Overexpression of p53 and bcl-2 proteins and the presence of HPV infection are independent events in head and neck cancer

The expression of p53 and bcl-2 proteins by immunohistochemistry and the identification of human papillomavirus (HPV) infection by a non-isotopic polymerase chain reaction (PCR)based method were investigated in 30 patients with head and neck cancer. Ten cases were HPV-positive (33%), mostly as double or multiple infections by high- or intermediate-risk types. Twenty-one patients were p53-positive (70%), 9/10 with HPV-positive tumours and 12/20 with HPV-negative tumours; this difference was not statistically significant. Only four cases were bcl-2-positive, irrespective of the presence of either HPV or p53. No correlation was found between these biological factors and tumour stage, differentiation grade, and alcohol or tobacco use. Our findings indicate that p53 is involved in the majority of cases, bcl-2 is rare, and high-risk HPV could play a key role, especially in tumours of tongue and tonsil. In conclusion p53 and bcl-2 protein expression and the presence of HPV infection are independent events in these malignancies.     

Overexpression of p53 protein in squamous cell carcinoma of buccal mucosa and tongue in Taiwan: an immunohistochemical and clinicopathological study

Sixty squamous cell carcinomas of tongue and buccal mucosa were examined for expression of p53 protein by using an immunohistochemical technique improved by an antigen retrieval method. Twenty-seven (45%) tumors demonstrated strongly positive staining. Thirteen of p53-positive tumors (48%) also exhibited Overexpression of p53 in immediately adjoining hyperplastic or pre-malignant epithelium. All 22 metastatic lymph nodes and 18 local recurrent lesions (except two) had an identical p53 immunophenotype to their corresponding primary sites. Mitotic indices were significantly higher in p53-positive tumors ( P < 0.01): however, no association of PCNA scores with p53 expression was found ( P > 0.1). There was no correlation between p53 Overexpression and tumor grade, size and staging, vascular invasion, lymph node metastasis, and early local recurrence. Overexpression of p53 was found to be relatively higher, although not statistically significant, in nonsmokers than in heavy smokers (66.7% vs. 42.9%). and in non-betel-quid chewers than in heavy chewers (62.5% vs. 34.2%). These data are consistent with the hypothesis that inactivation of p53 protein may occur in the early phases of oral tumorigenesis. It may not be a useful prognostic marker but could possibly be used for risk assessment and surveillance of local recurrence.     

p53 protein expression in sequential biopsies of oral dysplasias and in situ carcinomas

Immunohistochemically detectable levels of p53 may be seen early in the malignant transformation of some neoplasms. To determine if p53 is immunocytochemically detectable, and therefore presumptively abnormal, in oral dysplasias and in situ carcinomas, and to explore the natural history of p53 protein expression in these lesions, sequential biopsies from patients with lesions occurring in the same anatomic site were examined. Formalin-fixed, paraffin-embedded sections from 19 patients were evaluated immunohistochemically for p53 protein using antibody clones Pab1801 and BP53-12. With two exceptions, comparable results were observed with these antibodies. p53 protein was detected immunocytochemically in 6 of 13 patients with dysplasias; 3 of these progressed to p53-positive invasive carcinoma, one advanced to a more severe grade of p53-positive dysplasia, one developed into a p53-negative verrucous carcinoma, and one represented a p53-positive dysplasia developing five years after treatment of a p53-positive carcinoma. The p53-positive dysplasias, which were found in all subtypes (mild, moderate, severe), preceded histologic malignant change by months to years. p53 detection was evident in 4 of 6 patients with in situ lesions. Sequential biopsies of three of these lesions showed no change in lesion histology or p53 staining, and one lesion advanced to a p53-positive carcinoma. It is concluded that p53 protein may be detected early in the development of a subset of p53-positive oral squamous cell carcinomas. This phenomenon may be seen in dysplasias and in situ lesions, and it may have prognostic implications.     

Relationship of p53 overexpression to other cell cycle regulatory proteins in oral squamous cell carcinoma

Aberrations of the p53 gene and the overexpression of its protein are described in a variety of neoplasms, including oral and other head and neck cancers. Here we report the association of p53 (over)expression with a downstream cell cycle inhibitor p21/waf 1 in oral squamous cell carcinoma (SCC). The loss of expression of p16 and p27, two other cyclin-dependent kinase (cdk) inhibitors, was also examined. In this panel of tumours, 10/24 carcinomas were p53-immunopositive. Heterogeneous expression of p21 and p27 was seen in 10/24 SCC and 9/16 SCC, respectively, and this was not correlated to p53 status. The expression of p21 and p27 in these SCCs suggests the existence of mechanisms by which some growing tumour cells may tolerate these cell cycle inhibitors; eight SCCs lacked expression of both inhibitors but only two of these cancers overexpressed p53, suggesting that accumulation of p21/p27 can be independent of the functional status of the p53 gene. Data do not support a clear example of a phenotype that shows an overexpression of p53 with downregulation of p21 or p27 leading to cell cycle alterations. Furthermore, only three SCCs were p16-negative and p53-positive. This suggests that these two tumour suppressors may act in separate pathways.     

Low prevalence of expression of p53 oncoprotein in oral carcinomas from Sri Lanka associated with betel and tobacco chewing

Overexpression of p53 oncoprotein has been demonstrated in a wide range of human malignancies. We have examined the p53 expression amongst 38 Sri Lankan subjects with histologically confirmed oral squamous cell carcinomas. The mean age of the subjects was 59.4 years and betel chewing with tobacco was the most common habit (84%) with a high percentage of patients smoking (63%). Buccal mucosa was the most frequently affected site (68%) with a high proportion (79%) of well differentiated carcinomas. p53 expression was examined by standard immuno-histochemical methods on frozen sections using monoclonal antibodies PAb 1801, 240 and 421. Only 4 (11%) carcinomas showed nuclear reactivity mostly in random clusters of basal neoplastic cells. The low frequency of p53 expression could be due to deletion of both alleles or to premature truncated protein products due to nonsense mutations resulting in loss of antibody recognition sites. Alternatively the much lower prevalence than reported by others could be due to differences in aetiological agents and/or genetic predisposition of this population.     

p21WAF1/CIP1 expression is a marker of poor prognosis in oral squamous cell carcinoma

BACKGROUND: Previous research on the prognostic relevance of p21(WAF1/CIP1) in oral squamous cell carcinomas (OSCC) yielded inconclusive and contradictory data. OBJECTIVES: To investigate the prognostic significance of p21(WAF1/CIP1) expression, its relationship to p53 accumulation, proliferation-associated proteins Ki-67 and cyclin D1 in relation to survival and clinicopathological features in OSCC. METHODS: Surgical specimens taken from 106 randomly selected patients were studied by immunohistochemistry. Expression of the protein of interest was correlated with clinical data. RESULTS: p21(WAF1/CIP1) expression was found in 61.3% of OSCCs. Expression of p21(WAF1/CIP1) significantly correlated with tumor size (P = 0.005), lymph node involvement (P = 0.002), clinical stage (P < 0.001), and tumor site (P = 0.002). Patients with tumors showing p21(WAF1/CIP1) immunopositivity had decreased 2-year survival (P = 0.018). Expression of p21(WAF1/CIP1) was not related to age, gender, risk factors (tobacco, alcohol), dental status, or tumor differentiation grade. The p21(WAF1/CIP1) expression positively correlated with proliferation-related variables Ki-67 (P = 0.010) and cyclin D1 (P < 0.001), but not with p53 expression. CONCLUSIONS: The expression of p21(WAF1/CIP1) was found to be associated with poorer prognosis and tumor aggressivity in OSCC.     

Association of overexpression of p53 oncoprotein with the state of cell proliferation in oral carcinoma

p53 is a nuclear phosphoprotein recognised as important in the regulation of normal cell growth and proliferation, the wild-type protein suppressing cell division. Expression of presumptive mutant protein, detected by immunohistochemistry, is used increasingly as a diagnostic and prognostic marker in human neoplasms. A question arises as to whether or not p53 (over)expression in a lesion is any more or less informative than other markers of cell proliferation. Twenty well-differentiated oral squamous cell carcinomas which had earlier been examined for immunoreactivity against a panel of p53 antibodies were examined for the status of cell proliferation – both in islands of invading neoplastic cells and in the non-malignant epithelial margins. The status of epithelial cell proliferation was found to be significantly higher in p53-positive tumours when enumerated by Ki-67 antibody, both within the tumour as well as its margins. This may confer a growth advantage to these neoplasms and reflect a status of inactivated p53 protein, although the actual cause of the rapid proliferation may lie in activation/inactivation of other genes. The PCNA labelling indices, on the other hand, were closely similar in both p53-positive and -negative groups, suggesting that stabilisation of p53 protein does not influence the proliterative advantage in these carcinomas via a deregulation step of PCNA-related gene products.     

Cytoplasmic expression of p53 protein and its morphological features in salivary gland lesions

An immunohistochemical study of p53 protein was carried out on 45 salivary gland lesions using a monoclonal antibody, Bp53–12, raised to the intracellular domain of the p53 protein. p53 protein expression was found in 34.4% of 32 salivary gland carcinomas. Nuclear p53 expression was detected in tumor cells but not in non-neoplastic cells, except in one salivary duct carcinoma. The perinuclear cytoplasm of luminal duct cells was specifically positive for the antibody used here. Cytoplasmic p53 expression was observed mostly in non-neoplastic cells. There was a tendency for the Cytoplasmic staining of p53 protein to be observed in the normal cells adjacent to p53-positive carcinomas, but none of the normal cells were positive in the tissues surrounding p53-negative carcinomas. Cytoplasmic expression of p53 protein in salivary gland tissues seems to be correlated with tumorigenesis.     

The use of tobacco‐free betel‐quid in conjunction with alcohol/tobacco impacts early‐onset age and carcinoma distribution for upper aerodigestive tract cancer

J Oral Pathol Med (2011) 40 : 684–692 Background: Recognition of how risk factors affect the age when cancers are first diagnosed may help to establish more appropriate cancer screening and preventive strategies. Methods: To investigate the independent and synergistic effects of alcohol, tobacco‐free betel‐quid (TF‐BQ), and cigarette use on diagnosis age and dissemination of upper aerodigestive tract squamous cell carcinoma (UADT‐SCC), we recruited pathology‐proven 1522 patients with UADT‐SCC for study. Results: A 49‐, 53‐, 57‐, and 62‐year‐old stepwise older median age at carcinoma diagnosis was, respectively, found among patients with oral, pharyngeal, esophageal, and laryngeal cancer. Oral cavity (53.2%) and larynx (11.6%) were separately the dominant and recessive sites where the UADT‐SCC occurred. Although alcohol and tobacco bestowed increased risks of earlier tumor occurrence only for oral/pharyngeal and oral cancers, respectively, TF‐BQ was consistently observed to confer elevated age‐associated risks for each UADT‐SCC [adjusted hazard ratio (aHR) = 1.6–2.3]. Alcohol and TF‐BQ joint consumers experienced a stepwise increased cumulative risk (CR) of contracting carcinomas of the larynx (46.2%), esophagus (47.5%), pharynx (53.5%), and oral cavity (60.5–71.0%), with >68% of CRs found among drinkers who started chewing before age 20. Alcohol + Betel + Cigarette and Alcohol + Betel users exhibi‐ted earlier diagnosis ages than non‐users: 10 years ahead for oral cancer, 7, 17, and 12 years earlier for pharyngeal, esophageal, and laryngeal cancers. Noticeably, higher cumulative cancer risks regarding earlier tumor occurrence were correspondingly identified for these users aged 43, 49, 43, and 44 upward. Conclusions: Tobacco‐free betel‐quid, in conjunction with alcohol and/or tobacco consumption, impacts early cancer occurrence for specific UADT‐SCC and influences tumor site incidence pattern of these neoplasms.     

p53 alterations in betel quid- and tobacco-associated oral squamous cell carcinomas from Taiwan

Alterations of p53 have been explored in Taiwanese oral squamous cell carcinomas (OSCCs) consisting of a betel quid (BQ)/tobacco-related subgroup of 36 subjects and a tobacco-related subgroup of 13 subjects. Mutations in conserved exons were found in 12 tumors. Seven mutations were clustered in a hot-spot region mapped to a region between codons 273–282 in exon 8. The incidence of p53 mutation in BQ/tobacco tumors was 22% (8/36). The frequency of p53 allelic loss (21%, 3/14) in BQ/tobacco tumors approximates to the incidence of mutation. This is the first study demonstrating allelic deletion of p53 in such malignancies. Twenty-four of 43 samples showed positive p53 immunostaining. All tumors harboring mis-sense mutations of p53 in conserved exons exhibited nuclear protein accumulation. The incidence of mutation in conserved exons in BQ/tobacco-associated Asian OSCCs (15%) is significantly different from worldwide OSCCs (46%) related primarily to tobacco consumption ( P =0.00001).     

Assessment of p63 expression in the salivary gland neoplasms adenoid cystic carcinoma, polymorphous low-grade adenocarcinoma, and basal cell and canalicular adenomas

PURPOSE: The purpose of this study was to determine the extent of p63 immunoreactivity in the malignant salivary gland neoplasms adenoid cystic carcinoma (ACC) and polymorphous low-grade adenocarcinoma (PLGA) and to compare this to the expression of this marker in the benign salivary gland tumors canalicular adenoma and basal cell adenoma. Few studies on the expression of p63 in head and neck salivary gland tumors have been published to date. P63, a selective immunohistochemical marker of basal/stem cells of stratified epithelium and of myoepithelial cells, is a p53 homologue that plays an essential role in both morphogenesis of epidermis and limb development. P63 immunoreactivity has been demonstrated in squamous cell and urothelial carcinomas. It is generally absent in most nonsquamous cell carcinomas.Study design Formalin-fixed paraffin-embedded sections from 49 salivary gland neoplasms, representing 6 canalicular adenomas, 11 basal cell adenomas, 17 PLGA and 15 ACC accessioned from 1989 to 2002 by the Department of Pathology, Long Island Jewish Medical Center, New Hyde Park, NY, were stained with an anti-p63 monoclonal antibody. RESULTS: Nuclear p63 reactivity was uniformly positive in PLGA (17/17, 100%). Positive reactivity was also identified in the majority of cases of ACC (13/15, 87%), primarily in the nonluminal myoepithelial-like cells surrounding luminal cells. Canalicular adenoma did not exhibit any p63 immunoreactivity. All basal cell adenomas of parotid origin stained strongly for p63, with staining localized to the peripheral tumor cells situated adjacent to the connective tissue stroma. None of the basal cell adenomas originating in the upper lip stained with p63. In native adjacent salivary gland tissue, p63 reactivity was identified focally in the nuclei of myoepithelial and basal duct cells. CONCLUSIONS: P63 is strongly expressed in basal cell adenoma of parotid origin, and in ACC and PLGA. Canalicular adenoma did not demonstrate p63 staining, consistent with this tumor's putative luminal ductal cell differentiation. Our results suggest that the neoplastic cells in PLGA may represent either a population of p63-positive epithelial stem/reserve cells similar to the basal cells of stratified epithelium, or modified myoepithelial cells. Given the staining pattern of the tumors examined, p63 does not appear to be an ideal marker for distinguishing between ACC, PLGA, and basal cell adenoma.     

Expression and mutations of p53 in salivary gland tumours

A series of 219 salivary gland tumours (103 carcinomas and 116 benign tumours) were analysed for p53 protein expression using immunohistochemistry, and for mutations in p53 gene using non-radioactive single strand conformation polymorphism (SSCP). p53 expression was present in 36% (42/116) of the benign tumours and in 54% (56/103) of the carcinomas. The highest prevalence of p53 expression was found in adenoid cystic carcinomas (69%). followed by mucoepidermoid carcinomas (67%). Of the benign tumours, pleomorphic adenomas showed the highest prevalence of p53 positivity (41%). In malignant tumours, expression of p53 bore no correlation to local recurrence, metastatic disease or survival of the patients. Exons 5 through 9 were analysed and four mutations were found in 20 cases of p53-immunopositive tumours and two in 20 p53-negative tumours. Each of the exons 5.6 and 8/9 had two mutations, whereas no mutations were detected in exon 7.     

Expression of p53 protein correlates with decreased survival in patients with areca quid chewing and smoking-associated oral squamous cell carcinomas in Taiwan

Expression of p53 protein was examined in oral squamous cell carcinoma (SCC) from patients who were areca quid (AQ) chewers and/or tobacco smokers, using anti-p53 antibodies with an immunoperoxidase technique. Positive p53 stain was observed in 47 of 81 (58%) cases of oral SCC. p53 overexpression was found to higher in patients without AQ chewing and smoking habits than in patients with these two habits (80% vs 52%, P=0.076). No significant correlation was found between p53 expression and the patients' age, sex, cancer location, clinical staging, primary tumor TNM status, or histological differentiation of SCC. The Kaplan-Meier analysis showed that the prognosis for patients with p53-negative tumors was significantly better than that for patients with p53-positive tumors (P<0.05). A significant correlation was also observed between positive lymph node status and poor prognosis (P<0.05). These results suggest that p53 may serve as an adjuvant marker of poor survival in patients with oral SCCs in Taiwan.     

Potentially malignant and malignant lesions of the lip. Role of silver staining nucleolar organizer regions, proliferating cell nuclear antigen, p53, and c-myc in differentiation and prognosis

The cellular changes leading to carcinoma of the lip are still not completely understood. This study was carried out on 44 malignant and potentially malignant lesions of the lower lip [30 squamous cell carcinomas (SCC), 7 actinic cheilitis, 3 leukoplakias, and 4 nodal metastases from lower lip SCC]. Silver-stained nucleolar organizer regions (AgNORs) and the immunohistochemical expression of proliferating cell nuclear antigen (PCNA), p53, and c-myc were evaluated on formalin-fixed, paraffin-embedded sections. The results indicate that the size and numbers of AgNORs and the percentage of PCNA-positive cells are sensitive parameters for discriminating between potentially malignant lesions and SCC, and for the prognostic sub-typing of lower lip SCC. Furthermore, while p53 positivity was found more frequently in high-grade carcinomas, p53-positive cellular clones were also found in some potentially malignant lesions, a finding probably related to ultraviolet-related cellular damage. These p53-positive lesions could be considered at higher risk of progression to malignancy than the p53-negative ones, although there is no evidence for this as yet. c-myc positivity was found only in some high-grade carcinomas and metastases, and appeared correlated with the later phases of lip carcinogenesis. The combined evaluation of the proliferation status, together with the changes in p53 and c-myc oncoproteins, might constitute useful markers for the prognostic evaluation of potentially malignant, as well as malignant, lesions of the lip.     

Expression of p53 oncoprotein increases intratumoral microvessel formation in human salivary gland carcinomas

P53 protein and vascular endothelial growth factor (VEGF) expression, and mean intratumoral microvessel density (IMVD) were studied by immunohistochemistry in 31 salivary gland carcinomas, consisting of 11 adenoid cystic carcinomas (AdCCs), 10 mucoepidermoid carcinomas (MECs), 7 acinic cell carcinomas (AcCCs), and 3 squamous cell carcinomas (SCCs). Cases with p53 protein in more than 20% of tumor cells were detected in one AdCC, four MECs, one AcCC, and two SCCs. Both frequency of p53 and VEGF expression, and mean IMVD, were higher in the MECs and SCCs than in the AdCCs and AcCCs. Similarly, both VEGF expression and mean IMVD were significantly higher (P<0.05) in the eight p53-positive tumors than in the 23 negative tumors. Six cases with survival periods less than 5 years showed significantly higher frequency of p53 and VEGF expression and of mean IMVD than those with longer survival periods. These results indicate that p53 expression might partly correlate with VEGF expression and mean IMVD, and be a factor in the survival of patients with salivary gland carcinomas.     

Analysis of human papillomavirus DNA in oral squamous cell carcinomas

Evidence from several laboratories suggests that HPV plays a role in the etiology of squamous cell carcinomas of the oral cavity. A rnultifactorial risk factor profile for the development of oral cancer may include HPV in addition to well-established risk factors such as tobacco and alcohol use. The prevalence of oral carcinomas repotted to be associated with HPV has varied widely due to differences in the sensitivity of the assay used for HPV detection. The aims of this study were: (1) to ascertain the prevalence of HPV DNA in oral squamous cell carcinomas using the most sensitive technique available, the polymerase chain reaction; (2) to determine the type of HPV in the tumors; and 3) to correlate the virologic data with other risk factor data obtained from patients' records. Fourteen (78%) of 18 primary tumors, 6 (67%) of 9 normal epithelial tissues from the patients and 5 (100%) of 5 neck metastases were HPV DNA-positive. Of the 14 HPV DNA-positive primary tumors, specific typing revealed HPV 16 in 2, HPV 18 in 2, HPV 16 and IS in 5, HPV 6/11, 16 and 18 in 4, and HPV 6/11 in 1. HPV types in the normal or metaslatic tissue were usually the same as those in the respective primary tumor. There was no significant association between HPV presence and any of 12 factors or patient characteristics studied.     

The p53 molecule and its prognostic role in squamous cell carcinomas of the head and neck

Despite intense research, the 5-year survival rate for patients with squamous cell carcinoma of the head and neck (SCCHN) is still low. Several different factors have been studied in the search for one or more factors that give important prognostic information at the time of diagnosis. Many recent studies have focused on the TP53 tumour suppressor gene, analysing its gene status and protein status. When looking at p53 protein expression, using immunohistochemistry, no correlation to patient outcome has been seen for the whole group of SCCHN. However, a significant association between p53 expression and poor patient outcome was found when looking only at patients with laryngeal squamous cell carcinomas. Also, in oral premalignant lesions, expression of p53-positive cells in the suprabasal layers of the epithelium has been seen as an indication of impending malignant development. Concerning the prognostic significance of mutations in the TP53 gene, results differ. But when restricting analysis to tumours with mutations causing an obvious change in protein, TP53 mutation was found to be a strong and independent variable for prognosticating survival. This review article gives an up-to-date overview of the p53 molecule and evaluates its possible prognostic role in SCCHN. Today it is clear that the p53 pathway is very important in SCCHN biology and potentially in its treatment. The function and importance of a few other cell cycle proteins connected to p53 are also discussed.     

Role of human papillomavirus infection in carcinogenesis of oral squamous cell carcinoma with evidences of prognostic association

J Oral Pathol Med (2012) 41 : 9–15 Background: Betel nut chewing, cigarette smoking and alcohol drinking are thought to be major environmental risk factors responsible for the development of oral squamous cell carcinomas. Oncogenic human papillomavirus infections have a well‐established association with uterine cervical carcinoma. However, little is known about the exact role of human papillomavirus infections in oral squamous cell carcinomas. This study is designed to elucidate the role of human papillomavirus infections in cancer development and prognosis of oral squamous cell carcinomas. Methods: Molecular techniques including in situ hybridization and immunohistochemistry of p16^INK4A and p53 for evidences of human papillomavirus in tissue micro‐arrays were investigated. Results: Twenty‐four of 65 cases of oral squamous cell carcinomas were found positive for in situ hybridization and 14 were found positive for p16^INK4A. The majority of cases without the evidence of human papillomavirus were related to p53 over‐expression. There were statistically significant correlations between the results of human papillomavirus test and size or extent of the tumor (P = 0.003) or the stage of oral squamous cell carcinomas (P = 0.015). Kaplan–Meier plot analysis demonstrated a tendency of longer survival in cases of oral squamous cell carcinomas with the evidence of human papillomavirus or positive p16 ^INK4A. Conclusions: Human papillomavirus infections may play a unique role in oral carcinogenesis. Our data strongly suggest that human papillomavirus‐positive oral squamous cell carcinomas comprise a distinct clinical and pathological disease entity that appears related to a better outcome with longer survival and bears a causally associated relationship different from other carcinogenic mechanisms.     

Expression of p53 protein in oral squamous cell carcinomas and adjacent non-tumorous mucosa of the floor of the mouth: an archival immunohistochemical study using wet autoclave pretreatment for antigen retrieval

Routinely formalin-fixed and paraffin-embedded material of 22 squamous cell carcinomas of the floor of the mouth (T2NoMo, Ro), together with adjacent dysplastic or normal mucosa, were immunohistochemically investigated using a panel of four anti-p53 antibodies (CM1, PAbl801, DO7, PAb240) subsequent to wet autoclave pretreatment for antigen retrieval. p53 immunoreactivity was detected in 9/22 (40%) carcinomas with PAbl801 and DO7 antibodies, and in 8/22 cases using CM1 and PAb 240. p53-positive tumour cells accumulated predominantly at the invasive front of the carcinomas. A focal or scattered p53 immunoreactivity was observed in the adjacent normal and/or dysplastic mucosa in 17/22 (77%) cases using both CM1 and PAbl801 antibodies, in 10/22 with DO7, and in 8/22 with PAb240. This study has demonstrated examples of different p53 immunophenotypes in the non-tumorous and neoplastic oral mucosa of the same patient without significant correlation to the clinicopathological parameters studied.