Absence of caspase 3 activation in neoplastic cells of nasopharyngeal carcinoma biopsies predicts rapid fatal outcome.

Poor prognosis in nasopharyngeal carcinoma patients may result from resistance to the apoptosis-inducing effect of radio- and/or chemotherapy. Apoptosis depends on proper activation of caspase 3, resulting in cleavage of key proteins like PARP-1. To investigate whether disruption of the apoptosis pathway results in therapy-resistant tumour cells, we investigated whether absence of caspase 3 activation in tumour biopsies of nasopharyngeal carcinoma patients is related to poor clinical outcome. Moreover, we investigated whether absence of caspase 3 activation is related to loss of procaspase 3 expression or expression of the apoptosis regulators p53, bcl-2 and XIAP. We studied 36 Indonesian nasopharyngeal carcinoma patients without evidence of distant metastases who were treated with curative intent by radiotherapy only. Activation of caspase 3 and expression of the different markers were determined using specific antibodies. Levels of caspase 3 activation were determined by quantifying positively staining tumour cells. Nasopharyngeal carcinoma-derived C15 and C17 tumour cells were used as control. Absence of caspase 3 activation was strongly related to a poor clinical response to radiotherapy and to a higher T and N stage, resulting in a particularly poor clinical outcome with regard to progression-free (P<0.0001) and overall survival time (P<0.0001). Absence of caspase 3 activation was significantly correlated to loss of expression of procaspase 3 (P=0.04). In nasopharyngeal carcinoma patients treated with curative intent, absence of active caspase 3-positive neoplastic cells predicts rapid fatal outcome, and is associated with poor response to radiotherapy and high T and N stage at time of presentation.     

Correlation of clinical, pathological status, hormone receptor and C-erbB-2 oncoprotein in breast cancer patients

This study is to evaluate the correlation of some established prognostic factors, hormone receptor and C-erbB-2 expression of breast cancer patients in Yogyakarta, Indonesia. Beginning January 1997, 60 breast cancer patients who were treated either by mastectomy, breast conserving surgery or biopsy, were evaluated clinically in connection with age, menopausal status, stage, tumor size, nodes; also histologically regarding type, grade and mitotic index. Patients were evaluated for estrogen and progesterone receptor, as well as C-erbB-2 expression with immunohistochemical techniques. Median age was 47.5 years old, range from 28 to 80 years old. Most of them were premenopause (65.0%). One patient (1.7%) was a man. Most of the patients were stage IIB (25.0%), 51.7% with positive estrogen receptor and 65.9% with positive progesterone receptor. The type was mostly invasive duct carcinoma, high grade (70.0%). Most of the tumor size ranged between 2-5 cm (56.9%), with more than 3 nodes in 38.3% of patients. High mitotic index was found in 69.5% and positive C-erbB-2 in 71.7% patients. Correlation of C-erbB-2 and other prognostic factors showed that only stage, node and mitotic index had significant correlations (p = 0.016; 0.035 and 0.005, respectively). A significant correlation was also found between ER and PR level, PR and tumor size, stage and tumor size, stage and nodes, tumor size and nodes (p < 0.05), and a borderline correlation between ER and tumor size (p = 0.065) in conclusion, this preliminary study showed that breast cancer in Yogyakarta had an aggressive phenotype. C-erbB-2 positivity was correlated significantly with stage of the disease, number of nodes involved and mitotic index. Hormone receptors also correlated with some prognostic factors in breast cancer patients.     

Noninvasive diagnosis of nasopharyngeal carcinoma: nasopharyngeal brushings reveal high Epstein-Barr virus DNA load and carcinoma-specific viral BARF1 mRNA

Nasopharyngeal carcinoma (NPC) is the most prevalent ENT-tumour in Indonesia. We investigated the primary diagnostic value of Epstein-Barr virus (EBV) DNA load and mRNA detection in noninvasive nasopharyngeal (NP) brushings, obtained prospectively from consecutive Indonesian ENT-patients with suspected NPC (N=106) and controls. A subsequent routine NP biopsy was taken for pathological examination and EBER-RISH, yielding 85 confirmed NPC and 21 non-NPC tumour patients. EBV DNA and human DNA load were quantified by real-time PCR. NP brushings from NPC patients contained extremely high EBV DNA loads compared to the 88 non-NPC controls (p<0.0001). Using mean EBV DNA load in controls plus 3 SD as cut-off value, specificity, sensitivity, positive and negative predictive values were 98, 90, 97 and 91%, respectively. Epstein-Barr nuclear antigen 1 (EBNA1) and the carcinoma-specific BARF1 mRNA were detected by nucleic acid sequence based amplification and found in 86 and 74% of NP brushings, confirming NPC tumour cell presence. EBV RNA positivity was even higher in fresh samples stored at -80 degrees C until RNA expression analyses (88% for both EBNA1 and BARF1). EBV RNA-negative NP brushings from proven NPC cases had the lowest EBV DNA loads, indicating erroneous sampling. No EBV mRNA was detected in NP brushings from healthy donors and non-NPC patients. In conclusion, EBV DNA load measurement combined with detection of BARF1 mRNA in simple NP brushings allows noninvasive NPC diagnosis. It reflects carcinoma-specific EBV involvement at the anatomical site of tumour development and reduces the need for invasive biopsies. This procedure may be useful for confirmatory diagnosis in large serological NPC screening programs and has potential as prognostic tool.     

p63 cytoplasmic aberrance is associated with high prostate cancer stem cell expression

Introduction: Prostate cancer in Indonesia is the 3rd ranking cancer among males and the 5th rank for theircancer mortality. Prognostic markers that can identify aggressive prostate cancer in early stages and helpselect appropriate therapy to finally reduce the mortality are therefore urgently needed. It has been suggestedthat stem cells in the prostate gland have a role in initiation, progression, and metastasis of cancer, althoughcontroversy continues to exist. Maintenance of normal stem cell or reserve cell populations in several epitheliaincluding prostate has been shown to be regulated by p63 and alteration of p63 expression is considered to havean oncogenic role in prostate cancer. We hypothesize that the expression of cytoplasmic aberrance of p63 isassociated with high ALDH1A1 expression as a cancer stem cell marker, thus leading to progression of prostatecancer. Methods: Using a cross-sectional study during two years (2009-2010), a total of 79 paraffin embeddedtissues of benign prostatic hyperplasia, PIN prostatic intraepithelial neoplasia, low and high Gleason scoreprostate cancer were investigated using immunohistochemistry. Associations between cytoplasmic p63 andALDH1A1, as well as with pathological diagnosis, were analyzed by Chi-Square test using SPSS 15.0. Links ofboth markers with cell proliferation rate (KI-67) and apoptotic rate (cleaved caspase 3) were also analyzed byKruskal-Wallis test. Results: The mean age of patient at the diagnosis is 70.0 years. Cytoplasmic aberrance ofp63 was associated with ALDH1A1 expression (p<0.001) and both were found to have significant relationshipswith pathological diagnosis (including Gleason score), (p=0.006 and p<0.001 respectively). Moreover, it was alsofound that higher levels of cytoplasmic p63 were significantly associated with the frequency of proliferatingcells and cells undergoing apoptosis in prostate cancers (p=0.001 and p=0.016 respectively). Conclusion: p63cytoplasmic aberrance is associated with high ALDH1A1 expression. These components are suggested to havean important role in prostate cancer progression and may be used as molecular markers.     

High numbers of granzyme B/CD8-positive tumour-infiltrating lymphocytes in nasopharyngeal carcinoma biopsies predict rapid fatal outcome in patients treated with curative intent

This study determined whether tumour-infiltrating lymphocytes (TILs) in nasopharyngeal carcinomas (NPCs) include activated cytotoxic T lymphocytes (CTLs) and whether the numbers of activated CTLs in these biopsies are related to clinical outcome. Moreover, the study investigated whether the numbers of activated CTLs are associated with the expression of MHC class I proteins and the granzyme B antagonist PI-9 in the tumour cells. Forty-three Indonesian NPC patients (T(1-3), N(1-3), M(0)), who were treated with curative intent by radiotherapy only, were studied. Tumour-infiltrating activated CTLs were detected using antibodies against granzyme B, CD8, and CD56. Expression of MHC class I proteins and PI-9 was also determined by immunohistochemistry. Granzyme B-positive TILs were detected in all NPC biopsies. The presence of a high percentage (>25%) of granzyme B-positive TILs appeared to be a very strong predictor of a rapid fatal clinical outcome, independent of stage. Complete absence of MHC class I heavy chain expression in tumour cells was observed in 11 of 31 evaluable cases and low levels were observed in seven additional cases. No association between MHC class I expression and the numbers of granzyme B-positive TILs was observed. Expression of the granzyme B antagonist PI-9 in tumour cells was detected in three cases. It is concluded that the presence of many granzyme B-positive TILs in a selected group of Indonesian NPC patients is a strong and stage-independent marker for a rapid fatal clinical outcome.     

Diagnostic value of measuring Epstein-Barr virus (EBV) DNA load and carcinoma-specific viral mRNA in relation to anti-EBV immunoglobulin A (IgA) and IgG antibody levels in blood of nasopharyngeal carcinoma patients from Indonesia

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia and is strongly associated with Epstein-Barr virus (EBV). We investigated the primary diagnostic value of circulating EBV DNA and anti-EBV immunoglobulin G (IgG) and IgA levels in Indonesian NPC patients (n = 149). By a 213-bp Epstein-Barr virus nuclear antigen 1 (EBNA1)-based real-time LightCycler PCR, 72.5% of patients were positive for EBV DNA in whole blood, with 29.5% having levels above a previously determined clinical cutoff value (COV) of 2,000 EBV DNA copies/ml, the upper level in healthy carriers. In a 99-bp LightCycler PCR, 85.9% of patients were positive and 60.4% had levels above the COV. This assay quantified a significantly higher EBV load than the 213-bp PCR assay (P < 0.0001), suggesting that circulating EBV DNA is fragmented. Using data from 11 different studies, we showed a significant inverse correlation between PCR amplicon size and the percentage of patients positive for circulating EBV DNA (Spearman's rho = -0.91; P < 0.0001). EBV DNA loads were unrelated to anti-EBV IgG or IgA levels, as measured by VCA-p18 and EBNA1-specific synthetic peptide-based enzyme-linked immunosorbent assays. The presence of circulating tumor cells was assessed by amplification of BamHI-A rightward frame 1 (BARF1) mRNA, a viral oncogene abundantly expressed in EBV-carrying carcinomas but virtually absent from EBV-associated lymphomas. Despite high EBV DNA loads and the presence of EBNA1 and human U1A small nuclear ribonucleoprotein mRNA, BARF1 mRNA was never detected in blood. We conclude that amplicon size significantly influences EBV DNA load measurement in NPC patients. The circulating EBV DNA load is independent of serological parameters and does not reflect intact tumor cells. The primary diagnostic value of the EBV DNA load for the detection of NPC is limited.     

Single-assay combination of Epstein-Barr Virus (EBV) EBNA1- and viral capsid antigen-p18-derived synthetic peptides for measuring anti-EBV immunoglobulin G (IgG) and IgA antibody levels in sera from nasopharyngeal carcinoma patients: options for field screening

Assessment of immunoglobulin A (IgA) antibody responses to various Epstein-Barr virus (EBV) antigen complexes, usually involving multiple serological assays, is important for the early diagnosis of nasopharyngeal carcinoma (NPC). Through combination of two synthetic peptides representing immunodominant epitopes of EBNA1 and viral capsid antigen (VCA)-p18 we developed a one-step sandwich enzyme-linked immunosorbent assay (ELISA) for the specific detection of EBV reactive IgG and IgA antibodies in NPC patients (EBV IgG/IgA ELISA). Sera were obtained from healthy donors (n = 367), non-NPC head and neck cancer patients (n = 43), and biopsy-proven NPC patients (n = 296) of Indonesian and Chinese origin. Higher values of optical density at 450 nm for EBV IgG were observed in NPC patients compared to the healthy EBV carriers, but the large overlap limits its use for NPC diagnosis. Using either EBNA1 or VCA-p18 peptides alone IgA ELISA correctly identified 88.5% and 79.8% of Indonesian NPC patients, with specificities of 80.1% and 70.9%, whereas combined single-well coating with both peptides yielded sensitivity and specificity values of 90.1 and 85.4%, respectively. The positive and negative predictive values (PPV and NPV, respectively) for the combined EBNA1 plus VCA EBV IgA ELISA were 78.7% and 93.9%, respectively. In the Indonesia panel, the level of EBV IgA reactivity was not associated with NPC tumor size, lymph node involvement, and metastasis stage, sex, and age group. In the China panel the sensitivity/specificity values were 86.2/92.0% (EBNA1 IgA) and 84.1/90.3% (VCA-p18 IgA) for single-peptide assays and 95.1/90.6% for the combined VCA plus EBNA1 IgA ELISA, with a PPV and an NPV for the combined EBV IgA ELISA of 95.6 and 89.3%, respectively. Virtually all NPC patients had abnormal anti-EBV IgG diversity patterns as determined by immunoblot analysis. On the other hand, healthy EBV carriers with positive EBV IgA ELISA result showed normal IgG diversity patterns. By using EBV IgG immunoblot diversity as confirmation assay for EBV IgA ELISA-positive samples, the sensitivity and specificity for NPC diagnosis increased to 98% and 99.2%, respectively, in the Indonesian NPC samples. The use of these combined methods for seroepidemiological screening studies is proposed.     

Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects

Epstein-Barr virus (EBV)-specific immunoblot analysis was used to reveal the molecular diversity of immunoglobulin (Ig) G and IgA antibody responses against Epstein-Barr nuclear antigen (EBNA), early antigen (EA), and viral capsid antigen (VCA) in serum samples from patients with nasopharyngeal carcinoma (NPC) and control subjects, by use of immunofluorescence assay (IFA). Control donors (n=150) showed IgG responses to few EBV proteins--VCA-p18, VCA-p40, EBNA1, and Zebra--and sporadically weak IgA reactivity to EBNA1 and VCA-p18. Patients with NPC stage 1 (n=6) had similar response patterns. Patients with NPC stage 2-4 (n=132) showed significantly more diverse IgG and IgA responses to EA and VCA proteins--VCA-p18/-p40, EBNA1, Z-encoded broadly reactive activator, and EAd-p47/54, -DNAse, -thymidine kinase, and -p138. No correlation was found between IFA titers and the number of EBV proteins recognized by IgG or IgA. Our results reveal dissimilarity between EBV polypeptides recognized by IgG and IgA antibodies, which suggests independent B cell triggering events.     

Histological evaluation of rabbit gingival wound healing transplanted with human amniotic membrane

Human amniotic membrane has been used as a material to accelerate wound healing and reconstruct damaged organs. The aim of the present study was to assess histologically human amniotic membrane transplantation on rabbit's gingival wound. Three- to 4-month-old male rabbits were divided into 2 groups, i.e., control (group I) and amniotic membrane-transplanted animals (group II). Buccal gingival wounds were created by a punch-biopsy instrument and covered by a 5-layered human amniotic membrane for group II or left uncovered for group I. Gingival biopsies were taken at days 1, 3, 5, 7 and 10, processed for paraffin sections and stained with haematoxylin-eosin or von Gieson. Thickness of epithelial layer, the number of polymorphonuclear cells (PMN), fibroblasts and new blood vessels as well as density of collagen fibres were assessed. The results showed that the number of fibroblasts and new blood vessels, but not PMN, from group II was higher than that from group I (P < 0.05). Similarly, the epithelial thickness and density of collagen fibres from group II were significantly higher than those from group I (P < 0.05). The results of the present study indicate that amniotic membrane transplantation may induce rapid epithelialization and both granulation tissue and collagen formation but suppress inflammation, suggesting that amniotic membrane transplantation may promote rapid gingival wound healing in rabbits compared to secondary healing.