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.

Oral papillary squamous cell carcinoma in twelve dogs.

Papillary squamous cell carcinoma (PSCC) is a distinct histological subtype of oral squamous cell carcinoma (SCC), described in both dogs and man. In dogs, PSCC has long been considered a malignant oral tumour of very young animals, but it has recently been reported to occur in adult dogs as well. The aim of this study was to describe the major clinicopathological characteristics of canine oral PSCC (COPSCC). Twelve dogs diagnosed with COPSCC were included in this retrospective study (1990-2012). The majority (75%) of the dogs were >6 years of age (median age 9 years). All tumours were derived from the gingiva of dentate jaws, with 66.7% affecting the rostral aspects of the jaws. The gross appearance of the lesions varied, with one having an intraosseous component only. The majority (91.7%) of the tumours were advanced lesions (T2 and T3), but no local or distant metastases were noted. Microscopically, two patterns were seen: (1) invasion of bone forming a cup-shaped indentation in the bone or a deeply cavitating cyst within the bone (cavitating pattern), (2) histologically malignant growth, but lack of apparent bone invasion (non-cavitating pattern). The microscopical appearance corresponded to imaging findings in a majority of cases, with cavitating forms presenting with a cyst-like pattern of bone loss or an expansile mass on imaging and non-cavitating forms showing an infiltrative pattern of bone destruction on imaging. These features suggest two distinct biological behaviours of COPSCC.

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.

Stromal control of oncogenic traits expressed in response to the overexpression of GLI2, a pleiotropic oncogene.

Hedgehog signaling is often activated in tumors, yet it remains unclear how GLI2, a transcription factor activated by this pathway, acts as an oncogene. We show that GLI2 is a pleiotropic oncogene. The overexpression induces genomic instability and blocks differentiation, likely mediated in part by enhanced expression of the stem cell gene SOX2. GLI2 also induces transforming growth factor (TGF)B1-dependent transdifferentiation of foreskin and tongue, but not gingival fibroblasts into myofibroblasts, creating an environment permissive for invasion by keratinocytes, which are in various stages of differentiation having downregulated GLI2. Thus, upregulated GLI2 expression is sufficient to induce a number of the acquired characteristics of tumor cells; however, the stroma, in a tissue-specific manner, determines whether certain GLI2 oncogenic traits are expressed.

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.

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.

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.

Marsupialization as a definitive treatment for the odontogenic keratocyst.

PURPOSE: We sought to show that marsupialization can be a definitive treatment for the odontogenic keratocyst (OKC). MATERIALS AND METHODS: Ten patients (10 males and 4 females) between the ages of 11 and 64 with biopsy-proven OKC (8 mandibular and 2 maxillary) measuring between 2 and 8 cm were treated by marsupialization consisting of excision of the overlying mucosa and the opening of a 1-cm window into the cystic cavity and, where possible, suturing of the cyst lining to the oral mucosa. Immunohistologic determination of bcl-2 was done for all samples of cyst lining. The cavities were kept open either by vigorous use of a home syringe by the patient or by suturing into place the flange and short length of a nasopharyngeal airway. Once the cyst had largely filled in, histologic material was taken from the base of the residual depression and studied by light microscopy and bcl-2 expression. RESULTS: In the 10 patients, the OKCs completely resolved both clinically and radiographically. The time taken for resolution varied from 7 to 19 months. In all cases, the histologic material obtained after marsupialization showed normal epithelium only, with no signs of cystic remnants, daughter cysts, or budding of the basal layer of the epithelium. At initial biopsy, bcl-2 was expressed in the keratocyst lining, but not in the histologic material obtained after marsupialization. Follow-up time ranged from a minimum of 1.8 years to a maximum of 4.8 years. Teeth at the periphery of the cysts were observed to upright and erupt. CONCLUSIONS: All 10 OKCs resolved completely after marsupialization. Teeth within the cyst were found to be upright and erupt. Marsupialization requires a cooperative patient who will irrigate the cavity and keep it open. It appears that the cyst lining is replaced by normal epithelium during this treatment.

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.

Lingual nerve damage due to inferior alveolar nerve blocks: a possible explanation.

BACKGROUND: An explanation for the predominance of injuries to lingual nerves over those to inferior alveolar nerves as a result of inferior alveolar nerve blocks may be due to the nerves' fascicular pattern. A unifascicular nerve may be injured more easily than a multifascicular nerve. METHODS: The authors unilaterally dissected lingual and inferior alveolar nerves from 12 cadavers. They cut the specimens 2 millimeters above the lingula for both the lingual nerve and inferior alveolar nerve and opposite the site of the middle of the third molar for the lingual nerve, and they counted the number of fascicles at each site. RESULTS: For the lingual nerve at the lingula, the mean number of fascicles was three (range, one to eight). Four of the 12 nerves (33 percent) were unifascicular at this point. Opposite the third molar, the lingual nerve had a mean of 20 fascicles (range, six to 39). In every case, there were more fascicles in the third molar region than above the lingula in the same nerve. At the lingula, the inferior alveolar nerve had a mean of 7.2 fascicles (range, three to 14). CONCLUSION: This study may explain the observation that when an inferior alveolar nerve block causes permanent nerve impairment, the lingual nerve is affected about 70 percent of the time and the inferior alveolar nerve is affected only 30 percent of the time. In 33 percent of cases, the lingual nerve had only one fascicle at the lingula; a unifascicular nerve may be injured more easily than a multifascicular one. CLINICAL IMPLICATIONS: There is no known way to avoid the remote possibility of nerve damage resulting from an inferior alveolar nerve block. The lingual nerve may be predominantly affected because of its fascicular pattern.

Genetic mutations in certain head and neck conditions of interest to the dentist.

This article identifies certain syndromes of the head and neck, which a dentist may see in clinical practice, and relates these syndromes to their sites of mutation on involved genes. This paper is timely with the near completion of the Human Genome Project, the mapping of the entire human genetic material. Knowing the site of the genetic lesion is important in helping clinicians understand the genetic basis for these conditions, and may help in our future understanding of remedies and treatments.

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.

Oral cancer in the molecular age.

Oral cancer represents an accumulation of defects in the genes that encode key proteins associated with growth and development. Dysregulation of these proteins is central to malignant conversion. This appears to involve three major changes in cell function: 1. altered cell growth, death and longevity; 2. unencumbered cell movement; and 3. development of a new blood supply (angiogenesis). Specific genes, such as p53, p27, p16, and cyclin D-1, are altered in oral cancer through mutation, amplification, or deactivation. These genes are also frequently altered in many other malignancies. In oral mucosa, etiologic agents--especially tobacco and alcohol, and possibly some viruses--are known to induce alterations in the genes and gene functions associated with cell cycle regulation, contributing to the development of squamous cell carcinoma and epithelial dysplasias. Identification of the specific genes/proteins and the sequence in which they appear in the transformation of a normal cell to a malignant cell is necessary for the formulation of new treatment strategies, the development of early detection methods, and the prediction of patient outcome.

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.

Circadian expression of clock genes in human oral mucosa and skin: association with specific cell-cycle phases.

We studied the relative RNA expression of clock genes throughout one 24-hour period in biopsies obtained from the oral mucosa and skin from eight healthy diurnally active male study participants. We found that the human clock genes hClock, hTim, hPer1, hCry1, and hBmal1 are expressed in oral mucosa and skin, with a circadian profile consistent with that found in the suprachiasmatic nuclei and the peripheral tissues of rodents. hPer1, hCry1, and hBmal1 have a rhythmic expression, peaking early in the morning, in late afternoon, and at night, respectively, whereas hClock and hTim are not rhythmic. This is the first human study to show a circadian profile of expression for all five clock genes as documented in rodents, suggesting their functional importance in man. In concurrent oral mucosa biopsies, thymidylate synthase enzyme activity, a marker for DNA synthesis, had a circadian variation with peak activity in early afternoon, coinciding with the timing of S phase in our previous study on cell-cycle timing in human oral mucosa. The major peak in hPer1 expression occurs at the same time of day as the peak in G(1) phase in oral mucosa, suggesting a possible link between the circadian clock and the mammalian cell cycle.

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.

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.

p53 gene mutations in sequential oral epithelial dysplasias and squamous cell carcinomas.

Previous studies of oral cancer have suggested that alterations of the p53 tumour suppressor gene occur early in the precancerous stage of development. However, these observations have been based on cross-sectional assessment of abnormal p53 protein staining by immunohistochemistry and may not necessarily reflect gene changes. The purpose of this longitudinal study was to examine the changes in the p53 gene in progressive, sequential epithelial dysplasias and carcinomas from the oral cavity. The study analysed 24 formalin-fixed, paraffin-embedded tissue biopsies from ten patients with two or more temporally distinct lesions from the same site in the oral cavity with the diagnosis of hyperkeratosis, epithelial dysplasia, carcinoma in situ or squamous cell carcinoma. Exons 5-8 of the p53 gene were amplified from genomic DNA using intronic primers and directly sequenced using fluorescent-labelled primers. Standard immunohistochemistry with the DO7 monoclonal antibody was used to detect mutant and wild-type p53 protein. Mutations of the p53 gene were identified in 9 of 24 samples. Eight were missense mutations and one occurred at a splice site. In six patients, mutations of the p53 gene occurred late after the transformation of epithelial dysplasia to carcinoma. In two patients with progressive dysplasia, but who had yet to develop invasive carcinoma, p53 missense mutations occurred at the carcinoma in situ stage in one case and in a moderate dysplasia in the other. There was an inconsistent relationship between gene mutations and the level of p53 protein staining by immunohistochemistry. It is concluded that during oral carcinogenesis, p53 gene mutations seem to occur relatively late and are associated with transformation to the invasive phenotype.

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.