Heparin-LL37 complexes are less cytotoxic for human dental pulp cells and have undiminished antimicrobial and LPS-neutralizing abilities.

AIM: To investigate whether glycosaminoglycans (GAGs) binding to high-dose LL37 eliminates its cytotoxicity to dental pulp cells (hDPCs) whilst retaining undiminished antimicrobial and LPS-neutralizing abilities. METHODOLOGY: hDPCs were stimulated with varying concentrations of LL37, and their cell viability was analysed by MTT. Then, high-dose LL37 (10 µmol L-1 ) was bound to varying concentrations of three GAGs, heparin, chondroitin sulphate and hyaluronic acid, and their cytotoxic effects on hDPCs and antimicrobial effects were evaluated and compared. Furthermore, the LPS-neutralizing ability of heparin (5 µg mL-1 )-LL37 (10 µmol L-1 ) complexes, which were found to be less cytotoxic for hDPCs with undiminished antimicrobial ability, was investigated. Statistical analysis was performed using one-way analysis of variance (anova), followed by Dunnett's test. P values below 0.05 were considered significant. RESULTS: LL37 significantly reduced the cell viability of hDPCs in a dose-dependent manner (P < 0.01). LL37 (10 µmol L-1 ) binding to heparin within a limited concentration range (2~6 µg mL-1 ) eliminated the cytotoxicity for hDPCs (P < 0.01) whilst exerting potent antimicrobial effects against Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Aggegatibacter actinomycetemcomitans and Escherichia coli. LL37 (10 µmol L-1 ) binding to chondroitin sulphate exhibited similar functions (P < 0.01); however, the effective chondroitin sulphate concentration was highly restricted (3 µg mL-1 ). LL37 (10 µmol L-1 ) binding to hyaluronic acid was unable to abrogate the cytotoxicity of LL37 even at higher concentrations (10 and 100 µg mL-1 ). Moreover, exogenous addition of LPS dose-dependently reduced the amount of LL37 precipitated with the heparin-LL37 agarose beads (P < 0.01), and the released LL37 simultaneously neutralized the pro-inflammatory ability of LPS in macrophages (P < 0.01). CONCLUSIONS: Heparin-LL37 complexes generated at suitable concentration ratios are easy to make, are less cytotoxic and are broad-range antimicrobial materials that can neutralize LPS by providing LL37 in accordance with the amount of free LPS. They may be a potential treatment to save dental pulp tissue from the acute inflammation exacerbated by invading bacteria and the LPS they release.

Pleomorphic lobular carcinoma of the breast: a comparison of cytopathological features with other lobular carcinoma variants.

OBJECTIVE: Pleomorphic lobular carcinoma (PLC) is a subtype of breast cancer with unique morphological features, but it remains controversial whether PLC should be considered an independent disease entity. The aim of this study was to illustrate cytopathological characteristics of PLC in comparison with other lobular carcinoma variants. METHODS: We investigated clinicopathological features of PLC (n = 11) compared with those of other variants of invasive lobular carcinoma (ILC, non-PLC) (n = 32). Histological variants of the non-PLC group consisted of classic (n = 25), solid (n = 2), alveolar (n = 1) and a tubulolobular type (n = 4). A review of cytological reports and fine needle aspiration (FNA) smear samples was performed for the PLC (n = 9) and non-PLC (n = 27) groups. RESULTS: Patients with PLC were older, and had a higher nuclear grade and a higher incidence of axillary lymph node metastasis and triple negative phenotype than non-PLC patients (P = 0.007, P < 0.001, P = 0.02 and P < 0.001, respectively). Cytological findings in PLC included medium- to large-sized nuclei, prominent nucleoli, a moderate-to-severe degree of pleomorphism, apocrine change and background necrosis, none of which were evident in the smears of the non-PLC group (P < 0.001, P = 0.002, P < 0.001, P < 0.001, and P = 0.03, respectively). Despite these differences, patients with PLC and non-PLC showed similar clinical outcomes in our follow-up period. CONCLUSIONS: Based on our results, a cytological diagnosis of PLC should be proposed if there are moderate- to large-sized nuclei, prominent nucleoli, a moderate-to severe degree of nuclear pleomorphism, apocrine change and necrosis in the background in FNA biopsy samples.

Mucinous carcinoma of the breast: a comparative study on cytohistological findings associated with neuroendocrine differentiation.

OBJECTIVE: Mucinous carcinoma (MCA) may show neuroendocrine differentiation (ND), but the cytological features characteristic of ND remains elusive. We compared fine needle aspiration (FNA) findings of MCA between cases with high and low degrees of ND. METHODS: Histological sections of 37 MCA cases were immunohistochemically evaluated for expression of chromogranin A and synaptophysin, and were graded as 0 to 3+ degrees of ND. They were divided into low ND (grade 0 and 1+) and high ND (grade 2+ and 3+) groups. Pre-operative FNA samples of each group were assessed for cytological features. RESULTS: The mean age of the high ND group (n = 18) was higher than the low ND group (n = 19, P = 0.01). In FNA samples of the high ND group, 17 cases showed moderate to severe degrees of discohesiveness, but low ND cases mainly showed no or only mild discohesiveness (P < 0.001). Nine of the low ND cases displayed overlapped, cohesive cell clusters, whereas, in the high ND cases, the cells were arranged in a loose, flat and monolayered pattern (P = 0.045). Fourteen of the high ND cases had round nuclei, but oval nuclei were predominant in the low ND cases (P = 0.027). The nuclei were eccentrically located in 12 of the high ND cases but were centrally located in 14 of the low ND cases (P = 0.01). CONCLUSIONS: Mucinous carcinoma with high ND may be diagnosed by the presence of discohesiveness, a flat, monolayered pattern, and round or eccentrically located nuclei. Features of ND in carcinomas in other organs, such as intracytoplasmic granules and coarse chromatin, may not be reliable cytological features of ND in MCA.

Kosterlitz-Thouless-type transition in a charge ordered state of the layered organic conductor α-(BEDT-TTF)2I3.

The current-voltage characteristics in the charge order state of the two-dimensional organic conductor α-(BEDT-TTF)(2)I(3) exhibit power law behavior at low temperatures. The power law is understood in terms of the electric-field-dependent potential between electrons and holes, which are thermally excited from the charge order state. The power law exponent steeply changes from 1 to 3 in the range from 30 to 45 K with decreasing temperature, thereby suggesting the occurrence of a Kosterlitz-Thouless-type transition; many (few) unbound electron-hole pairs are thermally excited above (below) the transition. The effects of the finite size and interlayer coupling on the power law behavior are discussed.

Loss of IκBζ Drives Dentin Formation via Altered H3K4me3 Status.

Enforced enrichment of the active promoter marks trimethylation of histone H3 lysine 4 (H3K4me3) and acetylation of histone H3 lysine 27 (H3K27ac) by inhibiting histone demethylases and deacetylases is positively associated with hard tissue formation through the induction of osteo/odontogenic differentiation. However, the key endogenous epigenetic modulator of odontoblasts to regulate the expression of genes coding dentin extracellular matrix (ECM) proteins has not been identified. We focused on nuclear factor (NF)-κB inhibitor ζ (IκBζ), which was originally identified as the transcriptional regulator of NF-κB and recently regarded as the NF-κB-independent epigenetic modulator, and found that IκBζ null mice exhibit a thicker dentin width and narrower pulp chamber, with aged mice having more marked phenotypes. At 6 mo of age, dentin fluorescent labeling revealed significantly accelerated dentin synthesis in the incisors of IκBζ null mice. In the molars of IκBζ null mice, marked tertiary dentin formation adjacent to the pulp horn was observed. Mechanistically, the expression of COL1A2 and COL1A1 collagen genes increased more in the odontoblast-rich fraction of IκBζ null mice than in wild type in vivo, similar to human odontoblast-like cells transfected with small interfering RNA for IκBζ compared with cells transfected with control siRNA in vitro. Furthermore, the direct binding of IκBζ to the COL1A2 promoter suppressed COL1A2 expression and the local active chromatin status marked by H3K4me3. Based on whole-genome identification of H3K4me3 enrichment, ECM and ECM organization-related gene loci were selectively activated by the knockdown of IκBζ, which consistently resulted in the upregulation of these genes. Collectively, this study suggested that IκBζ is the key negative regulator of dentin formation in odontoblasts by inhibiting dentin ECM- and ECM organization-related gene expression through an altered local chromatin status marked by H3K4me3. Therefore, IκBζ is a potential target for epigenetically improving the clinical outcomes of dentin regeneration therapies such as pulp capping.

Progressive atrophy of the cerebrum in 2 Japanese sisters with microcephaly with simplified gyri and enlarged extraaxial space.

This is a case report that describes 2 sisters with microcephaly, simplified gyri, and enlarged extraaxial space. Clinical features of the cases include dysmorphic features, congenital microcephaly, failure of postnatal brain growth, neonatal onset of seizures, quadriplegia, and severe psychomotor delay. Neuroradiological imaging demonstrated hypoplasia of bilateral cerebral hemispheres with enlarged extraaxial spaces, simplified gyral patterns without a thickened cortex, hypoplastic corpus callosum, and enlarged lateral ventricles, with a reduction in gray and white matter volume during the prenatal and neonatal periods. Repeat MRI revealed progressive atrophy of the cerebral gray and white matter, with enlarged lateral ventricles, although the sizes of the bilateral basal ganglia, thalamus, and infratentorial structures were relatively preserved. These neuroradiological findings imply that this disease is caused by the gene involved in neuronal and glial proliferation in the ventricular zone and in tangential neuronal migration from the ganglionic eminence. The nature of the progressive degeneration of the hemispheric structures should be clarified.

Grf40, A novel Grb2 family member, is involved in T cell signaling through interaction with SLP-76 and LAT.

We molecularly cloned a new Grb2 family member, named Grf40, containing the common SH3-SH2-SH3 motif. Expression of Grf40 is predominant in hematopoietic cells, particularly T cells. Grf40 binds to the SH2 domain-containing leukocyte protein of 76 kD (SLP-76) via its SH3 domain more tightly than Grb2. Incidentally, Grf40 binds to linker for activation of T cells (LAT) possibly via its SH2 domain. Overexpression of wild-type Grf40 in Jurkat cells induced a significant increase of SLP-76-dependent interleukin (IL)-2 promoter and nuclear factor of activated T cell (NF-AT) activation upon T cell receptor (TCR) stimulation, whereas the COOH-terminal SH3-deleted Grf40 mutant lacked any recognizable increase in IL-2 promoter activity. Furthermore, the SH2-deleted Grf40 mutant led to a marked inhibition of these regulatory activities, the effect of which is apparently stronger than that of the SH2-deleted Grb2 mutant. Our data suggest that Grf40 is an adaptor molecule involved in TCR-mediated signaling through a more efficient interaction than Grb2 with SLP-76 and LAT.

The cytokine and chemokine profiles in rhabdomyolysis in a patient with Gaucher disease type II.

This report describes a patient with Gaucher disease type II who developed severe rhabdomyolysis. We treated him successfully and measured various cytokine and chemokine levels sequentially to elucidate the pathophysiology of rhabdomyolysis. The serum levels of interleukin-6, -8, -10, granulocyte colony-stimulating factor, interferon-gamma, and monocyte chemoattractant protein-1 were markedly elevated in the early phase of rhabdomyolysis. These findings indicate that cytokines and chemokines are related to the massive myolysis and regenerating process. A viral infection may have triggered rhabdomyolysis through exaggerated activation of macrophages in our patient. The profiles of cytokines and chemokines should be examined in further cases to increase our understanding of the pathophysiology of rhabdomyolysis.

Hydroxyurea-induced foot ulcer in a case of essential thrombocythaemia.

OBJECTIVE: To illustrate the association between hydroxyurea and the development of ulcers. METHOD: A case study is presented, in which histological changes, mean corpuscular volume (MCV) and transcutaneous oxygen tension (TcPO2) were all measured and analysed, both during hydroxyurea treatment and following it's discontinuation. RESULTS: Two months following the cessation of hydroxyurea therapy, the patient's ulcer had healed completely. Biopsy specimens taken before and after its discontinuation showed a considerable improvement in vascularity, with a capillary density 6.28 times higher after discontinuation of the drug. TcPO2 was just 8mmHg at the first measurement, and this increased to 65mmHg at the second. CONCLUSION: These findings suggest deficient neovascularisation and circulation during hydroxyurea treatment. Changes in MCV also appeared to have an effect on the progress of wound healing, which supports the hypothesis that macroerythrocytosis may be involved in the development of these rare ulcers, via impairment of the microcirculatory rheology.

Efficacy of continuous acyclovir infusion in neonatal herpes virus encephalitis.

We have successfully eliminated herpes simplex virus-2 from the central nervous system in a case of neonatal herpes simplex virus encephalitis with a continuous acyclovir infusion. A male infant delivered from a healthy 22-year-old woman without genital or systemic herpes symptoms around delivery began to develop fever and intractable seizures. He was started on intermittent intravenous acyclovir (20 mg/kg every 8 h) based on the diagnosis of herpes encephalitis. The virus was not eliminated with intermittent acyclovir and vidarabine, while continuous acyclovir was ultimately effective in eliminating herpes simplex virus from his central nervous system. This report demonstrates the efficacy of continuous acyclovir infusion in neonatal herpes simplex virus encephalitis.

Isolated Pulmonary Cryptococcosis Confused with Lung Tumor 5 Years After Kidney Transplantation: A Case Report.

BACKGROUND: In transplant recipients, due to the use of immunosuppressive therapy, it is occasionally difficult to distinguish between an infection and malignancy, especially in the case of a lung lesion. Here, we report a case of isolated pulmonary cryptococcosis after kidney transplantation that was difficult to distinguish from a lung tumor. CASE REPORT: A 52-year-old man underwent a kidney transplant from his mother when he was 44 years old. Immunosuppression was maintained with tacrolimus, methylprednisolone, and mycophenolate mofetil. His post-transplant course was uneventful and serum creatinine levels were maintained. Five years post-transplantation, a non-contrast computed tomography (CT) examination revealed a nodule measuring 3 mm in diameter in the middle lobe of the right lung. The nodule gradually increased to 12 mm in 2 years. Positron emission tomography/CT examination showed a maximum standardized uptake value of 0.5 for the nodule. Biochemical examination revealed no elevation in total leucocyte count and C-reactive protein levels. However, tumor markers were elevated: serum carcinoembryonic antigen, 5.9 ng/mL; pro-gastrin-releasing peptide, 84.6 pg/mL. Furthermore, the serum cryptococcus antigen was negative. Therefore, thoracoscopic partial lung resection was performed. Pathologically, a number of spherical fungi from the necrotic substance of the tumor were confirmed positive by periodic acid-Schiff and Grocott-Gomori staining. The patient was therefore diagnosed with pulmonary cryptococcosis. Two years later, the patient is alive and has shown no evidence of recurrence. CONCLUSIONS: In lung nodules after kidney transplantation, even if serum cryptococcus antigen is not identified, it is necessary to keep in mind the possibility of pulmonary cryptococcosis.

Effects of buthionine sulfoximine treatment on diaphragm contractility and SR Ca2+ pump function in rats.

The purpose of this study was to examine the effects of glutathione (GSH) depletion and cellular oxidation on rat diaphragm contractility and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) function in vitro under basal conditions and following fatiguing stimulation. Buthionine sulfoximine (BSO) treatment (n = 10) for 10 days (20 mM in drinking water) reduced (P < 0.05) diaphragm GSH content (nmol/mg protein) and the ratio of GSH to glutathione disulfide (GSH/GSSG) by 91% and 71%, respectively, compared with controls (CTL) (n = 10). Western blotting showed that Hsp70 expression in diaphragm was not increased (P > 0.05) with BSO treatment. As hypothesized, basal peak twitch force (g/mm(2)) was increased (P < 0.05), and fatigability in response to repetitive stimulation (350-ms trains at 100 Hz once every 1 s for 5 min) was also increased (P < 0.05) in BSO compared with CTL. Both Ca(2+) uptake and maximal SERCA activity (mumol.g protein(-1).min(-1)) measured in diaphragm homogenates that were prepared at rest were increased (P < 0.05) with BSO treatment, an effect that could be partly explained by a twofold increase (P < 0.05) in SERCA2a expression with BSO. In response to the 5-min stimulation protocol, both Ca(2+) uptake and maximal SERCA activity were increased (P < 0.05) in CTL but not (P > 0.05) in BSO diaphragm. We conclude that 1) cellular redox state is more optimal for contractile function and fatigability is increased in rat diaphragm following BSO treatment, 2) SERCA2a expression is modulated by redox signaling, and 3) regulation of SERCA function in working diaphragm is altered following BSO treatment.

Clinical pictures of 75 patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD).

We clarified the clinical features of NICCD (neonatal intrahepatic cholestasis caused by citrin deficiency) by retrospective review of symptoms, management and long-term outcome of 75 patients. The data were generated from questionnaires to paediatricians in charge of the patients. Thirty of the patients were referred to hospitals before 1 month of age because of positive results in newborn screening (hypergalactosaemia, hypermethioninaemia, and hyperphenylalaninaemia). The other 45, the screen-negative patients, were referred to hospitals with suspected neonatal hepatitis or biliary atresia because of jaundice or discoloured stool. Most of the screen-negative patients presented before 4 months of age, and 11 had failure to thrive. Laboratory data showed elevated serum bile acid concentrations, hypoproteinaemia, low levels of vitamin K-dependent coagulation factors and hypergalactosaemia. Hypoglycaemia was detected in 18 patients. Serum amino acid analyses showed significant elevation of citrulline and methionine concentrations. Most of the patients were given a lactose-free and/or medium-chain triglyceride-enriched formula and fat-soluble vitamins. Symptoms resolved in all but two of the patients by 12 months of age. The two patients with unresolved symptoms suffered from progressive liver failure and underwent liver transplantation before their first birthday. Another patient developed citrullinaemia type II (CTLN2) at age 16 years. It is important to recognize that NICCD is not always a benign condition.

[The efficacy of perioperative administration of steroid and erythromycin in the surgery for lung cancer complicated with interstitial pneumonia].

OBJECTIVES: We evaluated the efficacy of perioperative administration of steroid and erythromycin in surgery for lung cancer complicated with interstitial pneumonia (IP) to prevent postoperative acute exacerbation. PATIENTS AND METHODS: We operated on 21 lung cancer patients with IP for 10 years. The patients were given 400 mg of erythromycin over 1 week before surgery and re-administered on the 1st operative day. The patients were also given 125 mg of methylprednisolone intravenously just before operation and continued until the 2nd operative day. RESULTS: Lobectomy was performed in 16, segmentectomy or partial resection in 2 each, and completion pneumonectomy in 1. Three patients developed acute exacerbation of IP, but it occurred after the re-operation due to postoperative complications in 2. We experienced no operative death within 30 days, however, 2 died during the hospital stay due to multiple organ failure and sepsis. Seven of 21 patients had postoperative complications; air leakage over 1 week in 4, arrhythmia in 3, and atelectasis, postoperative bleeding, and pneumonia in 1 each, the morbidity rate was 33%. CONCLUSIONS: We conclude that the administration of steroid and erythromycin in surgery for lung cancer with IP was suspected the usefulness to prevent a postoperative acute exacerbation of IP.

Current Practices in Choosing Estimands and Sensitivity Analyses in Clinical Trials: Results of the ICH E9 Survey.

BACKGROUND: An addendum to the International Conference on Harmonisation E9 (ICH E9) guidance document (Statistical Principles for Clinical Trials) is currently under development. The aim of the addendum is to promote harmonized standards on the choice of estimand (a well-defined measure of the treatment effect that is being estimated) in clinical trials and to describe a consensual framework for planning, conducting, and interpreting sensitivity analyses of clinical trial data. METHODS: In order to help understand current practices relating to the choice of estimands and sensitivity analyses for clinical trials, the ICH E9 working group developing the addendum conducted a survey with a primary focus on clinical trials involving drugs, vaccines, and biologics. The survey was distributed electronically between May 19, 2015, and June 11, 2015, to various stakeholder groups within ICH, including industry, regulatory, and academic communities. A total of 1305 respondents participated. RESULTS: Of the 1305 respondents 547 (42%), 344 (26%) and 283 (22%) were from Europe, USA and Japan respectively. Over half of the respondents work in pharmaceutical companies, and approximately a quarter of respondents noted oncology as the primary therapeutic area they work in. Over half of the respondents (595, 55%) noted the treatment effect being estimated was 'in the entire target population of patients regardless of whether they will take treatment as instructed'. The most common methods for handling missing data in primary analyses were mixed-models repeated measures (555, 56% respondents) and last observation carried forward (549, 55% respondents). The majority of respondents (816, 83%) noted they conducted sensitivity analyses to estimate treatment effects in different ways compared to the primary analysis by using alternative assumptions (627, 78%) and/or using alternative statistical methods (616, 76%). CONCLUSIONS: The survey results have provided useful information to the ICH E9 working group on current practices on the choice of primary estimands for measuring treatment effects in confirmatory clinical trials, and approaches used to select sensitivity analyses.

Allelotype of non-small cell lung carcinoma--comparison between loss of heterozygosity in squamous cell carcinoma and adenocarcinoma.

We examined loss of heterozygosity (LOH) on all autosomal chromosomes in 53 non-small cell lung carcinomas. Frequent LOH was observed on the long arms of chromosomes 1 (37%), 2 (31%), 5 (30%), 8 (31%), and 13 (32%), and the short arms of chromosomes 3 (54%) and 17 (62%). LOH on chromosomes 3p and 17p was observed in all informative cases of squamous cell carcinoma, but was significantly less frequent in adenocarcinomas (P = 0.003 and 0.001, respectively). Similarly, LOH on chromosome 13q was observed frequently in squamous cell carcinomas (5 of 9 informative cases, or 56%), but in only 5 of 26, or 19%, of adenocarcinomas. In contrast, LOH on chromosome 2q was observed only in adenocarcinomas. In addition, this chromosomal arm was lost more frequently in poorly differentiated, compared to well differentiated adenocarcinomas. Furthermore, a correlation between fractional allelic loss and pathohistological grade was identified. These results implicate the presence of several tumor suppressor genes associated with development and/or progression of non-small cell lung carcinomas.

Induction of maturation in cultured human monocytic leukemia cells by a phorbol diester.

Suspension cultures of a human monocytic leukemia cell line, THP-1, were treated with 0.16 to 160 nM 12-O-tetradecanoylphorbol-13-acetate (TPA). In an original cell line, THP-1-O, cultured again from -80 degrees cryopreservation, more than 80% of the cells adhered to the glass substrate with marked morphological change within 3 hr of TPA treatment. Adherent cells became flat and amoeboid in shape, and many microvilli and flaps of the cell surface disappeared. Well-developed Golgi apparatus, rough endoplasmic reticula, and a large amount of free ribosomes were seen in the cytoplasm. On the other hand, in THP-1-R cells cultured continuously without cryopreservation for 26 months, approximately 80% of the cells adhered to the substrate 48 hr after TPA treatment. Round and ovoid shapes were kept in THP-1-R cells treated with TPA. Surface Fc receptors for immunoglobulin G were present on more than 90% of THP-1-O and THP-1-R cells and were little affected by treatment with TPA. Sixty to 70% of the TPA-treated THP-1-O and THP-1-R cells were able to phagocytize yeasts and immunoglobulin G-coated sheep erythrocytes. Less than 20% of the untreated THP-1 cells were able to phagocytize yeasts and immunoglobulin G-coated sheep erythrocytes. In histochemical staining, alpha-naphthyl butyrate esterase was enhanced after treatment with TPA. Lysozyme activity in culture supernatants was not affected by TPA treatment. When exposed to latex beads and TPA, increased 14CO2 production from [1-14C]glucose in THP-1-O cells was observed. These results indicate that, after treatment with TPA, human monocytic leukemia cells may be converted into mature cells with functions of macrophages.

Prognostic value of genetically diagnosed lymph node micrometastasis in non-small cell lung carcinoma cases.

The predictive value of lymph node micrometastasis, detected by immunohistochemical or genetic methods, is well appreciated in terms of prognosis. However, a major problem is high false-positive rates, because most methods focus on cytokeratin, which is a component not only of carcinoma but also normal epithelial and nonepithelial cells. Mutant allele-specific amplification (MASA) can detect DNAs derived from cancer cells itself, reportedly with high sensitivity. It was, therefore, used with nested-PCR using p53 or K-ras mutation for analysis of lymph node micrometastasis in non-small cell lung carcinoma (NSCLC) patients in the present study, in comparison with the immunohistochemical method using an anti-cytokeratin reagent for the same samples. Lymph nodes from 31 NSCLC patients with p53 and K-ras mutated tumors (30 and 1, respectively) staged as pathological (p)-T1-4 N0-1 and M0 were examined. Genetic and immunohistochemical methods demonstrated positive reactions in 34 (15%) and 61 (27%) of 229 lymph nodes, respectively (9 cases, 29%, and 24 cases, 77%). The concordance with the two methods was 77%, but 13 (39%) of 34 genetically positive lymph nodes could not be detected by immunohistochemistry (IHC). Of 22 cases with p-N0 disease, 6 (27%) were genetically positive in hilar and/or mediastinal lymph nodes, and 4 (67%) of them died after cancer relapse. In contrast, none of the patients without micrometastasis died of cancer (P < 0.001, log rank analysis). Of the same p-N0 patients, 17 (77%) were positive by IHC, and 4 (24%) of them died of cancer, whereas 5 negative patients did not suffer cancer relapse. Survival did not significantly differ between cases positive and negative (P = 0.246) by IHC. According to the g-N (N factor restaged by a genetic method), patients with g-N1 and g-N2 disease had a shorter survival than those with g-N0 disease (P = 0.042 and P < 0.001, respectively). However, no significant difference was observed with grading by IHC. Thus, detection of micrometastasis in regional lymph nodes with the MASA method, in other words with a carcinoma-specific marker, is of greater prognostic significance for early stage NSCLC patients than immunohistochemical results. This approach should facilitate selection of patients for whom postoperative adjuvant chemotherapy should be performed.

p53 null mutations undetected by immunohistochemical staining predict a poor outcome with early-stage non-small cell lung carcinomas.

The importance of p53 mutations in the pathogenesis of human lung carcinoma is well established, but it is still controversial whether the presence of p53 mutations or overexpression of p53 protein adversely affects an individual patient's chances of survival. The controversy may be partially due to the methodological differences in examination for p53 alterations: gene analysis or immunohistochemical staining. Furthermore, recent studies have suggested that different types of mutations of the p53 tumor suppressor gene confer different biological properties. To clarify the relationship between immunohistochemical staining and prognosis, we investigated mutations using single-strand conformation polymorphism followed by sequencing for exons 4-8 and 10 in 144 surgically treated non-small cell lung carcinoma patients with intensive clinical follow-up. Of 144 cases, 107 adenocarcinomas were examined for immunohistochemical staining with RSP53 antibody. p53 gene mutations were observed in 65 tumors (45%), including 44 missense and 21 null mutations, the latter comprising 7 nonsense mutations, 8 deletions, 2 insertions, and 4 splicing junction mutations. Presence of p53 mutations was an independent prognostic factor with a statistical trend (P = 0.14) in stage I patients but not in all cases. When examined by mutational pattern, null mutation was a significant indicator of poor outcome by multivariate analysis (P = 0.03) in stage I patients, whereas cases with missense mutations and without mutations did not differ (P = 0.76). Forty (37%) tumors demonstrated overexpression of the p53 protein but without any survival difference. Most tumors (76%) with missense mutations were immunopositive, but those with null mutations with one exception (93%) were not, and the concordance between the mutations and immunohistochemical staining was rather low at 65%. These data suggest that the type of p53 mutation is important for prediction of outcome in early-stage non-small cell lung carcinoma patients, whereas immunohistochemical staining for abnormal p53 gene products is nonpredictive. Furthermore, null mutations causing loss of function of the gene product may play more important roles than missense mutations in tumor progression.