Comparison between a new assay system, Elecsys® Anti-p53, and conventional MESACUP™ for the detection of serum anti-p53 antibodies: A multi-institutional study.

The sensitivity and specificity of a new automated electrochemiluminescence immunoassay system, Elecsys® Anti-p53 (Elecsys), were compared with that of the conventional serum anti-p53 antibody (s-p53-Ab) enzyme-linked immunosorbent assay kit [MESACUP anti-p53 test (MESACUP)]. Elecsys and MESACUP were used to analyze the levels of s-p53-Abs in patients with esophageal, colorectal and breast cancer. A total of 532 controls and 288, 235 and 329 patients with esophageal, colorectal and breast cancer, respectively, were enrolled. Additionally, the sera of patients with benign diseases of the esophagus, colorectal system and breast, patients with autoimmune diseases and healthy volunteers were analyzed as controls. Sensitivity and specificity were compared between the two assay systems. Positive agreement rates were 58.7% in all samples, 71.2% in esophageal samples, 73.6% in colorectal samples and 35.1% in breast samples. Negative agreement rates for the different cancer types were ≥97.1% and the overall agreement rates were ≥92.3%. When the specificities of the two assays were aligned for all samples, Elecsys demonstrated higher sensitivities for all types of analyzed cancer together, as well as for esophageal, colorectal and breast cancer, respectively. Although positive concordance between the two assay systems was low in terms of specificity, Elecsys had a higher sensitivity than the MESACUP.

New Assay System Elecsys Anti-p53 to Detect Serum Anti-p53 Antibodies in Esophageal Cancer Patients and Colorectal Cancer Patients: Multi-institutional Study.

BACKGROUND: Several recent studies suggest that serum anti-p53 antibodies (s-p53-Abs) may be combined with other markers to detect esophageal and colorectal cancer. In this study, we assessed the sensitivity and specificity of s-p53-Abs detection of a new electrochemiluminescence immunoassay (ECLIA; Elecsys anti-p53). METHODS: Elecsys anti-p53 assay was used to analyze the level of s-p53-Abs in blood sera from patients with esophageal or colorectal cancer taken before treatment. Control blood sera from healthy volunteers, patients with benign diseases, and patients with autoimmune diseases served as a reference. In addition, squamous cell carcinoma antigen (SCC-Ag) and cytokeratin 19 fragments (CYFRA21-1) were assessed in patients with esophageal cancer, and carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 were assessed in patients with colorectal cancer. RESULTS: Samples from 281 patients with esophageal cancer, 232 patients with colorectal cancer, and 532 controls were included in the study. The median value of s-p53-Abs in control samples was < 0.02 µg/mL (range < 0.02-29.2 µg/mL). Assuming 98% specificity, the cut-off value was determined as 0.05 µg/mL. s-p53-Abs were detected in 20% (57/281) of patients with esophageal cancer and 18% (42/232) of patients with colorectal cancer. In combination with SCC-Ag and CEA, respectively, s-p53-Abs detected 51% (144/281) of patients with esophageal and 53% (124/232) of patients with colorectal cancer. CONCLUSIONS: The new s-p53-Abs assay Elecsys anti-p53 was useful in detecting esophageal and colorectal cancers with high specificity. Adding s-p53-Abs to conventional markers significantly improved the overall detection rates.

Performance analysis of the anti-Ki67 antibody clone 30-9 for immunohistochemical staining of breast cancer.

BACKGROUND: Although Ki67 has important clinical relevance in breast cancer, its assessment results vary according to assay due to differences in both analytical and interpretation processes. We aimed to validate the performance of anti-Ki67 antibody clone 30-9 by comparison with clone MIB-1 and to investigate utility of the image analysis system in Ki67 assessment using clinical breast cancer samples. METHODS: A series of sequential tissue sections was prepared from formalin-fixed paraffin-embedded blocks of surgically resected breast cancer specimens from 50 patients. The tissue sections were stained immunohistochemically with anti-Ki67 antibodies, 30-9 and MIB-1, as well as with hematoxylin and eosin for morphological analysis. We scanned all the stained slides with Ventana iScan HT and selected the Ki67 counting areas based on morphological findings. Three pathologists independently studied images of the counting areas to determine Ki67-positive rates. In addition, the images of 30-9-stained slides were analyzed using the image analysis system, VENTANA Virtuoso. RESULTS: Ki67-positive rates by 30-9 showed a strong correlation with those by MIB-1 for all pathologists (pathologist #1: r = 0.985, pathologist #2: r = 0.987, pathologist #3: r = 0.982). Between 30-9 and MIB-1, there was no significant difference of CV%, showing variabilities of Ki67-positive rates among pathologists. Ki67-positive rates showed a strong correlation between the image analytical values and the pathologist-counted median values (r = 0.952). CONCLUSIONS: The performance of 30-9 is equivalent to that of MIB-1 in Ki67 assessment of breast cancer. The image analysis system can substitute for or support visual counting by a pathologist.

[Association between smoking and tuberculosis infection].

PURPOSE: Several reports show smoking as a risk factor of tuberculosis (TB) infection, especially in prisoners, emigrants, the homeless, or people in areas where TB is endemic. These reports mostly used the tuberculin test to detect TB. However, there is no report evaluating smoking as a risk factor of TB infection among people coming into contact with TB with the use of the Interferon-Gamma Release Assays (IGRA) test. MATERIAL & METHOD: We compared TB infection in smokers and non-smokers who came into contact with TB infection by using the IGRA test. We retrospectively collected information about people coming into contact with TB who visited the Daiichi Dispensary from July 1, 2011 to June 30, 2012. They were divided into 2 groups (IGRA positive or negative) and smoking (present/past or never). RESULT: Out of 390 subjects who came into contact with TB examined, 229 were male and 161 were female. The mean age was 39.0 years, 98 were present smokers, 69 were past smokers, and 223 were never-smokers. There were 19 IGRA-positive and 371 IGRA-negative subjects. The IGRA positive rate was 4.9%. Out of 19 IGRA-positive subjects, 13 were smokers or ever-smoker (68.4%). Out of 371 IGRA-negative subjects, 154 cases were smoker or ever-smoker (41.5%). Smoking experience (present and past) was statistically significant in the IGRA-positive group. There were no significant differences in sex, age, drinking habits, and level of contact. Multivariate analysis showed smoking was only one independent risk factor for being IGRA-positive (odds ratio 3.06, 95% confidence interval: 1.14-8.21, p = 0.027). DISCUSSION: Our results suggest that smoking experience in subjects coming into contact with TB is a risk factor for TB infection. TB cases in smokers are reported to be more severe and have delayed detection of disease. They are also more likely to infect those who come in contact with them. If TB source cases and their contacts are both smokers and co-exist in a narrow and limited area, the contacts might be at higher risk of exposure to TB-contaminated air than non-smokers.

[Liver dysfunction during treatment of latent tuberculosis infection].

PURPOSE: The indications for treatment for latent tuberculosis infection were revised in 2007 to reflect that any subject with a higher risk of tuberculosis regardless of age should be treated. We worried about the incidence of liver dysfunction due to isoniazid (INH) in patients older than 30 yrs. of age. We evaluated the frequency of liver dysfunction due to INH according to age and discussed the possibility of its prevention. METHODS: We reviewed the clinical records of 99 patients younger than 29 yrs. and 229 patients older than 30 yrs. who were treated for latent tuberculosis infection from August 2007 to December 2008 at our clinic. The liver function tests (AST and ALT) were performed before the treatment, one and a half months after the start of the treatment, and almost every month during the treatment. We defined liver dysfunction as an AST and/or ALT greater than 100 IU/L. RESULTS: Seven out of the 99 younger patients (7.1%) and 42 out of the 229 (18.3%) older patients developed liver dysfunction. The difference between the two age groups was statistically significant according to the chi-square test (p < 0.01). After the occurrence of liver dysfunction, 35 out of 49 patients (71%) completed the treatment by maintaining the same or a decreased dose of INH, while the medication was discontinued in 9 patients who were then followed up by chest X-ray examination. Two of these 49 patients discontinued the medication by themselves. CONCLUSIONS: The frequency of liver damage due to INH was higher in persons older than 30 yrs. In this group, 3 persons developed severe liver damage with ALT and/or AST higher than 1000 IU/L. Early detection is required to avoid serious damage. Thus, we decided to perform liver function tests more often, i.e., at 2 weeks after the onset of treatment and every month thereafter.

Correlating phosphatidylinositol 3-kinase inhibitor efficacy with signaling pathway status: in silico and biological evaluations.

The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in human cancers, and several agents targeting this pathway including PI3K/Akt/mammalian target of rapamycin inhibitors have recently entered clinical trials. One question is whether the efficacy of a PI3K pathway inhibitor can be predicted based on the activation status of pathway members. In this study, we examined the mutation, expression, and phosphorylation status of PI3K and Ras pathway members in a panel of 39 pharmacologically well-characterized human cancer cell lines (JFCR39). Additionally, we evaluated the in vitro efficacy of 25 PI3K pathway inhibitors in addition to conventional anticancer drugs, combining these data to construct an integrated database of pathway activation status and drug efficacies (JFCR39-DB). In silico analysis of JFCR39-DB enabled us to evaluate correlations between the status of pathway members and the efficacy of PI3K inhibitors. For example, phospho-Akt and KRAS/BRAF mutations prominently correlated with the efficacy and the inefficacy of PI3K inhibitors, respectively, whereas PIK3CA mutation and PTEN loss did not. These correlations were confirmed in human tumor xenografts in vivo, consistent with their ability to serve as predictive biomarkers. Our findings show that JFCR39-DB is a useful tool to identify predictive biomarkers and to study the molecular pharmacology of the PI3K pathway in cancer.

A new evaluation method for quantifying PI3K activity by HTRF assay.

PIK3CA, coding a catalytic subunit of PI3K p110alpha, is frequently mutated in cancer. In previous studies, p110alpha with hotspot mutations such as E545K and H1047R were shown to be gain-of-function mutations. However, quantitative evaluation of these mutants was not well established. Recently, a new method for measuring PI3K activity using homogeneous time-resolved fluorescence (HTRF) has been developed. Using this method, we constructed a quantitative evaluation system for PI3K activity. Serial dilutions of standard PIP3 were subjected to the PI3K-HTRF assay in order to establish a regression line for calibration. The recombinant FLAG-tagged p110alpha proteins were engineered together with a regulatory subunit p85alpha in human embryonic kidney 293T cells. Anti-FLAG-Ig immunoprecipitates were then subjected to the assay, which enabled us to quantitatively evaluate the activities of hotspot mutants of p110alpha. We believe this method will also be applicable to the evaluation of p110alpha having uncharacterized mutations found in cancer.

[Adverse effect other than liver dysfunction in treatment of latent tuberculous infection by isoniazid].

PURPOSE: To study the frequency and degree of adverse effect, other than liver dysfunction, of isoniazid (INH) preventive therapy in Japanese people. OBJECT AND METHOD: Chart review of Japanese persons who started isoniazid preventive chemotherapy in the two clinics in Tokyo, from 2003/1/1 to 2004/12/31. RESULT: There were 779 cases who did not transiently or completely stop INH preventive therapy because of adverse effect, and 20 cases who stopped INH transiently or completely because of adverse effect other than liver damage (total 799 cases). Of those cases, 153 cases (153/799=19.1%) experienced one or more adverse effect other than liver damage, and 20 cases (20/799=2.5%) of these 153 cases stopped INH transiently (12 cases) or completely (8 cases). For each category of adverse effect, digestive system symptoms were most frequent (5.9%), and then in frequency order, lethargy or easy-fatigability (4.6%), central nervous symptoms (4.5%), skin eruptions (2.6%), acne (2.5%), alcohol intolerance-like symptoms (2.5%), peripheral neuropathy (0.4%), arthralgia or limb pain (0.3%). Adverse effect requiring stopping INH transiently or completely were skin eruption (1.3%), digestive system symptoms (1.1%), central nervous symptoms (0.6%), acne (0.1%). Most of the adverse effect were not serious, and not required hospitalization. In isoniazid (INH) preventive therapy in Japanese people, adverse effect other than liver damage were not infrequent, but most of them are not serious, and do not disturb continuation of preventive therapy in most cases.

[Liver damage in treatment of latent tuberculous infection by isoniazid].

PURPOSE: To study the frequency and degree of liver damage as adverse effect of isoniazid (INH) preventive therapy in Japanese people. OBJECT AND METHOD: Chart review of Japanese persons who started isoniazid preventive chemotherapy in the two clinics in Tokyo, from 2003/1/1 to 2004/12/31. RESULT: There were 779 cases who did not transiently or completely stop INH preventive therapy because of adverse effect, and 26 cases who stopped INH transiently or completely because of liver damage as adverse effect (total 805 cases). In 371 cases, of those 779 cases, AST (asparate aminotransferase) and ALT (alanine aminotransferase) was measured after starting INH at least once. In 14.9% (59/397) of these 391 cases (= 371 + 26), liver damage as adverse effect was found. In 1.51% (6/397), liver damage with AST and/or ALT higher than 400 IU/L was found. Clinical hepatitis, associated with clinical symptom of hepatitis, was seen in 0.37% (3/805). Hepatitis with liver failure was seen in 0.12%. There was no death due to liver damage. CONCLUSION: Liver damage as adverse effect of isoniazid (INH) preventive therapy in Japanese people is not rare.

[Clinical studies on the pathogenetic factors of cavitary and nodular bronchiectatic types in pulmonary Mycobacterium avium complex disease].

We investigated the clinical, laboratory and radiological findings of 273 newly diagnosed cases of pulmonary Mycobacterium avium complex (MAC) disease, who were diagnosed in our hospital during 7 years from January 1996 to December 2002. Radiological findings of all cases were classified at the time of diagnosis into 2 patterns, the cavitary (Cav) type and the nodular bronchiectasis (NB) type. Clinical and laboratory findings at the time of diagnosis of 44 death cases were compared with those of 273 newly diagnoses cases, to analyze the prognostic factors of this disease. MAC disease cases showed a marked increase in number in recent years, but only in women. Mean age at the first visit was 65.7 years in men and 63.2 years in women, and when limited to fatal cases, it was 72.3 years in men and 69.4 years in women. Low body weight in terms of body mass index (BMI) and moderately low serum albumin level were found at the time of the first hospital visit in all the newly diagnosed and death cases. In the fatal cases, the peripheral blood lymphocyte counts revealed a relatively smaller number than the normal range, and the PPD skin test showed a negative reaction in 57.7% of all cases, suggesting the presence of lowered cell-mediated immunity at the time of diagnosis. Whether malnutrition occurs as a result of MAC disease or the individuals with lower nutrition level are easy to develop to MAC disease remains to be clarified. In regard to radiological findings, many cavitary (Cav) type cases were found in men and nodular bronchiectasis (NB) type in women among newly diagnosed cases, while the cavitary type was observed in many in both men and women fatal cases. The mean duration period from diagnosis to death was 28.3 months in men and 60.2 months in women, showing a longer survival after diagnosis, perhaps due to earlier hospital visits by women. The average age at death was 74.4 years old in men and 73.8 years old in women, and the two radiological patterns did not change throughout the entire disease course.

[Clinicopathologic study of 50 autopsy cases of idiopathic pulmonary fibrosis and non-diffuse usual interstitial pneumonia].

We investigated 50 autopsy cases of idiopathic pulmonary fibrosis (IPF) and non-diffuse usual interstitial pneumonia (UIP), and subgrouped them into three subtypes based on morphologic differences in the fibrosis (honeycomb): typical thick-walled honeycomb type (16 cases), atypical thin-walled honeycomb type (27 cases) and atelectatic indurated type (6 cases), with one undetermined case. In the thin-walled type, the percentage of males (93%), the percentage of smokers (89%), and the percentage of lung cancer cases (52%) were significantly higher than in the other two subtypes (p < 0.02, p < 0.001 and p < 0.05, respectively). However, in the thick-walled and indurated types there were significantly higher percentages of DAD (38% and 67%, respectively) than in the thin-walled type (15%) (P < 0.05). Accordingly, each subtype was thought to be closely related to its IPF clinical features, and showed differences in the development of acute exacerbation and lung cancer. This study proposes the existence of a UIP subset and suggests that this subgrouping can help in the management of the disease.