High frequency of mutation of epidermal growth factor receptor in lung adenocarcinoma in Thailand.

Recent reports have suggested influences of racial difference on the frequency of mutation of EGFR in lung cancer. We therefore sought to characterize the frequency and pattern of mutation of EGFR in lung adenocarcinoma in Thai patients. Overall, EGFR catalytic domain mutations were detected in 35/61 (57.4%). We found 29/60 (48.3%) of exon 19 deletions, 5/54 (9.3%) of exon 21 point mutations, and 1/54 (1.9%) of double-mutation of both exons. The presence of these mutations was significantly associated with non-smoking habit. In summary, we report a strikingly high prevalence of mutation of EGFR in Thai lung adenocarcinoma, which may explain the high response rate to the treatment with TKI among Asian populations.

Sequential increases in proliferating cell nuclear antigen expression in head and neck tumorigenesis: a potential biomarker.

BACKGROUND: Proliferating cell nuclear antigen (PCNA) is a 36-kd nuclear protein whose expression is associated with DNA synthesis and cell proliferation. Tumorigenesis in head and neck squamous cell carcinoma is proposed to be a multistep process; dysregulation of proliferation is a potential marker of this process. PURPOSE: PCNA dysregulation was analyzed in squamous cell carcinoma tissue samples containing premalignant lesions (hyperplasia and/or dysplasia) and in adjacent normal epithelium to better understand proliferative changes during head and neck tumor development. METHODS: Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded sections by using a monoclonal anti-PCNA antibody. PCNA expression was analyzed in 33 head and neck squamous cell carcinomas and in their adjacent premalignant lesions from different sites and compared with that in the control samples, which had never been exposed to first-hand tobacco smoke. PCNA expression was assessed by semiquantitative scoring (scale 0-3) in three epithelial layers (basal, parabasal, and superficial). The labeling index and the weighted mean index of PCNA expression were calculated. RESULTS: Normal epithelium adjacent to the tumor had much more proliferative activity than the controls: The weighted mean index of PCNA expression was four-fold higher in the basal layer and sixfold higher in the parabasal layer. PCNA expression increased as tissues progressed from adjacent normal epithelium to hyperplasia (P < .001), hyperplasia to dysplasia (P < .001), and dysplasia to squamous cell carcinoma (P = .065); the total increase in PCNA expression ranged from fourfold to 10-fold from adjacent normal epithelium to squamous cell carcinoma. PCNA expression was higher in the parabasal than in the basal layer at all premalignant stages (23 of 25 samples in adjacent normal epithelium, 12 of 13 in hyperplasia, and 17 of 22 in dysplasia). As the tissue progressed from normal through premalignant stages to squamous cell carcinomas, we observed not only incremental increases in the labeling index, but also incremental increases in PCNA expression per labeled cells. CONCLUSIONS: These results indicate that PCNA could be a useful biomarker for multistep carcinogenesis in head and neck cancer and may serve as an intermediate end point in chemopreventive trials.

Telomerase activity in oral leukoplakia and head and neck squamous cell carcinoma.

The expression of telomerase, a ribonucleoprotein complex, is necessary to overcome cellular senescence, and it is associated with immortal cells and cancer. However, its role in precancerous lesions such as oral leukoplakias is less known. The purpose of this study is to investigate the presence of telomerase activity in oral leukoplakia and the relationship between the enzyme and multistep tumorigenesis. Telomerase activity was detectable in 14 of 16 human head and neck squamous cell carcinomas and 10 of 26 oral leukoplakia tissues. We also showed that the expression of telomerase in the premalignant lesions was associated with phenotypic progression, the degree of dysplasia. These results indicate that telomerase is activated frequently during the late stage of oral premalignancy and may play a crucial role in head and neck squamous cell carcinogenesis.

Increased polysomies of chromosomes 7 and 17 during head and neck multistage tumorigenesis.

Head and neck cancer development has been proposed to represent a multistage process characterized by dysregulation of proliferation and differentiation and driven by an accumulation of genetic alterations in an anatomic field repeatedly exposed to carcinogens. To visualize the accumulation of genetic alterations during head and neck tumorigeneses and to determine the extent of the genetically altered field, we probed 25 squamous cell carcinomas of the head and neck and their adjacent premalignant lesions for numerical chromosome aberrations by nonisotopic, in situ hybridization using chromosome-specific centromeric DNA probes for chromosomes 7 and 17. Normal control oral epithelium from individuals free of cancer showed no chromosome polysomy (i.e., cells with > or = 3 chromosome copies), whereas histologically normal epithelium adjacent to the tumors showed squamous cells with polysomies for chromosomes 7 and 17. Moreover, the frequency of cells with polysomy increased as the tissues passed from histologically normal epithelium to hyperplasia to dysplasia to cancer. The finding of genotypic abnormalities in histologically normal and precancerous regions adjacent to the tumor supports the concept of field cancerization. The finding of progressive genetic changes as the tumor develops supports the concept of multistep carcinogenesis in the head and neck region. Such genotypic parameters could serve as biomarkers in the assessment of the risk of progression to malignancy and as intermediate end points in chemoprevention trials.

Epstein-Barr virus DNA in serum/plasma as a tumor marker for nasopharyngeal cancer.

Nasopharyngeal cancer (NPC) constitutes a type of carcinoma encountered frequently in Southern China, among Eskimos of the Arctic region, and to a lesser extent in Southeast Asia. Because EBV DNA present in plasma or serum of NPC patients has proven to represent a promising noninvasive tumor marker, the present study was designed to determine the incidence of serum/plasma EBV DNA by nested PCR during various disease management stages. By this method, we could detect EBV DNA in plasma/serum of 98 of 167 NPC patients prior to treatment, compared with 10 of 77 samples derived from healthy blood donors serving as controls, with a similar prevalence observed in plasma versus serum. Investigation of 13 patients subjected to radiotherapy revealed plasma EBV DNA to persist in the plasma of one case, whereas among the remaining patients, it had vanished during the early phase of treatment. Finally, with 52 samples derived from 37 NPC patients during follow-up, we established 100% specificity and 0% false-positive rate for plasma DNA detection by nested PCR. Moreover, we subjected 24 known EBV DNA-positive serum samples to DNase digestion prior to DNA extraction and amplification to differentiate between free and encapsulated viral DNA, which demonstrated complete absence of the human beta-globin genomic DNA in contrast to EBV DNA detectable in 14 samples. In conclusion, applying this noninvasive method, serum/plasma EBV DNA constitutes a reliable tumor marker prior to, during, and after treatment of NPC.

Telomerase assay for differentiating between malignancy-related and nonmalignant ascites.

The differential diagnosis between malignancy-related ascites (MRAs) and nonmalignant ascites (NMAs) has remained an essential problem in clinical practice. Our purpose was to determine the diagnostic value of ascitic fluid telomerase activity in discriminating these two categories compared with that of cytological examination. Twenty-five MRAs and 47 NMAs as the control group were enrolled in our study. In the MRA group, telomerase activity was detected in 13 of 16 (81.3%) cases of peritoneal carcinomatosis and in 6 of 9 (66.7%) cases of hepatocellular carcinoma (HCC)-associated ascites. Contrasting that, cytological examination was positive in only 9 of 16 (56.3%) and 1 of 9 (11.1%) cases, respectively. In the NMA group, telomerase-positive ascitic fluid samples were found in 2 of 47 (4.3%) cases, all belonging to subgroups that contained large numbers of lymphocytes in the ascites. In our study, the telomerase activity and cytological examination exhibited a sensitivity of 76% and 40% and a specificity of 95.7% and 100%, respectively. Regarding subgroups of MRAs, the telomerase activity and cytological examination demonstrated a sensitivity of 81.3% and 56.3%, respectively, in peritoneal carcinomatosis and a sensitivity of 66.7% and 11.1%, respectively, in HCC-associated ascites. In conclusion, telomerase activity is a more sensitive marker than cytological examination for differentiating between MRAs and NMAs. It may also serve as a useful indicator for detecting early i.p. metastasis in HCC-associated ascites.

Epstein-Barr viral DNA in serum of patients with nasopharyngeal carcinoma.

This study evaluated Epstein-Barr virus (EBV) DNA in sera of 42 patients with nasopharyngeal carcinoma (NPC) and 82 healthy individuals who had been infected previously with EBV. Thirteen of 42 NPC samples were positive for EBV DNA in their sera, whereas all 82 normal controls were negative. In addition, EBV typing between primary tumors and sera showed identical results, suggesting that serum EBV DNA represented tumor DNA. To evaluate the importance of the serum NPC DNA, clinical data and tumor phenotypes including age, sex, WHO type, EBV type, stage, tumor invasion, metastasis, and apoptosis were correlated with serum EBV DNA, and only apoptosis was found statistically significant. In conclusion, EBV DNA was detectable in the serum of some patients with NPC, represented tumor DNA, and might have clinical implications in the future.

Oncogene expression in cholangiocarcinoma and in normal hepatic development.

The expression of the proteins encoded by the ras, myc, and erb B-2 oncogenes was examined in 63 paraffin-embedded human cholangiocarcinomas of Thai and English origin using immunohistochemistry. The observed distributions were compared with oncogene expression in a series of human hepatocellular carcinomas. In an attempt to relate expression of these three oncogenes to specific stages of normal tissue differentiation, tissue sections of normal fetal, infant, and adult human livers were also examined. Of 63 cholangiocarcinomas, 59 (95%) expressed p62 c-myc, 47 (75%) expressed p21 c-ras, and 46 (73%) expressed p190 c-erbB-2. The expression of c-myc and c-ras but not of c-erb B-2 correlated directly with tumor differentiation as judged by morphologic criteria. No difference was observed in oncogene expression between intrahepatic and extrahepatic cholangiocarcinomas. Twelve of 14 hepatocellular carcinomas (86%) stained positively for all three oncoproteins. During normal liver development, expression of c-myc and c-ras was shown to occur from 18 weeks' gestation until 5 years of age, but not thereafter. Expression of c-myc, c-ras, and c-erbB-2 oncogenes may be used as immunohistochemical markers to distinguish cholangiocarcinoma from nonneoplastic biliary tissues, and may provide useful information concerning the cell biology of tumor differentiation.

Implantation metastasis of carcinoma after percutaneous fine-needle aspiration biopsy.

Implantation of malignant cells along the needle tract is an extremely rare but potential complication following percutaneous needle aspiration biopsy of malignant lesions. Percutaneous fine-needle aspiration biopsy (FNAB) has recently received more attention for cytologic diagnosis of bronchogenic carcinoma because of its high diagnostic yield, simplicity, and low morbidity. On the other hand, dissemination of cancer cells by needle aspiration biopsy can change a potentially resectable localized lung cancer to an unresectable one. We report two cases: one patient underwent FNAB of a metastatic left adrenal mass that seeded a paraspinal muscle implantation of malignant cells that subsequently developed a tumor mass, and the second patient had tumor cell implantation in the chest wall after FNAB of a pleural-based adenocarcinoma of the lung. The theoretical and practical importance of tumor cell spread along the needle tract is discussed. Because of its rare incidence, however, this complication should not affect the use of needle aspiration biopsy in bronchogenic carcinoma, although care should be undertaken during the procedure.

Human papillomavirus DNA in plasma of patients with cervical cancer.

BACKGROUND: Human papillomavirus (HPV) is a crucial etiological factor for cervical cancer (CC) development. From a diagnostic view-point, the consistent presence of HPV in CC allows the viral DNA to be used as a genetic marker. The aims of this study were to evaluate the presence, physical status and clinical significant of HPV DNA in circulation of CC patients. RESULTS: Whereas 6 out of 50 (12%) HPV positive CC patients revealed plasma HPV DNA, it was detected in none of 20 normal controls or 13 HPV negative CC cases. The plasma DNA exhibited an HPV type identical to the HPV in the primary tumors and the DNA from both sources was integrated into host genome. Interestingly, several findings suggested an association between plasma HPV DNA and metastasis. First, three of the HPV DNA positive cases were CC patients with clinical stage IVB or recurrence with distance metastases (P = 0.001, RR = 15.67). Second, the amount of plasma HPV DNA from metastatic patients to be three times more than three other patients without metastases. Finally, the later cases had tendency to develop recurrence distant metastases within one year after complete treatment when compared with other HPV associated CC patients with the same stage but without the present of plasma HPV DNA. CONCLUSIONS: The plasma HPV DNA originated from the CC, was associated with metastasis and could be used as a marker representing the circulating free CC DNA.

Cytochrome P450 2E1 polymorphism and nasopharyngeal carcinoma development in Thailand: a correlative study.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a rare tumor in most parts of the world but occurs at relatively high frequency among people of Chinese descent. The cytochrome P450 2E1 enzyme (CYP2E1) is responsible for the metabolic activation of nitrosamines, and has been shown to be a susceptibility gene for NPC development in Taiwan [RR = 2.6; 95%CI = 1.2-5.7]. Since there has been only one report of this link, it was decided to investigate the susceptibility of CYP2E1 to NPC development in other populations. Therefore, the correlation between the RsaI polymorphism of this gene and NPC was studied in-patients including Thai and Chinese in Thailand. The present study comprised 217 cases diagnosed with NPC and 297 healthy controls. RESULTS: Similar to the result found in Taiwanese, a homozygous uncut genotype demonstrated a higher relative risk both when all cases were analyzed [RR = 2.19; 95%CI = 0.62-8.68] or individual racial groups, Thai [RR = 1.51; 95%CI = 0.08-90.06] or Chinese [RR = 1.99; 95%CI = 0.39-10.87]. The ethnicity-adjusted odds ratio is 2.39 with 95%CI, 0.72-7.89. CONCLUSIONS: Though our finding was not statistically significant due to the moderate sample size of the study, similarity to the study in Taiwan with only a slight loss in precision was demonstrated. The higher RR found for the same genotype in distinct populations confirmed that CYP2E1 is one of several NPC susceptibility genes and that the RsaI minus variant is one mutation that affects phenotype.

Clinical significance of p53 antigen and anti-p53 antibodies in the sera of hepatocellular carcinoma patients.

BACKGROUND: To analyze the clinical significance of serum p53 protein and anti-p53 antibodies as serological markers for hepatocellular carcinoma (HCC). METHODS: We studied clinical data, i.e., age, sex, etiology, serum alpha-fetoprotein (AFP) level. TMN staging, and Okuda staging in 141 patients with HCC. The sera of these patients were analyzed for serum p53 protein and serum anti-p53 antibodies by enzyme-linked immunosorbent assay (ELISA). RESULTS: Serum p53 antigen and serum anti-p53 antibodies were detected in the sera of 32 of the 141 (22.7%) patients and 26 of the 141 patients (18.4%), respectively. Of note, the HCC patients who were positive for p53 antigen (32/141) had no circulating anti-p53 antibodies. When both these groups of patients were combined as a serum p53 status-positive group, the total number in this group was 58 (41.1 %). Positive status of p53 was not associated with age (P = 0.206), serum alpha-fetoprotein level (P = 0.851). Okuda staging (P = 0.243), or survival (P = 0.078), but was correlated significantly with TMN staging (P = 0.049). Interestingly, a shorter survival time (mean, 3.9 months) was noted in the serum p53 status-positive group. in comparison with the longer survival time (mean, 6.5 months) in the serum p53 status-negative group. CONCLUSIONS: Combination of the detection of serum p53 antigen and antibodies by ELISA may represent a suitable noninvasive investigation in assessing the clinical implications and prognoses of patients with HCC.

Vertebral osteomyelitis mimicking bone metastasis in breast cancer patients.

Vertebral osteomyelitis can be a diagnostic pitfall for physicians, since it is protean and often subtle in its clinical presentation. It can coexist with metastatic lesions or mimic vertebral bone metastasis. When it occurs in patients with breast cancer, who are prone to have bone metastasis, it can present perplexing diagnostic problems. Misdiagnosing vertebral osteomyelitis as bone metastasis or vice versa results in delayed diagnosis and inappropriate treatment and may cause serious morbidity. We emphasize this problem by presenting the cases of two patients with breast cancer whose clinical course was complicated by vertebral osteomyelitis. When the clinical course of breast cancer is different from this usual presentation, a different process should be suspected, and histologic diagnosis should be promptly sought. Fine-needle aspiration biopsy and culture of suspicious-appearing bony lesions is recommended as a rapid and reliable method of establishing a definite diagnosis in this circumstance.

Natural history of untreated primary hepatocellular carcinoma: a retrospective study of 157 patients.

Primary hepatocellular carcinoma (HCC) is a common malignancy with a dismal prognosis; new modalities of treatment as alternatives to surgery have been developed for unresectable patients. The authors obtain baseline data for the natural history of HCC so that the efficacy of new treatments may be evaluated. A retrospective study of 157 untreated patients with tissue-proven or serodiagnosed HCC was conducted. Clinical characteristics including laboratory investigation, treatment received, survival from the time of diagnosis, and prognostic factors were evaluated. There were 129 men and 28 women (ratio, 4.6:1). Median age was 50.9 years (range, 14.1-85.3 years). The most common symptoms and signs were weight loss (68.2%), abdominal fullness (62.5%), abdominal pain (51.6%), hepatomegaly (73.7%), ascites (45.2%), and jaundice (40.6%). Eighteen percent had extrahepatic metastases of which the lungs were the most common site. Seventy percent were hepatitis B virus related. Overall median survival was 8.7 weeks after the time of diagnosis. Survivals by stages were: TNM II, 16.6 weeks; TNM III, 7.3 weeks; TNM IVA, 9.7 weeks; TNM IVB, 7.6 weeks; Okuda II, 10.7 weeks; and Okuda III, 7.3 weeks. Multivariate analysis revealed serum total bilirubin and albumin as independent prognostic factors of survival. Common causes of death were upper gastrointestinal hemorrhage (34.1%), cancer-related causes (cachexia, HCC rupture, metastatic disease, 31.8%), and hepatic failure (25.0%). Patients with HCC were diagnosed at late stages of their disease and the advanced nature of the tumor precluded effective therapy. Earlier tumor detection at a time when patients are better candidates for treatment may be aided by an active surveillance program of high risk groups.

Telomerase assay and nested polymerase chain reaction from nasopharyngeal swabs for early noninvasive detection of nasopharyngeal carcinoma.

The main purpose of this study was to analyze sensitivity and specificity of combining nested polymerase chain reaction for detection of Epstein-Barr virus (EBV) genome and telomerase assay for identifying nasopharyngeal carcinoma (NPC). Eighty patients with NPC and 27 healthy control subjects were included in this study; 97. 5% and 94.9% of NPC patients were positive for EBV genome and telomerase activity, respectively. When nasopharyngeal swabs were tested, 95.7% presented the EBV genome and 85.5% were positive for telomerase expression. The sensitivity for counting either positive result of these two techniques was 100%. Among the 27 control subjects, only 6 and 5 cases were positive for EBV DNA and telomerase activity, respectively. This indicated a specificity of 92.6% when both positive results were included. At present, early diagnosis of NPC requires multiple biopsy specimens, especially to identify subclinical cases. Because this study showed a very high sensitivity for detecting NPC from swabs when combining the telomerase assay and nested polymerase chain reaction technique, this noninvasive technique may be a good candidate for screening of subclinical NPC, especially before multiple biopsy specimens are obtained.

Chemoprevention of head and neck cancer.

Tobacco-related upper aerodigestive tract cancer is a worldwide health problem. Clinical advances in surgery, radiation, and chemotherapy have shown only marginal improvement on the survival rate of these malignancies in the past two decades. The concept of multistage carcinogenesis and field cancerization, the high incidence of SPTs, and the promising results of chemopreventive agents especially 13 cRA to effectively reverse oral premalignancy and prevent SPTs indicate a new avenue to improve the outcome of these newplastic diseases. The study of second primary tumor prevention is too early to show any survival benefit in the treatment arm and toxicity of high dose 13 cRA is significant. The current prospective large scale phase III SPT prevention trial of low-dose 3 cRA may establish a new standard approach for the control of head and neck neoplasms. Identification of potentially useful panels of biomarkers as intermediate endpoints will undoubtedly reduce time and cost currently required to conduct chemoprevention trials.

Ruptured primary hepatocellular carcinoma at Chulalongkorn University Hospital: a retrospective study of 32 cases.

Rupture of Primary hepatocellular carcinoma (HCC) is relatively common in high incidence areas including Thailand. There have been attempts to establish a standard treatment to manage this phenomenon. We retrospectively reviewed the records of patients with HCC from January 1989 to June 1997, and ten per cent (32/306) had tumor rupture during the course of the disease. Overall median survival of the patients with tumor rupture was 2.7 months [95% confidence interval (CI), 0-5.9 months] that was not significantly different from that of the patients without rupture (median 6.6 months; 95% CI, 4.0-9.1 months) (P = 0.4605). Among the ruptured group, the patients treated with surgical intervention survived longer than those receiving supportive care alone (median = 15.5 months; 95% CI, 8.7-22.2 months and median = 0.4 months; 95% CI, 0.2-0.5 months, P = 0.0027). The resectional and non-resectional surgical subgroups also had better survival than the supportive group (P = 0.0300 and P = 0.0209, respectively). In conclusion, surgical intervention, if applicable, should be performed in managing ruptured HCC.

Molecular biology of head and neck tumorigenesis: the role of p53 expression and genetic instability.

Head and neck cancers progress as multistep tumorigenesis through accumulation of genetic instability. The p53 tumor-suppressor gene encodes a cell-cycle checkpoint protein that functions in the G1 phase of the cell cycle. When DNA damage is incurred, p53 transactivates a number of downstream genes whose products, with diverse biologic activities, contribute to the cellular response to DNA damage. One major p53-mediated function in response to DNA damage is to induce the G1 cell-cycle arrest, or delay, which probably allows time for the cell to repair DNA damage prior to S-phase entry. In cell lacking of p53 function, a condition of genetic instability results from checkpoint loss (Fig. 4.). These events occur early from ANL to SCC and increase gradually through multistep tumorigenesis. Due to the potential role of p53 expression and genetic instability, both might be useful biomarkers in assessing the risk of head and neck tumorigenesis.

Tumor angiogenesis.

Tumor angiogenesis is the growth of new blood vessels which is required for tumor growth and progression. Vascularization of the tumor occurs through a series of sequential steps before or during the multistep progression to neoplasia. Several events occur during the formation of new vessels including production of protease enzymes, unregulation of positive regulators of angiogenesis, and down regulation of negative regulators. In addition, tumor associated macrophage also influence angiogenesis by secreting enzymes, enzymes inhibitors and cytokines. Recent knowledge in tumor angiogenesis may have clinical implications in diagnosis and treatment. Quantification of microvessel density in tumor specimen correlates either metastasis or recurrence in many malignancies such as breast cancer and lung cancer. Therefore, assessment of tumor angiogenesis may serve as prognostic factors. Therapeutic applications include the development of new agents with antiangiogenic properties, vascular targeting drugs, antibody-based therapy, and gene therapy. Combination of antiangiogenic therapy with cytotoxic drugs may enhance antitumor activity. Moreover, the role of antiangiogenic therapy in adjuvant setting may provide and alternative approach to better cancer treatment in the near future.

CA 50: a tumor marker for gastrointestinal malignancies.

Efforts to find the ideal tumor marker, together with the advanced knowledge of the carbohydrate expression by cancer and the development of monoclonal antibody technology have facilitated the generation of many new tests used in clinical oncology. CA 50, a novel cancer-associated carbohydrate marker, is detected by the C 50 antibody that has been obtained by immunization of mice with a human colorectal adenocarcinoma cell line. This antibody that defines CA 50 reacts with both the afucosyl form of sialylated Lewis(a) carbohydrate moiety and sialylated Lewis(a) moiety which is also the antigenic epitope in the CA 19-9 assay. CA 50 is not organ-specific and its elevated levels in serum can be observed in a variety of malignancies, especially gastrointestinal cancers. In contrast to CA 19-9, high CA 50 levels can also be seen in malignant tumors outside the digestive tract. The expectation, that CA 50 might be positive in the Lewis negative patients who cannot synthesize CA 19-9, is supported by the histoimmunologic study. However, in serum determination close correlation between CA 50 and CA 19-9 has been observed even in patients who have Lewis negative phenotype. In clinical application, CA 50 is marginally beneficial for the diagnosis, but very useful for the follow-up of patients with pancreatic cancers. It gives results rather similar to CA 19-9. Moderately high serum levels of CA 50 can also be seen in benign hepatobiliary diseases, especially in jaundice cases. Therefore, this should be considered in order to obtain the most advantage of the marker. For other gastrointestinal cancers, CA 50 in combination with other previously defined markers may give additional information for the evaluation of some patients with colorectal, biliary, or gastric cancers. At present, there are many new emerging tumor markers used in clinical oncology. Increasing our knowledge about these markers, their capabilities and limitations will enable us to use them effectively in the evaluation of cancer patients.