Somboon Keelawat

Dissecting the Akt/Mammalian Target of Rapamycin Signaling Network: Emerging Results from the Head and Neck Cancer Tissue Array Initiative

Purpose: As an approach to evaluate the expression pattern and status of activation of signaling pathways in clinical specimens from head and neck squamous cell carcinoma (HNSCC) patients, we established the Head and Neck Cancer Tissue Array Initiative, an international consortium aimed at developing a high-density HNSCC tissue microarray, with a high representation of oral squamous cell carcinoma. Experimental Design: These tissue arrays were constructed by acquiring cylindrical biopsies from multiple individual tumor tissues and transferring them into tissue microarray blocks. From a total of 1,300 cases, 547 cores, including controls, were selected and used to build the array. Results: Emerging information by the use of phosphospecific antibodies detecting the activated state of signaling molecules indicates that the Akt-mammalian target of rapamycin (mTOR) pathway is frequently activated in HNSCC, but independently from the activation of epidermal growth factor receptor or the detection of mutant p53. Indeed, we identified a large group of tissue samples displaying active Akt and mTOR in the absence of epidermal growth factor receptor activation. Furthermore, we have also identified a small subgroup of patients in which the mTOR pathway is activated but not Akt, suggesting the existence of an Akt-independent signaling route stimulating mTOR. Conclusions: These findings provide important information about the nature of the dysregulated signaling networks in HNSCC and may also provide the rationale for the future development of novel mechanism-based therapies for HNSCC patients.

Rosiglitazone Effect on Radioiodine Uptake in Thyroid Carcinoma Patients with High Thyroglobulin but Negative Total Body Scan: A Correlation with the Expression of Peroxisome Proliferator–Activated Receptor-Gamma

BACKGROUND Thyroid carcinoma patients with high thyroglobulin (Tg) level but negative total body scan (TBS) are difficult to treat with radioiodine (RAI). The objective of this study was to determine if treatment with rosiglitazone (RZ), a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, was associated with an increase in RAI uptake in thyroid carcinoma patients with high serum Tg and negative TBSs. We also determined if there was a correspondence between the effect of RZ and the degree of staining for PPAR-gamma within thyroid cancer tissues. METHODS We prescribed 8 mg of RZ daily for 6 weeks in 23 patients with epithelial cell thyroid carcinoma who previously had negative posttherapeutic I-131 total body scans (post Rx TBSs) with high serum Tg concentrations. Diagnostic total body scans (Dx TBSs) before and 6 weeks after RZ treatment were compared. An ablative dose of I-131 was then given to all patients, and post Rx TBS was performed to evaluate RAI uptake. Immunohistochemical staining of PPAR-gamma expression in thyroid cancer biopsies was done to correlate this with possible effects of RZ on RAI uptake. RESULTS Seven patients had strong PPAR-gamma-positive staining in thyroid biopsies, nine patients had weakly positive staining, and seven patients had negative staining. Five of seven patients with strong staining had either positive post Rx TBS, or both Dx TBS and post Rx TBS. One of nine patients with weak staining had positive Dx TBS and post Rx TBS. In contrast, none of the seven patients with negative staining had positive TBS. CONCLUSIONS RZ can increase RAI uptake in thyroid tissue in the majority of patients with epithelial cell thyroid carcinoma whose previous posttherapeutic I-131 scans were negative provided they have high intensity and extent of PPAR-gamma expression in thyroid tissue. Few, if any, patients with weak or no PPAR-gamma expression in thyroid cancer tissue increase RAI uptake after RZ treatment.

High prevalence and incidence of high-grade anal intraepithelial neoplasia among young Thai men who have sex with men with and without HIV.

Background:Men who have sex with men (MSM) are at elevated risk of having anal cancer. However, the prevalence and incidence among MSM of high-grade anal intraepithelial neoplasia (HGAIN), the putative precursor of anal cancer, is understudied, particularly in Asians. Methods:A total of 123 HIV-positive and 123 HIV-negative MSM were enrolled at the Thai Red Cross AIDS Research Centre in Bangkok, Thailand, and followed for 12 months. Anal sample collection for human papillomavirus (HPV) genotyping and high-resolution anoscopy (HRA) with biopsies were performed at every visit. Results:Mean age at enrollment was 28.9 years. HIV-positive MSM were more commonly infected with high-risk HPV types in the anus than HIV-negative MSM (57.5 vs. 36.6%; P = 0.001). The prevalence of HGAIN was 18.9% in HIV-positive and 11.4% in HIV-negative MSM (P = 0.1). The incidence of HGAIN at 12 months was 29% in HIV-positive and 8% in HIV-negative MSM (P = 0.001). The hazard ratios for incident HGAIN in multivariate models were 5.16 [95% confidence interval (CI) 1.89–14.08, P < 0.001] in MSM with persistent HPV 16 and/or 18 infection and 2.62 (95% CI 1.04–6.61, P = 0.042) in HIV-positive MSM. Conclusions:Approximately one-third of HIV-positive MSM developed incident HGAIN within 12 months. Given the relative increased prevalence of HIV among MSM worldwide, local HGAIN data are needed to guide practitioners, policy makers, and communities in planning for strategies to screen for and treat HGAIN in this population.

LINE-1 and Alu hypomethylation in mucoepidermoid carcinoma

BackgroundMucoepidermoid carcinoma (MEC) can be classified into low-, intermediate-, and high-grade tumors based on its histological features. MEC is mainly composed of three cell types (squamous or epidermoid, mucous and intermediate cells), which correlates with the histological grade and reflects its clinical behavior. Most cancers exhibit reduced methylation of repetitive sequences such as Long INterspersed Element-1 (LINE-1) and Alu elements. However, to date very little information is available on the LINE-1 and Alu methylation status in MEC. The aim of this study was to investigate LINE-1 and Alu element methylation in MEC and compare if key differences in the methylation status exist between the three different cell types, and adjacent normal salivary gland cells, to see if this may reflect the histological grade.MethodsLINE-1 and Alu element methylation of 24 MEC, and 14 normal salivary gland tissues were compared using Combine Bisulfite Restriction Analysis (COBRA). Furthermore, the three different cell types from MEC samples were isolated for enrichment by laser capture microdissection (LCM), essentially to see if COBRA was likely to increase the predictive value of LINE-1 and Alu element methylation.ResultsLINE-1 and Alu element methylation levels were significantly different (p<0.001) between the cell types, and showed a stepwise decrease from the adjacent normal salivary gland to the intermediate, mucous and squamous cells. The reduced methylation levels of LINE-1 were correlated with a poorer histological grade. In addition, MEC tissue showed a significantly lower level of LINE-1 and Alu element methylation overall compared to normal salivary gland tissue (p<0.001).ConclusionsOur findings suggest that LINE-1 methylation differed among histological grade mucoepidermoid carcinoma. Hence, this epigenetic event may hold value for MEC diagnosis and prognostic prediction.

Use of Human Papillomavirus DNA, E6/E7 mRNA, and p16 Immunocytochemistry to Detect and Predict anal High-Grade Squamous Intraepithelial Lesions in HIV- Positive and HIV-Negative Men Who Have Sex with Men

Background Men who have sex with men (MSM) are at high risk of having anal cancer. Anal high-grade squamous intraepithelial lesion (HSIL) is the precursor of anal cancer. We explored the use of different biomarkers associated with human papillomavirus (HPV) infection and HPV-mediated cell transformation to detect and predict HSIL among HIV-positive and HIV-negative MSM. Methodology/Principal Findings A total of 123 HIV-positive and 123 HIV-negative MSM were enrolled and followed for 12 months. High-resolution anoscopy (HRA) with biopsies were performed at every visit along with anal sample collection for cytology, high-risk HPV DNA genotyping, HPV E6/E7 mRNA, and p16 immunocytochemistry. Performance characteristics and area under the receiver operator characteristics curve were calculated for these biomarkers at baseline, and Cox regression compared the usefulness of these biomarkers in predicting incident HSIL. High-risk HPV DNA, E6/E7 mRNA, and p16 immunocytochemistry each identified 43–46% of MSM whose baseline test positivity would trigger HRA referral. E6/E7 mRNA had the highest sensitivity (64.7%) and correctly classified the highest number of prevalent HSIL cases. With the exception of p16 immunochemistry, most tests showed significant increases in sensitivity but decreases specificity versus anal cytology, while the overall number of correctly classified cases was not significantly different. Baseline or persistent type 16 and/or 18 HPV DNA was the only test significantly predicting incident histologic HSIL within 12 months in models adjusted for HIV status and low-grade squamous intraepithelial lesions at baseline. Conclusions/Significance Countries with a high HIV prevalence among MSM and limited HRA resources may consider using biomarkers to identify individuals at high risk of HSIL. E6/E7 mRNA had the highest sensitivity for prevalent HSIL detection regardless of HIV status, whereas type 16 and/or 18 HPV DNA performed best in predicting development of incident HSIL within 12 months.

LINE-1 and Alu methylation patterns in lymph node metastases of head and neck cancers.

BACKGROUND The potential use of hypomethylation of Long INterspersed Element 1 (LINE-1) and Alu elements (Alu) as a biomarker has been comprehensively assessed in several cancers, including head and neck squamous cell carcinoma (HNSCC). Failure to detect occult metastatic head and neck tumors on radical neck lymph node dissection can affect the therapeutic measures taken. OBJECTIVE The aim of this study was to investigate the LINE-1 and Alu methylation status and determine whether it can be applied for detection of occult metastatic tumors in HNSCC cases. METHODS We used the Combine Bisulfite Restriction Analysis (COBRA) technique to analyse LINE-1 and Alu methylation status. In addition to the methylation level, LINE-1 and Alu loci were classified based on the methylation statuses of two CpG dinucleotides in each allele as follows: hypermethylation (mCmC), hypomethylation (uCuC), and 2 forms of partial methylation (mCuC and uCmC). Sixty-one lymph nodes were divided into 3 groups: 1) non-metastatic head and neck cancer (NM), 2) histologically negative for tumor cells of cases with metastatic head and neck cancer (LN), and 3) histologically positive for tumor cells (LP). RESULTS Alu methylation change was not significant. However, LINE-1 methylation of both LN and LP was altered, as demonstrated by the lower LINE-1 methylation levels (p<0.001), higher percentage of mCuC (p<0.01), lower percentage of uCmC (p<0.001) and higher percentage of uCuC (p<0.001). Using receiver operating characteristic (ROC) curve analysis, %uCmC and %mCuC values revealed a high level of AUC at 0.806 and 0.716, respectively, in distinguishing LN from NM. CONCLUSION The LINE-1 methylation changes in LN have the same pattern as that in LP. This epigenomic change may be due to the presence of occult metastatic tumor in LN cases.

Detection of subcentimeter metastatic cervical lymph node by 18F-FDG PET/CT in patients with well-differentiated thyroid carcinoma and high serum thyroglobulin but negative 131I whole-body scan.

PURPOSE This study aimed to evaluate the diagnostic value of 18F-FDG PET/CT and identify the best parameter to detect subcentimeter cervical nodal metastasis in patients with a well-differentiated thyroid carcinoma (WDTC), elevated serum thyroglobulin (Tg) levels, but negative findings in the 131I whole-body scan (WBS). MATERIALS AND METHODS We prospectively studied 30 consecutive patients with WDTC after standard surgery and radioiodine treatment. All patients had serum Tg greater than 10 ng/mL during thyroid hormone withdrawal but negative findings in the therapeutic 131I WBS. One whole-body CT scan and serial whole-body and neck PET scans were performed between 10 and 170 minutes after 18F-FDG injection. Parameters studied were SUVmax, percent change in SUVmax, SUV ratios of lesions to reference organs, and their percent change. Result in the PET/CT scan was correlated with histopathology and follow-up information. Patient-based and lesion-based (subcentimeter cervical lymph nodes) analyses were performed. Outcome of Tg level after lymph node resection was also analyzed. RESULTS The overall sensitivity, specificity, accuracy, and positive and negative predictive values were 100%, 78%, 93%, 91%, and 100%, respectively. In lesion-based analysis, the differential SUVmax between 2 time points did not provide higher sensitivity than the individual SUVmax at the 60th or 90th minute. A combined SUVmax at the 90th minute greater than 2.75 and a percent change of SUVmax between the 60th and 90th minute greater than -1.1% provides the best diagnostic value with sensitivity, specificity, accuracy, and positive and negative predictive values of 81%, 90%, 83%, 97%, and 56%, respectively. After surgery, patients with completely resected PET-positive nodes without distant metastasis showed reduction of suppressed Tg to less than 2 ng/mL. CONCLUSIONS Combined SUVmax at the 90th minute and the percent change of SUVmax between the 60th and 90th minute provides the best diagnostic value to differentiate benign from malignant conditions in subcentimeter lymph nodes.

TROP-2 immunohistochemistry: a highly accurate method in the differential diagnosis of papillary thyroid carcinoma

We aimed to evaluate the diagnostic utility of the novel immunohistochemical marker TROP-2 on thyroid specimens (226 tumours and 207 controls). Whole slide immunohistochemistry was performed and scored by automated digital image analysis. Non-neoplastic thyroid, follicular adenomas, follicular carcinomas, and medullary carcinomas were negative for TROP-2 immunostaining. The majority of papillary thyroid carcinoma (PTC) specimens (94/114, 82.5%) were positive for TROP-2; however, the pattern of staining differed significantly between the histopathological variants. All papillary microcarcinomas (mPTC), PTC classic variant (PTC cv), and tall cell variant (PTC tcv) were TROP-2 positive, with mainly diffuse staining. In contrast, less than half of the PTC follicular variant specimens were positive for TROP-2, with only focal immunoreactivity. TROP-2 could identify PTC cv with 98.1% sensitivity and 97.5% specificity. ROC curve analysis found that the presence of >10% of TROP-2 positive cells in a tumour supported a diagnosis of PTC. The study of intratumoural heterogeneity showed that low-volume cytological samples of PTC cv could be adequately assessed by TROP-2 immunostaining. The TROP-2 H-score (intensity multiplied by proportion) was significantly associated with PTC variant and capsular invasion in encapsulated PTC follicular variant (p<0.001). None of the baseline (age, gender) and clinical (tumour size, nodal disease, stage) parameters were correlated with TROP-2 expression. In conclusion, TROP-2 membranous staining is a very sensitive and specific marker for PTC cv, PTC tcv, and mPTC, with high overall specificity for PTC.

In vivo gene expression and immunoreactivity of Leptospira collagenase

Pulmonary hemorrhage is an increasing cause of death of leptospirosis patients. Bacterial collagenase has been shown to be involved in lung hemorrhage induced by various infectious agents. According to Leptospira whole genome study, colA, a gene suggested to code for bacterial collagenase has been identified. We investigated colA gene expression in lung tissues of Leptospira infected hamsters. Golden Syrian Hamsters were injected intraperitoneally with Leptospira interrogans serovar Pyrogenes. The hamsters were sacrificed on days 3, 5 and 7 post-infection and lung tissues were collected for histological examination and RNA extraction. Lung pathologies including atelectasis and hemorrhage were observed. Expression of colA gene in lung tissues was demonstrated by both RT-PCR and real time PCR. In addition, ColA protein was cloned and the purified protein could react with sera from leptospirosis patients. Leptospira ColA protein may play a role in Leptospira survival or pathogenesis in vivo. Its reaction with leptospirosis sera suggests that this protein is immunogenic and could be another candidate for vaccine development.

Epithelioid Sarcoma of the Parotid Gland of a Child

Epithelioid sarcoma is a rare soft-tissue tumor that usually occurs in young adults, with a median age of 26 years. This malignancy has been divided into distal and proximal types. The latter is found in proximal body sites including the head and neck region. We present a rare case of parotid proximal-type epithelioid sarcoma in a 1-year-old male child; this is the 4th reported case in the literature and the youngest in a pediatric patient to date. The tumor showed prominent rhabdoid features by light microscopy. Immunohistochemical studies revealed positive staining to cytokeratin (AE1/AE3), epithelial membrane antigen, vimentin, and BAF47. Thus, while the tumor resembled a malignant rhabdoid tumor, the positive staining for BAF47 supported instead a diagnosis of epithelioid sarcoma, according to our current understanding of these 2 tumor types. Also, the clinical course was not the typical aggressive behavior of a rhabdoid tumor. The patient underwent radical parotidectomy without adjuvant therapy and remained disease-free at follow-up, 14 months after surgery.