A 3'-UTR KRAS-variant is associated with cisplatin resistance in patients with recurrent and/or metastatic head and neck squamous cell carcinoma.

BACKGROUND: A germline mutation in the 3'-untranslated region of KRAS (rs61764370, KRAS-variant: TG/GG) has previously been associated with altered patient outcome and drug resistance/sensitivity in various cancers. We examined the prognostic and predictive significance of this variant in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS: We conducted a retrospective study of 103 HNSCCs collected from three completed clinical trials. KRAS-variant genotyping was conducted for these samples and 8 HNSCC cell lines. p16 expression was determined in a subset of 26 oropharynx tumors by immunohistochemistry. Microarray analysis was also utilized to elucidate differentially expressed genes between KRAS-variant and non-variant tumors. Drug sensitivity in cell lines was evaluated to confirm clinical findings. RESULTS: KRAS-variant status was determined in 95/103 (92%) of the HNSCC tumor samples and the allelic frequency of TG/GG was 32% (30/95). Three of the HNSCC cell lines (3/8) studied had the KRAS-variant. No association between KRAS-variant status and p16 expression was observed in the oropharynx subset (Fisher's exact test, P = 1.0). With respect to patient outcome, patients with the KRAS-variant had poor progression-free survival when treated with cisplatin (log-rank P = 0.002). Conversely, KRAS-variant patients appeared to experience some improvement in disease control when cetuximab was added to their platinum-based regimen (log-rank P = 0.04). CONCLUSIONS: The TG/GG rs61764370 KRAS-variant is a potential predictive biomarker for poor platinum response in R/M HNSCC patients. CLINICAL TRIAL REGISTRATION NUMBERS: NCT00503997, NCT00425750, NCT00003809.

Female Maylandia zebra prefer victorious males.

Females of a widespread species of the rock-dwelling haplochromine cichlids of Lake Malawi, Maylandia zebra, show preference for males that successfully evict intruding males from their territory. This behaviour, experimentally induced by the investigators in a laboratory setting, was also preferred over males that were not permitted to interact with any other individual.

Molecular biology of squamous cell carcinoma of the head and neck.

Squamous cell carcinoma of the head and neck (HNSCC) is a heterogeneous but largely preventable disease with complex molecular abnormalities. It arises from a premalignant progenitor followed by outgrowth of clonal populations associated with cumulative genetic alterations and phenotypic progression to invasive malignancy. These genetic alterations result in inactivation of multiple tumour suppressor genes and activation of proto-oncogenes, including p16(ink4A), p53, cyclin D1, p14(ARF), FHIT, RASSF1A, epidermal growth factor receptor (EGFR), and Rb. Intramucosal migration and clonal expansion of transformed cells with formation of abnormal genetic fields appear to be responsible for local recurrences and development of second primary tumours.

Patterns of CDKN2A gene loss in sequential oral epithelial dysplasias and carcinomas.

The CDKN2A gene locus encodes two different proteins derived from alternative splicing. p16 (exons 1alpha, 2, and 3) acts as a G1 cell cycle regulator, and p14ARF (exons 1beta, 2, and 3) acts to modulate MDM2-mediated degradation of p53. Inactivation of p16 is a common finding in many cancers; however, there is little data on CDKN2A gene abnormalities in oral precancer. In this longitudinal study, we examined changes in the CDKN2A gene locus in sequential epithelial dysplasias and oral carcinomas from 11 patients. Genomic DNA was extracted from laser-microdissected lesional tissue, and exons 1alpha, 1beta, and 2 were analyzed by duplex PCR. Immunohistochemistry was done to identify p16 and p14ARF protein expression. Two adjacent polymorphic microsatellite markers were used for allelotyping. Homozygous deletion of exon 1alpha was identified in 2 of 17 (12%) precancerous lesions. Loss of either exon 1alpha, exon 2, or both was seen in seven of nine (78%) carcinomas. In five of these carcinomas, there was loss of only exon 1alpha. No case showed deletion of exon 1beta. In 5 of 11 patients, microsatellite markers showed differing patterns of allelic imbalance in the precancerous lesions and the subsequent carcinoma, suggesting a complex genetic pattern of progression from dysplasia to carcinoma. We conclude that during oral carcinogenesis homozygous deletion of exon 1alpha of the CDKN2A gene is common but that deletion of exon 2 and 1beta is less frequent. Moreover, our results suggest that the progression from oral precancer to cancer, in some cases, is more complex genetically than predicted by linear models of carcinogenesis.

Identification of a mononucleotide repeat as a major target for mitochondrial DNA alterations in human tumors.

Mitochondrial DNA (mtDNA) mutations scattered through coding and noncoding regions have been reported in cancer. The mechanisms that generate such mutations and the importance of mtDNA mutations in tumor development are still not clear. Here we present the identification of a specific and highly polymorphic homopolymeric C stretch (D310), located within the displacement (D) loop, as a mutational hotspot in primary tumors. Twenty-two % of the 247 primary tumors analyzed harbored somatic deletions/insertions at this mononucleotide repeat. Moreover, these alterations were also present in head and neck preneoplastic lesions. We further characterized the D310 variants that appeared in the lung and head and neck tumors. Most of the somatic alterations found in tumors showed deletion/insertions of 1- or 2-bp generating D310 variants identical to constitutive polymorphisms described previously. Sequencing analysis of individual clones from lymphocytes revealed that patients with D310 mutations in the tumors had statistically significant higher levels of D310 heteroplasmy (more than one length variant) in the lymphocyte mtDNA as compared with the patients without D310 mutations in the tumor mtDNA. On the basis of our observations, we propose a model in which D310 alterations are already present in normal cells and achieve homoplasmy in the tumor through a restriction/amplification event attributable to random genetic drift and clonal expansion.

Patterns of p53 gene mutations in head and neck cancer: full-length gene sequencing and results of primary radiotherapy.

p53 gene alterations are common in head and neck cancers, but their prognostic value has not been clearly established. Despite evidence in other cancers that sequencing of the entire p53 coding region provides prognostic information, full-length p53 gene sequencing has rarely been performed in head and neck cancers. In this study, p53 was assessed in a series of 42 pretreatment biopsies from patients with laryngeal carcinomas by full-length gene sequencing and by immunohistochemistry (IHC). Associations among p53 genotype, protein expression, and local recurrence were assessed in 35 irradiated patients followed for a minimum of 5 years. DNA was extracted from formalin-fixed, paraffin-embedded biopsies, and exons 2-11 of the p53 gene were individually amplified by PCR and then directly sequenced. IHC was performed to detect mutant and wild-type p53 protein using the DO7 monoclonal antibody. p21 protein expression was assessed using the EA1 monoclonal antibody. Twenty genetic alterations were observed in 42 tumors (48%). Four of these alterations (20%) occurred outside exons 5-8. There was a significant association between p53 gene and protein status (chi2 = 4.18, P = 0.04), although the correlation was weak (phi coefficient = -0.327). Although local relapse following radiation was significantly associated with nodal status, no correlations were observed between p53 status (gene or IHC) and local recurrence following radiation therapy, based on the Kaplan-Meier method. These results show that p53 mutations are common in laryngeal carcinomas and that a proportion occur outside traditionally examined regions. The lack of correlation between p53 status and local control suggests that this marker is not as powerful as traditional prognostic factors, such as lymph node status.

Expression pattern of CK7, CK20, CDX-2, and villin in intestinal-type sinonasal adenocarcinoma.

BACKGROUND: Intestinal-type sinonasal adenocarcinoma (ITAC) is an uncommon neoplasm, which resembles adenocarcinoma of the gastrointestinal tract. ITAC occurs sporadically or in association with occupational exposure to hardwood dust and other agents. AIMS: To investigate the phenotype and possible pathogenetic mechanisms of primary sinonasal and nasopharyngeal adenocarcinomas by staining for cytokeratin 7 (CK7), CK20, CDX-2, and villin. METHODS: Twelve sporadic sinonasal and nasopharyngeal adenocarcinomas were stained with monoclonal antibodies to CK7, CK20, CDX-2, and villin. The ITACs were classified as papillary, colonic, solid, mixed, or mucinous types. RESULTS: The diagnosis of ITAC was confirmed in 10 cases: five were colonic type and five were papillary. One was a sinonasal papillary low grade adenocarcinoma, and one a papillary nasopharyngeal adenocarcinoma, and these tumours were CK7 positive, but CK20, CDX-2, and villin negative. All ITACs were positive for CK20, CDX-2, and villin, and six were CK7 positive. One ITAC had a focus of intestinal metaplasia away from the invasive carcinoma. CONCLUSIONS: Sinonasal ITACs have a distinctive phenotype, with all cases expressing CK20, CDX-2, and villin. Most ITACs also express CK7, although a proportion of tumours are CK7 negative. ITAC seems to be preceded by intestinal metaplasia of the respiratory mucosa, which is accompanied by a switch to an intestinal phenotype. Although ITACs are morphologically similar, differences in cytokeratin expression patterns suggest two distinct types. The expression pattern of CK7, CK20, CDX-2, and villin positive may be useful in separating these tumours from other non-ITAC adenocarcinomas of the sinonasal tract and nasopharynx.

Expression of mismatch repair proteins, beta catenin, and E cadherin in intestinal-type sinonasal adenocarcinoma.

BACKGROUND: Despite their histological resemblance to colorectal adenocarcinomas, there is little information about the molecular events involved in the pathogenesis of intestinal-type sinonasal adenocarcinomas (ITACs). AIMS: To evaluate the possible role of DNA mismatch repair (MMR) gene defects or disruptions of the E cadherin-beta catenin complex in ITAC by investigating the immunohistochemical expression of the MMR gene products, beta catenin, and E cadherin in a group of sporadic ITACs. METHODS: Ten sporadic cases of ITAC were stained with antibodies against MLH1, MSH2, MSH3, MSH6, beta catenin, and E cadherin. RESULTS: Nine cases showed strong nuclear expression of MLH1, whereas one case showed moderate staining. All 10 cases were strongly positive for MSH2 and MSH3. MSH6 was strong in nine cases, and moderate in one. Membranous beta catenin expression was strong in all 10 cases, and no case showed cytoplasmic or nuclear staining. E cadherin was strong in seven cases, and moderate in three cases. CONCLUSIONS: The preserved nuclear expression of MLH1, MSH2, MSH3, and MSH6 suggests that mutations or promoter methylation of MMR genes do not play a role in the pathogenesis of ITAC. The strong membranous staining for E cadherin and beta catenin and lack of abnormal cytoplasmic or nuclear expression is in keeping with the preservation of E cadherin-beta catenin complexes and beta catenin pathways.

High prevalence of B-cell monoclonality in labial gland biopsies of Japanese Sjögren's syndrome patients.

Patients with Sjögren's syndrome (SS) have an increased risk of developing malignant lymphoma. Although some clinical parameters may herald the imminent onset of lymphoma, few reliable markers are available to predict the progression to a malignant lymphoproliferative disorder. Although there are a number of immunological and serologic features that distinguish SS in Japanese patients, in common with their Western counterparts these patients also have an increased risk of lymphoid neoplasia. Recently we have reported finding a high prevalence (17%) of monoclonal immunoglobulin (Ig) heavy chain gene rearrangements in labial salivary gland (LSG) biopsies of Western SS using the polymerase chain reaction (PCR). In many cases this finding was predictive for the subsequent development of lymphoma. In this study LSG from 50 Japanese SS patients were examined for Ig heavy chain gene monoclonality using PCR to amplify the VDJ region and identified in 7 of 50 (14%) cases. Three patients with monoclonality in the LSG had evidence of lymphoma at extra-salivary gland sites. In one of these the diagnosis of lymphoma was made subsequent to lip biopsy. In the other two lymphoma at extra-salivary gland sites was diagnosed prior to LSG biopsy. The results suggest that the prevalence of Ig heavy chain gene monoclonality in LSG of Japanese SS patients is similar to that in the West, and that neoplastic cells can be identified in LSG as a component of more widely disseminated disease.

Scoring oral mucositis.

Oral mucositis is a common, dose limiting and potentially serious complication of both radiation and chemotherapy. Both these therapies are non-specific, interfering with the cellular homeostasis of both malignant and normal host cells. An important effect is the loss of the rapidly proliferating epithelial cells in the oral cavity, gut and in the bone marrow. Within the mouth, the loss of these cells leads to mucosal atrophy, necrosis and ulceration. Although post-treatment healing is generally uneventful, severe mucositis can be life threatening, especially if complicated by dehydration or secondary infection. Accurate and reproducible evaluation of oral mucositis is important in order to monitor patient toxicity during therapy, to document the toxicity of conventional therapy and to critically assess the effects of alternative therapies. A number of oral toxicity scoring systems have been described, but direct comparisons have rarely been undertaken and little data exist regarding inter- and intra-user reliability. This paper reviews a number of oral mucositis scoring systems that are commonly used and will also discuss, briefly, the biological basis of its development and management.

Frequent cyclin D1 gene amplification and protein overexpression in oral epithelial dysplasias.

Amplification of the cyclin D1 gene has been identified in 17-55% of head and neck squamous cell carcinoma. In some tumors, this alteration has been associated with decreased survival and increased recurrence rates. In precancerous lesions of the mouth, the frequency of cyclin D1 gene amplification is not known. In addition, it is unknown whether amplification of the gene translates to overexpressed cyclin D1 protein in these lesions. We examined 59 formalin-fixed, paraffin embedded tissue biopsies of oral epithelial dysplasias (OED) and 25 oral squamous cell carcinoma (SCC) from the floor of the mouth for cyclin D1 gene and protein levels. Genomic DNA was extracted from laser microdissected lesional tissue and a duplex, quantitative PCR assay was used to determine the amplification of the cyclin D1 gene relative to interferon-gamma. Cyclin D1 protein expression was determined using immunohistochemistry and counting positive nuclei by computer image analysis. We found cyclin D1 gene amplification in 41% of mild, 45% of moderate and 24% of severe OEDs. Cyclin D1 was amplified in 36% of SCC. Overexpression of cyclin D1 protein was identified in 29% of mild, 47% of moderate, 29% of severe OED's, and in 32% of SCC. Overexpression of cyclin D1 protein was identified in similar proportions of all grades of dysplasia and SCC. There were statistically significant correlations identified between gene and protein levels in all categories of disease. We concluded that amplification of the cyclin D1 gene is frequent in OED and that duplex, quantitative polymerase chain reaction is a reliable method to detect this change in routinely processed biopsies. The strong correlation between cyclin D1 gene amplification and protein levels suggests that this method may be suitable to assess cyclin D1 gene status in tissues not suitable for protein analysis.

Effects of ECT given two vs. three times weekly.

The benefits and side effects of electroconvulsive therapy (ECT) given two vs. three times per week were examined in depressed inpatients. Twenty subjects were randomly assigned to one of two treatment conditions (unilateral ECT two or three times weekly). Examiners without knowledge of treatment condition rated depression and psychiatric status and administered tests of memory and visual-motor problem solving; subjects also provided self-ratings of depression. Measures were collected before treatment and 2 and 4 weeks after treatment began. Both schedules of treatment produced significant and equivalent improvements in psychiatric symptomatology, but visual memory impairment was significantly lower in the twice-weekly group.

Development of an orofacial model of acute inflammation in the rat.

An appropriate model was created by the paraperiosteal injection of mustard oil (20% allyl isothiocyanate dissolved in mineral oil) into the periarticular temporomandibular tissue of anaesthetized rats. Inflammation was assessed by the plasma extravasation of Evans' blue dye bound to plasma protein. This was confirmed visually and compared spectrophotometrically with the contralateral untreated control site (p less than 0.0005). A time-course study of the effect of mustard oil on Evans' blue extravasation revealed a gradually increasing effect that was maximal at 30 min after administration, with no further increase at 60 min. A dose-response study showed that giving 30 microliters of 20% mustard oil produced the maximal effect, with no further increase from 50 microliters. To confirm induction of inflammation, polymorphonuclear neutrophil infiltration was assessed morphometrically and found to increase in the treated tissue compared with the contralateral untreated control (p less than 0.001).

Overexpression of cyclin A and cyclin B1 proteins in astrocytomas.

BACKGROUND: Cyclins are proteins that are expressed during the progression of a normal cell through the cell cycle. In a number of cancers, overexpression of cyclin A and cyclin B1 proteins has been reported, and in some instances the levels of expression correlated well with the grades of malignancy. The expression of cyclin A and cyclin B1 proteins in astrocytoma may be linked to the histologic grade or proliferative activities. OBJECTIVE: To study the expression of cyclin A and cyclin B1 proteins in astrocytomas and correlate the labeling indices (LIs) of cyclin A and cyclin B1 with histologic grade and Ki-67 LI. DESIGN: The surgical biopsy specimens from 65 adults with astrocytomas were reviewed and divided into grades based on the World Health Organization system. The paraffin sections were immunostained using primary antibodies against Ki-67, cyclin A, and cyclin B1. The LIs of these astrocytomas for the 3 different antibodies were determined by computerized image analysis. RESULTS: The cyclin A LI showed good correlation with astrocytoma grade and Ki-67 LI. Both the nuclear and cytoplasmic cyclin B LIs correlated well with the tumor grade but showed poor correlation with Ki-67 LI. CONCLUSIONS: This study suggests that although both cyclin A and B protein expression are related to the grade of malignancy in astrocytomas, cyclin A levels more generally reflect the proliferative state of these tumors. We also provide indirect evidence that cyclin B1 is associated with the aberrant progression through the G2-M phase checkpoint in astrocytomas.

Observer agreement in the grading of oral epithelial dysplasia.

OBJECTIVES: To determine the extent of observer agreement in diagnosis of oral epithelial dysplasia (OED). Published studies of OED examiner agreement report relatively low agreement levels; however, these studies were limited by the methodologies employed. METHODS: For this study, 64 slides were each independently examined twice by three oral pathologists. Consistency was assessed by determining intra- and interexaminer agreement. Conformity was assessed by using the modal diagnosis as a gold standard. RESULTS: The group showed moderate interobserver agreement when grading the presence or absence of OED with a group-simple kappa (Ks) of 0.51 (95% CI = 0.42-0.61), and substantial agreement when using a 5-point ordinal scale with a group-weighted kappa (Kw) of 0.74 (95% CI = 0.64-0.85). The group showed fair to substantial intraexaminer agreement when assessing the presence or absence of OED, with Ks ranging from 0.22 to 0.78, and showing almost a perfect agreement using a 5-point ordinal scale, with Kw ranging from 0.82-0.96. Conformity with the comparison standard modal diagnosis was almost perfect, with pairwise Kw ranging from 0.81 to 0.92. CONCLUSION: Overall, there was substantial intra- and interobserver consistency and almost perfect conformity in the grading of OED. Appropriate statistical methods are necessary to determine the degree of observer agreement.

Lymphoma in Sjögren's syndrome. From histopathology to molecular pathology.

A number of autoimmune diseases predispose to the development of neoplasia. A particularly well-recognized association is the development of lymphoma in Sjögren's syndrome. Although this risk has been estimated to be 44 times that of the general population, few reliable prognostic indexes exist for individual patients. Recent advances in molecular biology have improved our understanding of Sjögren's syndrome and permitted better characterization of the generalized lymphoproliferation associated with the condition. This article reviews the histopathology of the major and minor salivary gland lesions of Sjögren's syndrome and discusses advances in molecular biology that have permitted more accurate prediction of lymphoma development in this group of patients.

Solitary angiokeratoma of the oral cavity.

Angiokeratoma is a rare, cutaneous disorder that typically occurs at multiple sites and is often associated with a number of metabolic disorders. Although solitary cutaneous forms have been reported, to date localized lesions within the oral cavity have not been described. We report on an 82-year-old man with a history of squamous cell carcinoma of the ear and scalp who also had a solitary pigmented lesion of the oral cavity. Excisional biopsy of the buccal mucosal lesion showed features consistent with angiokeratoma. We report the first intra-oral solitary angiokeratoma and review the clinical and pathologic features of this unusual condition.

Giant cell angiofibroma of the oral cavity: report of a new location for a rare tumor.

Giant cell angiofibroma is a rare, soft tissue tumor that was first described in the orbit. Since then, several case reports have described this tumor in a number of extra-orbital sites, suggesting a wider anatomic distribution than is generally recognized. The tumor typically acts in a benign fashion with only rare local recurrences but no tendency to metastatic disease. Here, we report the first case of a giant cell angiofibroma in the oral cavity. The tumor presented as a soft tissue nodule on the buccal mucosa of a 60-year-old man. The histologic differential diagnosis included a number of other uncommon soft tissue neoplasms, including giant cell fibroblastoma, solitary fibrous tumor, and pleomorphic lipoma. The histologic and immunohistochemical features of this tumor and differentiation from other histologically similar soft tissue neoplasms are briefly discussed.

Multiple canalicular adenomas: a case report and review of the literature.

The canalicular adenoma is an uncommon, benign salivary gland tumor that most frequently occurs in the upper lip. Rarely, it manifests itself clinically and histologically as a multifocal lesion, a feature not generally seen with other intraoral salivary gland tumors. Here we report a case of canalicular adenoma that manifested itself with 13 clinically discrete tumor masses involving the upper lip and anterior buccal mucosa. In addition to the clinical nodules, there were microscopic foci of tumor cells in the adjacent normal-appearing salivary gland tissue surrounding the tumors. This article also reviews previously reported multifocal canalicular adenomas and discusses their features, emphasizing differences in the reported growth patterns of this unusual tumor.

Advanced diagnostic methods in oral and maxillofacial pathology. Part I: molecular methods.

The practice of pathology is currently undergoing significant change, in large part due to advances in the analysis of DNA, RNA, and proteins in tissues. These advances have permitted improved biologic insights into many developmental, inflammatory, metabolic, infectious, and neoplastic diseases. Moreover, molecular analysis has also led to improvements in accuracy of disease diagnosis and classification. It is likely that, in the future, these methods will increasingly enter into the day-to-day diagnosis and management of patients. The pathologist will continue to play a fundamental role in diagnosis and will likely be in a pivotal position to guide the implementation and interpretation of these tests as they move from the research laboratory into diagnostic pathology. The purpose of this 2-part series is to provide an overview of the principles and applications of current molecular biologic and immunologic tests. Part I will discuss the biologic fundamentals of DNA, RNA, and proteins and the methods that are currently available or likely to become available to the pathologist in the next several years for their isolation and analysis in tissue biopsies.