Identifying a sphenoid sinus fungus ball using a nomogram model.

BACKGROUND: Sphenoid sinus fungus ball (SSFB) is a rare entity and usually presents with non-specific symptoms. SSFB could potentially lead to serious orbital and intracranial complications. Computed tomography (CT) scan is usually the first imaging test of the diagnostic workup in patients with specific clinical symptoms. This study aimed to compare the clinical characteristics and CT features between SSFB and unilateral (non-fungus ball) chronic sphenoid rhinosinusitis (USRS) and help differentiate between these two most common inflammatory diseases of the sphenoid sinus. METHODS: By retrospective database review, 66 patients with a histopathologic diagnosis of isolated SSFB were recruited for analysis. Fifty-four patients who underwent endoscopic sinus surgery with clinical and histopathological diagnoses of USRS were enrolled as the control group. Clinical characteristics and CT features were evaluated. RESULTS: Headache, rhinorrhoea, nasal obstruction, postnasal dripping, and hyposmia were the most common symptoms in both groups. In the univariate analysis, older age, lower white blood cell counts, irregular surface, bony dehiscence, lateral wall sclerosis, and intralesional hyperdensity (IH) were significant predictors for SSFB. Older age, irregular surface, and IH remained statistically significant in the multivariate analysis. Based on the results of the regression analysis, a nomogram for predicting the probability of SSFB was plotted. CONCLUSIONS: We developed a nomogram model as a novel preoperative diagnostic tool for identifying SSFB according to the predictors both in clinical characteristics and on CT features. This could help the clinicians in predicting the probability of SSFB, to reduce ineffective or delayed treatment and occurrence of complications.

Application of Anlotinib Combined With Neoadjuvant Chemotherapy in Primary EWS/PNET of Lung: A Case Report.

Primary pulmonary EWS/PNET(PPES) is extremely rare and is associated with a poor prognosis. Tumor angiogenesis plays an important role in tumor, so it has become a hot topic in molecular targeted therapy. Anlotinib is a new oral small molecular multi-targeted receptor tyrosine kinase (RTK) inhibitor. This report describes a 20 year-old man with PPES. After 4 neoadjuvant chemotherapy cycles (VACwith alternating IE) combined with anlotinib, the left total pneumonectomy was performed. Then maintenance anlotinib monotherapy was continued, no sign of recurrence to date as an outcome. To our knowledge, this is the first demonstration of anlotinib combined with neoadjuvant chemotherapy efficacy in PPES.

Enhanced O-succinyl-l-homoserine production by recombinant Escherichia coli ΔIJBB*TrcmetL/pTrc-metAfbr -Trc-thrAfbr -yjeH via multilevel fermentation optimization.

AIMS: Constructing a strain with high yield of O-succinyl-l-homoserine (OSH) and improving the titre through multilevel fermentation optimization. METHODS AND RESULTS: OSH high-yielding strain was first constructed by deleting the thrB gene to block the threonine biosynthesis. Single-factor experiment was carried out, where a Plackett-Burman design was used to screen out three factors (glucose, yeast and threonine) from the original 11 factors that affected the titre of OSH. The Box-Behnken response surface method was used to optimize the fermentation conditions. Through gene editing and medium optimization, the titre of OSH increased from 7·20 to 8·70 g l-1 in 500 ml flask. Furthermore, the fermentation process and fed-batch fermentation conditions including pH, temperature, feeding strategy and feeding medium were investigated and optimized. Under the optimal conditions, the titre of OSH reached 102·5 g l-1 , which is 5·6 times higher than before (15·6 g l-1 ). CONCLUSIONS: O-succinyl-l-homoserine fermentation process was established and the combination of response surface methodology and metabolic pathway analysis effectively improved the titre of OSH. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, the titre of OSH reached the needs for industrial production and the metabolic pathway of OSH was demonstrated for further optimization.

Long noncoding RNA NNT-AS1 functions as an oncogene in breast cancer via repressing ZFP36 expression.

Breast cancer is one of the most common cancers in women. This study focuses on the effects of Long non-coding RNAs (lncRNAs) NNT-AS1 on breast cancer cell growth and metastasis. Fifty-six pairs of breast cancer (BC) tissues and matched paracarcinoma tissues were obtained. The BC cell lines and normal human breast cell line were employed. NNT-AS1 in BC cells was knocked down by shRNA. Cell counting kit-8 assay (CCK-8), colony formation assay, cell cycle analysis, cell apoptosis analysis, cound healing assay, Transwell assay, cioinformatics analysis, Western blot analysis and Xenograft model were used. Quantitative real-time polymerase chain reaction (qRT-PCR) assay indicated that expression of NNT-AS1 was obviously upregulated in breast cancer tissues compared with adjacent tissues (n=56). Knockdown of NNT-AS1 could attenuate breast cancer cell viability, proliferation, invasion and migration, as well as promote cell apoptosis and induce cell cycle arrest at G0/G1 phase. ZFP36 was directly combined with NNT-AS1, and silencing of ZFP36 could rescue tumor suppression role by downregulating NNT-AS1 on cell proliferation and metastasis. Knockdown of NNT-AS1 could suppress cell growth and metastasis via interacting with ZFP36 in vivo. This study demonstrated that knockdown of NNT-AS1 had tumor-suppressive effect on breast cancer progression and metastasis via interacting with ZFP36 in vitro and in vivo, which provides a new insight into the treatment and prognosis evaluation of breast cancer.

Aberrant hedgehog signaling is responsible for the highly invasive behavior of a subpopulation of hepatoma cells.

Hepatoma exhibits a series of heterogeneous subpopulations in its cell surface markers, tumorigenicity, invasion and metastatic capability. We previously demonstrated that the CD133(-)/EpCAM(-) hepatoma subpopulation was more metastatic than its counterpart; however, the controlling mechanisms are unexplored. The present study aimed to delineate the significance of aberrant hedgehog (Hh) signaling in the mediation of metastases. Fluorescence-activated cell sorting-enriched CD133(-)/EpCAM(-) (double negative, DN), Huh-7 cells underwent a transwell selection for metastatic cells (transwell-selected, TS). The TS cells displayed much greater metastatic activity as evidenced by an increased invasion rate, extremely upregulated expression of matrix metalloproteinase (MMP)-1/2/9 genes compared with DN and double-positive (DP) subpopulations. In contrast to DP cells, TS cells lost E-cadherin and were all vimentin-positive as shown by immunocytochemistry. There was a transitional increase in Gli-1/2 gene expression levels from DP, DN to TS subpopulations, which was consistent with elevated Gli-1/2 or Twist-1 protein levels in the nuclear fraction. Furthermore, truncated Gli-1 (tGli-1), which transactivates molecules involved in metastasis, was detected in the highly invasive Huh-7 cell subpopulation, but not in less metastatic hepatoma cells or normal hepatocytes. The enhanced metastatic features with increased expression of MMPs as well as the presence of twist and snail genes in TS Huh-7 cells were reversed by LDE225, a potent Smoothened antagonist. In conclusion, the highly metastatic capability of a unique TS subpopulation was highly attributed to significant epithelial-mesenchymal transition, enhanced Hh activity and aberrant occurrence of a tGli-1 variant, which appears to be responsible for the highly invasive behavior.

Overexpression of miR-98 inhibits cell invasion in glioma cell lines via downregulation of IKKε.

OBJECTIVE: MicroRNAs (miRNAs) function as negative regulators for the expression of genes involved in cancer metastasis. The aim of this study was to investigate the potential role of miR-98 in gliomas and validate its regulatory mechanism. PATIENTS AND METHODS: Cell viability assays are used to measure proliferation of cell. mRNA expression is measured by qRT-PCR. Western blot analysis is used to measure protein expression. RESULTS: Functional studies showed that miR-98 overexpression inhibited glioma migration and invasion, but had no effect on the cell viability. An enhanced green fluorescent protein reporter assay, quantitative RT-PCR, and a western blot analysis confirmed that miR-98 suppressed the expression of IκB kinase (IKKε) by directly targeting its 3'-untranslated region, also, the NF-κB p65 nuclear translocation and matrix metalloproteinase (MMP)-9 expression were significantly arrested in glioma cells treated with miR-98 mimics. Accordingly, the overexpression of IKKε or NF-κB p65 can restore cell migration and invasion after being inhibited by miR-98, and can restore NF-κB p65 nuclear translocation as well as increase MMP-9 expression. CONCLUSIONS: These findings demonstrated that miR-98 functions as a tumor suppressor in gliomas. Furthermore, miR-98 may act as a potential therapeutic biomarker for glioma patients.

A small-molecule inhibitor of UBE2N induces neuroblastoma cell death via activation of p53 and JNK pathways.

Neuroblastoma (NB) is the most common extracranial neoplasm in children. In NB, loss of p53 function is largely due to cytoplasmic sequestration rather than mutation. Ubiquitin-conjugating enzyme E2 N (UBE2N), also known as Ubc13, is an E2 ubiquitin-conjugating enzyme that promotes formation of monomeric p53 that results in its cytoplasmic translocation and subsequent loss of function. Therefore, inhibition of UBE2N may reactivate p53 by promoting its nuclear accumulation. Here, we show that NSC697923, a novel UBE2N inhibitor, exhibits potent cytotoxicity in a panel of NB cell lines evidenced by its ability to induce apoptosis. In p53 wild-type NB cells, NSC697923 induced nuclear accumulation of p53, which led to its increased transcriptional activity and tumor suppressor function. Interestingly, in p53 mutant NB cells, NSC697923 induced cell death by activating JNK pathway. This effect was reversible by blocking JNK activity with its selective inhibitor, SP600125. More importantly, NSC697923 impeded cell growth of chemoresistant LA-N-6 NB cell line in a manner greater than conventional chemotherapy drugs doxorubicin and etoposide. NSC697923 also revealed in vivo antitumor efficacy in NB orthotopic xenografts. Taken together, our results suggest that UBE2N is a potential therapeutic target in NB and provide a basis for the rational use of UBE2N inhibitors like NSC697923 as a novel treatment option for NB patients.

miR-1207-5p and miR-1266 suppress gastric cancer growth and invasion by targeting telomerase reverse transcriptase.

hTERT is the catalytic subunit of the telomerase complex. Elevated expression of hTERT is associated with the expansion and metastasis of gastric tumor. In this study, we aimed to identify novel tumor suppressor miRNAs that restrain hTERT expression. We began our screen for hTERT-targeting miRNAs with a miRNA microarray. miRNA candidates were further filtered by bioinformatic analysis, general expression pattern in different cell lines, gain-of-function effects on hTERT protein and the potential of these effects to suppress hTERT 3' untranslated region (3'UTR) luciferase activity. The clinical relevance of two miRNAs (miR-1207-5p and miR-1266) was evaluated by real-time RT-PCR. The effects of these miRNAs on cell growth, cell cycle and invasion of gastric cancer cells were measured with CCK-8, flow cytometry and transwell assays. Finally, the ability of these miRNAs to suppress the transplanted tumors was also investigated. Fourteen miRNAs were identified using a combination of bioinformatics and miRNA microarray analysis. Of these fourteen miRNAs, nine were expressed at significantly lower levels in hTERT-positive cell lines compared with hTERT-negative cell lines and five could downregulate hTERT protein expression. Only miR-1207-5p and miR-1266 interacted with the 3' UTR of hTERT and the expression levels of these two miRNAs were significantly decreased in gastric cancer tissues. These two miRNAs also inhibited gastric tumor growth in vitro and in vivo. Altogether, miR-1207-5p and miR-1266 were determined to be hTERT suppressors in gastric cancer, and the delivery of these two miRNAs represents a novel therapeutic strategy for gastric cancer treatment.

USP7 inhibitor P22077 inhibits neuroblastoma growth via inducing p53-mediated apoptosis.

Neuroblastoma (NB) is a common pediatric cancer and contributes to more than 15% of all pediatric cancer-related deaths. Unlike adult tumors, recurrent somatic mutations in NB, such as tumor protein 53 (p53) mutations, occur with relative paucity. In addition, p53 downstream function is intact in NB cells with wild-type p53, suggesting that reactivation of p53 may be a viable therapeutic strategy for NB treatment. Herein, we report that the ubiquitin-specific protease 7 (USP7) inhibitor, P22077, potently induces apoptosis in NB cells with an intact USP7-HDM2-p53 axis but not in NB cells with mutant p53 or without human homolog of MDM2 (HDM2) expression. In this study, we found that P22077 stabilized p53 by inducing HDM2 protein degradation in NB cells. P22077 also significantly augmented the cytotoxic effects of doxorubicin (Dox) and etoposide (VP-16) in NB cells with an intact USP7-HDM2-p53 axis. Moreover, P22077 was found to be able to sensitize chemoresistant LA-N-6 NB cells to chemotherapy. In an in vivo orthotopic NB mouse model, P22077 significantly inhibited the xenograft growth of three NB cell lines. Database analysis of NB patients shows that high expression of USP7 significantly predicts poor outcomes. Together, our data strongly suggest that targeting USP7 is a novel concept in the treatment of NB. USP7-specific inhibitors like P22077 may serve not only as a stand-alone therapy but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact USP7-HDM2-p53 axis.

USP4 targets TAK1 to downregulate TNFα-induced NF-κB activation.

Lys63-linked polyubiquitination of transforming growth factor-ß-activated kinase 1 (TAK1) has an important role in tumor necrosis factor-α (TNFα)-induced NF-κB activation. Using a functional genomic approach, we have identified ubiquitin-specific peptidase 4 (USP4) as a deubiquitinase for TAK1. USP4 deubiquitinates TAK1 in vitro and in vivo. TNFα induces association of USP4 with TAK1 to deubiquitinate TAK1 and downregulate TAK1-mediated NF-κB activation. Overexpression of USP4 wild type, but not deuibiquitinase-deficient C311A mutant, inhibits both TNFα- and TAK1/TAB1 co-overexpression-induced TAK1 polyubiquitination and NF-κB activation. Notably, knockdown of USP4 in HeLa cells enhances TNFα-induced TAK1 polyubiquitination, IκB kinase phosphorylation, IκBα phosphorylation and ubiquitination, as well as NF-κB-dependent gene expression. Moreover, USP4 negatively regulates IL-1ß-, LPS- and TGFß-induced NF-κB activation. Together, our results demonstrate that USP4 serves as a critical control to downregulate TNFα-induced NF-κB activation through deubiquitinating TAK1.

Genetic predisposition of white matter infarction with protein S deficiency and R355C mutation.

BACKGROUND: The association between protein S deficiency (PSD) and ischemic stroke is controversial and warrants further investigation. METHODS: We conducted a genotype and MRI correlation study in a Chinese family in which hereditary PSD cosegregated with premature ischemic strokes. Six out of 11 family members inherited PSD type III in an autosomal dominant manner. RESULTS: Among all PSD members, a novel missense mutation 1063C→T in exon 10 of protein S alpha (PROS1) was identified, which encoded a substitution of arginine to cysteine at position 355 (R355C) in the first globular domain of laminin A of protein S. Wild-type PROS1 sequences were retained in non-PSD members. MRI detected deep white matter infarctions predominantly distributed in the borderzone regions. The infarct topography was homogeneous in all adult mutant carriers. By contrast, cerebral infarction was absent in nonmutant carriers. Extensive investigation in the family did not reveal any confounding stroke risk. Haplotype analysis with high-density single nucleotide polymorphism markers revealed a 6.1-Mb minimally rearranged region (rs12494685 to rs1598240) in 3q11.2, lod = 3.0. Among the 7 annotated genes in this region, PROS1 is known to be associated with thrombotic disorders. MRI screening in an additional 10 PSD families without R355C showed no cerebral infarction. CONCLUSIONS: PROS1 R355C mutation cosegregated with PSD type III and premature white matter infarctions in the index family. The findings substantiate an association between PSD and stroke. Study of the mechanism underlying this association may improve our understanding of premature cryptogenic white matter infarction.

TSG101 is not mutated in lung cancer but a shortened transcript is frequently expressed in small cell lung cancer.

TSG101 is a candidate tumor suppressor gene whose deletion in NIH3T3 cells leads to spontaneous lung metastases in nude mice. Aberrant transcripts of TSG101 have been identified in 47% of primary breast carcinomas, without evidence of intragenic deletions at the TSG101 locus on 11p15. To investigate the possible role of TSG101 in lung cancer, which often shows 11p allele loss, we performed transcript analysis and mutational analysis of TSG101 in lung cancer cell lines. Reverse transcriptase RT-PCR and Northern analysis detected a common TSG101 transcript, shortened because of an internal deletion, which was expressed simultaneously with the wild-type transcript in 89% of small cell lung cancer (SCLC) lines. In contrast, the wild-type transcript was expressed alone in normal tissues, primary non-small cell lung cancer (NSCLC) specimens, and the majority of NSCLC cell lines. Sequence of the shortened SCLC transcript was identical to that of the most common aberrant transcript identified in breast cancer, consisting of a deletion of exons 2-4 and part of 1 and 5. Southern analysis of SCLC lines expressing the shortened transcript did not detect any intragenic deletions. Single strand conformational polymorphism (SSCP) analysis and direct sequencing of TSG101 cDNAs also identified no mutations or deletions. These results suggest that TSG101 is not mutated in lung cancer but that aberrant splicing of TSG101 occurs in SCLC.

Overexpression of cdc25A and cdc25B is frequent in primary non-small cell lung cancer but is not associated with overexpression of c-myc.

Cyclin-dependent kinases can be activated by cdc25, which removes inhibitory phosphates from tyrosine and threonine residues. At least three cdc25 genes (cdc25A, cdc25B, and cdc25C) have been identified in humans. Accumulating evidence indicates that cdc25A and cdc25B possess oncogenic properties. Recently, overexpression of cdc25A and of cdc25B was found in many breast and head and neck cancers. To determine potential roles of cdc25s in non-small cell lung cancer (NSCLC), we analyzed primary tumors and corresponding normal lung tissues from 40 patients with NSCLC for relative expression levels of these genes by multiplex reverse transcription PCR (RT-PCR). cdc25A was overexpressed in 60% (24 of 40) of the tumors and cdc25B in 45% (18 of 40) of the tumors, whereas cdc25C was not overexpressed in any of the tumors analyzed. Because c-myc can increase cdc25A and cdc25B expression, it may be a factor in cdc25 overexpression. We found that c-myc was overexpressed in only 18% (7 of 40) of the tumors. We found no association between overexpression of c-myc and cdc25A or cdc25B. We also investigated whether the cdc25B gene was amplified in NSCLC and found this was true in 40% (8 of 20) of the tumors tested. However, this amplification was not correlated with gene expression status. Interestingly, among 24 tumors with cdc25A overexpression and 18 with cdc25B overexpression, 42% (10 of 24) and 44% (8 of 18) were poorly differentiated histological type. In contrast, well or moderately differentiated tumors had lower frequencies of cdc25A and cdc25B overexpression [19% (3 of 16) and 23% (5 of 22), respectively]. These data indicate that overexpression of cdc25A and cdc25B is frequent and that it may play an important role in NSCLC. However, it is unlikely that this overexpression is caused by c-myc stimulation or cdc25B gene amplification.

Characterization of the ETO and AML1-ETO proteins involved in 8;21 translocation in acute myelogenous leukemia.

The AML1 and ETO genes are disrupted by the nonrandom chromosomal translocation t(8;21) in acute myelogenous leukemia (AML). While the AML1 gene encodes a transcription factor indispensable for definitive hematopoiesis, the biological function of ETO is unknown. To understand the role of ETO and AML1-ETO in the pathogenesis of AML, the full length cDNAs of ETO and AML1-ETO were cloned and antibodies against AML1 and ETO proteins have been developed in our laboratory. Western blot analysis showed that ETO and AML1-ETO were identified as 70 kDa and 94 kDa proteins, respectively, and that both proteins, like AML1, were associated with the nuclear matrix. To examine whether the t(8;21)-positive AMLs expressed a 94-kDa AML1-ETO, protein fractions isolated from leukemia blasts of 10 patients with t(8;21)-positive AML and the Kasumi-1 cells were analyzed by Western blotting. The 94 kDa AML1-ETO fusion protein was detected in all samples. However, this fusion protein was not detectable in all 40 patients with t(8;21)-negative AMLs. The biological significance of AML1-ETO was examined in K562 cells, which stably overexpress AML1-ETO. We found that AML1-ETO blocked the erythroid differentiation of K562 cells induced by low doses of Ara-C. Thus, t(8;21)-positive AMLs appear to overexpress the AML1-ETO fusion protein, which may be responsible for differentiation block and leukemogenesis in AML.

Cell-cycle regulation of DNA damage-induced expression of the suppressor gene PML.

The promyelocytic leukemia (PML) gene, which encodes a growth- and transformation-suppressor, has been identified at the non-random chromosomal translocation break point t(15; 17)(q22; q12) of acute promyelocytic leukemia. To elucidate if PML may play a role in cellular response to DNA damage, PML expression was analyzed by immunofluorescence staining in HeLa cells treated with ionizing radiation (IR) and cisplatin. Our studies demonstrated IR at 20Gy, and cisplatin at 6 micrograms/ml caused more than 5-10 fold increases in PML protein expression in the PML Oncogenic Domain (POD) by immunofluorescent staining. Northern blotting showed that there was no gross increase in mRNA levels indicating that the induction is a post-transcriptional event. Flow cytometry showed that HeLa cells treated with IR were progressively arrested in G1, which correlates with the optimal expression of PML in the cell cycle. To determine if PML expression was under the control of the tumor suppressor p53, which is known to arrest cells in G1, HeLa cells were transfected with the wild-type p53 gene. PML expression in p53 transduced cells were 5-10 fold higher than the control, indicating that the enhanced expression of PML is apparently dependent on the p53 pathway. These data also indicate that PML may play an important role in cellular response to DNA damage such as DNA repair or apoptosis during G1 arrest.

Clonal genetic alterations in the lungs of current and former smokers.

BACKGROUND AND PURPOSE: Genetic damage has been identified at multiple chromosomal sites (i.e., loci) in lung cancer cells. We questioned whether similar damage could be detected in the bronchial epithelial cells of chronic smokers who do not have this disease. METHODS: Biopsy specimens from six different bronchial regions were obtained from 54 chronic smokers (40 current smokers and 14 former smokers). The presence of squamous metaplasia and dysplasia (abnormal histologic changes) in the specimens was documented by examination of hematoxylin-eosin-stained sections, and a metaplasia index ([number of biopsy specimens with metaplasia/total number of biopsy specimens] x 100%) was calculated for each subject. Loss of heterozygosity (i.e., loss of DNA sequences from one member of a chromosome pair) involving microsatellite DNA at three specific loci-chromosome 3p14, chromosome 9p21, and chromosome 17p13-was evaluated by means of the polymerase chain reaction. Fisher's exact test and logistic regression analysis were used to assess the data. Reported P values are two-sided. RESULTS: Data on microsatellite DNA status at chromosomes 3p14, 9p21, and 17p13 were available for 54, 50, and 44 subjects, respectively. The numbers of individuals who were actually informative (i.e., able to be evaluated for a loss of heterozygosity) at the three loci were 36 (67%), 37 (74%), and 34 (77%), respectively. DNA losses were detected in 27 (75%), 21 (57%), and six (18%) of the informative subjects at chromosomes 3p14, 9p21, and 17p13, respectively. Fifty-one subjects were informative for at least one of the three loci, and 39 (76%) exhibited a loss of heterozygosity. Forty-two subjects were informative for at least two of the loci, and 13 (31%) exhibited losses at a minimum of two loci. Loss of heterozygosity at chromosome 3p14 was more frequent in current smokers (22 [88%] of 25 informative) than in former smokers (five [45%] of 11 informative) (P = .01) and in subjects with a metaplasia index greater than or equal to 15% (21 [91%] of 23 informative) than in subjects with a metaplasia index of less than 15% (six [46%] of 13 informative) (P = .003). In five informative individuals among nine tested nonsmokers, a loss of heterozygosity was detected in only one subject at chromosome 3p14 (P = .03), and no losses were detected at chromosome 9p21 (P = .05). CONCLUSIONS: Genetic alterations at chromosomal sites containing putative tumor-suppressor genes (i.e., 3p14 and the FHIT gene, 9p21 and the p16 gene [also known as CDKN2], and 17p13 and the p53 gene [also known as TP53]) occur frequently in the histologically normal or minimally altered bronchial epithelium of chronic smokers.

Frequent inactivation of p16INK4a in oral premalignant lesions.

Head and neck carcinogenesis is believed to be a multistep process, whereby genetic events accumulate in the carcinogen-exposed field at risk, resulting in distinct phenotypic premalignant changes that eventually evolve into invasive cancer. Frequent loss of heterozygosity (LOH) at the chromosome 9p21 region and inactivation of p16(INK4a) by different mechanisms have been described in head and neck squamous cell carcinoma (HNSCC). Recently, we reported that loss of 9p21 is also frequent in oral premalignant lesions. To investigate potential inactivation of p16(INK4a) in these premalignant lesions, we analysed 74 biopsies from 36 patients by immunohistochemistry (IHC) for expression of the p16 protein. Loss of p16 expression was found in 28 (38%) of the lesion biopsies from 17 patients (47%). LOH at the D9s171, a marker in the 9p21 region, was observed in 19 lesion biopsies from 12 cases and correlated with absence of p16 by IHC in 11 (92%) of the 12 comparable cases and 15 (79%) of 19 lesion biopsies. By direct sequencing of ten lesion biopsies from ten individuals with LOH at D9s171 for p16(INK4a) exon 2, one non-sense mutation at codon 88 (GGA-->TGA) was identified. Our data suggest that inactivation of p16(INK4a) may play an important role in early head and neck cancer development.

Indirect validation of benzene exposure assessment by association with benzene poisoning.

We present a validation study of a quantitative retrospective exposure assessment method used in a follow-up study of workers exposed to benzene. Assessment of exposure to benzene was carried out in 672 factories in 12 cities in China. Historical exposure data were collected for 3179 unique job titles. The basic unit for exposure assessment was a factory/work unit/job title combination over seven periods between 1949 and 1987. A total of 18,435 exposure estimates was developed, using all available historical information, including 8477 monitoring data. Overall, 38% of the estimates were based on benzene monitoring data. The highest time-weighted average exposures were observed for the rubber industry (30.7 ppm) and for rubber glue applicators (52.6 ppm). Because of its recognized link with benzene exposure, the association between a clinical diagnosis of benzene poisoning and benzene exposure was evaluated to validate the assessment method that we used in the cohort study. Our confidence in the assessment method is supported by the observation of a strong positive trend between benzene poisoning and various measures, especially recent intensity of exposure to benzene.