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Flow cytometric DNA ploidy in salivary gland tumours
Abstract
This study on 279 tumours of the salivary glands was conducted to analyse whether the assessment of DNA ploidy by flow cytometry may assist histopathology in discriminating benign from malignant types of tumours. The group of benign tumours included 164 pleomorphic adenomas, 51 Warthin's tumours, 7 basal cell adenomas, 2 lipomas as well as 5 other different tumours. All of the 229 benign tumours were diploid. The malignant tumours consisted of 18 adenoid cystic adenomas, 10 mucoepidermoid carcinomas, 5 acinic cell carcinomas, 5 carcinoma in pleomorphic adenoma as well as of 12 other malignancies belonging to 7 different tumour entities. Twelve of 50 malignant salivary gland tumours were aneuploid. There was no significant relationship between the DNA ploidy status and histopathological grading, lymph node metastasis and local recurrence development, respectively. In three cases which initially were taken for pleomorphic adenomas by routine histological examination, aneuploid cell populations exposed by DNA flow cytometric analysis gave rise to a closer inspection of the suspect lesions. Examination of consecutive slides actually revealed small assemblies of carcinoma cells that required a final diagnosis as non-invasive carcinoma in pleomorphic adenoma. The most obvious value of DNA flow cytometry in salivary gland tumours is thus its contribution to assist histopathology in identifying potentially malignant lesions.
... Abnormal DNA content has been related to aggressive behavior in Mucoepidermoid Carcinomas (MEC), Adenoid Cystic Carcinomas (AdCC), Acinic Cell Carcinomas (ACC) and oncocytomas (Onc) [8]. S-phase value is correlated with the behavior of the tumors [13,14]. ...
... Aneuploidy in the present Egyptian study was higher than that reported by other researchers in SGTs [4,7,13]. This may be due to late diagnosis of these tumors in our patients as most cases diagnosed at advanced stage and the sections were taken from deep tumor tissue to increase the number of tumor cells in our samples. ...
... This may be due to the few numbers of cases involved in this study. This result was also observed by other investigators [8,13], who reported that there was no significant relationship between DNA ploidy status and histopathological grading. Other studies in oral cancer [23,24] reported that there is no relationship between the histopathological grade of oral epithelial dysplasia or oral squamous cell carcinoma and the ploidy status. ...
Aim: The aim of this study is to investigate the DNA ploidy and S-Phase Fraction (SPF) of some Salivary Gland Tumors (SGTs) in Egyptian patients and to investigate the correlation between these two biological parameters and the presumptive behavior of these neoplasms.
Methods: Flow cytometric analysis of DNA pliody and S-Phase Fraction (SPF) was done in 50 fresh tumor tissue sections of SGTs which diagnosed as 15 benign and 35 malignant tumors.
Results: 93.3% of the benign SGTs tumors were diploid, while only 34% of malignant tumors were diploid and 66% were aneuploid. The malignant SGTs had higher SPF than the benign tumors but with no significant difference. There was no significant correlation of ploidy status or SPF with the tumor grades of mucoepidermoid carcinomas.
Conclusion: DNA aneuploidy may be a key indicator for tumors activity and malignancy in SGTs, while the SPF has a minor role in the evaluation of SGTs activity.
... Abnormal DNA content has been related to aggressive behavior in Mucoepidermoid Carcinomas (MEC), Adenoid Cystic Carcinomas (AdCC), Acinic Cell Carcinomas (ACC) and oncocytomas (Onc) [8]. S-phase value is correlated with the behavior of the tumors [13,14]. ...
... Aneuploidy in the present Egyptian study was higher than that reported by other researchers in SGTs [4,7,13]. This may be due to late diagnosis of these tumors in our patients as most cases diagnosed at advanced stage and the sections were taken from deep tumor tissue to increase the number of tumor cells in our samples. ...
... This may be due to the few numbers of cases involved in this study. This result was also observed by other investigators [8,13], who reported that there was no significant relationship between DNA ploidy status and histopathological grading. Other studies in oral cancer [23,24] reported that there is no relationship between the histopathological grade of oral epithelial dysplasia or oral squamous cell carcinoma and the ploidy status. ...
Aim: The aim of this study is to investigate the DNA ploidy and S-Phase Fraction (SPF) of some Salivary Gland Tumors (SGTs) in Egyptian patients and to investigate the correlation between these
two biological parameters and the presumptive behavior of these neoplasms.
Methods: Flow cytometric analysis of DNA pliody and S-Phase Fraction (SPF) was done in 50 fresh tumor tissue sections of SGTs which diagnosed as 15 benign and 35 malignant tumors.
Results: 93.3% of the benign SGTs tumors were diploid, while only 34% of malignant tumors were diploid and 66% were aneuploid. The malignant SGTs had higher SPF than the benign tumors but
with no significant difference. There was no significant correlation of ploidy status or SPF with the tumor grades of mucoepidermoid carcinomas.
Conclusion: DNA aneuploidy may be a key indicator for tumors activity and malignancy in SGTs, while the SPF has a minor role in the evaluation of SGTs activity.
... DNA content can be assessed by flow or image cytometry, which are able to detect gross genomic aberrations (17,18). According to some authors, flow cytometry (FCM) of solid tumours has drawbacks because of the process of cell separation and that fractions of aneuploid cells could be lost (19). ...
... There are few large series published in the English language focusing on ploidy analysis for the prediction of tumour aggressiveness, prognosis, or to distinguish benign and malignant salivary gland tumours (12,17,18,23,28). Some authors reported that DNA diploidy may be seen in both benign and malignant lesions, but aneuploidy is mainly seen in malignant lesions (12,24). ...
... Fifty per cent of CEPA (n = 8) were aneuploid, which coincides with our IC results. Driemel et al. (18) reported three aneuploid CEPA (60%, n = 5), which had been initially diagnosed as PA, but the ploidy results aided the oral pathologists to reclassify them to CEPA. In our study, two out of five aneuploid CEPA were intracapsular, which validates the use of ploidy analysis in these tumours. ...
To determine whether DNA ploidy by image cytometry is a good diagnostic tool to distinguish benign and malignant salivary gland tumours.
A total of 62 salivary gland tumours were studied. Cases were histologically diagnosed [haematoxylin and eosin (H&E)]. According to the World Health Organization (WHO) classification, there were 14 mucoepidermoid carcinomas (MEC), 11 adenoid cystic carcinomas (ACC), 10 pleomorphic adenomas (PA), 10 carcinoma ex PA (CEPA), 9 acinic cell carcinomas (ACCa), 3 polymorphous low-grade adenocarcinomas (PLGA), 2 papillary cystadenocarcinomas (PC), 1 myoepithelial carcinoma (MC), 1 undifferentiated carcinoma (UC) and 1 mucinous adenocarcinoma (MA). Paraffin sections (40 microm) were micro-dissected to isolate tumour areas; cell nuclei were extracted and Feulgen-stained cytospin monolayers were analysed using a DNA image cytometry system. For each case, DNA index (DI) was calculated relative to internal controls (lymphocytes; DI=1.0). Cases were categorized as diploid or aneuploid and the proportion of cells over 5c was also calculated.
Fifty-three of 62 salivary gland tumours were uniformly diploid. Only nine cases were aneuploid: five CEPA, one low-grade MEC, one PC, one UC and one MA.
The vast majority of salivary gland tumours were diploid. High-grade malignancies may be aneuploid, and ploidy may be useful to identify malignant change in atypical PA. Further, larger studies are needed to confirm our results and to further evaluate the usefulness of the technique in high-grade lesions.
... Capsular characteristics include completeness of the capsule, formation of satellite nodules or pseudopodia, rupture of the tumor, and positive resection margins 2,13,14 . Multinodularity is generally found in pleomorphic adenoma after recurrence 15,16 . According to a study by Naeim et al. 17 , nodularity is due to infiltration of the stroma, leading to low cellularity. ...
Introduction:
This study investigates the relationship between cellularity and capsular characteristics of pleomorphic adenoma and its influence on operative strategies.
Material and methods:
The capsular characteristics and clinical data of patients with pleomorphic adenomas were reviewed according to Seifert's definition: (1) classic type with balanced amount of cells and stroma, (2) myxoid type with abundant ground substance, interspersed spindle cells, and (3) cellular type with predominance of ductal trabecular structures and little stroma. The immunoreactivity of cellular proliferation (Ki-67) was semi-quantitatively measured using immunohistochemistry. Variables were analyzed using Fisher's test and one-way ANOVA, with (p < 0.05) considered statistically significant.
Results:
The duration of presence was associated with cellularity (p = 0.01). In terms of capsular characteristics, satellite nodules and positive resection margins were not related to cellularity, except for incomplete capsules (p = 0.03). There was no difference in the staining scores of Ki-67 (p = 0.12).
Conclusion:
Lower cellularity reflects higher probability of an incomplete capsule, requiring more consideration for operative strategies to prevent recurrence.
... Synthesis Phase Fraction (SPF) is also correlated with tumors behavior. Generally, tumors with high SPF have demonstrated more aggressive behavior and poorer prognosis (10) . The DNA ploidy analysis and S-phase fraction measurement can be easily performed on both fresh and formalin-fixed tissue specimens, and have become a useful as diagnostic and prognostic tools to be applied on solid malignant tumors in general and particularly on oral cancer (11) . ...
Introduction: Cancer is a serious public health problem in many parts of the world; oral squamous cell carcinoma was the 11 th most common malignant neoplasm in human papulation. The hamster cheek pouch carcinogenesis model is a well-known animal system that closely mimics the development of premalignant and malignant lesions in human oral cancer. The expressions of cancerization process in this model using DNA ploidy and S-phase fraction is the point of interest.
... Synthesis Phase Fraction (SPF) is also correlated with tumors behavior. Generally, tumors with high SPF have demonstrated more aggressive behavior and poorer prognosis (10) . The DNA ploidy analysis and S-phase fraction measurement can be easily performed on both fresh and formalin-fixed tissue specimens, and have become a useful as diagnostic and prognostic tools to be applied on solid malignant tumors in general and particularly on oral cancer (11) . ...
... Synthesis Phase Fraction (SPF) is also correlated with tumors behavior. Generally, tumors with high SPF have demonstrated more aggressive behavior and poorer prognosis (10) . The DNA ploidy analysis and S-phase fraction measurement can be easily performed on both fresh and formalin-fixed tissue specimens, and have become a useful as diagnostic and prognostic tools to be applied on solid malignant tumors in general and particularly on oral cancer (11) . ...
Introduction: Cancer is a serious public health problem in many parts of the world; oral squamous cell carcinoma was the 11 th most common malignant neoplasm in human papulation. The hamster cheek pouch carcinogenesis model is a well-known animal system that closely mimics the development of premalignant and malignant lesions in human oral cancer. The expressions of cancerization process in this model using DNA ploidy and S-phase fraction is the point of interest.
... The DNA ploidy status has been previously investigated in salivary gland tumors and showed that only malignant tumors are aneuploid (6)(7)(8). However, the frequency of aneuploidy in this group of lesions is lower than in other solid tumors (9). ...
Chromosomal instability, leading to aneuploidy, is one of the hallmarks of human cancers. USP44 (ubiquitin specific peptidase 44) is an important molecule that plays a regulatory role in the mitotic checkpoint and USP44 loss causes chromosome mis-segregation, aneuploidy and tumorigenesis in vivo. In this study, it was investigated the immunoexpression of USP44 in 28 malignant salivary gland neoplasms and associated the results with DNA ploidy status assessed by image cytometry. USP44 protein was widely expressed in most of the tumor samples and no clear association could be established between its expression and DNA ploidy status or tumor size. On this basis, it may be concluded that the aneuploidy of the salivary gland cancers included in this study was not driven by loss of USP44 protein expression.
... Grande parte de nossa amostra corroborando com Ohtaké et al. 16 também apresentou áreas de células atípicas que poderiam representar um foco de malignização ou um carcinoma ex-adenoma pleomorfo em sua fase inicial, sendo necessária a atenção à presença destes achados nos adenomas pleomorfos. Driemel et al. 7 acreditam que a citometria de fl uxo do DNA representa uma importante forma de identifi car neoplasias malignas e neoplasias benignas de glândulas salivares. ...
O adenoma pleomorfo (AP) é o tumor de glândula salivar mais comum e caracteriza-se pela diversidade morfológica. Sua transformação maligna origina o carcinoma ex-adenoma pleomorfo (CAP). O objetivo deste trabalho foi traçar o perfi l clínico e morfológico de todos os APs e CAPs dos arquivos do Serviço de Anatomia Patológica da Faculdade de Odontologia de Bauru - USP compreendendo o período de 1963 até 2003 e estabelecer correlações entre os aspectos encontrados e os já descritos na literatura. A metodologia adotada foi a revisão de 58 APs e três CAPs de glândulas salivares através de análise de fi chas clínicas e cortes microscópicos corados em HE dos arquivos do departamento. Constatamos que o gênero mais afetado pelo AP e CAP foi o feminino, com faixa etária da 3ª a 5ª e 6ª a 7ª décadas de vida, respectivamente. A localização mais freqüente foi no palato para ambos, e as características microscópicas representativas foram o estroma fibro-hialino, as estruturas epiteliais em ilhotas, cordões e ductos para os adenomas e nos carcinomas a metaplasia escamosa, e as células claras em estruturas ductais e acinares. A literatura permitiu uma discussão ampla com relação aos diversos aspectos abordados neste estudo de revisão clínica e morfológica dos APs e CAPs. É importante avaliar cada minúcia destas lesões para o estabelecimento do diagnóstico preciso e seguro de AP ou CAP.
... Flow-citometria În cazurile de tumori ale glandelor salivare, valoarea citometriei (numărarea şi măsurarea celulelor sanguine) în secreţia salivară constă în sprijinul histopatologiei prin decelarea posibilelor tumori maligne. Flow-citometria s-a dovedit a fi utilă şi în evaluarea prognosticului în cazurile carcinoamelor adenoidchistice, prin determinarea numărului de cromozomi din ADN-ul celulelor tumorale [11]. Această informaţie pare a fi corelată cu prognosticul general şi supravieţuirea fără boală pe termen lung. ...
Malignant salivary gland tumors make up 6% of all head and neck tumors. Several types of cancer can develop in these glands. Only malignant tumors of the salivary glands are discussed in this paper. The diagnosis and treatment of salivary gland malignancies remain complex, with challenging problems for maxillo-facial surgeon. Scientists have found few risk factors that make a person more likely to develop salivary gland cancer. Salivary gland cancer is uncommon, and there is no widely recommended screening schedule for this cancer. CT scanning or MRI is useful for determining the extent of large tumors, for evaluating extraglandular extension, for determining the actual depth of parotid tumors, and for discovering other tumors in one gland or in the controlateral gland. Fine-needle aspiration biopsy (FNAB) is a valuable diagnostic adjunct in evaluation of head and neck masses but its role in evaluation of salivary gland tumors is controversial. There are also discussed some histologic findings about mucoepidermoid carcinoma, adenoid cystic carcinoma, acinic cell carcinoma, carcinoma ex-pleomorphic adenoma, squamous cell carcinoma and adenocarcinoma developed in salivary glands. Carefully planned and executed surgical excision is the primary treatment for all malignant tumors of the salivary glands. The principles of surgery vary with the site of origin. The extent of surgery is based on the size of the tumor, local extension and neck metastases. Staging of malignant salivary gland tumors is important for predicting prognosis and for accurate comparison of treatment results.
... Die bislang an Speicheldrüsentumoren durchgeführten Untersuchungen sind weit weniger aufschlussreich, was im Wesentlichen auf zwei Ursachen zurückgeführt werden kann: Sie beschränken sich meist auf eine geringe Fallzahl und die DNA-Analysen beruhen häufig auf der Verwendung von archiviertem Tumormaterial, womit in der Regel nur eine unzureichende Messpräzision erreicht werden kann. Da Teilergebnisse dieser mit 279 Fällen umfangreichsten Studie bereits an anderer Stelle veröffentlicht wurden [9, 10, 23], soll hier ein kritischer Überblick über die klinische Bedeutung hoch-auflösender DNA-Durchflusszytophotometrie für die Dignitätsdiagnostik bei Tumoren der Speicheldrüsen im Vordergrund stehen. ...
DNA ploidy and S-Phase fraction (SPF) of 279 salivary gland tumours were analysed using high-resolution DNA flow cytometry. All 229 benign neoplasms were diploid while 12 of 50 malignant tumours showed cell populations with aneuploid DNA content. The SPF values of diploid malignancies were significantly higher if compared with pleomorphic adenomas but did not differ from that of the zystadenolymphoma (Warthin tumour) group. While aneuploidy represents a distinct indicator of malignancy SPF values are of minor relevance for dignity assessment in salivary gland tumours.
... According to some authors ploidy analysis could help to distinguish atypical or recurrent PA from CEPA (34,35). We have studied the ploidy status of 10 CEPA and found 50% to be aneuploid (36). ...
Recent studies have proposed that minichromosome maintenance (Mcm) proteins may be sensitive proliferation markers and may serve as novel biomarkers for prognostication and diagnosis of various pre-malignant and malignant lesions. The aims of this study were to determine the expression of Mcm-2, Ki-67 and geminin in salivary gland (SG) tumours, and to evaluate their usefulness for diagnosis or for prediction of tumour behaviour.
Tissue from 62 SG tumours was assembled in tissue microarray format. There were 13 adenoid cystic carcinomas (ACC), 10 carcinoma ex pleomorphic adenomas (CEPA), 10 mucoepidermoid carcinomas (MEC), 10 polymorphous low-grade adenocarcinomas (PLGA), 10 pleomorphic adenomas (PA) and nine acinic cell carcinomas (AcCC). Clinicopathological data were collected retrospectively and immunohistochemical analyses of Mcm-2, Ki-67 and geminin were performed on all lesions. Labelling index (LI) for each marker was determined by counting the percentage of positive cells in six random fields from three arrays per case.
Mcm-2 expression was higher than Ki-67 and geminin in all tumours studied. Mcm-2 LI was higher in ACC (28.2 +/- 19.2%) than in CEPA, AcCC, MEC, PA and PLGA (5.3 +/- 4.1%, P = 0.001). Mcm-2 LI was higher in CEPA (20.4 +/- 5.0%) than in PA (6.9 +/- 5.0%, P = 0.001). LI did not correlate to tumour grade or outcome for any of the markers or tumour types.
The findings suggest that Mcm-2 may be a sensitive proliferation marker in SG tumours and may be useful for differential diagnosis between PA and CEPA, and ACC and PLGA. Further studies are warranted to assess the value of Mcm-2 as a predictor of recurrence and survival.
Mithilfe hoch-auflösender DNA-Durchflusszytophotometrie wurden DNA-Ploidie und S-Phasenfraktion (SPF) bei 279 Speicheldrüsentumoren bestimmt. Alle 229 benignen Neoplasien waren diploid; 12 von 50 malignen Tumoren wiesen Zellpopulationen mit aneuploidem DNA-Gehalt auf. Die SPF-Werte der diploiden Malignome waren signifikant höher als bei pleomorphen Adenomen, unterschieden sich jedoch nicht von denen der Zystadenolymphome (Warthin-Tumoren). Während Aneuploidie ein Malignitätsmerkmal darstellt, ist der SPF-Wert für die Dignitätsdiagnostik bei Speicheldrüsentumoren nur von eingeschränktem Nutzen.
Überleben und Lebensqualität bei den Krebserkrankungen im Kopf-Hals-Bereich hängen direkt von der Größe bei Erstdiagnose ab. Zusätzlich zur Primärprävention, bei der weitere Anstrengungen erforderlich sind, müssen die Möglichkeiten der Früherkennung verbessert werden. Allerdings fehlt es zum einen in Deutschland an der für ein erfolgreiches Screening erforderlichen Infrastruktur, zum anderen haben die bisher etablierten Untersuchungsverfahren eine zu niedrige Sensitivität und Spezifität. Die Fortschritte, die aktuell im Bereich der Endoskopietechnik, der Nachweismethoden und der Probengewinnung erzielt wurden, eröffnen jedoch Perspektiven im Bereich der Sekundärprävention. Mittels Chromoendoskopie und Narrowband Imaging können Schleimhautveränderungen leichter erkannt werden, mittels konfokaler Endomikroskopie und optischer Kohärenztomografie lassen sich optische „Schnitte” durch das Gewebe legen, und das hyperspektrale Imaging erlaubt eine Gewebsklassifikation anhand spektraler Signaturen. Diese Verfahren ermöglichen somit eine „optische Biopsie”. Auch die Palette der quantitativen und objektiven Parameter, die an minimal-invasiv gewonnenen Proben gemessen werden können, wurde größer und könnte die Grundlage für eine verlässliche Klassifikation der Schleimhautveränderungen sein (multiparametrische Zytometrie, quantitative Histologie). Schließlich könnten Ansätze der Proteomics und der Lab-on-the-Chip-Technologie eine bessere Identifikation der Hochrisikopatienten gewährleisten. Sensitivität und Spezifität all dieser Verfahren wurden jedoch noch nicht geprüft. Vor diesem Hintergrund und angesichts des ungeklärten Nutzen-Risiko-Verhältnisses sowohl für den Einzelnen als auch für die Solidargemeinschaft ist es jedoch bis zu einem Bevölkerungs-basierten Screeningprogramm noch ein weiter Weg.
Survival and quality of life in head and neck cancer are directly linked to the size of the primary tumor at first detection. In order to achieve substantial gain at these issues, both, primary prevention and secondary prevention, which is early detection of malignant lesions at a small size, have to be improved. So far, there is not only a lack in the necessary infrastructure not only in Germany, but rather worldwide, but additionally the techniques developed so far for early detection have a significance and specificity too low as to warrant safe implementation for screening programs. However, the advancements recently achieved in endoscopy and in quantitative analysis of hypocellular specimens open new perspectives for secondary prevention. Chromoendoscopy and narrow band imaging (NBI) pinpoint suspicious lesions more easily, confocal endomicroscopy and optical coherence tomography obtain optical sections through those lesions, and hyperspectral imaging classifies lesions according to characteristic spectral signatures. These techniques therefore obtain optical biopsies. Once a "bloody" biopsy has been taken, the plethora of parameters that can be quantified objectively has been increased and could be the basis for an objective and quantitative classification of epithelial lesions (multiparametric cytometry, quantitative histology). Finally, cytomics and proteomics approaches, and lab-on-the-chip technology might help to identify patients at high-risk. Sensitivity and specificity of these approaches have to be validated, yet, and some techniques have to be adapted for the specific conditions for early detection of head and neck cancer. On this background it has to be stated that it is still a long way to go until a population based screening for head and neck cancer is available. The recent results of screening for cancer of the prostate and breast highlight the difficulties implemented in such a task.
Our aim was to evaluate the DNA content in malignant salivary gland tumours using image cytometry and its possible relationships with clinical and morphologic findings, disease course and prognosis.
The study sample comprised 31 patients diagnosed and treated for primary malignant salivary gland tumours. Formalin-fixed, paraffin-embedded surgical specimens of all patients were Feulgen-stained for DNA content analysis by image cytometry. Statistical analysis was used to investigate possible relationships between DNA content variables and clinical and histological findings, disease course and patient survival.
Seventeen (55%) cases of our sample were graded as DNA diploid, four (13%) as DNA aneuploid and 10 (32%) as DNA multiploid. In 15 (48%) cases, the 5c exceeding rate (5cER) was higher than 1.7%. DNA ploidy correlated with N stage and tumour size. DNA ploidy and 5cER had a statistically significant prognostic influence on overall and disease-free survival in univariate analysis. However, in multivariate analysis, stage classification was the only parameter with an independent prognosis value.
Abnormal DNA content is a common finding in salivary gland cancers. Our results suggest an important role of DNA content analysis in the evaluation of these tumours.
DNA ploidy studies on salivary gland tumours have shown that the proportion of aneuploid cases, although confined to the malignant entities, is considerably lower than for other solid malignancies. To analyse whether the S-phase fraction (SPF) may contribute to discrimination of diploid malignant from benign tumours, DNA flow cytometric data from 45 different malignant salivary gland tumours was compared with that of 121 pleomorphic adenomas. All benign tumours were diploid. Twelve malignant tumours contained aneuploid cell populations. The SPF values for diploid malignancies ranged between 0.9% and 11.0% (mean 3.9%), and between 0.5% and 7.9% (mean 2.7%) for pleomorphic adenomas. A 4% cut-off value gained statistical significance for discriminating diploid malignant tumours from pleomorphic adenomas (P<0.01). The sensitivity for SPF>4% was 46% and the positive predictive value was 40%. A sensitivity of 60% and a positive predictive value of 54% was achieved by combining aneuploidy and SPF>4%. These results show that DNA flow cytometry may contribute to diagnostic assessment in salivary gland tumours.
The mutagenic ranges of aneuploidy, an abnormal number of chromosomes, and gene mutation are analyzed for their abilities to cause the dominant phenotypes of cancer. In the cell, activating gene mutations are buffered because virtually all gene products are kinetically linked into biochemical assembly lines and thus functionally controlled by upstream and downstream enzymes working at their native rates. Inactivating mutations are also buffered, because (i) they are oversupplied with substrate from unmutated upstream enzymes, (ii) are functionally complemented by a second un-mutated allele, and (iii) because in the cell all enzymes work far below saturation. Therefore, gene mutations are typically recessive and thus unable to generate dominant phenotypes. The argument, that all hypothetically carcinogenic gene mutations are exceptional dominants, is hard to reconcile with their failure to transform cells in vitro and in transgenic animals. By contrast,. numerical variations of chromosomes, encoding complete biochemical assembly lines, inevitably generate dominant phenotypes, consider the chromosomes that determine sex or Down syndrome., Thus aneuploidy above an as yet poorly defined threshold emerges as the only plausible mutation to cause the dominant phenotypes of cancer. The aneuploidy hypothesis also explains the exceedingly long latent periods, years to decades, between carcinogen and carcinogenesis. Since aneuploidy destabilizes mitosis by unbalancing mitosis proteins, it catalyzes karyotype evolution that eventually generates carcinogenic karyotypes. Three predictions of the hypothesis have been confirmed experimentally, (i) that human cancer cells, reportedly generated by 'three defined genetic elements', are aneuploid, (ii) that an 'immortal' liver cell line, reportedly safe for human transplantation, is aneuploid and thus preneoplastic, (iii) that the high mutation rates of cancer cells to drug and multidrug-resistance are due to chromosome reassortments.
This prospective study on 348 patients with oral squamous cell carcinoma who underwent radical surgery established a close association between the DNA ploidy status of the primary tumor and the risk of local recurrence development. Nine percent of patients with flow cytometrically diploid tumors developed a recurrence compared to 46% of those with aneuploid tumors. This correlation held true even if evaluated with respect to tumor stage or histological grade. Thirteen percent of the diploid and 59% of the aneuploid group showed lymph node metastasis. These results provide substantial evidence that cytogenetic events that underlie aneuploidy formation from initially diploid progenitor cells are functionally linked to the development of tumor cell populations that have the capability to establish independently growing colonies in foreign tissues.
Numeric aberrations in chromosomes, referred to as aneuploidy, is commonly observed in human cancer. Whether aneuploidy is a cause or consequence of cancer has long been debated. Three lines of evidence now make a compelling case for aneuploidy being a discrete chromosome mutation event that contributes to malignant transformation and progression process. First, precise assay of chromosome aneuploidy in several primary tumors with in situ hybridization and comparative genomic hybridization techniques have revealed that specific chromosome aneusomies correlate with distinct tumor phenotypes. Second, aneuploid tumor cell lines and in vitro transformed rodent cells have been reported to display an elevated rate of chromosome instability, thereby indicating that aneuploidy is a dynamic chromosome mutation event associated with transformation of cells. Third, and most important, a number of mitotic genes regulating chromosome segregation have been found mutated in human cancer cells, implicating such mutations in induction of aneuploidy in tumors. Some of these gene mutations, possibly allowing unequal segregations of chromosomes, also cause tumorigenic transformation of cells in vitro. In this review, the recent publications investigating aneuploidy in human cancers, rate of chromosome instability in aneuploidy tumor cells, and genes implicated in regulating chromosome segregation found mutated in cancer cells are discussed.
We studied 12 histologically malignant salivary tumors that showed complete encapsulation or only limited microscopic invasion. Most cases were histologically characterized by atypical and mitotically active luminal cells forming dilated, angular, variably sized glands in the subcapsular region, varying proportions of nonluminal tumor cells, and a background of central fibrosed hyalinized stroma. The appearance is that of a low-grade carcinoma. Focal higher grade carcinoma was superimposed on this histologic data in three cases. Neither recurrences nor metastases were seen in 11 of 12 patients after surgical resection with a follow-up of 1.2 to 13 yrs (mean, 4.2 years). Ploidy studies were performed on the paraffin-embedded tissue in 11 cases and yielded results for 7 cases. Aneuploid cell populations were found in five tumors ; two had normal diploid populations ; and the ploidy results are not predictive of tumor behavior. This type of salivary gland tumor fits diagnostically within the category of noninvasive and minimally invasive carcinoma ex pleomorphic adenoma (also referred to as in situ and low-grade malignant mixed tumors), a class that requires additional awareness and precise recognition as it signifies a good prognosis after surgical resection.
BACKGROUND
Salivary duct carcinoma (SDC) is a high grade aggressive malignancy of the major salivary glands. Clinical and pathologic features that may be predictive of survival are not well delineated. The microscopic features of SDC are remarkably similar to those of mammary ductal carcinoma, raising the question of whether these tumors share antigenic or hormonal features.METHODS
We reviewed the clinical and pathologic characteristics of 26 cases of SDC treated at the Mayo Clinic from 1960 to 1989. Immunoperoxidase studies and flow cytometry were performed in 25 and 24 cases, respectively.RESULTSThe study population consisted of 22 men and 4 women (mean age, 66 years). The parotid gland was involved in 23 patients and the submaxillary gland in 3. Five of 24 tumors studied were diploid (21%), and 19 (79%) were nondiploid. Nine tumors (35%) recurred locally and 16 (62%) metastasized distantly; 20 patients (77%) died of disease at a mean interval of 3 years after diagnosis. Female sex was the only significant negative prognostic factor analyzed, but positive nodal status approached significance. Paraffin-section immunostaining showed positive reactions for epithelial membrane antigen (100%), keratin (AE1/AE3) (88%), -lactalbumin (88%), GCDFP-15 (76%), and carcinoembryonic antigen (72%); S-100 protein was rarely detected (4%). Stains for estrogen receptor were uniformly negative, but one tumor was positive for progesterone receptors.CONCLUSIONS
The prognosis for SDC is dismal, and clinically useful prognostic factors were not found. Our results do not confirm hormonal concordance between SDC and breast carcinoma. Cancer 1996;77:223-30.
Cytogenetic findings of 21 benign salivary gland tumors, including 14 pleomorphic adenomas, 5 Warthin's tumors, 1 myoepithelioma, and 1 cystadenoma, are reported. The present study confirms that pleomorphic adenomas characteristically have highly specific rearrangements involving only a few chromosome regions (3p21, 8q12 and 12q13–15) which suggests their specific role in the mixed tumors genesis. Warthin's tumors also show nonrandom numerical and structural alterations that were concurrent in one of the cases studied. To our knowledge no cytogenetic data are available in myoepitheliomas and cystadenomas. The former reveals a normal karyotype and the latter shows only clonal numerical alterations (gain of chromosomes 2 and 18). © 1995 Wiley-Liss, Inc.
BACKGROUND
The authors studied a series of 97 consecutive cases of salivary gland tumors to investigate the correlation between the biologic parameters DNA ploidy and S-phase fraction (SPF) and the presumptive behavior of the neoplasms, as well as their potential clinical utility.METHODS
Histopathologic classification and grading of the tumors were evaluated according to 1991 World Health Organization criteria. DNA analysis was performed by flow cytometry in fresh material after propidium iodide staining. Clinical data and follow-up information were obtained from the clinical charts.RESULTSAll the 71 benign salivary tumors showed a DNA diploid pattern. Seven carcinomas (7.2%) exhibited DNA aneuploidy. Eleven (42.3%) of 26 malignant tumors were considered low grade carcinomas, all of them being DNA diploid. Of the remaining 15 tumors, classified as high grade carcinomas, 7 showed DNA aneuploidy. SPF values ranged from 0.6% to 27.7%. A statistically significant difference was found between the mean SPF values of benign and malignant tumors, diploid and aneuploid tumors, and low grade and high grade carcinomas. When a cutoff value of 3% was used to discriminate histopathologic subgroups with prognostic impact, a significant difference was found between benign and malignant salivary tumors, high grade and low grade carcinomas, and high grade and benign tumors (P < 0.001).CONCLUSIONS
The data from this study confirm the low incidence of DNA aneuploidy in salivary gland tumors and suggest the potential utility of SPF estimation in evaluating the clinical behavior of these neoplasms. Cancer 1999;85:273–81. © 1999 American Cancer Society.
52 salivary adenocarcinomas of the years 1965–1980 from the files of the Salivary Gland Registry, Institute of Pathology, University of Hamburg, were evaluated retrospectively with regard to clinical follow up and cytochemically assessed nuclear DNA content. The age distribution showed a peak from the 6th to 8th decade (range 3 to 87 years). The mf ratio was 11.36, the mean age was 59.3 years. Over 80% of the tumours were located in the major salivary glands. The clinical course was characterized by metastases present at initial diagnosis (16 cases), subsequent development of metastases (9 cases), local recurrence (15 cases) or death from tumour (10 cases) and was related to differentiation, grade 3 tumours showing the worse clinical courses. In 37 cases, nuclear DNA content was determined by a single scanning cytophotometry device. 28 cases were diploid, 9 were atypical. The clinical course was significantly related to the histogram type, atypical tumours showing a dismal prognosis.
Benign metastasizing pleomorphic adenoma (BMPA) is a rare tumor of the salivary glands. Despite benign histopathologic features, it can metastasize and is sometimes lethal. No chromosomal data have been reported for this tumor type. We have by chromosome banding and fluorescence in situ hybridization analysis examined the short-term cultures of three skeletal metastases from a BMPA and identified two related hypodiploid clones: 44,XX,dic(3;22)(p11;q13) or der(3)t(3;22)(p11;q?) add(22)(q?),der(9;21)(q10;q10),der(13)t(1;13)(q11;p13)/45,XX,-3,der(9;21 ) (q10;q10),der(13)t(1;13)(q11; p13),?der(22)t(3;22)(q22;q13), +mar. The karyotypic features of this BMPA thus differ from the characteristic cytogenetic findings in pleomorphic adenomas and carcinomas ex pleomorphic adenoma.
A total number of 317 consecutive patients with squamous cell carcinoma of the oral cavity were analysed by DNA flow cytometry. The proportion of tumors consisting exclusively of diploid cells decreased from 75% in T1G1 carcinomas to less than 10% in advanced tumors in favor of cases characterized by the presence of cell lines with aberrant DNA content. This observation indicates a genetic evolution of tumor clones with severe karyotype aberrations from diploid progenitors which obviously represents an ultimate event in the pathogenesis of oral carcinomas. Aneuploidy formation clearly contributes to the acquisition of progressive malignant behaviour as is underlined by a dramatic difference of the survival outcome of patients with diploid tumors (5-year overall survival: 88%) as compared to those who had aneuploid tumors (36%; P<0.001), a relationship which held true even if identical tumor stages were considered. There were no indications of successive genetic changes of the cellular DNA contents during tumor progression which would explain the wide range of variation of the individual DNA values. This observation supports the hypothesis of an aneuploidy formation by a single cytogenetic event, such as an abnormal mitosis. Although there were indications of an even worse survival outcome in the group of patients with peri-diploid tumor cell lines (P<0.1), a significant influence of different DNA contents on the prognosis could not be detected.
This prospective DNA flow cytometric study on 386 primary squamous cell carcinomas of the oral cavity showed that only 18% of the patients with diploid primary tumors had lymph node metastasis on admission compared to 52% of those with aneuploid carcinomas. The aneuploid group without evidence of lymph node involvement at the time of primary tumor treatment carried a 3-fold increased risk for developing late metastasis (23%) compared with the diploid group (8%). The clinical manifestation of occult metastasis in patients with diploid carcinomas was delayed by about two years compared to the aneuploid group. These ploidy-specific differences of the metastatic behaviour held true even if stratified with respect to tumor stage, histological grade and tumor localization. These results provide substantial evidence that cells with gross DNA content aberrations have a significantly higher probability of successfully producing a metastatic colony than flow cytometrically diploid tumor cells. An excellent 5-year survival rate of 90% in the diploid NO group in contrast to 52% in aneuploid NO cases and an even worse survival rate of 21% in patients with lymph node involvement at presentation underline the clinical importance of these findings.
This prospective study on 348 patients with oral squamous cell carcinoma who underwent radical surgery established a close association between the DNA ploidy status of the primary tumor and the risk of local recurrence development. Nine percent of patients with flow cytometrically diploid tumors developed a recurrence compared to 46% of those with aneuploid tumors. This correlation held true even if evaluated with respect to tumor stage or histological grade. Thirteen percent of the diploid and 59% of the aneuploid group showed lymph node metastasis. These results provide substantial evidence that cytogenetic events that underlie aneuploidy formation from initially diploid progenitor cells are functionally linked to the development of tumor cell populations that have the capability to establish independently growing colonies in foreign tissues.
Adenoid cystic carcinoma is an unusual but capricious tumor of salivary gland origin. Characteristically, these tumors follow a relentless clinical course, although some patients experience prolonged disease-free survival. Tumor size, site, and grade have been shown to correlate with tumor behavior. Recent investigation by others has suggested that DNA ploidy as determined by flow cytometry may provide an additional biologic marker of tumor behavior. This study was undertaken to investigate the relationship of DNA ploidy to tumor grade, biologic behavior, and patient outcome. A retrospective comparison of flow cytometric evaluations of paraffin-embedded formalin-fixed tumor specimens with patient outcome and histopathologic grade was undertaken. Follow-up of 4 to 7 years in 26 patients confirmed that the presence of a solid histologic component in the tumor correlated with the presence of recurrent or persistent disease (p = 0.04). Twenty-two of 28 tumors (78%) were found to be aneuploid on at least one section. Comparison of DNA ploidy with either patient outcome or the presence of a solid component did not achieve statistical significance, although a trend was suggested. This study confirms previous studies demonstrating the validity of histopathologic evaluation of tumor grade in the prediction of the biologic behavior of adenoid cystic carcinoma. However, our findings suggest that DNA ploidy has only limited value as an additional marker of tumor behavior in this patient population.
The nuclear DNA content in the tumor cells of 17 pleomorphic adenomas (PA) and 26 adenoid cystic carcinomas (ACC) was assayed by cytofluorometry to clarify the etiology of the differences in their biological nature. Aneuploidy was found in only two solid-pattern ACC samples; all the other samples had diploid stemlines. With respect to the polyploid cell rate and the S + G2M fraction, the differences between the four histological patterns of PA were not significant. In the ACC, the S + G2M fraction was significantly higher in solid pattern than in cribriform or trabecular pattern tumors, which may indicate that proliferative activity is higher in the solid pattern than in the other two. The polyploid cell rate in ACC was significantly higher in the solid pattern than in the cribriform pattern carcinomas. There was no significant difference in the mean polyploid cell rate between PAs and ACCs. The mean S + G2M fraction was significantly higher in all histological patterns of ACCs than in PAs. These results suggest that proliferative activity is greater in any one of the histological patterns of ACC than in PA. These findings may largely explain the biological differences between the two tumors.
This chapter discusses the use of 4', 6-diamidino-2-phenylindole (DAPI) for flow cytometry (FCM). DAPI has been used from many years. It proved to be a specific, highly fluorescent stain, very well suited for FCM of DNA in whole cells, in nuclei, and in chromosomes. The chapter presents the staining protocol that has been developed for use in cell suspensions after fixation with 70% ethanol. It is applicable in specimens that have to be stored before measurement. The procedure consists of a pretreatment of cells with citric acid plus Tween 20 to produce a suspension of isolated nuclei, followed by the addition of a solution of disodium phosphate or trisodium citrate with DAPI, which raises the pH and stains the DNA. This protocol is recommended for the accurate and reproducible measurement of the nuclear DNA, especially for the assessment of DNA content variations in cell populations affected by chemical or physical mutagens and for the discrimination of spermatogenic cells.
Acinic cell carcinomas of the Salivary Gland Registry, Institute of Pathology, University of Hamburg, West Germany, from 1965 to 1980 (n = 55) were evaluated retrospectively with respect to histologic, cytophotometric, and clinical data. The majority of the tumors (92.8%) were located in the parotid gland. Two thirds of the patients were female; one third were male. Mean age at primary diagnosis was 55.4 years. The tumors were graded into highly differentiated (76%) or less differentiated forms (24%) according to classic histologic and cytologic criteria. The clinical course was characterized by no recurrence in 15 cases; in 17 cases, recurrences developed, and 12 patients died of their tumor, some as late as 240 months after primary diagnosis. Differentiation showed a weak correlation with the clinical course. In 35 cases, nuclear DNA content of tumor cells was assessed cytochemically. The tumors were "diploid" or "near-diploid" in 34 cases; DNA content showed no correlation to the clinical course. As a result of long-term follow-up, it becomes evident that acinic cell carcinoma is prone to develop recurrences and metastases. Complete tumor removal during the primary operation seems to be important for controlling the disease inasmuch as the ostensible prognostic predictors evaluated here proved to be unreliable.
The cytogenetical observations in eight successfully cultured human adenolymphomas are reported. When the results were considered with those of two previously reported cases, three main stemline groups could be distinguished: (a) one with a normal karyotype and noted as a primary or secondary stemline in all hitherto studied tumors; (b) a second group with only numerical changes, either loss of the Y chromosome or trisomy or monosomy 5; and (c) a third group with only structural changes, as a rule with one or two reciprocal translocations. With regard to the last group, studies of many more cases are necessary to decide whether distinctive subgroups exist. Analyses using molecular methods are also urgently needed to clarify whether the normal stemline cells contain submicroscopic changes.
The clonal DNA content of tumor biopsy specimens of 110 patients with primary and untreated squamous cell carcinoma of the oral cavity was determined by flow cytometric study. The ploidy status was compared with tumor size and histologic grading. Thirty tumors (27.3%) were diploid; in 80 cases (72.7%) cell lines with abnormal DNA content were detected. The portion of aneuploid tumors increased with decreasing degree of histologic differentiation (P less than 0.001) from G1 (38.1%) to G2 (76.6%) and G3 (92.0%). Only one of 13 T1 carcinomas (7.6%) showed abnormal clonal DNA content, but 76.9% of T2 and 90.6% T3 tumors (P less than 0.001) did. The emergence of aneuploid clones obviously represents a marker of malignancy progression in oral carcinoma.
Fifteen acinic cell carcinomas from an equal number of patients were analysed for their DNA content and proliferative (S-phase) index by flow cytometry from archival tissues. Seven of the carcinomas manifested a diploid DNA content. None of the patients with diploid acinic cell carcinomas died of their carcinomas and none developed metastases in follow-up periods extending for 10 or more years. Four of eight patients with aneuploid acinic cell carcinomas have died because of their malignancies within a 10 year period after the first surgical removal of the carcinoma. Five of the eight patients exhibited metastases. Although the number of cases does not permit strong correlations between histopathological features, abnormalities in DNA content and outcome of patients, it was noted that carcinomas with prominent necrosis, tubuloductal differentiation and 'dedifferentiated' areas displayed more aggressive biological courses.
Flow cytometric study has been used to measure the cellular DNA content of solid tumors for the last decade, and of paraffin-embedded tumor specimens for the last 5 years. Ploidy and proliferative activity are the two properties commonly measured by DNA content flow cytometric study. The ability to study archival, paraffin-embedded tumors has hastened an appreciation of the prognostic utility of this assay. Either abnormal ploidy or elevated proliferative activity predict a worsened disease-free or overall survival in most common adult malignancies. Both abnormalities are associated with poor outcome in locoregional breast, non-small cell lung, and colorectal cancers, and in all stages of ovarian cancer. Abnormal ploidy is also a dire prognostic indicator for cancers arising from the kidney, bladder, prostate, and endometrium. Clinical management of patients with these diseases may be aided by studying their tumors for these objective markers of biological aggressiveness.
52 salivary adenocarcinomas of the years 1965-1980 from the files of the Salivary Gland Registry, Institute of Pathology, University of Hamburg, were evaluated retrospectively with regard to clinical follow up and cytochemically assessed nuclear DNA content. The age distribution showed a peak from the 6th to 8th decade (range 3 to 87 years). The m:f ratio was 1:1.36, the mean age was 59.3 years. Over 80% of the tumours were located in the major salivary glands. The clinical course was characterized by metastases present at initial diagnosis (16 cases), subsequent development of metastases (9 cases), local recurrence (15 cases) or death from tumour (10 cases) and was related to differentiation, grade 3 tumours showing the worse clinical courses. In 37 cases, nuclear DNA content was determined by a single scanning cytophotometry device. 28 cases were diploid, 9 were atypical. The clinical course was significantly related to the histogram type, atypical tumours showing a dismal prognosis.
Forty-five patients with adenoid cystic carcinoma (ACC) of salivary glands were retrospectively studied to determine the prognostic use of DNA ploidy analysis compared with clinicopathologic features. Nuclear suspensions were prepared from 37 of these tumors by the Hedley technique on paraffin-embedded material. The DNA content was analyzed by flow cytometry after propidium iodide staining. Thirty-five tumors were diploid and 2 were tetraploid. Mean survival was 117 and 52 months for the diploid and tetraploid patients, respectively. The median S-phase fraction (Spf) of the 35 diploid tumors was 4.45%. Of the 17 diploid patients who died of disease, there were 11 tumors with high Spf (greater than 4.45%) and 6 tumors with a low Spf (P less than 0.05 chi-square test). The presence of more than 30% of a solid pattern in the tumor was strongly associated with poor median survival in Kaplan-Meier survival curve analysis (P less than 0.05 log rank test). Grade, stage, recurrence, and metastases were also found to be important prognostic factors. Because few tumors were nondiploid, these results suggest that S-phase fraction analysis may be a more useful prognostic indicator than ploidy classification.
The present review considers the three main issues of the cytogenetic analysis of human solid tumors: the technical limitations, the difficulty in interpreting the available results, and the uncertainty affecting any hypothesis about the role of chromosome changes in tumorigenesis. Information is given on the recent improvements in the field of tumor cell culturing and karyotyping, with a critical discussion of more than 1300 cytogenetic studies from 18 different tumors, and clues for a synthetic understanding of the cytogenetics of malignancy.
Karyotypic progression may be viewed in at least two ways. One approach seeks evidence for increasing and progressive deviation from the normal chromosome pattern in tumors. The clearest examples, found in some leukemias, are those in which successive karyotypic changes are superimposed on an already aberrant cell population. Evidence of chromosomal progression within solid tumors is far less frequent, possibly because the tumors themselves are at a relatively late stage in their evolution. An alternative approach, therefore, attempts to correlate the extent of karyotypic deviation with other aspects of tumor progression. Recent data, based on classical cytogenetic analyses and flow cytometry, are presented to determine relationships between karyotype and specific origin and morphology of tumors. The predominant theme which emerges, not surprisingly, is that the more deviant chromosome patterns are associated with other measures of increased biologic malignancy. What is surprising is the degree to which these properties are expressed in primary tumors and the relative lack of evidence for further karyotypic evolution with recurrence or metastasis.
Examples of genetic instability, evolution through polyploidy, gene amplification, and selection for specific chromosomal rearrangement are found in populations of premalignant and malignant human cells. There is increasing recognition of the importance of tumor-specific chromosome aberrations in the stepwise progression from the normal to the fully neoplastic cell.
Thirty-one salivary gland epithelial-myoepithelial carcinomas from 26 patients were studied by DNA flow cytometry, and immunostaining for Ki-67 and HER-2/neu oncogene product. The results were correlated with clinicopathologic factors and patient outcome. The tumor most commonly involved the parotid gland, and mainly affected patients in their 6th to 8th decades. The clinical course was characterized by a high incidence of local recurrence (50%) and not infrequent distant metastasis (25%). None of the patients in this cohort died of disease. DNA content analysis revealed 21 neoplasms with DNA diploidy and 5 tumors with DNA aneuploidy; all aneuploid cases were near-diploid (hyperdiploidy) and showed low proliferative activity. All aneuploid and 60% of the diploid neoplasms developed recurrences and/or metastases. Immunohistochemical analysis of Ki-67 proliferation markers also showed low overall growth fractions. Interestingly, Ki-67 immunoreactivity was largely restricted to myoepithelial cells, suggesting a central role for this cell in the development of these tumors. HER-2/neu oncogene analysis failed to demonstrate overexpression in any of the tumors examined.
This study indicates that epithelial-myoepithelial carcinoma is a low grade malignant neoplasm with a high propensity for recurrence. IIER- 2/neu oncogene and Ki-67 offer no additional advantages over current factors in the biologic evaluation of these neoplasms. DNA aneuploidy may allow for the identification of a subset of tumors that is more prone to recurrence and metastasis, but further studies with extended followup are needed.
Twelve cases of salivary duct carcinoma were examined clinically, pathologically and by flow cytometry to quantify their histological features as well as attempt to identify factors predictive of patient outcome. All of the tumours arose in the parotid gland. Eight of the twelve patients were male. Four patients died of disease (median survival 12.5 months); three are alive with disease; and five are alive with no evidence of disease (mean follow-up of 50 months). Two tumours arose in a pre-existing pleomorphic adenoma. Positive lymph nodes were present in eight of ten patients sampled; patients with two or more positive lymph nodes tended to die of their disease or be alive with metastases. Comedo necrosis, perineural invasion and vascular invasion were common findings by light microscopy. Ten of the twelve tumours were aneuploid. Neither clinical stage, tumour size, aneuploidy nor histological features correlated with patient outcome. This study confirms the aggressive nature of salivary duct carcinoma.
Extensive chromosome and DNA content heterogeneity within and between human solid tumors has been observed using both classical karyotype and DNA cytometry. Experimental evidence suggests, at least in some tumor types, that DNA stemline heterogeneity in tumor progression is according to a three-compartment model with diploidy shifting to tetraploidy and then to hypotetraploidy.
The human colorectal adenoma-carcinoma sequence appears as one of the most potentially informative systems for the study of DNA stemline heterogeneity in human tumors since adenomas, adenomas with early cancer, and adenocarcinomas in nontreated patients represent clear morphologically distinct stages of tumor progression. The quantitative measurement of DNA content in the G0.1 phase of the cell cycle was performed by high resolution flow cytometry in a large number of cases using multiple fresh or frozen samples.
The distribution of the degree of DNA aneuploidy values, also known as DNA index, (DI not equal to 1) among 467 human precancer and cancer colorectal lesions was clearly nonrandom and showed modes at DI = 0.9, 1.2, 1.5, 1.8, and 2.2 with a clear valley at DI = 1.3. Whereas DNA aneuploid subclones within early lesions were up to about 80% near-diploid (DI < or = 1.3), DNA subclones within advanced cancer were in the vast majority with DI = 1.5-1.8 and, in a small fraction, with DI > 2. In addition, in adenomas with early cancer, which represent a link in colorectal tumor progression, early and late DNA stemlines often coexisted.
The natural history of the colorectal adenoma-carcinoma sequence appears to be characterized by near-diploid subclones as early events and by late-stage hypotetraploidy. A new model is proposed that predicts the origin of the near-diploid subclones by "loss of symmetry" in cell division and their evolution (in particular hypodiploid) to the late-stage hypotetraploidy by tetraploidization. This model agrees with recent data associating molecular biology events, cytogenetic data, and DNA stemline heterogeneity in colorectal and other tumor systems.
A retrospective study was performed on 51 patients with adenoid cystic carcinomas to see whether DNA ploidy, tumor stage, and histopathologic grading correlated with prognosis.
Histopathologic grading was performed according to Szanto et al and DNA content was estimated from archived material using the technique by Hedley et al.
Thirty-nine tumors were DNA diploid and 12 were DNA aneuploid. Histologic grade III was more often associated with DNA aneuploidy than the lower grades (p = 0.011). DNA ploidy also correlated with clinical stage (p = 0.011). Log-rank analysis and Cox regression analysis of treatment failures revealed significant findings for S-phase value and DNA ploidy.
Our results indicate that DNA ploidy estimations, S-phase value, and histologic grading are prognostic factors in adenoid cystic carcinomas. These examinations should therefore be incorporated in the evaluation of patients with adenoid cystic carcinomas.
We used flow cytometry in a retrospective study of pleomorphic adenoma and carcinoma arising in pleomorphic adenoma, using paraffin-embedded tissue, to assess the relationship among proliferative activity, ploidy, and recurrence or malignant transformation. Twenty-four specimens obtained from 22 tumors were acceptable for analysis (co-efficient of variation, < or = 7.0), including multiple samples from two tumors. Fourteen tumors (13 benign and one malignant) were diploid. Six tumors were aneuploid: four benign pleomorphic adenomas and two carcinomas arising in pleomorphic adenoma. Two tetraploid tumors were malignant recurrences from the same patient. Of the recurrent tumors (nine benign and four malignant), 54% were aneuploid. The highest S-phase fractions were observed in recurrent and malignant pleomorphic adenomas. Immunostaining with p105, a nuclear proliferation antigen, revealed increased proliferative activity in a majority of pleomorphic adenomas. Increased proliferative activity and aneuploidy occurred in benign pleomorphic adenomas.
The cytogenetic findings in 13 cultured Warthin's tumors (papillary cystadenoma lymphomatosum) are reported. Only one case showed an abnormal stemline. This was pseudodiploid and characterized by three different reciprocal translocations and one deletion. Small abnormal sidelines, however, were seen in three additional cases. All except one of the 12 tumors with a normal stemline contained variant cells. These showed a variety of numerical and/or structural aberrations. From this study it is obvious that to determine whether or not cytogenetically distinctive subgroups actually exist in cystadenolymphomas (as in pleomorphic adenomas) the number of cases must be greatly amplified.
Malignant salivary gland tumors are rare, constitute a heterogeneous group and are often difficult to diagnose histologically. This is borne out by the fact that in the present study 43.2% of 118 salivary gland tumors originally diagnosed as mucoepidermoid, acinic cell and adenoid cystic carcinomas had their original diagnosis altered upon reclassification. Patients with confirmed adenoid cystic carcinomas had a much worse prognosis than those with mucoepidermoid and acinic cell carcinomas.
DNA flow cytometry showed that very few of the above mentioned three types of malignant neoplasms revealed aneuploid DNA stemlines, indicating that this is not a relevant prognostic tumor marker within the groups. However, several of the tumors that had their diagnosis changed, mostly to undifferentiated adenoor squamous cell carcinomas, showed aneuploid DNA stemlines. The survival time of patients with aneuploid tumors was considerably reduced compared to those with diploid tumors. Among confirmed acinic cell, mucoepidermoid and adenoid cystic carcinomas the S-phase fraction was a significant prognostic factor, as it was among all tumors examined. This indicates that DNA aneuploidy and S-phase fractions are potential prognostic factors for malignant salivary gland tumors, and that DNA flow cytometry may assist the characterization of such tumors.
To test the prognostic ability of flow cytometry and counts of silver-enhanced intranuclear nucleolar organizing regions (AgNORs) in acinic cell carcinoma.
Using statistical methods to establish if analysis of DNA content, percentage DNA synthetic (S)-phase, and the AgNOR counts on 45 cases of acinic cell carcinoma with clinical follow-up ranging from 10 to 379 months are predictors of time to recurrence or time to death due to acinic cell carcinomas.
Whether tumors with low AgNOR counts and diploid DNA are favorable acinic cell carcinomas and the converse, aneuploid DNA and elevated AgNOR count, predict unfavorable neoplasms.
Tumors with a diploid DNA content had as unfavorable a clinical course as aneuploid acinic cell carcinomas. Similarly, S-phase and AgNOR count results showed considerable overlap when separated into carcinomas with or without local recurrence, metastasis, or death due to tumor. Statistical evaluation also failed to provide predictors of clinical course based on ploidy, percentage S-phase, or AgNOR counts.
The results, although negative, are important in showing that data on DNA content, cell cycle, and nuclear limits useful in other neoplasms are of limited practical application in establishing predictors of time to recurrence or time to death in acinic cell carcinomas. Solving the enigmas, for clinicians and pathologists, associated with acinic cell carcinomas will require further information about the biology of this neoplasm.
Salivary duct carcinoma is an uncommon and relatively unknown clinically aggressive adenocarcinoma of salivary origin that histologically demonstrates a remarkable resemblance to invasive carcinoma of the breast. We report the clinicopathologic features of 13 cases that were also examined by image analysis for DNA ploidy. The results were then analyzed collectively with the less than 100 cases of salivary duct carcinoma reported in the English-language literature to define the characteristics of this unusual neoplasm. The 12 men and one woman averaged 68 years of age (range, 49 to 90 years). All tumors arose in the parotid (10 cases) or submandibular glands (three cases). Nine tumors were aneuploid, three diploid, and one was indeterminate because of insufficient tissue. Follow-up (median, 24 months) was available in 12 cases: three patients died of disease, six were alive without disease, and three died of other causes. Combining our cases with those in the literature, a total of 104 cases, confirms that salivary duct carcinoma is a highly malignant neoplasm with distinctive clinical and pathologic features. It arises almost exclusively in the major salivary glands (96% of cases), is three times more common in men, and usually occurs in patients over 50 years of age (range, 22 to 91 years). One-third of patients experience local recurrences, 59% develop positive regional lymph nodes, 46% have systemic metastases (lungs and bones), and 65% die of their disease, usually within 4 years of diagnosis. Determination of tumor ploidy has no prognostic significance. The presence of distant metastasis was the only clinicopathologic feature that was statistically associated with prognosis (p = 0.02); all patients with systemic metastasis died of disease.
Intratumor DNA heterogeneity was investigated by flow cytometric analysis of multiple samples taken from different sites of 8 benign and 16 malignant primarily resected salivary gland tumors. All benign tumors had diploid DNA content. The overall incidence of DNA diploidy in 16 malignant cases examined was 50%. Intratumor differences in DNA ploidy were observed in four malignant tumors (25%); 2 of these 4 heterogeneous tumors contained both aneuploid and diploid cell clones. The remaining 12 tumors showed a homogeneous DNA content in the different specimens; 8 were diploid, 3 aneuploid, and 1 was polypoid. The DNA nondiploid tumors were clinically more advanced than the DNA diploid ones (p < 0.01). The tumor proliferation rate (fraction of cells in S-phase) was higher in DNA nondiploid samples than in diploid ones (p < 0.01). The DNA nondiploid tumors seemed to recur more often than DNA diploid ones did. The data emphasize the usefulness of DNA measurements for the characterization of malignant salivary gland tumors but also the importance of adequate sampling in assessing their DNA ploidy.
Based on the hypothesis that three main cytogenetic subtypes of salivary gland pleomorphic adenomas can be distinguished which may also represent different etiologic entities, we investigated whether these subtypes correspond to clinical, histologic, or biologic features of 220 tumors karyotyped (including 117 tumors with detailed clinical history and histologic subtyping). The following results were obtained. As compared with the group of patients showing salivary gland pleomorphic adenomas with an apparently normal karyotype, the patients in the "8q12-group" were significantly younger (51.1 years versus 39.3 years, p < 0.001). The distribution of histologic subtypes also showed highly significant differences between the groups. Whereas the breakpoint in the 8q12 group was always mapped to a single band, no exact localization of the breakpoint in the group of tumors showing chromosome number 12 abnormalities was possible. In most cases, however, the breakpoints were clustered to 12q15. Finally, all tumors with 8q12 breakpoints showed a characteristic in vitro cellular morphology which was also observed in a few tumors with an apparently normal karyotype but in none of the tumors with the 12q13-15 breakpoint.
The occurrence of abnormal nuclear DNA content in major salivary gland adenomas is not well known and its correlation with tumor recurrence has not been documented previously. From 1987 to 1991, 119 consecutive major salivary gland adenomas were operated on at Turku University Central Hospital. These tumors were analyzed by flow cytometry and 100 (84%) were found to be diploid, 12 (10%) near-diploid and 7 (6%) aneuploid with DNA indexes > 1.15. The mean proliferation rate measured as a percentage of cells in the S-phase fraction was 2.5 +/- 1.6%. The histological slides were then blindly reclassified according to current World Health Organization classification. As a result histological classification was changed in 3 tumors: malignant cells were found in 2 aneuploid tumors and 1 diploid neoplasm. Preoperative cytological fine-needle aspiration biopsy had been considered as possibly malignant in 2 of these cases. Among all case material 10 specimens were recurrent tumors; although the tendency to recur depended on the extent and adequacy of the surgery performed, multiple recurrences were associated with non-diploid tumors.
In this study, 32 pleomorphic adenomas (PAs) and seven adenoid cystic carcinomas (ACCs) were analysed for the evaluation of proliferating cell nuclear antigen (PCNA) indices and flow cytometric variables. Our aim was to assess any possible relationship between these parameters and the clinico-pathological variables and to clarify their histogenesis and reasons for their biological differences. The tumours were divided into three groups, mainly epithelial (E), myxoid (M) and chondroid (C); PCNA labelling index (LI) and weighted mean index (WI) and the WI/LI ratio were analysed in the predominant components; a single PCNA index, weighted by the percentage of each component, was also calculated. Only WI/LI was found to be significantly different in the three components, while PCNA single index did not show either significant differences by sex, age, site and size, or any correlation with the S phase fraction. A significant difference was found between PAs and ACCs by site (P < 0.01) and DNA ploidy (P < 0.05); furthermore, all PCNA indices (single index) were significantly lower in PAs than in ACCs.
The aim of the present study is to characterise the cell kinetics of pleomorphic adenoma of the parotid gland by assessing DNA content and proliferating cell nuclear antigen (PCNA) positivity. In 22 parotid adenomas, DNA content was measured by densitometry in histological serial sections stained with Feulgen's method and PCNA positivity was determined by immunohistochemistry with the monoclonal antibody PC10. To assess the proliferative activity, DNA index and PCNA index were evaluated. It was possible to distinguish two types of adenoma. In Group I there was a prevalence of diploid cells with a low PCNA index. Group II is represented by adenomas with a large percentage of triploid cells and a PCNA index significantly higher than that of Group I. Our findings suggest that the possibility of recurrence or malignant transformation depends on intrinsic biological properties of each adenoma.
We studied 12 histologically malignant salivary tumors that showed complete encapsulation or only limited microscopic invasion. Most cases were histologically characterized by atypical and mitotically active luminal cells forming dilated, angular, variably sized glands in the subcapsular region, varying proportions of nonluminal tumor cells, and a background of central fibrosed hyalinized stroma. The appearance is that of a low-grade carcinoma. Focal higher grade carcinoma was superimposed on this histologic data in three cases. Neither recurrences nor metastases were seen in 11 of 12 patients after surgical resection with a follow-up of 1.2 to 13 yrs (mean, 4.2 years). Ploidy studies were performed on the paraffin-embedded tissue in 11 cases and yielded results for 7 cases. Aneuploid cell populations were found in five tumors; two had normal diploid populations; and the ploidy results are not predictive of tumor behavior. This type of salivary gland tumor fits diagnostically within the category of noninvasive and minimally invasive carcinoma ex pleomorphic adenoma (also referred to as in situ and low-grade malignant mixed tumors), a class that requires additional awareness and precise recognition as it signifies a good prognosis after surgical resection.
To evaluate and correlate morphologic features, glial fibrillary acidic protein (GFAP) reactivity and DNA content parameters in 32 pleomorphic adenomas of the salivary glands.
The adenomas were subclassified according to the proportion of stroma and type of stromal differentiation. DNA flow cytometry was carried out on paraffin-embedded material. GFAP reactivity was determined immunohistochemically and evaluated as the percentage of positive cells. Follow-up ranged from 17 to 71 months; no recurrences were observed.
Seven cases were aneuploid, 10 peridiploid and 15 diploid. Nondiploid tumors had a significantly higher S-phase fraction. Nondiploid adenomas were significantly associated with a greater percentage of stroma, while S-phase fraction showed only a trend toward being higher in tumors with a greater quota of stroma. Ploidy type and S-phase fraction were unrelated to sex, age, tumor diameter or site. GFAP reactivity was unrelated to subtype or S-phase fraction; a higher frequency of diploid tumors was seen among cases with a greater number of reactive cells.
Aneuploidy is present in a significant percentage of typical cases. It is unrelated to tumor bulk and appears to have no effect on recurrence as long as surgical excision is complete.
We correlated MR imaging findings with those from flow cytometry and determined prognostic factors of patients with major salivary gland lesions.
DNA ploidy (11 aneuploid, 35 diploid lesions) and S-phase fraction (SPF) (12 high-SPF, 28 low-SPF lesions) percentages as determined by flow cytometric technique in 46 major salivary gland lesions were correlated with MR findings and signal-intensity ratios of lesion to muscle on fast spin-echo T2-weighted images, unenhanced spin-echo T1-weighted images, and gadolinium-enhanced fat-suppressed spin-echo T1-weighted images. SPF percentages were correlated with the three signal-intensity ratios by stepwise regression analysis. Prognostic indicators of disease-free survival were assessed with Cox multivariate analysis (range of follow-up, 2-50 months; mean, 16.6 months).
Signal-intensity ratios for all pulse sequences were significantly smaller in aneuploid lesions than in diploid lesions. Incidence of ill-defined margins (p < .001), invasion (p = .014), and hypointensity to the gland on T2-weighted images (p = .047) was significantly higher in aneuploid lesions than in diploid lesions. Of these, signal-intensity ratios on enhanced T1-weighted images were most accurate for predicting aneuploidy. A threshold of 1.55 for signal-intensity ratios on enhanced T1-weighted images yielded the highest accuracy (86%) for aneuploidy. Signal-intensity ratios on T2-weighted images (p = .025) and enhanced T1-weighted images (p < .001) were significantly smaller in high-SPF lesions than in low-SPF lesions. A threshold of 1.73 for signal-intensity ratios on enhanced T1-weighted images yielded 73% accuracy for high-SPF lesions, which was inferior to the prediction possible from ill-defined margins (80% accuracy). Aneuploidy (p = .008), ill-defined margins (p = .036), and signal-intensity ratios on unenhanced T1-weighted images (p = .008), related significantly and negatively to disease-free survival. A signal-intensity ratio of 1.22 or less for unenhanced T1-weighted images indicated a high risk of developing recurrence (100% sensitivity).
MR findings and signal-intensity ratios can reflect DNA ploidy and SPF status and can predict prognoses for patients with major salivary gland lesions.
Thirty-four mucoepidermoid carcinomas were studied retrospectively with regard to histological and clinical parameters. In 28 of the tumors DNA patterns were also assessed using flow cytometry. Twenty-two of the 28 tumors (79%) were DNA diploid and 6 (21%) DNA aneuploid. Two tumors (7%) showed intratumoral DNA as indicated by different stemlines in specimens investigated from different parts of the tumor. DNA ploidy correlated significantly with cervical lymph node status (P < 0.01), but not with tumor size or histological grade. The mean S-phase value was 2.7% and was significantly higher in aneuploid samples than in diploid ones (P < 0.05). The recurrence rate was significantly lower for patients with stage I and II tumor compared with those with stage III and IV disease (P < 0.01). Five aneuploid tumors showed significantly higher recurrence rates (5/6) than the diploid ones (1/22) (P < 0.01). In univariate analysis for survival, only N stage tumor (P < 0.05) and tumor DNA ploidy (P < 0.0003) had significant prognostic influence. Thus, DNA ploidy seems to be a valuable parameter for evaluating the biological behavior of mucoepidermoid carcinomas of the salivary glands.
Twenty-eight examples of papillary cystadenoma lymphomatosum (Warthin's tumour) of the parotid gland were analysed by high-resolution DNA flow cytometry. The mean coefficient of variation was found to be 1.19% (SD: 0.41). All tumours were DNA diploid. These results did not correspond with expected deviations based on published chromosomal studies. Also, the homogeneously low S-phase fractions (mean: 4.8%; SD: 2.7) found did not support the hypothesis of etiologically distinctive subgroups in these tumours.
We report the cytogenetic, fluorescence in situ hybridization (FISH), and DNA ploidy analyses of a high grade carcinoma ex-pleomorphic adenoma of the submandibular gland. Our overall combined analyses showed a marked DNA aneuploidy and numerical abnormalities involving all chromosomes. Cytogenetic analysis revealed a near tetraploid modal chromosomal number with tetraploid loss of chromosomes Y, 1, 6, 9, 11, 14, 15, 17, and 19-21 and hypertetraploid gain of chromosomes 7, 8, and 22. The structural abnormalities included der(1;14)(q10;q10), del(6)(q15q34), +del(6)(q15q34), +der(8) t(1;8)(q12;q12.2),der(9;19)(q10;q10),add(14)(p11.2),i(20)(q10),der(21) t(8;21)(q11.2;q22.3),+der(21)t(8;21) (q11.2;q22.3). Interphase FISH of the primary and short-term cultured cells using directly labeled pericentromeric probes for chromosomes 6-12, 17, 18, and Y resulted in alterations corresponding to the cytogenetic findings. DNA ploidy analysis of both the primary and cultured tumor cells showed a hyperdiploid stemline with DNA indices of 2.6. The results indicate that: (1) marked numerical, structural chromosomal, and DNA content abnormalities are present in this tumor; and (2) alteration at 8q and 6q regions, together with previous results, suggest an association between these events and the development and/or progression of this tumor.
Whether and how human tumours are genetically unstable has been debated for decades. There is now evidence that most cancers may indeed be genetically unstable, but that the instability exists at two distinct levels. In a small subset of tumours, the instability is observed at the nucleotide level and results in base substitutions or deletions or insertions of a few nucleotides. In most other cancers, the instability is observed at the chromosome level, resulting in losses and gains of whole chromosomes or large portions thereof. Recognition and comparison of these instabilities are leading to new insights into tumour pathogenesis.
Intestinal metaplasia identifies Barrett's esophagus (BE) and is associated with an increased risk for esophageal adenocarcinoma. Dysplasia occurs as an intermediate step. However, progression from metaplasia to neoplasia without the demonstration of dysplasia has been described. The role of dual-parameter flow cytometry (FC) as a predictor of neoplastic risk in dysplasia-free cases was evaluated. DNA/protein FC and histology were performed on 362 samples from 30 dysplasia-free BE patients, followed up since 1985 once every 1-2 years. Nine cases were aneuploid, five of which (group IV) were frankly aneuploid; in the other four cases (group III), aneuploidy was detectable by dual-parameter analysis only. Twenty-one patients were diploid. Twelve (group II) also had an abnormally high G1-phase protein content compared to group I (nine patients), which were diploid with a low-moderate protein content. In three patients of group IV an adenocarcinoma in situ was diagnosed, after 5, 6, and 10 years, respectively. In two patients of group III, a low- and a high-grade dysplasia were observed at 3 and 6 years follow-up, respectively. One patient of group I first acquired a high protein content, then an aneuploid DNA content, and then progressed to adenocarcinoma (12 years). None of the still diploid patients (17 cases) have progressed to dysplasia or cancer compared with 6 of 13 presently aneuploid patients (P < 0.01). In conclusion, DNA/protein FC is a marker of increased malignant potential and thus may be used to detect patients at higher risk in dysplasia-free BE and assist in understanding the various stages of malignant transformation in long-term follow-up studies.
The authors studied a series of 97 consecutive cases of salivary gland tumors to investigate the correlation between the biologic parameters DNA ploidy and S-phase fraction (SPF) and the presumptive behavior of the neoplasms, as well as their potential clinical utility.
Histopathologic classification and grading of the tumors were evaluated according to 1991 World Health Organization criteria. DNA analysis was performed by flow cytometry in fresh material after propidium iodide staining. Clinical data and follow-up information were obtained from the clinical charts.
All the 71 benign salivary tumors showed a DNA diploid pattern. Seven carcinomas (7.2%) exhibited DNA aneuploidy. Eleven (42.3%) of 26 malignant tumors were considered low grade carcinomas, all of them being DNA diploid. Of the remaining 15 tumors, classified as high grade carcinomas, 7 showed DNA aneuploidy. SPF values ranged from 0.6% to 27.7%. A statistically significant difference was found between the mean SPF values of benign and malignant tumors, diploid and aneuploid tumors, and low grade and high grade carcinomas. When a cutoff value of 3% was used to discriminate histopathologic subgroups with prognostic impact, a significant difference was found between benign and malignant salivary tumors, high grade and low grade carcinomas, and high grade and benign tumors (P < 0.001).
The data from this study confirm the low incidence of DNA aneuploidy in salivary gland tumors and suggest the potential utility of SPF estimation in evaluating the clinical behavior of these neoplasms.
A total number of 116 clinically neck-negative patients with squamous cell carcinoma of the oral cavity who underwent radical primary tumour surgery without simultaneous neck treatment were entered into this prospective study. The 5 year overall survival rate was 87% for patients with flow cytometrically diploid tumours and 58% for the aneuploid group (P < 0.05). By multivariate survival analysis, tumour stage (P < 0.05) and DNA ploidy (P < 0.05) were significantly associated with the outcome. The cumulative 3 year rate of delayed clinical manifestation of lymph node metastasis to the previously untreated neck was 12.6% for patients with flow cytometrically diploid tumours and 41.3% for the aneuploid group (P < 0.01). By multivariate analysis, the DNA ploidy status of the primary tumour was the only factor among tumour stage, localization and degree of histological differentiation predictive of occult metastasis development (P < 0.05). Also, patients with T1 tumours who frequently are not considered to benefit from elective neck dissection were at high risk of subclinical lymph node involvement if the primary tumours were aneuploid (47%), whereas only 10% of the diploid T1 sample showed occult neck disease. Particularly in patients with less extensive oral carcinomas, DNA aneuploidy is therefore an important decisive factor in elective neck dissection.
The clinical significance of flow cytometric DNA content in head and neck squamous cell carcinoma is still controversial.
We prospectively investigated the prognostic importance of tumor size (T), lymph node involvement at presentation (N), degree of histologic differentiation (G) and DNA ploidy (DNA) in 429 surgically treated patients with primary oral squamous cell carcinoma using multivariate statistics.
T, G and DNA were independent predictors of metastasis to the neck. N and DNA were independent prognostic factors of loco-regional recurrence development. N and T were prognostic of overall survival. N and DNA were predictive of recurrence-free survival. For the neck negative group, DNA remained as the only predictor of overall and relapse-free survival.
DNA flow cytometry adds significant prognostic information beyond that provided by established prognostic factors.