DNA analysis of malignant salivary gland carcinomas: comparison of different tissue preparations and measuring techniques.

This study correlates static and flow-cytometric analysis on paraffin-embedded and fresh material of salivary gland carcinomas. Nuclear suspensions for DNA analysis were prepared from paraffin-embedded and fresh material. Comparison of the results of static and flow cytometry on paraffin-embedded material revealed no significant correlation between DNA ploidy and S-phase value. The coefficients of variation were significantly lower for static than for flow cytometry (p < 0.05). Flow-cytometric analysis on fresh and paraffin-embedded material correlated well concerning DNA ploidy, but not for the S-phase. The coefficients of variation were significantly lower for the fresh than for the paraffin-embedded material (p < 0.001). Cytometric analysis on paraffin-embedded material of malignant salivary gland carcinomas should be critically evaluated. There is a need for flow-cytometric investigations on fresh material to clarify further the prognostic value of this method.

Tumour growth fraction and apoptosis in salivary gland acinic cell carcinomas. Prognostic implications of Ki-67 and bcl-2 expression and of in situ end labelling (TUNEL).

bcl-2 protein and Ki-67 (MIB-1) were studied in 32 acinic cell carcinomas (ACCs), all with a minimum of 5 years' clinical follow-up. Tumour apoptosis was evaluated by TdT dUTP nick end labelling (TUNEL) and by morphological criteria. Five patients died of their disease. Patients with stage I tumours had significantly better survival compared with other stages (P < 0.05). Patients with MIB-1-negative tumours had significantly better survival than patients with MIB-1-positive tumours (P = 0.05). This study confirms a previous report that MIB-1 is an independent prognostic factor for survival in patients with ACC. Stage I tumours had high expression of bcl-2 protein, but there was no difference when compared with other stages. TUNEL positivity was most prevalent in stage I tumours, compared with stages II, III, and IV (P < 0.05), probably indicating more apoptosis. This could imply a capacity of stage I tumours ('early tumours') for early selection of tumour cells for elimination by apoptosis. There was no significant difference between expression of bcl-2 and TUNEL, between these parameters and clinical outcome, or between any parameter and morphological subclassification. We conclude that MIB-1 has prognostic value in ACC. Clinical staging, bcl-2, and TUNEL are also potentially useful as prognostic markers.

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.

Primary radiotherapy of T1 and T2 glottic carcinoma--analysis of treatment results and prognostic factors in 223 patients.

Treatment results in 223 patients with T1 and T2 glottic carcinoma were analysed. A multivariate analysis was performed to evaluate the prognostic significance of factors related to tumour, patient and treatment. Locoregional control after radiotherapy was 90% for 129 patients with T1 tumours and 73% for 94 with T2 tumours. Disease-specific survival was 96% and 81% for patients with T1 and T2 tumours, respectively. In the multivariate analysis of locoregional control, subglottic extension contributed prognostic information to T-stage. In the univariate analysis, number of involved tumour sites, cord mobility and treatment interruption had a significant influence, which was lost in the multivariate analysis. Age gave additional prognostic information in the multivariate analysis of disease-specific survival. Significant adverse effects of radiotherapy were found in 9 patients (4%). Forty-nine patients (22%) had a second malignancy, 11 (5%) diagnosed before the glottic carcinoma.

Adenoid cystic carcinoma: significance of DNA ploidy.

BACKGROUND: DNA ploidy pattern is sometimes used as a prognostic factor. Heterogeneity of a tumor could, however, give false information when a single analysis is performed. METHODS: Twenty-eight patients with adenoid cystic carcinomas were retrospectively studied with regard to clinico-histologic parameters, and in 24 of these the DNA pattern was assessed using flow cytometry, with multiple analysis from different tumor parts, to determine prognostic factors. RESULTS: Of the carcinomas, 33% (8/24) were DNA aneuploid, and 17% (4/24) of the tumors showed intratumoral heterogeneity of DNA content; two of them with mixture of diploid and aneuploid stemlines. The DNA aneuploid tumors were clinically more advanced and demonstrated a higher frequency of solid architecture than did diploid tumors (p < 0.05). The S-phase values were significantly higher in aneuploid samples than in diploid ones (p < 0.05). The recurrence rate was significantly higher in patients with aneuploid tumors (75%) than with diploid ones (19%) (p < 0.05). The cumulative survival was worse for patients with aneuploid tumors than for those with diploid ones (p < 0.05). CONCLUSIONS: Our findings suggest a potentially important role for flow cytometry in evaluation of adenoid cystic carcinoma. It is of interest to observe that in some tumors both diploid and aneuploid stemlines can co-exist. If one sample is analyzed and demonstrates diploid cells, there is a 3% chance that the tumor is also heterogeneous with aneuploid stemlines. If one sample demonstrates aneuploid cells, there is a 7% chance for heterogeneity with diploid cells, as well. Two samples from different tumor parts can be considered representative.

Salivary duct carcinoma.

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.

Postural control after propofol anaesthesia in minor surgery.

There is, not only from an economic perspective, a continuous search for surgical and anaesthetic procedures that allow out-patient surgery to be conducted. Reliable estimators of the patient's street fitness are of the utmost importance. Balancing capacity is one aspect that should be considered. Clinical methods like Romberg's are subjective and imprecise. Dynamic posturography comprises a movable support surface and visual surround that are either stable or referenced to the patient's sway, with eyes open or closed. In this study the recovery from propofol anaesthesia was studied in 8 patients who underwent surgery for minor microlaryngoscopic procedures. Dynamic posturography who performed before, and 2 and 4 h after cessation of anaesthesia. All patients who were awake had normal balancing ability already at 2 h after anaesthesia. Further studies may supply additional information of other aspects of discharge after anaesthesia.

Heterogeneity of salivary gland tumors studied by flow cytometry.

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.

Prognostic factors in oral cavity carcinomas.

This retrospective study of 176 patients with oral cavity carcinoma showed that nearly 60% of the patients presented with an advanced stage of disease. The duration of symptoms was shorter in patients with a large tumour than with a small (p less than 0.001) and in patients with metastases than in those without (p less than 0.01). Lymph node metastases were more common in large tumours (p less than 0.01), in tumours with high malignancy grading (p less than 0.05) and in non-diploid tumours (p less than 0.001). The presence of lymph node metastases (p less than 0.001), tumour size (p less than 0.01) and tumour DNA ploidy (p less than 0.005) were the only parameters that significantly influenced survival (Cox regression analysis).

Polymorphous low-grade adenocarcinoma: a 'new' salivary gland tumour.

We describe the clinicopathological features of polymorphic low-grade adenocarcinoma. The hallmarks of cytologic uniformity, remarkable histologic diversity, and favourable prognosis are emphasized. Polymorphous low-grade adenocarcinoma is not listed in the existing WHO classification, but recent reports indicate that it may be the second most common malignant intraoral salivary gland tumour. Both pathologists and ENT surgeons, who are not specialized in salivary gland tumours, may not be familiar with this tumour entity, and polymorphous low-grade adenocarcinoma therefore merits a detailed description.

Heterogeneity of squamous cell carcinomas of the oral cavity studied by flow cytometry.

The DNA pattern was determined by flow cytometry in 76 samples from 16 squamous cell carcinomas of the oral cavity to assess intratumour DNA heterogeneity. Heterogeneous DNA content was found in 2 tumours (12%); both containing DNA diploid and DNA aneuploid cell clones. The remaining 14 tumours showed a homogeneous DNA distribution in the different specimens; 9 (56%) were diploid, 3 (19%) aneuploid and 2 (12%) were polyploid. The DNA non-diploid tumours were clinically more advanced than the DNA diploid ones (p less than 0.05). The tumour proliferation rate (fraction of cells in S-phase) was higher in aneuploid tumours than in diploid ones (p less than 0.01).

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 Linköping 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 in oral cavity carcinomas, with special reference to prognosis.

Single-cell DNA cytofluorometry was performed on paraffin-embedded tissue of 140 patients with squamous cell carcinomas of the oral cavity. Half of the tumors (71 of 140) were DNA nondiploid. Well-differentiated carcinomas were more often DNA diploid than moderately well-differentiated ones (P less than 0.001; chi-square). The aneuploid tumors responded better to preoperative radiotherapy than did the DNA diploid (P less than 0.001) and polyploid tumors (P less than 0.05; chi-square). Using the multivariate Cox's regression analysis multiploid type tumor, age of the patients and presence of lymph node metastases were the only significant factors influencing survival. DNA diploid tumors in stages I and II had a better prognosis than DNA nondiploid (P less than 0.01; Kaplan-Meier). The reverse was true for stages III and IV, where DNA nondiploid tumors had a better prognosis (P less than 0.05; Kaplan-Meier). Tumor stages (P less than 0.001; Kaplan-Meier) and especially lymph node metastases (P less than 0.0005; Kaplan-Meier) were major prognostic factors. Tumor DNA ploidy may be a complement to clinical and morphologic parameters as a prognostic predictor in squamous cell carcinoma of the oral cavity.

DNA content, malignancy grading and prognosis in T1 and T2 oral cavity carcinomas.

Microscopic malignancy grading using the 8-factor system proposed by Jakobsson et al. (1973), the 4-factor system set up by Glanz and Eichhorn (1985), and DNA cytofluorometry were applied to thirteen T1 and thirty-seven T2 squamous cell carcinomas of the oral cavity, 9 with and 41 without metastases. There was a significant correlation between the presence of lymph node metastases (N1) and the malignancy scores (P less than 0.05) and tumour DNA ploidy (P less than 0.01, chi-square). The total number of patients with initial and late lymph node metastases correlated significantly with polyploid nuclei (P less than 0.05) and with malignancy scores (P less than 0.001), which also correlated with regional recurrences (P less than 0.01, chi-square). No remaining tumour after preoperative radiotherapy indicated less risk for local recurrence than if tumour persisted (P less than 0.01, chi-square). The cumulative survival (Kaplan-Meier) was worse for patients with nodal involvement (N1) than for those without (N0) (P less than 0.01), and for patients with poorly differentiated tumours compared with moderately well differentiated (P less than 0.05) and to well differentiated (P less than 0.001). The prognosis was worse for patients with high malignancy scores than those with low (P less than 0.001). DNA diploid tumours had a better prognosis than DNA non-diploid, but the difference was not significant.

Malignancy grading and cytophotometric evaluation of T1 lip carcinomas.

Histological malignancy grading using the score system set out by Jakobsson et al. in combination with static DNA cytofluorometry was applied to 20 T1 squamous cell carcinomas of the lower lip, 11 with and 9 without lymph node metastases. The mean malignancy score was 18.0 for tumours with metastases and 13.0 for those without (P less than 0.05, chi square test). Nuclear polymorphism, mode of invasion, vascular invasion, and cellular response were the single factors with the strongest influence. All but 3 carcinomas were DNA diploid, but DNA ploidy and proliferative activity (S-phase) yielded no prognostic information. However, hypertetraploid cells were found in all DNA diploid carcinomas with metastases, but in only half of those without (P less than 0.05).

DNA pattern in oral cavity carcinomas in relation to clinical stage and histological grading.

Cytofluorometric DNA analysis was performed in 88 squamous cell carcinomas of the oral cavity. 48% (42/88) of the tumours were DNA non-diploid. The frequency of DNA non-diploid tumours seemed to correlate with the increasing size of the tumour, and the decrease of histological grading, and certainly with the presence of lymph node metastases (p less than 0.001). DNA non-diploid tumours often had shorter duration of symptoms. The S-phase level seemed to increase the less differentiated the tumour but did not correlate either to tumour size or to duration of symptoms. Polyploid nuclei were more common in poorly differentiated tumours (9/10) as compared to well differentiated ones (17/33) (p less than 0.05) and seemed to be more common in tumours with metastases (20/26) than in those without (40/62).

Preoperative irradiation in oral cavity carcinoma. A study with special reference to DNA pattern, histological response and prognosis.

The relation between DNA pattern and response to preoperative radiotherapy was studied in 51 patients with oral squamous cell carcinomas operated 4 weeks after irradiation. Small tumors (T1 and T2) showed more pronounced response to radiotherapy than larger ones (T3 and T4), as did DNA aneuploid tumors. Eight of 11 DNA aneuploid tumors showed no remaining tumor in the operation specimen, compared to 6 of 21 DNA polyploid and 2 of 19 DNA diploid tumors. None of 16 patients without demonstrable remaining cancer in the operation specimen had local recurrence. The presence of lymph node metastases was the most important prognostic factor.

Cervical lymph node metastases with occult primary.

Patients with cervical metastases from an unknown primary tumour present both diagnostic and therapeutic problems. Twenty patients with a metastatic lesion in the neck, in whom the primary tumour was not found despite extensive, diagnostic procedures, were treated in the Department of Otolaryngology, Linköping University Hospital, during a 14-year period (1971-1984). Since 1975 tonsillectomy has been performed regularly on the affected side as a part of the diagnostic procedure. Eight patients without any clinical signs of a tonsillar primary have been diagnosed to have carcinoma by tonsillectomy and careful examination of the serially sectioned tonsillectomy specimen. The diagnosis of the metastatic nodes was based on histopathological examination in 8 patients and on fine needle aspiration cytology in 12. Four of 9 patients with squamous cell carcinoma survived 3 years and so did 4 of 5 with anaplastic carcinoma. All 5 patients treated surgically are alive and 6 of 12 who were irradiated. Ten of 12 patients with mobile unilateral nodes (N1) were alive at follow-up after 1-13 years but only 3 of 8 patients with fixed nodes (N3) at follow-up of 1-3 years.