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Background: Cinnamon aldehydes found in cinnamon-flavored gums can incite mucosal alterations at points of contact with the oral mucosa. These alterations may include inflammation and epithelial proliferation, but as a rule, the changes are reversible and promptly resolve when gum-chewing activity is discontinued., Methods: The authors report a case of a 24-year-old woman who developed a squamous cell carcinoma of the tongue following persistent and prolonged exposure to cinnamon-flavored gum., Results: Several social, clinical, and histopathologic features point to the cinnamon-flavored chewing gum as a possible causal factor in the development of the patient's oral carcinoma., Conclusions: Prompt withdrawal of cinnamon products is encouraged in heavy gum chewers who develop cinnamon-related oral lesions. For those lesions which do not promptly resolve upon cinnamon withdrawal, diagnostic biopsy should be considered to exclude the possibility of a squamous cell carcinoma.

Atypical alveolar hyperplasia (AAH) is a potential precursor lesion from which lung adenocarcinomas arise and may be a good target for studying the early events of lung tumorigenesis. We have previously shown that AAHs are neoplastic epithelial proliferations that often harbor activating mutations of the K-ras oncogene. In the current study, we examined a spectrum of AAHs to determine the frequency and timing of p53 alterations in lung tumorigenesis. We analyzed 37 AAHs and their paired overt lung neoplasms for p53 protein accumulation using the monoclonal antibody DO7. DNA sequence analysis of the p53 gene was performed on those cases demonstrating p53 protein accumulation. AAHs were classified as low-grade, high-grade, or AAH-like carcinoma based on cytoarchitectural features. Accumulation of the p53 protein was found in none (0%) of 20 low-grade AAHs, in 1 (9%) of 11 high-grade AAHs, and in three (50%) of six AAH-like carcinomas. There was a statistically significant trend toward p53 accumulation with increasing grade of the AAHs. A missense mutation in exon 7 of the p53 gene was found in 1 AAH-like carcinoma, whereas mutations in exons 5 through 8 could not be detected in the other three AAHs with p53 protein accumulation. Three of the paired overt carcinomas harbored p53 mutations that were not present in the AAHs. Alterations of p53 do not appear to be common events in AAHs, especially when these lesions exhibit low-grade cytoarchitectural features. Alterations of p53, however, are more frequent at the level of AAH-like carcinoma and may be associated with the transition from a benign to a malignant proliferation of pneumocytes.

We previously demonstrated that loss of 13q occurred in greater than 50% of primary head and neck squamous cell carcinomas. Although the minimal region of loss at 13q14.1-14.3 included the Rb1 gene, Rb1 inactivation was found to be rare. To further investigate possible targets of 13q loss in head and neck cancer, 32 primary tumors were fine mapped by microsatellite analysis with the use of 10 new markers spanning the minimal region. Twenty-one (66%) of 32 tumors displayed loss of heterozygosity and once again the minimal region of loss was confirmed to be 13q14.1-14.3. In addition to 6 monosomies, 11 tumors had regions of loss that either selectively deleted (one tumor) or stretched into the BRCA2 region 13q12-13 (10 tumors). Therefore we pursued investigation of the BRCA2 gene by a coupled transcription and translation assay of its largest exon (exon 11; 4932 base pairs) to detect functional truncations indicative of mutation in 37 primary head and neck tumors with 13q loss. These tumors included 17 of the 32 tumors recently analyzed and 20 other tumors with 13q loss from a previous study. Additionally, we performed the transcription and translation assay on five head and neck cancer cell lines. We found no alterations in exon 11 of the BRCA2 gene in any of the tumors or cell lines suggesting that perhaps another tumor suppressor gene on 13q is involved in head and neck cancer.

Background: In patients with head and neck squamous cell carcinoma (HNSCC), a squamous cell carcinoma (SCC) in the lung represents either another primary tumor or a metastasis. This distinction greatly influences patient prognosis and could guide treatment strategies, but the nature of a solitary lung nodule is often difficult to discern by use of standard clinical and histologic parameters. Comparison of genetic alterations in the tumors could resolve this dilemma., Methods: We compared paired tumors from 16 patients with HNSCC and a solitary lung SCC for loss (i.e., deletion) of loci on chromosomal arms 3p and 9p. Losses at these loci occur early during neoplastic transformation of the respiratory tract. DNA from microdissected tumors and normal tissues was subjected to polymerase chain reaction-based microsatellite analysis. An effort was also made to distinguish primary lung cancers from lung metastases on the basis of clinical and histopathologic features., Results: In most cases, comparison of genetic alterations clarified the relationship between the lung tumor and the primary HNSCC. The paired tumors from 10 patients had concordant patterns of loss at all loci suggesting metastatic spread, whereas three paired tumors had discordant patterns of loss at all loci suggesting independent tumor origin. These observations were supported by the clinical and pathologic findings., Conclusions/implications: In patients with HNSCC and a solitary SCC in the lung, microsatellite analysis provides a rapid genetic approach for discerning clonal relationships. In such patients, we found that a solitary SCC in the lung more likely represents a metastasis than an independent lung cancer. Microsatellite analysis could potentially be applied to any patient with multiple tumors, where tumor relationships are not clear on clinical, radiographic, or even histopathologic grounds.

Follicular and Hürthle cell carcinomas of the thyroid cannot be differentiated from adenomas by either preoperative fine needle aspiration or intraoperative frozen section examination, and yet there exist potentially significant differences in the recommended surgical management. We examined, by PCR-based microsatellite polymorphism analysis, DNA obtained from 83 thyroid neoplasms [22 follicular adenomas, 29 follicular carcinomas, 20 Hürthle cell adenomas (HA), and 12 Hürthle cell carcinomas (HC)] to determine whether a pattern of allelic alteration exists that could help distinguish benign from malignant lesions. Alterations were found in only 7.5% of informative PCR reactions from follicular neoplasms, whereas they were found in 23.3% of reactions from Hürthle cell neoplasms. Although there were no significant differences between follicular adenoma and follicular carcinoma, HC demonstrated a significantly greater percentage of allelic alteration than HA on chromosomal arms 1q (P < 0.001) and 2p (P < 0.05) by Fisher's exact test. The documentation of an alteration on either 1q or 2p was 100% sensitive and 65% specific in the detection of HC (P < 0.0005, by McNemar's test). In conclusion, PCR-based microsatellite polymorphism analysis may be a useful technique in distinguishing HC from HA. Potentially, the application of this technique to aspirated material may allow this distinction preoperatively and thus facilitate more optimal surgical management. Consistent regions of allelic alteration may also indicate the locations of critical genes, such as tumor suppressor genes or oncogenes, that are important in the progression from adenoma to carcinoma. Finally, this study demonstrates that Hürthle cell neoplasms, now considered variants of follicular neoplasms, differ significantly from follicular neoplasms on a molecular level.

Expression of the invasion/metastasis suppressor, E-cadherin, is diminished or lost in thyroid carcinomas. Yet, mutational inactivation of E-cadherin is rare. Herein, we show that this loss is associated with hypermethylation of the E-cadherin 5' CpG island in a panel of human thyroid cancer cell lines. This aberrant methylation is evident in 83% of papillary thyroid carcinoma, 11% of follicular thyroid carcinoma, 40% of Hurthle's cell carcinoma, and 21% of poorly differentiated thyroid carcinomas. Contrary to previous reports, the majority of these poorly differentiated thyroid carcinomas express E-cadherin, but often within the cytoplasm rather than at the cell surface. Together, our data indicate that the invasion/metastasis suppressor function of E-cadherin is frequently compromised in human papillary, Hurthle's cell, and poorly differentiated thyroid carcinoma by epigenetic and biochemical events.

Although the frozen section is widely used to identify tissue type during parathyroid exploration in patients with hyperparathyroidism, questions regarding its accuracy have been raised. Frozen section error has been identified as a significant factor contributing to surgical failure. The purpose of this study was to establish the accuracy of frozen section in this setting and to identify pitfalls underlying frozen section error. The final pathologic diagnoses were compared with the paired frozen section diagnoses for all patients who underwent parathyroid exploration at this institution in the period between 1984 and 1997. For those cases in which a discrepancy was identified, the original histopathology slides and the medical records were reviewed. Of the 1579 frozen sections, a definitive and accurate diagnosis could not be determined in 20 cases (1.3%); in 7 (0.4%), the frozen section diagnosis was deferred, and in the other 13 cases, the frozen section diagnosis was incorrect. Overall accuracy rate was 99.2% after deferred cases were excluded. Frozen section artifact, sampling error, and judgmental error contributed to deferred or incorrect diagnoses. Several features confounded the distinction between parathyroid and thyroid tissue in 10 cases: the coexistence of parathyroid and nodular thyroid disease; intrathyroidal parathyroid glands showing conspicuous follicle formations or abundant oncocytic cells; and thyroid nodules with fatty stroma. The frozen section is a highly reliable means of identifying tissue type during parathyroid exploration. In exceptional cases, however, the distinction between parathyroid and thyroid tissue may not be possible owing to a striking overlap seen at the clinical, gross, and microscopic levels.

A 59-year-old white male presented with a two-month history of dry cough and shortness of breath. At bronchoscopy, a 1 cm mass on the laryngeal surface of the epiglottis was found. Immunohistochemical stain of the biopsy specimen revealed a monoclonal proliferation of plasma cells. Serological tests revealed normal serum immunoglobulin levels, and a bone marrow aspirate and biopsy was also normal; no abnormalities were found on serum or urine electrophoresis. He received radiotherapy (50 Gy), and was doing well three years following therapy without evidence of disease.

We examined 82 cases of primary non-small cell lung cancer (NSCLC) for loss of heterozygosity (LOH) at the chromosome 9p21-24 region using 16 microsatellite markers. A total of 52 tumors (63%) displayed LOH, and 25 of these cases displayed LOH for all markers. Two cases had small hemizygous losses confined to the p16 gene and more distal markers, whereas 3 cases had loss proximal to p16 and extended through marker D9S126. This latter region has recently been described as another minimal region of loss at 9p21 in lung cancer. However, homozygous deletion of the p16 gene was observed in 18 of 85 cases, with only 5 cases having large deletions extended into the D9S126 region. Furthermore, we did not observe homozygous deletion at the 9p21 region that excluded the p16 gene. Fluorescence in situ hybridization (FISH) analysis using genomic probes spanning either the p16 or Hel-N1 (located at D9S126) gene was performed in 14 tumors. The results from FISH correlated with the chromosomal mapping data, suggesting that the p16 region is the major target of deletion at chromosome 9p21 in primary NSCLC.

Background: Although fine-needle aspiration (FNA) is 90% sensitive in the detection of papillary carcinoma (PC) of the thyroid, its specificity has been reported as low as 52%. Consequently, patients who have an FNA suspicious for PC may undergo operation for a benign process. The ribonucleoprotein telomerase has been noted to be activated in a wide variety of carcinomas. We examined 30 PCs for telomerase activity to determine whether this would be a useful adjunct to FNA in the diagnosis of lesions suspicious for PC., Methods: Standard telomere repeat amplification protocol assays were performed on fresh frozen tissue samples from 30 PCs, 3 benign nodules, and 10 normal thyroids., Results: Telomerase activity was documented in 20 of 30 (67%) of the PCs, 0 of 3 benign nodules, and 0 of 10 normal thyroids. In all, 11 of the 20 PCs had FNA cytology that was nondiagnostic of PC, and 2 of the benign nodules had FNA that was suspicious for PC., Conclusions: The telomerase assay appears useful in the distinction of benign from malignant thyroid lesions that have FNA suspicious for but not diagnostic of PC. On the basis of these findings, a prospective trial examining telomerase activity in FNAs suspicious for thyroid cancer has been initiated.

Background: Adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene (tk) is one of the most effective gene therapy strategies for solid tumors in experimental animal studies. Foundational animal studies in an oral cancer model have demonstrated significant antitumor effects and improved animal survival using this treatment strategy., Objective: To assess the safety of adenovirus-mediated transfer of the herpes simplex virus tk gene for the treatment of oral cancer., Design: Oral tumors were established in C3H/HeJ mice and were treated with tk followed by systemic ganciclovir administration. Polymerase chain reaction amplification techniques were used to screen local surrounding tissues and distant organs for the presence of the adenoviral construct. Microscopic examination of the tissues was performed to determine the cytopathic effects of the vector. Blood samples were obtained from the animals to test for liver, renal, and bone marrow function after treatment., Results: The adenoviral vector was present in the livers, lungs, and kidneys of animals treated with the maximal single injection dose of 2 x 10(9) plaque forming units (pfu). No vector was noted systemically after delivery of an equally effective low dose of 1 x 10(8) pfu. Microscopic examination revealed no cytopathic effects in distant organs despite the presence of vector. Results of liver and renal function tests revealed no differences between treated and control animals. There was no statistical difference in white blood cell count, hematocrit, or platelet count between animals treated with ganciclovir and control animals., Conclusions: Based on these results, the direct delivery of adenovirus-tk followed by ganciclovir administration appears both efficacious and safe in an animal model. However, serum evaluation for adenovirus vector and screening organ function studies should be included in human protocols using this gene therapy scheme.

Fine-needle aspiration (FNA) of the thyroid is the sine qua non in the preoperative evaluation of thyroid nodules. Despite this, cytological examination of FNA cannot differentiate malignant from benign Hürthle cell neoplasms. We have previously shown that Hürthle cell carcinomas harbor more genetic alterations on chromosomal arms 1q and 2p than Hürthle cell adenomas, and that all Hürthle cell neoplasms have a significantly higher frequency of alterations on chromosomal arm 1p compared with normal thyroid. To determine if these genetic alterations could be detected in FNA samples, we examined DNA from FNAs that were available from eight Hürthle cell neoplasms. Polymerase chain reaction (PCR) amplification of DNA demonstrated either direct correlation with alterations seen in the tumor samples or in some instances, additional chromosomal alterations. We conclude that PCR-based microsatellite DNA analysis of preoperative FNA samples from Hürthle cell neoplasms can potentially distinguish Hürthle cell carcinomas from adenomas and that with further validation and perfection, this technique may allow more optimal surgical management of patients with these lesions.

Because of its propensity to spread along epithelial surfaces, colonic adenocarcinoma can mimic other neoplasms. For example, colonic adenocarcinoma can grow along the surface of the urinary bladder and can simulate primary bladder neoplasia, and metastatic colonic adenocarcinoma can grow along alveolar walls and can mimic primary lung neoplasia. Intraepithelial spread along bile ducts, however, is not a well-recognized behavior of hepatic metastases. Indeed, dysplastic change in the epithelium lining the biliary tract is sometimes used to discriminate primary biliary neoplasms from metastatic adenocarcinoma. We report on eight cases of colonic adenocarcinoma metastatic to the liver that demonstrated prominent spread throughout the biliary tree along intact basement membranes. Morphologically, this pattern closely resembled high-grade dysplasia (i.e., carcinoma in situ) of the extrahepatic and intrahepatic bile ducts. Clinically, two of the tumors were mistaken for primary biliary neoplasia because of the common radiologic finding of intrabiliary masses with distended bile ducts. A definite diagnosis of metastatic carcinoma was established by careful attention to the medical history, thorough evaluation of the morphologic features, and histologic comparison with the primary colon cancer. For patients with a history of colonic adenocarcinoma, consideration of a liver metastasis is appropriate even when certain histologic and radiographic features point to a neoplasm of biliary origin.

Thyroid cell growth and function are regulated by hormones and growth factors binding to cell surface receptors that are coupled via G proteins, Gs and Gq, to the adenylyl cyclase and phospholipase C signal transduction systems, respectively. Activating mutations of the TSH receptor and G alpha s have been documented in subsets of thyroid neoplasms. To test the oncogenic potential of activated G alpha s in transgenic mice, we used the cholera toxin A1 subunit that constitutively activates G alpha s and used the rat thyroglobulin gene promoter for targeting this transgene (TGCT) to thyroid follicular cells. Three (M1392, F1358, and F1286) of six founders identified were able to transmit the transgene to their offspring and thyroid glands from these mice contained elevated levels of cAMP. Concentrations of serum thyroxine were elevated as early as 2 months of age (M 1392 and F 1286). F1358 mice were euthyroid until 8 months of age, at which time they developed hyperthyroidism. All three TGCT lines developed thyroid hyperplasia independent of their thyroxine levels. DNA image analysis of thyroid follicular cells from both the hyper and euthyroid mice showed that DNA index and "S+G2/M" phase were increased compared with normal, changes similar to that seen in poor prognosis human carcinomas. These data suggest that the G alpha s-adenylyl cyclase-cAMP pathway has an important role in thyroid hyperplasia and the transgenic mouse models reported herein will allow further examination of the role of this pathway in thyroid oncogenesis.

Novel oncogene mutation detection techniques have demonstrated that standard histopathological examination may fail to detect clinically significant metastatic cancer cells. Recently, telomerase activity has been detected in most immortal cell lines and human tumors, potentially providing a novel diagnostic marker. We compared standard histopathological examination with the telomeric repeat amplification protocol assay and either a p53 plaque hybridization or a K-ras mutation ligation assay in the lymph nodes of 12 patents with surgically resectable non-small cell lung cancer. Telomerase activity was detected in 10 of 10 (100%) evaluable tumors. Eight of 9 (89%) histopathologically positive lymph nodes were telomerase positive, and 26 of 48 (54%) histopathologically negative lymph nodes were telomerase positive. In comparison, oligonucleotide plaque hybridization detected metastases in all 3 histopathologically positive nodes and in 3 of 27 histopathologically negative nodes. Similarly, the K-ras mutation ligation assay detected metastases in all 6 histopathologically positive lymph nodes examined and in 1 of 21 histopathologically negative lymph nodes. Thus, most of the "positive" nodes by telomerase assay did not harbor occult neoplastic cells that shared the same genetic alteration as the primary tumor. The high rate of false positives associated with the telomeric repeat amplification protocol assay limits its role in staging lymph nodes in patients with non-small cell lung cancer.

The inability to distinguish microinvasive follicular thyroid cancer from benign follicular tumors preoperatively presents an important surgical dilemma. We examined 44 follicular tumors and found telomerase activity in all 11 follicular carcinomas and in 8 of 33 benign follicular tumors. It was undetectable in 22 normal thyroid tissues adjacent to the tumors. Telomerase activity may thus provide a diagnostic marker distinguishing benign from malignant follicular thyroid tumors. The ability to identify invasive follicular thyroid tumors could avert over 14,000 thyroidectomies annually in the United States, thereby significantly decreasing morbidity and health care costs.

Background: Fine-needle aspiration cytology is sensitive for detecting malignancies such as papillary carcinoma of the thyroid gland. Because fine-needle aspiration specificity for papillary carcinoma of the thyroid is variable, routine intraoperative frozen section is often advocated., Study Design: To define the roles of fine-needle aspiration and frozen section in papillary carcinoma of the thyroid gland, we reviewed data from 82 patients who underwent thyroidectomy between August 1989 and August 1995 for papillary carcinoma of the thyroid cytology. Results of fine-needle aspirations were grouped into three categories: diagnostic of papillary carcinoma of the thyroid; diagnostic of follicular-variant of papillary carcinoma of the thyroid; or suspicious for papillary carcinoma of the thyroid. Definitive diagnoses were made on permanent histology., Results: A fine-needle aspiration revealing papillary carcinoma of the thyroid was 98 percent specific for cancer or 100 percent specific for follicular-variant of papillary carcinoma of the thyroid. A fine-needle aspiration that was suspicious for papillary carcinoma of the thyroid (n = 24) was only 54 percent specific for cancer. On the basis of gross intraoperative findings, 5 of these 24 patients underwent total thyroidectomy without frozen section, and all had carcinoma. The other 19 had frozen section analysis. Of the 5 patients with cancer detected by frozen section, 4 had cancer on permanent histology. Findings on frozen section demonstrated a follicular neoplasm in the other 14 patients, of which 4 ultimately were cancer., Conclusions: When papillary carcinoma of the thyroid or follicular-variant of papillary carcinoma of the thyroid is definitively diagnosed on fine-needle aspiration, the surgeon can perform definitive thyroidectomy without frozen section because of the high specificity for cancer. If the fine-needle aspiration is suspicious for papillary carcinoma of the thyroid, the incidence of cancer is 54 percent, and patients with these conditions should undergo surgery with frozen section. When either gross findings or frozen sections suggest malignancy, definitive thyroidectomy can be performed because 90 percent of such cases will be cancer. If frozen section is not diagnostic of malignancy, a thyroid lobectomy/isthmusectomy is recommended because 71 percent have a benign lesion. This systematic approach to papillary carcinoma of the thyroid will obviate unnecessary frozen sections while maintaining excellent diagnostic specificity.

We screened 73 primary head and neck squamous cell carcinoma (HNSCC) specimens for loss of heterozygosity (LOH) on chromosome 14q. Analysis of 20 polymorphic microsatellite markers identified 29 (40%) HNSCCs exhibiting LOH of 14q in at least one locus. Six tumors had probable monosomy of 14q, displaying allelic loss for all informative markers tested, and 23 demonstrated partial losses on 14q. Fine mapping with 1-10 additional markers revealed two poorly defined regions of loss (4-7 cM) at 14q13-21 and 14q31-32.1 in seven tumors. In 53 patients with previously untreated tumors treated with curative intent, LOH of 14q in these tumors correlated with poor survival. Compared to patients with tumors that retain heterozygosity of 14q, those with 14q LOH had a 3-fold increased risk of death in multivariate analysis (hazards ratio, 3.2; 95% confidence interval = 1.2-8.4). These data have confirmed a high frequency of chromosome 14q loss in HNSCC and suggest that LOH of any region on chromosome 14q is an indicator of poor outcome.

Although the ability to transmit high-resolution images of histopathological sections could have a profound impact on the practice of pathology, the application of video microscopy to the daily activities of surgical pathology has not been rigorously evaluated. In particular, certain aspects of video microscopy relating to frozen section evaluation have not been adequately assessed. We conducted a retrospective analysis of 48 excisional skin biopsy specimens encompassing a spectrum of benign and malignant lesions. To simulate an actual frozen section evaluation, only original frozen section slides were evaluated. Fields were selected and digitized (Roche Image Analysis System) by a pathology resident. Two sets of diagnoses were subsequently rendered by a surgical pathologist, the first set based on the digitized images and the second based on direct microscopic examination of the histological slides. The two sets of diagnoses were compared, and the concordance rates were as follows: malignant diagnoses, 100%; benign diagnoses, 100%; positive margins, 96%; negative margins, 99%. One (4%) of the 25 positive margins was indexed as negative by image analysis. Conversely, one (1%) of the 121 negative margins was indexed as positive by image analysis. In both of these cases, error was attributable to selection and digitization of an inappropriate field. We conclude that telepathology of static images is an accurate method of evaluating frozen sections of skin lesions. Potentially, this technology could be applied to the frozen section evaluation of other lesions as well. Static image analysis is, however, susceptible to errors induced by inappropriate field selection, emphasizing the need for trained and skillful personnel on both sides of the video camera.

Although papillary carcinoma accounts for approximately 70% of all thyroid cancers, preliminary studies of allelic loss have thus far not identified any areas of chromosomal deletion. We evaluated 30 papillary thyroid carcinomas for chromosomal loss/allelic imbalance by testing at least 2 microsatellite markers from every autosomal arm. Fifteen of the 30 tumors tested exhibited loss of heterozygosity/allelic imbalance (LOH/AI) at one or more loci. Chromosomal arms with frequent LOH/AI included 4q, 5p, 7p and 11p. An average of 1.1 chromosomal arms displayed LOH/AI in each individual tumor. Therefore, 4q, 5p, 7p and, to a lesser extent, 11p display significant LOH/AI in papillary thyroid cancer, which indicates the presence of putative tumor-suppressor gene loci at these chromosomal arms.

Telomerase is a ribonucleoprotein that maintains telomere length and whose activity is associated with escape from cellular senescence. Telomerase activity has been found in germline, immortalized, and malignant tumor cells. Using a modified PCR-based assay for telomerase activity, 26 of 35 (80%) primary, fresh, head and neck squamous cell cancer specimens and 3 of 6 head and neck squamous dysplastic lesions possessed telomerase activity. In addition, 14 of 44 (32%) oral rinses from a separate group of head and neck squamous cell cancer patients contained detectable telomerase activity, whereas 1 of 22 (5%) oral rinses from normal control patients exhibited telomerase activity. Telomerase activity in oral rinses was compared with corresponding activity in paired primary tumor samples for 19 cases: 7 of 19 demonstrated activity in both tumor and oral rinse, 2 of 19 lacked activity in both tumor and oral rinse, 10 of 19 tumors demonstrated activity that could not be detected in corresponding oral rinses, and there were no examples of positive oral rinses with corresponding negative tumors. Although currently limited in its sensitivity, analysis of telomerase activity in oral rinses represents a novel method to detect the presence of cancer cells shed in the upper aerodigestive tract.

Background: The p53 gene (also known as TP53) may be the most common genetic target involved in the malignant transformation of human cells. Direct sequence analysis has demonstrated that alteration of this gene occurs in approximately 45% of head and neck squamous cell carcinomas. The consequences of p53 mutations in these cancers with respect to tumor behavior and patient survival have not been rigorously determined., Purpose: We evaluated the implications of p53 mutations in relation to the control of locoregional disease and overall survival following radiation therapy., Methods: Data from 110 consecutive patients with invasive disease who were treated with primary radiation therapy (given with curative intent) or with adjuvant radiation therapy (following complete surgical extirpation of gross disease) were included in the analysis. A 1.8-kilobase fragment of the p53 gene encompassing exons 5-9 was amplified from the DNA of stored (frozen) tumor specimens; the amplified DNA was cloned and sequenced by use of standard techniques. Overall survival and locoregional disease-free survival after the completion of radiation therapy were estimated by the Kaplan-Meier method; survival comparisons were made by use of the logrank test or proportional hazards regression models. Reported P values are two-sided., Results: Fortyeight (44%) of the 110 tumors had cells bearing p53 mutations. The risk of locoregional recurrence following either primary or adjuvant radiation therapy was significantly greater (i.e., the time to recurrence was shorter) for patients whose tumors contained mutant p53 genes (univariate model hazard ratio [HR] for p53 mutation versus wild-type = 2.2; 95% confidence interval [CI] = 1.2-4.1; P = .02). The presence of regional lymph node metastases (presence versus absence, HR = 2.0; 95% CI = 1.0-4.2; P = .05) and treatment type (primary radiation therapy versus surgery plus adjuvant radiation therapy, HR = 2.3; 95% CI = 1.2-4.3; P = .01) were also associated with greater risks of locoregional failure. The presence of p53 mutations and lymph node metastases and treatment with primary, as opposed to adjuvant, radiation therapy remained significant risk factors in multivariate regression analysis. No relationship was demonstrated between p53 status and overall survival (mutant versus wild-type, HR = 1.1; 95% CI = 0.6-2.1; P = .66); however, a relationship was shown for tumor stage and overall survival (stages III and IV [more advanced] versus stages I and II [less advanced], HR = 3.3; 95% CI = 1.0-10.8; P = .05). Mutation of the p53 gene was not associated with patient age, sex, tumor stage, primary tumor site, regional lymph node status, degree of tumor cell differentiation, or treatment method., Conclusions: Mutation of the p53 gene is associated with an increased risk of locoregional failure in patients with invasive head and neck squamous cell carcinoma who are treated with radiation therapy.

The tumor suppressor gene p16 (CDKN2/MTS-1/INK4A) can be inactivated by multiple genetic mechanisms. We analyzed 29 invasive primary head and neck squamous cell carcinomas (HNSCC) for p16 inactivation with immunohistochemistry utilizing a new monoclonal antibody (mAb), DCS-50. p16 staining of the primary lesions was correlated with genetic analysis including: (a) detailed microsatellite analysis of markers at the p16 locus to detect homozygous deletion; (b) sequence analysis of p16; and (c) Southern blot analysis to determine the methylation status of the 5' CpG island of p16. Twenty-four of 29 (83%) head and neck squamous cell carcinoma tumors displayed an absence of p16 nuclear staining using immunohistochemistry. Of these 24 tumors, we found that 16 (67%) harbored homozygous deletions, 5 (21%) were methylated, 1 displayed a rearrangement at the p16 locus, and 1 displayed a frameshift mutation in exon 1. These data suggest that: (a) inactivation of the p16 tumor suppressor gene is a frequent event in squamous cell carcinomas of the head and neck; (b) p16 is inactivated by several distinct and exclusive events including homozygous deletion, point mutation, and promoter methylation; and (c) immunohistochemical analysis for expression of the p16 gene product is an accurate and relatively simple method for evaluating p16 gene inactivation.

A genetic progression model of head and neck squamous cell carcinoma has not yet been elucidated, and the genetic basis for "field cancerization" of the aerodigestive tract has also remained obscure. Eighty-seven lesions of the head and neck, including preinvasive lesions and benign lesions associated with carcinogen exposure, were tested using microsatellite analysis for allelic loss at 10 major chromosomal loci which have been defined previously. The spectrum of chromosomal loss progressively increased at each histopathological step from benign hyperplasia to dysplasia to carcinoma in situ to invasive cancer. Adjacent areas of tissue with different histopathological appearance shared common genetic changes, but the more histopathologically advanced areas exhibited additional genetic alterations. Abnormal mucosal cells surrounding preinvasive and microinvasive lesions shared common genetic alterations with those lesions and thus appear to arise from a single progenitor clone. Based on these findings, the local clinical phenomenon of field cancerization seems to involve the expansion and migration of clonally related preneoplastic cells.

Patients with head and neck cancers have a high (2-3%/year) incidence of second primary lesions. Clinically, these new lesions are identified either simultaneously with the primary lesion (synchronous) or after a period of time (metachronous). This observation has been attributed to the concept of "field carcinogenesis," which is based on the hypothesis that prolonged exposure to carcinogens leads to the independent transformation of multiple epithelial cells at several sites. An alternative theory is based on the premise that any transforming event is rare; following initial transformation, the progeny of the transformed clone spread through the mucosa and give rise to geographically distinct but genetically related tumors. We analyzed the pattern of X-chromosome inactivation in multiple primary tumors from eight female patients with head and neck cancer. In addition, we used microsatellite analysis to examine the pattern of allelic loss on chromosomes 9p and 3p, identified as early events in the progression of head and neck malignancies. In four of four cases, multiple tumors demonstrated the same pattern of X-chromosome inactivation. In the remaining four cases, X-chromosome deletions prevented interpretation (n = 3), or the androgen receptor locus was noninformative (n = 1). In three of nine patients, multiple tumors displayed the same pattern of loss of heterozygosity, two with identical breakpoints on chromosome 9p. In one patient, there was an identical microsatellite alteration at a 3p locus, definitive evidence that these tumors arose from the same clone. Our findings suggest that in at least a proportion of patients with head and neck cancers, multiple primary tumors arise from a single clone.

Atypical alveolar hyperplasia (AAH) is a potential precursor lesion from which lung adenocarcinomas arise and may be a good target for studying the early events of lung tumorigenesis. A common genetic alteration in lung adenocarcinomas is mutational activation of K-ras. To determine the timing of K-ras activation, we evaluated formalin-fixed and paraffin-embedded tissue samples of 41 AAHs and their paired lung neoplasms from 28 patients for codon 12 point mutations of the K-ras oncogene. K-ras codon 12 mutations were detected using PCR followed by allele-specific oligonucleotide hybridization. Mutations were found in 16 (39%) of the 41 AAHs, 8 (42%) of the 18 adenocarcinomas, and none (0%) of the 5 lung neoplasms that were not adenocarcinomas. Of the 18 patients with both an AAH and a synchronous lung adenocarcinoma, 6 had K-ras mutation in the adenocarcinoma but not in the AAH, 6 had mutations in the AAH but not in the adenocarcinoma, 4 did not harbor mutations in either the AAH or the adenocarcinoma, and 2 had mutations in both their AAH and their synchronous adenocarcinoma. In just 1 of the 18 patients was the same K-ras mutation present in the AAHs and adenocarcinoma of the patient. The detection of independent activating point mutations in a cancer-causing gene points to the neoplastic nature of AAH and suggests that glandular neoplasms of the lung arise from a background of field cancerization.

Tumour hypoxia is well recognised as a major factor contributing to radioresistance. This article examines the role of hypoxia in influencing the treatment outcome following radiotherapy (RT), and reviews the rationale and results of clinical trials that utilise hypoxic sensitizers or cytotoxins in the treatment of head and neck carcinoma. Histologic evidence for tumour hypoxia in human neoplasms was first reported in 1955. Since then, direct measurement by microelectrodes has revealed heterogeneity in intratumoural oxygen concentrations, and low oxygen concentrations are associated with poor local-regional control by RT. These findings coupled with the result of nuclear imaging studies employing radiolabelled imidazoles, provide strong evidence for the existence of tumour hypoxia which influences RT treatment outcome. Hyperbaric oxygen (HBO) trials for head and neck cancer, conducted in the early 1970s, demonstrated that HBO improved local control and survival rates in patients with head and neck cancer receiving radiotherapy (RT). Since the mid-1970s, clinical research in overcoming tumour hypoxia was mainly centred on the use of nitro-imidazoles as hypoxic cell sensitizers. However, the results from several major clinical trials remain inconclusive. Specifically, the Radiation Therapy Oncology Group (RTOG) misonidazole head and neck trial (298 patients) showed no benefit. The Danish misonidazole trial (626 patients) showed no overall benefit, however positive results were observed in a subgroup (304 pharyngeal cancer patients). Although the European Organisation for Research and Teaching of Cancer (EORTC) misonidazole trial with hyperfractionated RT showed no benefit, the Danish nimorazole trial demonstrated an overall benefit in survival as well as local control. The European etanidazole (ETA) trial (374 patients) showed no advantage of adding the drug to RT. The RTOG ETA trial (504 patients) showed no global benefit. However, positive results were observed in a subset of patients with early nodal disease (197 patients). In addition, a recent meta-analysis by Overgaard, utilising pooled results in the literature demonstrated that modification of tumour hypoxia significantly improved local-regional control in head and neck cancers with an odds ratio of 1.23 (95% confidence limits 1.09 to 1.37). Hypoxic cytotoxins, such as tirapazamine, represent a novel approach in overcoming radioresistant hypoxic cells. Tirapazamine is a bioreductive agent which, by undergoing one electron reduction in hypoxic conditions, forms cytotoxic free radicals that produce DNA strand breaks causing cell death. In vitro and in vivo laboratory studies demonstrate that tirapazamine is 40 to 150 times more toxic to cells under hypoxic conditions as compared to oxygenated conditions and that tirapazamine is superior to ETA in enhancing fractionated irradiation in mouse SCCVII and other tumour types with an enhancement ratio of 1.5 to 3.0. Phase I studies demonstrated that therapeutic doses of tirapazamine can be given safely. A multi-institutional phase II trial using tirapazamine with concurrent RT for head and neck cancer is now in progress.

Mel-CAM (previously MUC18) is an integral membrane glycoprotein involved in heterophilic intercellular adhesions. Mel-CAM is expressed specifically in certain normal mesenchymal tissues, including smooth muscle, endothelium, and Schwann cells. As a member of the immunoglobulin supergene family of cell adhesion molecules (CAMs), Mel-CAM may play a pivotal role in the normal differentiation and functional activity of these tissues. To determine the distribution of Mel-CAM in mesenchymal neoplasms and to investigate its potential role as a factor in tumor progression, we evaluated a spectrum of mesenchymal neoplasms by immunohistochemistry using a Mel-CAM-specific polyclonal antibody on formalin-fixed tissues. Mel-CAM positivity was observed in 5 (100%) of 5 leiomyomas, 29 (91%) of 32 leiomyosarcomas, 5 (100%) of 5 hemangiomas, 5 (100%) of 5 angiosarcomas, 3 (100%) of 3 Kaposi's sarcomas, 8 (100%) of 8 schwannomas, 10 (100%) of 10 neurofibromas, 0 (0%) of 8 malignant peripheral nerve sheath tumors, 2 (15%) of 13 malignant fibrous histiocytomas, 0 (0%) of 8 fibrosarcomas, 0 (0%) of 7 synovial sarcomas, and 0 (0%) of 6 liposarcomas. These results show that Mel-CAM is expressed consistently in neoplasms of smooth muscle and vascular origin, and that immunostaining for Mel-CAM may serve as a useful adjunct in differentiating leiomyosarcomas, angiosarcomas, and Kaposi's sarcomas from other spindle cell neoplasms. Furthermore, the observation that Mel-CAM is expressed consistently in schwannomas and neurofibromas but not in malignant peripheral nerve sheath tumors implicates Mel-CAM as a potential modulator of malignant transformation in peripheral nerve tumors.

To elucidate the genetic alterations that occur in salivary gland tumors, we screened every autosomal arm (and the X-chromosome) of 29 primary human salivary gland neoplasms (11 pleomorphic adenomas, 10 adenoid cystic carcinomas, 5 mucopidermoid carcinomas, and 3 carcinomas ex-mixed tumors) for allelic loss using 86 microsatellite markers. A minimum of two microsatellite markers were used per chromosomal arm to achieve informativity of at least 60% (excluding X). The pleomorphic adenomas demonstrated few areas of allelic loss; the most prominent chromosomal arm involved was 12q, lost in more than 35% of informative cases. The most significant allelic losses in adenoid cystic carcinoma were 1p, 2p, 6q, 17p, and 20p (>20% of informative cases) and 19q (40% of informative cases). Mucoepidermoid carcinoma showed 50% or greater loss at 2q, 5p, 12p, and 16q. Although losses at 9p, 3p, and 17p are common in squamous cell carcinoma of the head and neck, only the carcinoma ex-mixed tumors demonstrated loss at these loci, consistent with progression to a more aggressive phenotype. Salivary gland tumors display allelic loss patterns different from many other tumor types, suggesting distinct genetic pathways in the progression of these tumors.

Many tumour types have been reported to have deletion of 9p21 (refs 1-6). A candidate target suppressor gene, p16 (p16INK4a/MTS-1/CDKN2), was recently identified within the commonly deleted region in tumour cell lines. An increasing and sometimes conflicting body of data has accumulated regarding the frequency of homozygous deletion and the importance of p16 in primary tumours. We tested 545 primary tumours by microsatellite analysis with existing and newly cloned markers around the p16 locus. We have now found that small homozygous deletions represent the predominant mechanism of inactivation at 9p21 in bladder tumours and are present in other tumour types, including breast and prostate cancer. Moreover, fine mapping of these deletions implicates a 170 kb minimal region that includes p16 and excludes p15.

AIDS is associated with a high risk of certain malignancies, notably Kaposi's sarcoma (KS) and B-cell non-Hodgkin's lymphoma (NHL). The pathogenesis of these malignancies is not fully understood. One mechanism of malignant transformation recently described in colon tumorigenesis results from defects in DNA mismatch repair, manifest as widespread microsatellite instability. We demonstrate a high rate of microsatellite instability in KS and aggressive lymphomas obtained from HIV-infected patients, whereas there is no evidence of instability in similar lesions from HIV-negative patients. Further elucidation of the underlying mechanisms responsible for HIV-associated instability in primary tumours may provide insight into the pathogenesis of these AIDS-related neoplasms.

The distinction of solitary fibrous tumors (SFTs) from histologically similar neoplasms relies heavily on a characteristic microscopic appearance. No discriminating ultrastructural or immunohistochemical features are known. We evaluated 22 SFTs and 118 other tumors often considered in the differential diagnosis for immunoreactivity using a monoclonal antibody directed against the human hematopoietic progenitor cell antigen, CD34. All the SFTs (22 of 22, 100%) demonstrated strong CD34 immunoreactivity, irrespective of tumor site and histologic grade. Strong and generalized CD34 positivity was also found in most dermatofibrosarcoma protuberans (11 of 12, 92%) and occasional smooth-muscle tumors (leiomyomas 2 of 11, 18%; leiomyosarcomas 2 of 11, 18%). Variable numbers of CD34 positive cells were present in all neurofibromas (9 of 9, 100%) and focally present in most schwannomas (8 of 9, 89%). Some of the hemangiopericytomas (7 of 16, 44%) exhibited CD34 immunoreactivity, however, generally with weak intensity and patchy distribution. CD34 immunoreactivity was not observed in mesotheliomas (0 of 20, 0%), synovial sarcomas (0 of 13, 0%), fibrosarcomas (0 of 12, 0%), or spindle-cell thymomas (0 of 5, 0%). We conclude that CD34 immunoreactivity is a sensitive marker for SFT and, in conjunction with an appropriate immunohistochemical panel, may be useful in discriminating SFTs from other histologically similar neoplasms. The observation that some mesenchymal stromal cells and SFTs share a CD34-positive immunophenotype suggests a histogenetic relationship. The inclusion in this study of two cases of SFTs arising in the orbit establishes another site of origin for this tumor and provides further support for a mesenchymal histogenesis.

Our recent allelic analysis of head and neck squamous cell carcinomas identified a high incidence of chromosomal loss on 13q. To further define an area of minimal loss, we tested 60 primary head and neck squamous cell carcinomas in 59 patients for loss of heterozygosity (LOH) by using 10 polymorphic microsatellite markers spanning the long arm of chromosome 13. We examined the same primary tumors for inactivation of the retinoblastoma (Rb) gene by immunohistochemical analysis of paraffin-embedded specimens. Thirty-one of 60 (52%) tumors demonstrated LOH in at least one 13q marker. Twenty-nine of 31 (94%) lost a portion of 13q that included D13s133, which lies just telomeric to the Rb gene at 13q14.3. However, immunohistochemical staining revealed absence of Rb protein in only 6 of these 31 tumors (19.4%) with LOH. All but one tumor without LOH on 13q displayed normal Rb protein staining. Although Rb may be inactivated by an unusual mechanism in some head and neck squamous cell carcinomas, our data suggest that another tumor suppressor gene locus at 13q14 is likely to be involved in head and neck tumor progression.

A 55-year-old woman with painful splenomegaly underwent surgical exploration and resection of the spleen. Pathologic examination revealed a neoplasm of the spleen exhibiting malignant stromal and epithelial components with müllerian features. In the absence of a recognized neoplasm elsewhere, the carcinosarcoma most likely arose as a primary splenic tumor and may represent an extrauterine malignant mixed müllerian tumor arising in the spleen.

Mutations in the p53 tumor suppressor gene are frequently observed in primary lung adenocarcinomas, suggesting that these mutations are critical events in the malignant transformation of airway cells. These mutations are often associated with stabilization of the p53 gene product, resulting in the accumulation of p53 protein. In this study, 70 formalin-fixed, paraffin-embedded primary lung adenocarcinomas resected for potential cure were examined for p53 overexpression. These 70 lung adenocarcinomas were obtained from a series of patients with well-documented clinical histories, and all 70 carcinomas had been previously evaluated for point mutations in codon 12 of the K-ras oncogene. Overexpression of the p53 protein was detected using an antigen retrieval system (Target Unmasking Fluid) and the anti-p53 antibody CM-1. CM-1 is a polyclonal antibody directed against the wild-type p53 protein. Overexpression of the p53 protein was found in 23 (33%) of the 70 lung adenocarcinomas. In all 23 cases, overexpression was confined to neoplastic cells. Overexpression of the p53 protein correlated with cigarette smoking: 10 (56%) of the 18 adenocarcinomas from patients who were current smokers overexpressed p53 compared with 13 (33%) of the 40 adenocarcinomas from patients who had quit smoking and 0 (0%) of the 12 adenocarcinomas from patients who had never smoked (p = 0.002, trend test). Overexpression of the p53 protein was also related to the degree of histologic differentiation: 48% of the p53 negative carcinomas were well differentiated, whereas only 13% (p = 0.003) of the carcinomas in which p53 was overexpressed were well differentiated. Overexpression of the p53 protein did not correlate with point mutations in codon 12 of the K-ras oncogene, nor did it correlate with tumor stage or patient survival. These findings indicate that p53 protein is frequently overexpressed in primary lung adenocarcinomas. Furthermore, the association of tobacco smoking with this overexpression suggests that the p53 gene is a target of specific mutagens in tobacco smoke.

Dilated cardiomyopathy is a frequent and serious complication of idiopathic hemochromatosis. The mechanism by which disordered iron metabolism induces heart failure is not entirely understood, but myocardial dysfunction appears to be intimately related to the deposition of iron in myocytes. Cardiac function characteristically worsens or improves in proportion to the degree of iron accumulation in cardiac myocytes. The authors report the case of a 47-year-old man with idiopathic hemochromatosis and cirrhosis who developed symptoms of congestive heart failure and was found to have dilated cardiomyopathy 7 months after receiving a liver transplant. An initial endomyocardial heart biopsy demonstrated severe iron deposition in myocytes. The patient's heart failure worsened in the next 3 years and he eventually required a heart transplant. Examination of the explanted heart revealed dilated cardiomyopathy, but the previously demonstrated iron deposits in the cardiac myocytes were depleted. This "uncoupling" of cardiac function and cardiac iron load suggests that a threshold may be reached at which point the metabolic and ultrastructural derangements of iron deposition are no longer reversible, even with the removal of the inciting agent. Furthermore, displacement of myocyte iron stores after liver transplantation implicates altered hepatic iron metabolism as a primary or contributing mechanism in the pathophysiology of idiopathic hemochromatosis.