Prediction of recurrence by microsatellite analysis in head and neck cancer

We examined the possibility of using microsatellite alterations as markers to detect clonal tumor-derived cell populations in histopathologically negative surgical margins and cervical lymph nodes from head and neck cancer (HNC) patients. We used polymerase chain reaction (PCR)-based microsatellite analysis DNA to analyze primary tumors, paired surgical margins, and cervical lymph nodes from 41 HNC patients. Samples were scored for alterations as defined by the presence of new alleles (shifts) or loss of heterozygosity (LOH) at each of 10 markers. We identified 25 (61%) patients with primary HNC who appeared to have had a complete resection on the basis of the histopathological assessment and who were informative regarding microsatellite alterations in tumor tissue. In 11 of these 25 (44%) cases, PCR analysis of surgical margins showed the same microsatellite alterations as in the primary tumors. In 7 of these 11 patients, the carcinoma recurred locally, as compared with 1 out of 14 patients with negative margins (log rank test, P = 0.0049). Conversely, we were unable to detect clonal neoplastic cells in histopathologically negative lymph nodes examined by molecular analysis. Cox regression analysis showed that molecular positive margins were an independent prognostic factor (P = 0.04) for recurrence. This study demonstrates that microsatellite analysis may be a valuable tool for evaluating the risk of local recurrence.     

Cytological evaluation of head and neck tumors in children--a pattern analysis

A total of 135 pediatric head and neck tumors diagnosed in our institute were reviewed with a view to elucidate the overall cytological patterns and analyze the important cytological features. Ninety-four tumors (69.6%) were aspirated for a primary diagnosis, and in 41 (30.4%) cases, fine-needle aspiration cytology was performed to document relapse, recurrence or a metastasis. Among the 94 tumors aspirated for a primary diagnosis, 66 cases (70.2%) were accurately diagnosed, in 22 cases (23.4%) a broad working diagnosis was offered, and 6 cases (6.4%) were misdiagnosed. The accuracy rate was higher (79.3%) when relapse-recurrent and metastatic tumors were included. The smears were broadly divided into six patterns, viz. round cell, epithelial, anaplastic, giant cell, mixed inflammatory, and spindle cell patterns. The round cell pattern was the most frequent one encountered in this group. The cytological features that stood the test of variability were lymphoglandular bodies and a noncohesive cell population in hematolymphoid malignancies, pale chromatin and cytoplasmic vacuoles in primitive neuroectodermal tumor/Ewing's sarcoma (PNET/ES), neuropil and rosettes in neuroblastoma, and plasmacytoid rhabdomyoblasts in rhabdomyosarcoma. A fairly good accuracy was seen in the diagnosis of metastatic undifferentiated carcinoma and anaplastic lymphoma, but the giant-cell and spindle-cell tumors continued to pose a problem in diagnosis. Ancillary techniques such as immunocytochemistry and electron microscopy applied in limited cases helped evaluate Langerhans cell histiocytosis, alveolar rhabdomyosarcoma, and the PNET/ES family of tumors.     

Molecular genotyping of Candida parapsilosis group I clinical isolates by analysis of polymorphic microsatellite markers

Candida parapsilosis, a pathogenic yeast, is composed of three newly designated genomic species that are physiologically and morphologically indistinguishable. Nosocomial infections caused by group I C. parapsilosis are often associated with the breakdown of infection control practices and the contamination of medical devices, solutions, and indwelling catheters. Due to the low levels of nucleotide sequence variation that are observed, an investigation of the size polymorphisms in loci harboring microsatellite repeat sequences was applied for the typing of C. parapsilosis group I isolates. PCR primer sets that flank the microsatellite repeats for seven loci were designed. Following amplification by PCR, the size of each amplification product was determined automatically by capillary electrophoresis. A total of 42 C. parapsilosis group I isolates were typed by microsatellite analysis, and their profiles were compared to the hybridization profiles obtained by use of the Cp3-13 DNA probe. A high degree of discrimination (discriminatory power = 0.971) was observed by microsatellite analysis. The number of different alleles per locus ranged from 14 for locus B to 5 for locus C. Microsatellite analysis detected 30 different microsatellite genotypes, with 24 genotypes represented by a single isolate. Comparison of the genotypes obtained by microsatellite analysis and those obtained by analysis of the Cp3-13 hybridization profiles showed that they were similar, and these methods were able to identify related and unrelated isolates. Some discrepancies were observed between the methods and may be due to higher mutation rates and/or homoplasy by microsatellite markers. Identical results were observed between microsatellite analysis and Cp3-13 DNA hybridization profile analysis for C. parapsilosis isolates obtained from two patients, demonstrating the reproducibilities of the methods in vivo. Identical microsatellite profiles were observed for isolates displaying different phenotypic switching morphologies. Indistinguishable Cp3-13 DNA hybridization profiles were observed for six epidemiologically related isolates; however, only three of six primary isolates had identical microsatellite profiles. Size variation at a single locus was observed for three of six isolates obtained either after the outbreak period or from a different body site, suggesting the potential of the method to detect microevolutionary events. Interestingly, for most loci a single allele per strain was observed; in contrast, two alleles per locus were observed for some strains, and consistent with the findings for natural isolates, some isolates may be aneuploid. Due to the potential for high throughput, reproducibility, and discrimination, microsatellite analysis may provide a robust and efficient method for the genotyping of large numbers of C. parapsilosis group I isolates.     

HPV status in head and neck tumors

HPV infection has been associated with head and neck carcinoma (HNSCC). In the present study, a tumor collective from Middle Germany was analyzed using two HPV-detection methods, i.e., PCR followed by agarose gel electrophoresis and microarray chip hybridization. In addition, the predictive value of tumor morphology was assessed. In total, 76 HNSCCs from 52 patients were examined, including 23 pairs of primaries and corresponding metastases. The highest rate of positive tumors was observed in the tonsils, with 76% of the patients and 81% of tumor samples being positive for high-risk HPV. DNA chip analysis detected significantly more HPV-positive cases than did agarose gel electrophoresis. Except for one HPV-33-positive case, all others tumors harbored HPV 16. There was a good concordance between the HPV status of the primary tumor and its corresponding metastasis (21/23 cases, 91%). In two cases, HPV was found only in the primary tumor but not in the metastasis. Our results clearly confirmed the high prevalence of HR-HPV in tonsillar carcinomas with a rate that was even 20% higher than those reported in the literature. Morphology is a valuable indicator for an HPV association that needs to be confirmed by molecular tests.     

Molecular analysis of the candidate tumor suppressor gene ING1 in human head and neck tumors with 13q deletions

The candidate tumor-suppressor gene ING1 encodes p33(ING1), a nuclear protein which physically interacts with TP53. It has been shown that p33(ING1) acts in the same biochemical pathway as TP53, leading to cell growth inhibition. Interestingly, a rearrangement of the ING1 gene was found in a neuroblastoma cell line, supporting its involvement in tumor development. Because ING1 resides on the long arm of chromosome 13 (13q34) (a region frequently deleted in many tumor types), we sought to characterize its role in head and neck squamous-cell carcinoma (HNSCC). We first analyzed 44 primary tumors for loss of heterozygosity (LOH) at 13q, using four widely spaced microsatellite markers (13q14, 13q14.3-q22, 13q22, and 13q34). Twenty (48%) of the tumor samples showed LOH in all of the informative markers tested, including D13S1315 at 13q34. Two of the tumors displayed partial losses restricted to one marker (D13S118 at 13q14 in tumor 1164, and D13S135 at 13q14.3-q22 in tumor 1398). We then determined the genomic structure of the ING1 gene and sequenced the entire coding region in 20 primary tumors showing 13q LOH and in five head and neck cancer cell lines. A single germline polymorphism was detected in 10 of the tumors analyzed (T to C change) located 110 nucleotides upstream of the starting methionine. No somatic mutations were found in any of the samples, suggesting that ING1 is not a tumor suppressor gene target in head and neck cancer. Genes Chromosomes Cancer 27:319-322, 2000.     

No microsatellite instability using Bethesda panel and revised markers in uterine leiomyomas

Uterine leiomyomas are benign tumors of the uterus that arise clonally from smooth muscle cells of the myometrium and are very common reason for hysterectomy. The aim of this study was to evaluate microsatellite instability (MSI) in uterine leiomyomas using a set of MSI markers by Promega Corporation (Madison, WI, USA) and the Bethesda guideline. DNA was isolated from paired normal and tumoral tissues in 50 patients with uterine leiomyomas and MSI was analyzed by using seven microsatellite markers. Our result showed that microsatellite stability was found in all uterine leiomyomas. These data confirm the genetic status of uterine leiomyomas for the first time in Korean populations, and suggest that uterine leiomyomas have genetic stability in Korean.     

Cytostatic and immunologic treatment of head and neck tumors

Advanced head and neck tumors have a very low survival rate when treated by classical methods of irradiation and surgery. This is why in the last ten years cytostatics and immunotherapy have been applied. Clinical experience speaks in favour of polychemotherapy in comparison to monochemotherapy. In head and neck tumors bleomycin, methotrexate, 5-fluorouracil, vincristine and cis-Platinum have been used most often. They can be applied as initial therapy, additional therapy after surgery and irradiation, and finally as palliative therapy. Present experiences have shown the best results with initial therapy in 2-3 cycles, before surgery and irradiation. Toxicity of cytostatics depends on the dose and the sensitivity of the patient. The interval between cycles is 2-3 weeks. According to clinical experience, active specific immunotherapy with tumor vaccine did not prove successful. The investigations have thus been directed towards the means which would mobilise the immunological forces of the organism and remove obstacles in reaction of cytotoxic drugs and tumor cells. Such biological response modifiers are interferon, hormone of the thymus, substances which increase, change or restore immunological reactivity, lymphocytes, tumor preventive agents, NK cells, lymphokines, monokines and allokines, plasmophoresis and indomethacin.     

Peripheral nerve sheath tumors of the head and neck

The head and neck region is a common location for benign peripheral nerve sheath tumors (PNST) and a rare site for malignant PNST. The diagnostic distinction between schwannoma (neurilemmoma) and benign neurofibroma remains clinically and prognostically important. Most benign PNST are schwannomas, and these do not have a recognized malignant potential. Malignant PNST arise de novo or from benign neurofibromas. The definitive diagnosis of malignant PNST may be difficult or impossible using only routine light microscopy. Electron microscopy and immunohistochemistry are special techniques that may be helpful. The prognosis in patients with malignant PNST depends heavily on the extent of surgical excision, size of the primary tumor, and presence or absence of von Recklinghausen's neurofibromatosis.     

Validation of gefitinib effectiveness in a broad panel of head and neck squamous carcinoma cells

Recently improved understanding of the pathogenesis of human head and neck squamous cell carcinoma (HNSCC) has led to the development of new, molecular-based therapeutic strategies, one of the more promising is the utilisation of tyrosine kinase (TK) inhibitors, targeting epidermal growth factor receptor (EGFR). In this study, we tested for gefitinib effectiveness in a broad panel of 12 newly established HNSCC cell lines, investigating its ability to reduce cell growth, to induce apoptosis and to modulate cell cycle and various EGFR pathway-related targets. Gefitinib IC50 values ranged between 0.064 and 33 microM, its capability to induce apoptosis and cell accumulation in G0/G1 phase was cell line-specific, and the main EGFR-related pathway involved in gefitinib activity was PI3K/Akt/mTor. We characterised our in vitro panel extensively, with the aim to identify predictive factors for gefitinib effectiveness; all cell lines were free of human papillomavirus infection, two were positive for Fhit expression, four expressed wild-type p53, and all of them variously expressed the other two p53 family members, p63 and p73. The comparison between the targets analysed and gefitinib effectiveness evidenced the absence of a clear relationship, excluding them as predictive factors for gefitinib efficacy. Our results confirmed the in vitro efficacy of an anti-EGFR approach, but other targets than those analysed here should be characterised in order to identify valid predictive factors for gefitinib utilisation.     

Multiple head and neck tumors frequently originate from a single preneoplastic lesion

The development of second primary tumors has a negative impact on the prognosis of head and neck squamous cell carcinoma. Previously, we detected genetically altered and tumor-related mucosal lesions in the resection margins in 25% of unselected head and neck squamous cell carcinoma patients (Tabor MP, Brakenhoff RH, van Houten VMM, Kummer JA, Snel MHJ, Snijders PJF, Snow GB, Leemans CR, Braakhuis BJM: Persistence of genetically altered fields in head and neck cancer patients: biological and clinical implications. Clin Cancer Res 2001, 7: 1523-1532). The aim of this study was to determine whether first and second primary tumors are clonally related and originate from a single genetically altered field. From 10 patients we analyzed the first tumor of the oral cavity or oropharynx, the >3-cm remote second primary tumor, and the mucosa from the tumor-free margins from both resection specimens. We compared TP53 mutations and loss of heterozygosity profiles using 19 microsatellite markers at chromosomes 3p, 9p, 13q, and 17p. In all patients, genetically altered mucosal lesions were detected in at least one resection margin from both first and second primary tumor. Evidence for a common clonal origin of the first tumor, second primary tumor, and the intervening mucosa was found for at least 6 of 10 patients. Our results indicate that a proportion of multiple primary tumors have developed within a single preneoplastic field. Based on different etiology and clinical consequences, we propose that independent second primary tumors should be distinguished from second field tumors, that arise from the same genetically altered field the first tumor has developed from.     

An update on mesenchymal tumors of the head and neck

The purpose of this review is to offer a brief reappraisal of several soft tissue neoplasms germane to the head and neck region. Specifically, this paper is intended to draw attention to the recently described low-grade sarcoma with neural and myogenic features. In addition, other tumors that are largely specific to the head and neck region, including nasopharyngeal angiofibroma, sinonasal hemangiopericytoma and spindle cell/pleomorphic lipoma, will be reviewed in light of recent advances in our understanding of their pathophysiology and diagnosis.     

Distinctive soft tissue tumors of the head and neck.

Among soft tissue tumors as a whole, those in the head and neck region are relatively uncommon, and the proportion of all soft tissue sarcomas that arise in this region is 相似文献   

采用新一代测序技术分析头颈部鳞状细胞癌的分子异质性

依据与高危亚型人类乳头状瘤病毒(HPV16、18)的相关性,头颈部鳞状细胞癌( HNSCC)被分为两种不同的临床亚型组。近年来,在某种程度上由于与 HPV 感染有关的HNSCC在逐渐增加,且其在预后和分子组织学方面的差异引起学者们的注意,然而,如何分类这些肿瘤,其潜在的作用机制和详尽的基因组织学仍然未被阐明。为了阐明伴和不伴HPV感染的HNSCC致癌途径,我们采用定向的新一代测序技术检测50个癌相关基因的单核苷酸多态性( SNPs),在伴和不伴HPV感染的HNSCC组织标本中,发现其中25个基因(25/50)可以检测到SNPs;在这25个基因中又有5个基因无论有无HPV感染,其变异模式是相似的。基因从酪氨酸蛋白激酶受体和其相关通路中,较大数量的序列变异优先出现在 HPV +的标本中。在基因相关肿瘤抑制功能方面,SNPs普遍存在HPV-的HNSCC标本中。这些观察结果可能帮助我们阐明两种临床上不同亚型HNSCC的具体分子发病机制。无论是HPV+,还是HPV-的HNSCC,SNPs的过度表现可能成为靶向治疗HNSCC的潜在变异序列。     

恶性肿瘤合并头颈部真菌感染的临床与病理学观察

目的 探讨恶性肿瘤合并头颈部真菌感染的临床与病理特点.方法回顾分析21例恶性肿瘤合并头颈部真菌感染患者的临床和病理资料;用HE染色、过碘酸-雪夫(PAS)染色和环六亚甲基四胺银(GMS)染色显示组织病变特点及真菌的形态特征,用黏蛋白5B(MUC5B)抗体免疫组织化学EnVision法染色标记真菌;13例组织标本进行了真菌培养.结果患者发病年龄12～72岁(中位年龄48岁),男性17例,女性4例.病理学检查证实合并侵袭性真菌性鼻窦炎(IFS)有8例(38.1%),其中波及眶内者6例(28.6%),侵入颅内1例;原发疾病为白血病(7例)及鼻咽癌(1例);病原真菌为接合菌(5例)和曲霉菌(3例),均有化疗或放疗及使用抗生素的病史.其余13例真菌感染发生于鼻腔鼻窦、咽喉及腭部原发肿瘤的坏死组织内,病原真菌主要为曲霉菌(6例)和念珠菌(4例),7例有放疗等治疗史.真菌培养结果9例阳性(9/13).随访病例14例,死亡6例.结论 恶性肿瘤可合并头颈部真菌感染,以IFS最为多见,好发于白血病化疗后,易累及眼眶,预后较差;病原真菌的类别在IFS以接合菌和曲霉菌多见;病理学检查仍然是尽快确定诊断及初步鉴别常见接合菌和曲霉菌类别的一个重要手段.     

头颈部鳞癌的微卫星不稳定性和杂合性丢失的初步研究

目的:探讨头颈部鳞癌的微卫星不稳定性(MSI)及杂合性丢失(LOH)。方法:选择来自3、5、6、8、9、13、17和18号染色体的15个微卫星标志对36例头颈部鳞癌标本和相应的外周血进行微卫星分析。结果:36例头颈部鳞癌中,27.8%(10/36)分别有1-8个位点存在MSI,MSI发生率较高的位点为:D17S520(22.9%)、D6S105(16.7%)和D8S264(13.9%)。在9p21-p22和3p14等处存在一定的LOH。微卫星异常的检出率与肿瘤分期、分级无相关性。结论:提示MSI是头颈部鳞癌中较为常见的遗传学变化,染色体9p21-p22和3p14区域可能存在与头颈部鳞癌有关的抑癌基因。     

Immunologic and cytogenetic markers expressed in non-Hodgkin lymphoma of head and neck

Very often, the first doctor who examines a patient with malignant lymphoma is an ENT specialist, because non-Hodgkin lymphoma is five times more frequent than Hodgkin disease in the head and neck region. Approximately 25% of extranodal lymphoma occurs in the head and neck and extranodal presentation is twice as frequent as nodal presentation. This paper present a study of the patients from ENT, Head & Neck Surgery Clinic of Coltea Clinical Hospital, Bucharest, Romania, diagnosed with malignant lymphoma. We developed a specific scheme for collecting data about patients, together with pathology details, immunology and cytogenetic markers. We tried to establish a relation between immunologic and cytogenetic markers and the clinical evolution of non-Hodgkin lymphoma. For this study, we analyzed data regarding CD10, CD5, CD20, Bcl-2, Bcl-6, Ki67 expression obtained from 58 patients with follicular lymphoma. An attempt was made to correlate the presence of certain immunohistochemical and cytogenetic markers with the evolution and aggressiveness of the disease, and we can say that Bcl-2 is positive in all tumor subtypes, being associated relatively frequently with CD5 expression, and is a marker of poor prognosis, while Bcl-6 is positive especially in the tumor forms associated with the predominance of small cells and is a marker of favorable prognosis.     

Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP)

The association of human telomerase activity with an indefinite replicative capacity of cells in vitro and advanced tumors in vivo is gaining wide support. The increasing interest in studying various aspects of telomerase expression in cancer required the development of a sensitive and reliable protocol for the extraction and detection of telomerase activity in cell culture material, and from small tissue samples obtained from biopsy, surgical reaction of tumors, and autopsy. Recently a novel procedure for the extraction and detection of telomerase activity was developed (Science 1994; 266: 2011–2015) which resulted in an estimated 10⁴ fold improvement in detectability compared with previous methods. The described procedures not only dramatically increased sensitivity but also allowed fast and efficient detection of telomerase activity in a large number of samples. A number of technical aspects which are of eritical importance for reproducibility and reliability of this assay using clinical material are addressed in this report. In addition, new methods to perform telomerase assays without the use of radioisotopes are described.Abbreviations AEBSF 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochlorine - BCA bieinchoninic acid - CCD camera charged coupled device camera - CHAPS 3-[(3-cholamidopropyl)-dimethyl-ammonio]-l-propanesulfonate - DEPC diethyl pyrocarbonate - DPBS Dulbecco's phosphate buffered saline - EGTA ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid - HPLC high pressure liquid chromatography - HPV human papillomavirus - PDA piperazine diacrylamide - PCR polymerase chain reaction - T4g32 protein T4 gene 32 protein - TRAP telomeric repeat amplification protocol - TRF terminal restriction fragment