Nuclear DNA cytofluorometry of normal human laryngeal epithelia and squamous cell carcinoma

Normal human laryngeal epithelia and laryngeal squamous cell carcinoma were assayed by Feulgen DNA cytofluorometry using free cell nuclei isolated from carnoy-fixed, paraffin-embedded specimens. In all of the 12 normal specimens, the epithelium showed typical diploid cell clones with low proliferative activity. Polyploid cells were seen in only two specimens from subjects aged 61 and 69 years respectively, and the number of polyploid cells seen in these two specimens was only two. Fifteen cancer cases were divided into three groups: an untreated group (5 cases), a chemotherapy group (5 cases) and a group of cases with recurrence after radiation therapy (5 cases). Among these three groups the DNA ploidy patterns were compared. In the untreated group, all cases showed a two-peak diploid pattern and a high proliferative activity, and polyploid cells were present. In the chemotherapy group, a wide one-peak histogram extending from 2C to about 5C was noted in 4 cases, and an aneuploid pattern in one case. Thus, the DNA ploidy pattern in the chemotherapy group differed from that in the untreated group. Of the 5 cases with recurrence after radiation therapy, one had a tetraploid pattern, but the remaining 4, a two-peak diploid pattern similar to that seen in the untreated group. Polyploid cells were observed in all these cancer cases. However, because they were also seen in some normal subjects, the finding of polyploid cells is not considered to be conclusive of cancer diagnosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear ploidy in neuroendocrine neoplasms of the larynx.

Seventeen neuroendocrine neoplasms of the larynx were examined for DNA ploidy. Twelve tumours were atypical carcinoids, four paragangliomas and one a small cell neuroendocrine carcinoma. Seven atypical carcinoids were aneuploid, one diploid and one tetraploid, with metastasis being aneuploid. In three cases only skin metastases were available for study, which were aneuploid. The small cell neuroendocrine carcinoma was aneuploid. Two of the paragangliomas were aneuploid and two diploid. Like other sites, neuroendocrine neoplasms of the larynx have a high rate of aneuploidy. Determination of DNA ploidy did not provide useful diagnostic or prognostic information.     

A clinical study of 104 patients with tongue cancer and the relationship between DNA ploidy and prognosis in 41 cases

One-hundred and four patients with previously untreated tongue cancer seen in our department between 1986 and 1998 were enrolled in a clinical study. The DNA ploidy patterns observed in fresh frozen specimens obtained from 41 patients were analyzed, and prognostic factors were investigated. According to the TNM classification (UICC 1997), 43 patients had stage I tumors, 29 had stage II tumors, 17 had stage III tumors, and 15 had stage IV tumors. The 5-year cause-specific survival rates for each stage were 94.7%, 64.4%, 50.0% and 45.7%, respectively. The most frequent cause of death associated with the original disease was the recurrence of the disease in cervical lymph nodes (19/27, 70.4%). The occurrence of late cervical metastasis was high among patients with a T2N0 disease. Patients with stage II disease should undergo elective neck dissection or be carefully monitored using ultrasonography. Among the 41 cases in which the DNA ploidy pattern was analyzed, diploid patterns were found in 30 cases and aneuploid patterns were found in 11. The 5-year cause-specific survival rate and the 5-year locoregional control rate were significantly lower for the aneuploid cases (18.2%, 38.9%) than for the diploid cases (66.5%, 69.8%) (p = 0.0003, p = 0.0339). The incidence of distant metastasis was significantly higher among the aneuploid cases (6/11, 54.5%) than among the diploid cases (3/30, 10.0%) (p = 0.0058). The ploidy pattern, as determined by flow cytometric DNA analysis, may reflect the malignancy grade of tongue cancers.     

Predicting radioresistance in early glottic squamous cell carcinoma by DNA content

Nuclear DNA content has been implicated as a prognostic factor in an increasing number of tumor types. Current data on the role of DNA content in head and neck carcinoma are conflicting and incomplete. To evaluate the role of DNA content in predicting radioresistance, 29 patients with T1N0M0 squamous cell carcinoma of the glottic larynx who had undergone uniform curative radiotherapy and whose clinical outcome was known had flow cytometric analysis for DNA content performed on their tumors with paraffin-embedded archival tissues. Five aneuploid lesions and 24 diploid lesions were identified. All aneuploid lesions occurred in radioresistant tumors. The probability of an aneuploid tumor failing radiotherapy was highly significant at p = .016. No DNA discordance was found in a sampling of half of the radioresistant lesions' pretreatment and recurrent specimens, for a 100% predictive value of moderate statistical power. On the basis of these findings, patients with aneuploid T1 glottic lesions should be referred for primary surgical therapy.     

Nucleolar organizer regions in glottic carcinomas: Comparison of DNA cytofluorometry and clinicopathological analysis

The significance of nucleolar organizer regions (NORs) and nuclear DNA content in 73 glottic carcinomas was assessed for proliferative activity and tumor progression. NORs stained with silver colloid were counted, and nuclear DNA content was assayed by cytofluorometry. The cytofluorometric study demonstrated that the percentage of tumors with aneuploidy tended to increase as histological differentiation decreased. Survival rates of patients with diploid and aneuploid tumors were not significantly different. AgNOR staining revealed that mean AgNOR numbers rose as histological differentiation of tumors decreased. Moreover, as T and N categories and stages showed advancing malignancy, mean AgNOR numbers tended to rise. However, there was no significant difference in survival rates between tumors with low and with high AgNOR counts. These studies indicate that while AgNOR staining is better than DNA cytofluorometry for determining histological differentiation of glottic carcinoma, neither is of prognostic value at the present time.     

Brief report: Prognostic importance of cellular DNA content in T1-2 N0 laryngeal squamous cell carcinomas treated with radiotherapy

One hundred fifty-two unselected, consecutive patients with T1-2N0 laryngeal squamous cell carcinoma received radical radiation therapy at the Division of Radiotherapy, Centro di Riferimento Oncologico, Aviano, Italy. Thirty-one (20.4%) of the patients showed disease recurrence or persistence (R/P) after radiotherapy. Flow-cytometric DNA ploidy measurements were performed in 72 cases; 20 had tumor R/P and 52 did not. Tumor R/P occurred more frequently (in 17 [85%] of 20 cases) in patients with diploid tumors. The hazard ratio of recurrence in diploid tumors as compared with aneuploid tumors, after inclusion of all the other significant prognostic factors in a Cox proportional hazards model, was 8.9 (P<.01). Therefore DNA ploidy seems to be an important marker of tumor R/P in patients with T1-2N0 laryngeal carcinoma after radiotherapy.     

Ploidy in head and neck cancer: a review and meta-analysis

A review is presented of all the series reporting ploidy in squamous cell carcinoma of the head and neck. A total of 1984 patients have been reported in 26 different series: 37% of tumours were diploid, 54% aneuploid and 11 % polyploid. Thus 64% of tumours were non-diploid. The mean age of patients with diploid and aneuploid tumours was very similar (60.9 and 60.3 years respectively) but patients with polyploid tumours had a mean age of 54 years. Although men were 5% more likely than women to have a non-diploid tumour the difference was not significant. Data relating ploidy to performance status are not available. The incidence of non-diploid tumours did not vary between sites, nor with stage grouping, but non-diploid tumours increased in frequency with diminishing degree of differentiation and with the presence of lymph node metastases. There was no difference in ploidy pattern between the primary tumour and node metastases. In the entire series the survival was better for diploid tumours than for non-diploid tumours. Subgroup analysis showed this effect to be due to mouth cancers, whereas ploidy did not affect the outcome in laryngeal cancer. Also, recurrence was more likely in non-diploid tumours. Patients with end-stage cancer treated by chemotherapy had a better survival if their tumour was non-diploid. Ploidy did not influence response to radiotherapy. When a tumour recurred after radiotherapy it was more likely to be diploid than a previously treated tumour. Non-diploid tumours had a greater S-phase fraction and a greater growth fraction than diploid tumours.     

DNA pattern of human pituitary tumors

The tumor tissue from 62 consecutive patients with pituitary adenomas was analyzed with regard to ploidy and percentage of cells in different phases of the cell cycle by use of flow-cytofluorometry. In six of 62 cases, two tumor-cell populations were identified; therefore the study material comprised 68 cell lines. Approximately half of the cell lines were diploid (33 of 68, or 49 per cent); five of 68 (7 per cent) were hypodiploid, and 30 of 68 (44 per cent) were hyperdiploid-aneuploid. A low occurrence of aneuploid cell lines appeared in hormonally nonsecreting tumors (22 per cent). An aneuploid DNA pattern was predominantly found in prolactinomas (70 per cent). In acromegaly, tumors secreting growth hormone had only an aneuploid DNA pattern in 41 per cent of the cases, whereas 67 per cent of the tumors with concomitant secretion of growth hormone and prolactin were aneuploid. The latter group also comprised the largest number of adenomas with two cell lines and all but one hypodiploid tumor. Most tumors with an aggressive clinical course were either aneuploid or diploid but with a high percentage of proliferating cells.     

Proliferative activity and cytometric characteristics in polyps of the nasal cavity and paranasal sinuses

Although several investigations have revealed the influence of cytokines, allergy, and environmental factors in polyp development, the etiology of nasal polyps is still unknown. To estimate the biology of this common disease the operative specimens of 50 patients who underwent surgery for polyps of the nasal cavity and the paranasal sinuses were examined; of these, 10 patients had recurrent disease and 23 patients had an allergy. The investigations included routine histology and quantitative DNA measurements, along with immunohistochemical identification of proliferation markers (i.e., MIB-1; proliferating cell nuclear antigen, PCNA). Histologically, most polyps revealed an infiltration with lymphocytes, eosinophilic granulocytes, and plasma cells. Twenty-five percent had a squamous metaplasia of the respiratory epithelium. Quantitative DNA analysis demonstrated diploid stemlines and lack of aneuploid cells with a DNA content exceeding 5c in most cases. Immunohistochemical detection of proliferation markers showed low proliferation rates in all cases. In 27 polyps no MIB-1 expression was detected, and in 7 polyps no PCNA expression was detected. The polyps of the 23 patients with proven allergic diathesis did not reveal higher scores for the parameters of DNA analysis (i.e., ploidy status and percentage of aneuploid cells) and proliferation scores. Nasal polyps of 10 patients with recurrent disease displayed higher scores for proliferation markers, and in five cases aneuploid cells with 5c exceeding rate (5cER) of 1.5-11.7% were detected. According to these results, polyps of the nasal cavity and paranasal sinuses showed low proliferation scores and were diploid. The data demonstrated that there was no increase of proliferation activity or ploidy shift toward aneuploidy in patients with allergy. Nevertheless, in recurrent disease some increase in proliferation activity and some changes in the parameters of the DNA analysis occurred, indicating more aggressive behavior of recurrent polyps in single cases.     

Prognostic significance of DNA ploidy in mucoepidermoid carcinoma

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.     

Flow cytometric analysis of DNA content in paraffin-embedded tissue in head and neck cancer (the second report)--primary lesions of laryngeal cancer

Flow cytometric analysis of DNA content using paraffin-embedded materials has become an important diagnostic, as well as prognostic, method for clinical pathology and investigative oncology. DNA content as measured in paraffin-embedded materials is closely related with that obtained from fresh specimens. This method also permits retrospective analysis on a lot of cases and studying different specimens of a tumor for intratumor heterogeneity. Flow cytometry was used to investigation of the DNA distribution in biopsy specimens from 30 patients with squamous cell carcinoma of the larynx. These patients had initially treated in our institute in the period from 1986 to 1988 and followed for two to four years. A modification of Hedley's method was used to prepare paraffin-embedded materials. DNA histogram were assessed in terms of DNA index (DI). The coefficient of variation for determination of DI ranged 4.2 to 13.7% (mean 7.8 +/- 2.4%). Using infiltrated lymphocytes and mesenchymal cells as an internal standards, aneuploid DNA histogram were found in 13 of 30 specimens (43.3%). DI ranged from 1.05 to 1.22. There was no significant correlation between DNA content and sex, age, tumor size, staging or treatment. The aneuploidy was found in 2 of 17 cases with well differentiated tumor (11.8%), 10 of 12 with moderately differentiated tumor (83.3%), one with poorly differentiated tumor (100%). 11.8% of patients with diploid tumor cells had recurrence during the interval of observation, compared to 38.5% of those with aneuploid tumor cells (p less than 0.05). In T1 lesions of glottic cancer aneuploid group had a significantly lower local control rate than diploid group (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Vagal paragangliomas: a clinical, pathological, and DNA assessment

Ten vagal paragangliomas were studied by image analysis and the results correlated with clinicopathologic features to determine if the DNA ploidy pattern could be used to separate benign from malignant paragangliomas. The tumours occurred in 8 women and 2 men ranging in age from 23 to 75 years (average 54 years). Follow-up was available in all 10 patients and ranged from 3 months to 27 years (average 7.8 years). Of the 10 tumours examined for DNA, 5 were diploid, 4 diploid-tetraploid, and 1 aneuploid. Two patients experienced local recurrences. One of these had a diploid tumour that recurred 22 years after excision and the other had an aneuploid tumour which recurred 4 years 4 months later and was associated with cervical lymph node metastasis. Two patients had malignant tumours with histologically confirmed metastases to noncontiguous cervical lymph nodes. One of the malignant tumours was diploid and the other aneuploid. This study concludes that DNA abnormalities are common in vagal paragangliomas and that tumour ploidy can not be used to assess malignant potential.     

Intratumoral DNA content heterogeneity in laryngeal squamous cell carcinoma.

Multiple tissue samples obtained from sections cut by Michaels and Gregor's method obtained from 21 consecutive total laryngectomies for squamous cell carcinoma were studied for intratumoral DNA content heterogeneity or homogeneity. Concordant DNA ploidy was manifested in all samples of five (23.8%) carcinomas (two diploid and three aneuploid), while 16 carcinomas (76.2%) demonstrated a variable DNA ploidy (diploid and aneuploid). Analysis of cellular proliferative activity demonstrated remarkable intratumoral stability in both concordant and discordant carcinomas. These data indicate there is a considerable heterogeneity of DNA ploidy but the proliferative rate is relatively stable within the carcinomas. Clinical implications of our findings are also presented.     

Ploidy status and the response of T1 glottic carcinoma to radiotherapy

Flow cytometric DNA ploidy measurements were performed on formalin fixed paraffin embedded tumour specimens from patients with a T₁ glottic laryngeal carcinoma in order to evaluate the role of DNA content in relation to local control. From 1980 to 1987, a consecutive series of 90 patients with a T₁ glottic laryngeal carcinoma were treated by radiotherapy with curative intent. Biopsies from 44 of these patients were readily available for DNA flow cytometry. In this group aneuploidy was associated with a significantly higher risk (P= 0.018) of local recurrence within 2 years after completion of radiotherapy (38% vs. 9% in the diploid group).     

Squamous cell carcinoma of the oral cavity. A review of 176 cases with application of malignancy grading and DNA measurements

This retrospective study comprised 176 patients with squamous cell carcinoma of the oral cavity treated at The Link?ping University Hospital over a 19-year period. Clinical parameters, microscopic malignancy grading (according to Jakobsson et al. and Glanz and Eichhorn), DNA cytofluorometry, analysis of therapeutic modalities and statistics regarding survival and prognosis are reported. The mean age was 70 years with a male: female ratio of 1.3:1 One hundred and four patients had T1 or T2 tumours and 109 an N0 neck. Cervical lymph node metastases were more frequent in patients with larger tumours (T3 + T4) than in those with smaller (T1 + T2) (P less than 0.01), in tumours with a high malignancy grading compared to those with a low (P less than 0.05) and in DNA non-diploid tumours compared to diploid ones (P less than 0.001). The aneuploid tumours responded better to preoperative radiotherapy than did diploid (P less than 0.01) or polyploid (P less than 0.05) tumours. Eighty-nine per cent of the recurrences occurred within 1 year of initial therapy. Secondary treatment was successful in 15 of 37 (41%) patients in whom the tumour recurred either at the primary site or in regional lymph nodes, but only in 1 of 8 (12%) with recurrences in both locations. Surgery alone or combined with radiotherapy resulted in equivalent survival rates for tumours in stages I and II. In advanced stages combined radiotherapy and surgery gave better survival figures than either modality alone (P less than 0.01; Kaplan-Meier). The presence of lymph node metastases (P less than 0.001), tumour size (P less than 0.01) and tumour ploidy (P less than 0.005) were the only clinical and histological parameters that significantly influenced survival (Cox regression analysis). Twenty-four patients developed a secondary primary malignancy; 21 of these were located in the aerodigestive tract.     

DNA ploidy status as a prognostic marker and predictor of lymph node metastasis in laryngeal carcinoma

In patients with laryngeal carcinoma, nodal metastasis, recurrence after radiotherapy, and prognosis are important factors in clinical decision-making. Parameters such as tumor stage are considered insufficient for predicting these important items. The DNA ploidy status of the tumor may be a useful additional marker. The DNA ploidy status of 38 laryngeal cancers was determined by flow cytometry. Correlations were studied with TNM stage, differentiation, survival rate, relapse risk, recurrence after radiotherapy, and nodal metastasis. A positive correlation of DNA ploidy status with the development of lymph node metastases was found for diploid and peridiploid versus aneuploid tumors (DNA index, <1.4 versus > or = 1.4; p = .007). No correlation was found between ploidy status and recurrence after radiotherapy. The overall survival rate (p = .01), but not the disease-specific survival rate or the relapse risk, showed a correlation with the ploidy status. The DNA ploidy status may be a useful marker for metastatic behavior in head and neck squamous cell carcinoma and may therefore be helpful in decision-making concerning elective treatment of the neck.     

Hürthle cell tumors of the thyroid. A flow cytometric DNA analysis

Twenty-five Hürthle cell tumors of the thyroid gland, histopathologically classified into three groups--adenomas, carcinomas, and indeterminant--have been studied by DNA flow cytometry using archived, paraffin-embedded tissues. Tumor ploidy characteristics were correlated with patient follow-up and survival with the conclusions that (1) nuclear DNA ploidy alone does not distinguish benign from malignant Hürthle cell tumors; (2) diploid DNA Hürthle cell carcinomas behave far less aggressively than aneuploid Hürthle cell carcinomas; and (3) all patients with aneuploid carcinomas died of their disease or are alive with persistent carcinoma.     

The measurement of DNA content and ploidy analysis in thyroid neoplasms

It is clear that the DNA content of endocrine cells is influenced by factors other than neoplastic change and transformation. Although it can be concluded that, in general, the DNA content of neoplasms is increased, it is less clear whether this increase in DNA content is the cause or the effect of neoplastic transformation. The actual consequences of an increased DNA content are still largely unknown. However, based on a substantial body of data on the measure of nuclear DNA content in thyroid neoplasms, several conclusions appear to be reasonable. First, the measurement of nuclear DNA content and ploidy analysis are not sufficiently reliable parameters upon which to distinguish a benign from a malignant thyroid neoplasm. Therefore, this parameter has failed to live up to the expectation that it would be a powerful diagnostic tool. Second, the measurement of nuclear DNA content is useful after a histomorphologic diagnosis has been made since it correlates very well with the prognosis and clinical outcome of the patient. It is clear that aneuploid thyroid carcinomas are responsible for earlier recurrence, an increased likelihood of distant and diffuse metastases, and an increased incidence of death compared with diploid thyroid carcinomas. Except for the rare occasion, diploidy implies a uniformly long-term survival whereas aneuploidy is associated with a variable clinical course. Irrespective of histomorphology, lethal lesions of the thyroid are invariably aneuploid, whereas lesions associated with prolonged survival or a favorable outcome can be either diploid or aneuploid. Aneuploidy in well-differentiated thyroid carcinoma is more likely in older patients, in less well-differentiated neoplasms, and in neoplasms infiltrating beyond the thyroid capsule. Age, type of neoplasm, extrathyroidal extension, and recurrent disease all appear to be more important prognostic variables than is nuclear DNA content. However, nuclear DNA content can increase the prognostic power of these variables and consequently may come to be increasingly useful in the management of some patients with thyroid neoplasms. After a histomorphologic diagnosis has been made, the measurement of nuclear DNA content and a determination of the DNA ploidy may have significant prognostic value.     

Cellular DNA amounts of squamous cell carcinomas of the head and neck region in relation to prognosis

Cytophotometric measurements of Feulgen DNA content were made in individual tumor cells from 45 patients with squamous cell carcinomas of the head and neck region. Eleven carcinomas were found to have a predominantly diploid distribution of DNA values, while the remaining 34 carcinomas were non-diploid. In 11 of the tumors — not entirely identical with the diploid tumors — 50% or less of the malignant cells had DNA values exceeding the modal diploid value (DNAG1 + 2 S.D.), whereas 34 had higher DNA values. The non-diploid carcinomas and those where >50% of the cells had DNA values >DNAG1 + 2 S.D. showed more advanced clinical stage, due to the presence of regional metastases. They were also associated with a poorer prognosis than the diploid carcinomas or those with ≤50% of the cells with DNA values >DNAG1 + 2 S.D. Cellular DNA amounts seemed to be of greater prognostic value than type of ploidy. The study indicates that DNA analysis may contribute important information on the biological behavior and prognosis of squamous cell carcinomas of the head and neck region.     

Carotid body paragangliomas. A clinicopathologic and DNA analysis of 13 tumors

The clinical and pathological features of 13 carotid body paragangliomas from 12 patients were examined and correlated with the DNA ploidy pattern as determined by image analysis. These tumors occurred in 7 women and 5 men aged 19 to 62 years (average, 42 years). All presented with a slowly enlarging, usually asymptomatic mass of 2 weeks' to 25 years' duration. Two patients were related and had a family history of paragangliomas. The tumors ranged from 2 to 6 cm. All contained scattered chief cells with pleomorphic nuclei, two exhibited mitoses, and three showed perineural and three vascular invasion. Follow-up was available in all 12 patients and ranged from 15 months to 28 years (average, 7.3 years). None of the tumors recurred locally, but one did metastasize to a single cervical lymph node that was apparent at the time of diagnosis. Of 13 carotid body paragangliomas examined for DNA, 4 were diploid, 3 diploid-tetraploid, 3 tetraploid, 2 aneuploid, and 1 polyploid. The only malignant tumor was polyploid. From these observations, we conclude that abnormalities in DNA content of carotid body paragangliomas are common and that tumor ploidy cannot be used to assess malignant potential. We also found no apparent relationship among nuclear pleomorphism, mitotic activity, perineural invasion, or vascular invasion and clinical behavior. Perineural and vascular invasion, however, were observed only in tumors with abnormal DNA histograms.