological significance of p53 inactivation in the development of SCLC, tetracycline (Tc)-inducible p53 expression plasmids were introduced into a SCLC cell line, N417, in which the p53 gene as well as the RB gene was inactivated. In the absence (induced) of Tc, cells transfected with the wild-type p53 gene formed colonies in 29-58% of those with a mutant p53 gene. However, wild-type p53 genes were expressed in 0 of 43 transfectants, whereas mutant p53 genes were expressed in 75% (36/48) of the transfectants, suggesting that the growth of SCLC cells was suppressed by the expression of the wild-type p53 gene. Thus, wild-type p53-inducible clones were further established by transfection in the presence (repressed) of Tc. The in vitro growth was significantly suppressed by the induction of wild-type p53 expression, and apoptosis but not G1 arrest was observed within 24 h of p53 induction. These results strongly suggest that the restoration of the p53 function is sufficient to suppress the growth of SCLC cells in which other genetic alterations remain uncorrected, and that growth suppression by p53 is due to induction of apoptosis but not due to induction of G1 arrest through the RB pathway.

Comparative allelotype of early and advanced stage non-small cell lung carcinomas.

To identify chromosomal loci of tumor suppressor genes involved in the genesis and progression of non-small cell lung carcinoma (NSCLC), comparative allelotype analysis was performed in 23 stage I primary lung tumors and in 22 metastatic lung tumors to the brain. In total, 84 loci on all 22 autosomal chromosomes were examined for loss of heterozygosity (LOH) by restriction fragment length polymorphism (RFLP) analysis with 40 polymorphic DNA probes and polymerase chain reaction (PCR)-LOH analysis of 44 polymorphic loci. LOH on chromosome arms 3p, 13q, and 17p was detected frequently (> 60%) in both stage I primary lung tumors and brain metastases, whereas the incidence of LOH on chromosome arms 2q, 5q, 9p, 12q, 18q, and 22q was more than 60% only in brain metastases. In particular, the incidence of LOH on chromosome arms 2q, 9p, 18q, and 22q in brain metastases was significantly higher than that in stage I primary lung tumors (P < 0.05). These results indicate that tumor suppressor genes on chromosome arms 3p, 13q, and 17p are involved in the genesis of NSCLC, whereas those on several chromosome arms, especially on 2q, 9p, 18q and 22q, play an important role in the progression of NSCLC.

DNA damage-inducible gene p33ING2 negatively regulates cell proliferation through acetylation of p53.

The p33ING1 protein is a regulator of cell cycle, senescence, and apoptosis. Three alternatively spliced transcripts of p33ING1 encode p47ING1a, p33ING1b, and p24ING1c. We cloned an additional ING family member, p33ING2/ING1L. Unlike p33ING1b, p33ING2 is induced by the DNA-damaging agents etoposide and neocarzinostatin. p33ING1b and p33ING2 negatively regulate cell growth and survival in a p53-dependent manner through induction of G(1)-phase cell-cycle arrest and apoptosis. p33ING2 strongly enhances the transcriptional-transactivation activity of p53. Furthermore, p33ING2 expression increases the acetylation of p53 at Lys-382. Taken together, p33ING2 is a DNA damage-inducible gene that negatively regulates cell proliferation through activation of p53 by enhancing its acetylation.

Comparison of pathogenic factors expressed by group A Streptococci isolated from patients with streptococcal toxic shock syndrome and scarlet fever.

Streptococcal toxic shock syndrome (STSS) is an illness with high mortality. To obtain clues to understanding the pathogenesis of STSS, we investigated the expression of several pathogenic factors in ten group A streptococcus (GAS) isolates from ten patients with STSS in Japan, in comparison with ten GAS isolates from children with scarlet fever. The ten scarlet fever-derived GAS isolates were equally low in lethality and anti-phagocytic activity in mice and in the production of streptolysin O (SLO), and equally high in production of superantigenic exotoxins (SAGTs) and cysteine proteinase. By comparison, the ten STSS-derived GAS isolates were heterogeneous in the expression of the above pathogenic factors, which ranged from low to high values. Most of the ten STSS-derived isolates were higher in lethality and anti-phagocytic activity and production of SLO, and lower in the production of SAGTs and cysteine proteinase than the ten scarlet fever-derived isolates. The results suggest that the lethality and anti-phagocytic activity examined in mice and SLO may be involved mainly in the development of most of the ten STSS cases.