Core classification of head and neck squamous cell carcinomas: Correlations between morphology,DNA ploidy and HPV infection

Aims

The study intended to reveal whether HPV infection is reflected by nuclear morphology and DNA cytometry parameters in head and neck squamous cell carcinomas (HNSCC).

Methods

In total, 39 HNSCC were selected for reanalysis by histomorphology applying the core classification, DNA cytometry and HPV detection. For the core classification, HE sections were assessed by a score system to evaluate the nuclear size, the mitosis size, their variabilities and the presence of tripolar or tetrapolar mitoses. HPV was analyzed by consensus PCR followed by a hybridization method for virus typing. Static DNA cytometry was applied on single cell suspension focusing particularly on the parameters DNA modal value, DNA index peak, DNA index mean, 2c deviation index and 5c exceeding rate. Statistical analysis was done by T-test or Fisher's exact test.

Results

The analysis revealed that HPV positive HNSCC had significantly smaller nuclei than HPV negative cases. Increasing values of the nuclear size and mitosis size were significantly associated with higher indices of the DNA cytometry analysis.

Conclusions

The study confirms that the core classification can provide information on the ploidy of HNSCC and that HPV positive tumors represent a distinct morphological and genetic carcinoma subtype.     

DNA ploidy and chromosomal alterations in head and neck squamous cell carcinoma

In head and neck squamous cell carcinomas (HNSCC) the prognostic factors that are routinely considered when deciding therapeutic strategies are still stage and site of the primary tumour, and the presence of nodal or distant metastases. However, it is recognised that these clinical predictors are limited since they do not satisfactorily reflect the biological behaviour of the individual tumour. With the evolving understanding of the genetic and molecular basis of human malignancies, there are an increasing number of factors being claimed to provide prognostic information even in HNSCC. Here we review own and published data on DNA ploidy, karyotyping and molecular cytogenetic changes and its relevance in HNSCC carcinogenesis. The survey suggests that the induction of aneuploidy is a very early event in tumour development being detectable already in non-dysplastic leukoplakia and highly predictive for the subsequent development of a carcinoma. Moreover, specific chromosomal imbalances are associated with different stages of cancer progression and patient's survival, which we have compiled into a progression model of HNSCC.     

Human papillomavirus detection in head and neck squamous cell carcinomas

Squamous cell carcinomas of the head and neck (HNSCC) are a frequent diagnosis in anatomic pathology practice. Tobacco use and heavy alcohol consumption are known risk factors for HNSCC but in other cases human papillomavirus (HPV) is linked to carcinogenesis. HPV proteins E6 and E7 promote oncogenesis by blocking the action of p53 and pRB, respectively. An absence of p53 mutations in addition to expression of p16 are part of the distinct molecular profile identified in the subset of HNSCCs because of HPV. Various methods are available for HPV detection but polymerase chain reaction and in situ hybridization techniques are commonly used. Both methods are amenable for testing formalin-fixed paraffin-embedded tissue that is a sample type readily available to the pathologist. HPV is detectable in approximately a quarter of all HNSCCs, and is particularly prevalent in the oropharynx in which the positivity rates approach 40%. A vast majority of HPV-related HNSCCs are owing to HPV type 16 with types 18, 31, and 33 accounting for almost all of the remaining cases. HPV-related HNSCCs are associated with better prognosis for both recurrence and survival. This group of tumors has also been shown to respond well to radiation treatment. As the clinical relevance of HPV in HNSCCs continues to emerge, anatomic pathologists are likely to receive increasing requests for testing. Herein, the authors review the biological and clinical aspects of HPV-associated HNSCC and review techniques for HPV detection.     

Recurrent cytogenetic abnormalities in squamous cell carcinomas of the head and neck region

We characterized the breakpoints, gains, and losses of chromosome material in squamous cell carcinomas of the head and neck region from 29 patients. Cell lines were karyotyped in 1/3 of cases, direct preparations or early in vitro harvests in 1/3, and both in 1/3 of cases. GTG-banding was employed in all cases, as were C-banding and RBG- and AgNOR-staining in most. Some tumors were near-diploid and others near-tetraploid, but many had mixed populations, with diploid, tetraploid, and octoploid subclones representing essentially the same karyotypic pattern. The most frequent changes were deletions. Losses affecting 3p13-p24, 5q12-q23, 8p22-p23, 9p21 -p24, and 18q22-q23 ranged in frequency from 40% to 60% of tumors. Loss of the short arm of the inactive X occurred in 70% of tumors from female patients, and loss or rearrangement of the Y occurred in 74% of tumors from male patients. Loss of 18q appeared to be associated with short survival, as did the presence of multiple deletions. There was gain (2-5 extra copies) of 3q21 -qter, 5p, 7p, 8q, and 11q 13-q23 in 28-38% of tumors. Three tumors had an hsr involving 11q13-q21. Gain of material at 11q13 is postulated to be associated with amplification of the PRADI/CCND gene at that locus. A translocation between proximal 1 p and either an acrocentric short arm or proximal 8p or 9p was observed in squamous cell carcinomas of the head and neck region but not in female genital tract tumors. No other abnormalities appeared to be site specific, suggesting a pattern of genetic evolution in squamous cell carcinoma that is independent of anatomic site. Genes Chrom Cancer 9:192-206 (1994). © 1994 Wiley-Liss, Inc.     

Genic alterations in oral and head and neck squamous cell carcinomas: analysis of international literature

The development of oral and head and neck squamous cell carcinomas occurs in relation with multiple events including mainly: loss of cycle cell control, evasion from apoptosis, telomerase reactivation. Complex interactions between a set of molecules, cell cycle proteins, tumour suppressor genes, oncogenes and the telomerase, occur in the multiple step process of carcinogenesis. The 2 main ways of control of the cell cycle rely on 2 tumour suppressor genes: the P53 gene and the retinoblastoma gene or RB gene. One of the regulation pathways or the 2 regulation pathways are disabled during the development of oral and head and neck squamous cell carcinomas. Most of the time, the inactivation of the P53 pathway results from a loss of function of the p53 protein, secondary to mutation and/or deletion of the P53 gene; It may also result of the amplification of the MDM2 gene and of the inactivation of the arf protein. The RB pathway leads to cell proliferation by loss of the p16 protein, by amplification of the cyclin D1 gene and less frequently by mutation of the RB gene or loss of the retinoblastoma protein. In India and South-East Asia, the activation of RAS and MYC oncogenes appears to be related with the presence of specific carcinogens in snuff and tobacco. By blocking apoptosis, the Bcl2 protein seems to increase the resistance of tumours to radiotherapy and chemotherapy.     

Expression of protein tyrosine kinases in head and neck squamous cell carcinomas

Protein tyrosine kinases (TKs) are overexpressed in many carcinomas and sarcomas. We studied the expression of the following TKs in head and neck squamous cell carcinoma (HNSCC): platelet-derived growth factor receptor (PDGFR), c-kit, epidermal growth factor receptor (EGFR), and a serine-threonine kinase, Akt. Formalin-fixed, paraffin-embedded tumor blocks from 44 consecutive patients with primary HNSCC and 5 specimens of benign pharyngeal and laryngeal mucosa were retrieved for immunohistochemical analysis. Of the specimens, 38 had enough material to stain for all 4 antibodies. The study included 21 pharyngeal (base of tongue, 14; tonsil, 6; soft palate, 1), 16 laryngeal, and 1 floor of the mouth carcinoma. All 4 kinases in the tumor samples were expressed highly (PDGFR, 95%-100%; EGFR, 38%-43%; c-kit, 50%-86%; p-Akt, 57%-81%), with EGFR, c-kit, and p-Akt significantly higher than in benign samples. None of the kinase expressions correlated with disease-free survival. The expression of the kinases raises the possibility of treatment of HNSCC by tyrosine and serine-threonine kinase inhibitors.     

Karyotypic evolution and tumor progression in head and neck squamous cell carcinomas

Cytogenetic analysis was performed on primary tumors, and paired recurrent or metastatic lesions, in 14 patients with head and neck squamous cell carcinomas (HNSCC), in order to identify chromosomal aberrations associated with tumor initiation and progression. Abnormal karyotypes were found in 12 of the 14 patients, with distinctive karyotypic similarities shown in all informative pairs. For individual patients, the degree of karyotypic complexity was similar for the primaries and paired recurrent or metastatic lesions. All 22 samples with clonal chromosomal aberrations displayed complex karyotypes with multiple numerical and unbalanced structural rearrangements, resulting in extensive genomic imbalances. The pathway of clonal evolution could be traced in a few patients, supporting the notion that some aberrations or imbalances, particularly partial or entire loss of 3p, i(8q), and homogeneously staining regions commonly mapping to 11q13, were early genetic events in the initiation of HNSCC.     

Expression of integrins and E-cadherin in squamous cell carcinomas of the head and neck

Integrins and cadherins are cell adhesion molecules suggested to play an important role in malignant progression and tumour differentiation. Our aim was to characterise the pattern of expression and the relations between integrin beta1, beta4, beta6 and E-cadherin and the different histopathological features important when judging tumour differentiation, using a well-defined scoring system. Formalin-fixed paraffin-embedded pre-irradiation biopsies from 85 patients with head and neck squamous cell carcinomas (HNSCC) were stained and evaluated for the expression of integrin beta1, beta4 and beta6 and E-cadherin. The integrins were upregulated in carcinomas compared to the adjacent mucosa and E-cadherin was downregulated. However, differences were found within the tumour: Expression of E-cadherin was lost and the three integrins were upregulated at the tumour borders, compared to central parts of the tumour biopsy. Expression of the integrins did not correlate with tumour or histopathological parameters, whereas expression of E-cadherin was correlated with high degree of keratinisation, high nuclear maturation and few mitoses - factors that characterise well-differentiated carcinomas -and E-cadherin can therefore be considered as a marker of differentiation. Furthermore, loss of adhesion expressed by low E-cadherin and integrin beta4 correlated with the presence of nodal metastases at the time of diagnosis.     

Cytogenetic analysis of head and neck carcinomas.

Ten primary squamous cell carcinomas (SCC) of the head and neck were evaluated cytogenetically after 10-14 days of in vitro culture. Addition of 3% L-glutamine was essential for consistent epithelial growth of these carcinomas. Outgrowth of cells from tissue explants contained a mixture of chromosomally normal and abnormal cells; the abnormal cells had extensive changes including translocations, marker chromosomes, inversions, deletions, and duplications. In addition, all carcinomas contained cells with pulverization and double minute chromosomes (dmin). Chromosomes 11, 13, and 14 had "hotspots" of rearrangements.     

Karyotypic heterogeneity and clonal evolution in squamous cell carcinomas of the head and neck

Head and neck squamous cell carcinomas (HNSCC) are often characterized by complex karyotypic changes, and a substantial proportion of the reported tumors have shown intratumor heterogeneity in the form of cytogenetically related (40%) or unrelated clones (20%). In order to study intratumor heterogeneity and to distinguish the temporal order of chromosome rearrangements in these tumors, two or more samples from different areas of the same tumor were separately examined in 19 HNSCC, yielding karyotypes from a total of 42 tumor samples. Intrasample heterogeneity was observed in 16 samples. Two samples displayed both related and unrelated multiple clones, four samples showed only multiple unrelated clones, and the remaining 10 samples had only related subclones. Intersample heterogeneity was detected in all but one tumor. Five tumors showed both cytogenetically related and unrelated multiple clones, 11 were found to have only related subclones, and the remaining two tumors showed only unrelated clones. Clonal evolution could be assessed in 13 tumors. A comparison of chromosome imbalances in different subclones from these tumors suggests that partial or entire loss of 3p, 8p, 9p, and 18q and gain of genetic material from 3q and 8q are likely to be early genetic events. In contrast, loss of 1q, 6p, 7q, and chromosome 10, as well as gain of chromosome arms 5p and 7p, are most probably later genetic events. One of the examined tumors contained two highly complex clones that were cytogenetically unrelated, indicating that this tumor had a multicellular origin.     

Protein microarrays for the detection of biomarkers in head and neck squamous cell carcinomas

Protein microarrays are of increasing importance for high-throughput screening of fresh tissues. In our study, protein microarrays were generated by printing antibodies onto membranes to characterize protein profiles expressed by head and neck squamous cell carcinomas (HNSCCs). Cellular proteomes of 30 matched normal squamous epithelial cells and carcinoma specimens were analyzed after tissue microdissection using microarrays composed of 83 different antibodies. As controls, Western blot analysis and tissue microarrays (TMAs) containing 98 HNSCC specimens were used. Of the 83 proteins examined, 14 showed differential expression between HNSCCs and normal epithelium. The protein microarray approach revealed an upregulation of 8 proteins and a downregulation of 6 proteins. Bag-1, Cox-2, Hsp-70, Stat3, pescadillo, MMP-7 (matrilysin), IGF-2, and cyclin D1 were identified to be significantly upregulated, whereas suppressor of cytokine signaling 1, thrombospondin, TGF-beta1, Jun, Fos, and Fra-2 were downregulated. The differential expression of these proteins was confirmed using Western blot and TMA. Upon correlation of differentially regulated proteins with the clinicopathologic data of our patients, MMP-7 (matrilysin) was found to be associated with survival in univariate, but not multivariate, analysis. These data indicate that our protein arrays provide protein information in a systematic, reproducible, and also high-throughput fashion.     

HPV genotypes and their prognostic significance in head and neck squamous cell carcinomas

Background

Human papillomavirus (HPV) has been reported in up to 50% of head and neck squamous cell carcinomas (HNSCCs). Presence of HPV in HNSCC has been associated with more favorable prognosis.

Objectives

This study was designed to disclose HPV genotype distribution in head and neck squamous cell carcinomas (HNSCC) and their role in disease outcome. In addition, role of herpesviruses 1 and 2 (HSV-1 and -2) and cytomegalovirus (CMV) as co-factors was elucidated.

Study design

HPV-genotyping of 106 HNSCC was done with Multimetrix^®-kit. Luminex-based-method was used to detect HSV-1 and -2 and CMV.

Results

In males, 50% of HNSCC were HPV DNA positive and 25% of these were multiple HPV-types infections and in women, 72% and 31%, respectively. Low-risk (LR) HPV-types were found in 20.5% and co-infection with HSV-1 in 6.6%. Patients with HPV-positive and -negative HNSCC had similar survival. Patients not treated with chemoradiotherapy and co-infected with HSV-1 and HPV had a worse outcome. Similarly patients with LR-HPVs treated with radiotherapy had a poor prognosis.

Discussion

Radiotherapy for HNSCC in patients with either the presence of LR-HPV-types or a co-infection with HPV and HSV-1 may result in poor outcome.     

Genome-wide analyses on loss of heterozygosity in head and neck squamous cell carcinomas

Head and neck squamous cell carcinoma (HNSCC) is a frequent malignancy with a poor survival rate. Identifying the tumor suppressor gene (TSG) loci by genomic studies is an important step to uncover the molecular mechanisms involved in HNSCC pathogenesis. We therefore performed comprehensive analyses on loss of heterozygosity (LOH) using a genome-wide panel of 191 microsatellite markers in 22 HNSCC samples. We found 53 markers with significantly high LOH (>30%) on 21 chromosomal arms; the highest values of those were observed on 3p, 9p, 13q, 15q, and 17p, corresponding to D3S2432 (67%), D9S921-D9S925 (67%) and GATA62F03 (86%), D13S1493 (60%), D15S211 (62%), and D17S1353 (88%), respectively. Fifteen hot spots of LOH were defined in 13 chromosomal arms: 2q22-23, 4p15.2, 4q24-25, 5q31, 8p23, 9p23-24, 9q31.3, 9q34.2, 10q21, 11q21-22.3, 14q11-13, 14q22.3, 17p13, 18q11, and 19q12 as loci reported previously in HNSCCs. Furthermore, we identified five novel hot spots of LOH on three chromosomal arms in HNSCC at 2q33 (D2S1384), 2q37 (D2S125), 8q12-13 (D8S1136), 8q24 (D8S1128), and 15q21 (D15S211). In conclusion, our comprehensive allelotype analyses have unveiled and confirmed a total of 20 possible TSG loci that could be involved in the development of HNSCC. These results provide useful clues for identification of putative TSGs involved in HNSCC by fine mapping of the suspected regions and subsequent analysis for functional genes.     

Human papillomavirus DNA in head and neck squamous cell carcinomas in the Free State,South Africa

Human papillomaviruses (HPVs) have been associated with a subset of head and neck squamous cell carcinomas (HNSCCs). The aim of this study was to determine the prevalence of HPV DNA in archived formalin-fixed paraffin-embedded tissue from patients with histologically confirmed HNSCCs in a South African cohort. A nested PCR was used for the detection of HPV DNA targeting the L1 gene. Positive samples were confirmed using an in-house hemi-nested PCR targeting the E6 gene and genotyped by sequence determination of amplicons. HPV DNA was detected in 57/780 (7.3%) samples, with the highest prevalence being in the sinonasal tract (16.0%) and oropharynx (10.8%). HPV16 was the most frequently detected type, being found in 26/57 (45.6%) positive samples. The prevalence of HPV DNA in HNSCCs found in this study was lower than that found in developed countries     

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。     

头颈部鳞癌中抑癌基因PTEN的研究进展

The FTEN, having a dual specificity phosphatase activity, is the first tumor suppressor gene that possess phosphatase activity hitherto. Many researches have suggested that FTEN play a major role in the tumorgenesis. In clinical, the head and neck squamous cell carcinoma(HNSCC) is one of the most common ma-lignant tumors. In this review, advances in the research of FTEN and the relationship between the PTEN and HNSCC are discussed.