Acinic cell carcinoma of the salivary glands: the prognostic relevance of DNA cytophotometry in a retrospective study of long duration (1965-1987).

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.

Methodological aspects on cytochemical DNA assessment of adenocarcinoma of the endometrium by means of image and flow cytometry using conventionally formalin-fixed and paraffin-embedded specimens.

Sometimes widely diverging results have been reported as regards the nuclear DNA ploidy pattern of adenocarcinomas of the endometrium. Since such discrepancies might be due to differences in the techniques applied, it seemed worthwhile to investigate this possibility in conventional uterine curetted specimens. In order to obtain a high incidence of tumours with cancer cell nuclei showing "aneuploid" DNA distribution pattern, a selection was made, so that only those adenocarcinomas that had led to a fetal outcome of the neoplastic disease were examined. The results of two image cytometric (ICM) techniques for cytochemical nuclear DNA assessments were compared. One was direct photographic cytometric measurements on Feulgen-stained sections; the other was densitometric assessments on isolated tumour cell nuclei of deparaffinised and disintegrated specimens. In 39 cases out of 43 the DNA ploidy pattern was the same by means of the two techniques. However, about half the numbers of the specimens (40 out of 83) were lost during the deparaffinisation and disintegration procedure. As far as could be found from a limited study on 20 (out of the 43) selected cases, these losses of specimens became even greater when the flow-cytometric (FCM) technique was applied on the deparaffinised specimens; about one third of these specimens were not possible to evaluate. In addition, in those where assessments by means of FCM could be made, the DNA ploidy pattern obtained differed from that of the two ICM techniques in not less than 80% of the cases. Broad peaks and high amounts of counts in the background in the DNA histograms indicated that most of the DNA assessments made by means of FCM on archival material of the present kind of curetted specimens of endometrial adenocarcinomas gave no reliable results. Consequently, differences in the techniques applied in cytochemical assessments of the nuclear DNA distribution pattern in endometrial carcinomas can explain the more or less controversial results reported from different laboratories.

Methodological aspects of DNA image cytometry in formalin-fixed paraffin-embedded material from pancreatic adenocarcinoma.

Deparaffinized and disintegrated material from conventionally formalin-fixed and paraffin-embedded surgical specimens of 100 cases of ductal adenocarcinoma of the pancreas was Feulgen-stained, and the cytochemical DNA distribution patterns of at least 100 single tumour cells and 50 "control" cells (fibrocytes) were assessed by means of image cytometry (ICM). In 77 cases a sufficient number of neoplastic cells could be obtained for these DNA assessments. The fairly high number (23) of cases that had to be excluded due to too small amounts of disintegrated cells or cell nuclei may be explained by the high content of connective tissue stroma in these pancreatic adenocarcinomas. The tumour cell nuclei in 76 of these 77 cases showed cytochemically a clear-cut "non-diploid" DNA distribution pattern. This observation reflects the well-known highly malignant growth potential of this carcinoma. Despite the fact that about 1/4 of the tumours had to be excluded, the main result of our methodological study is, after all that conventionally formalin-fixed paraffin-embedded specimens of most pancreatic adenocarcinomas can be successfully used for the deparaffinization-disintegration procedure preceding the nuclear DNA assessments by means of ICM. Additional studies are, however, required to obtain the diagnostic and prognostic impact of the results of such cytochemical analyses of the DNA distribution pattern in adenocarcinomas of the pancreas.

DNA evaluation in growth hormone producing pituitary adenomas: flow cytometry versus single cell analysis.

DNA patterns were analysed in 26 GH-producing pituitary adenomas by flow cytometry as well as by microspectrophotometry. Twelve tumours (46%) were diploid according to both methods, whereas 5 tumours (19%) showed aneuploid DNA patterns. Nine tumours were classified differently by the two methods: flow cytometry resulted in diploidy in 2 and aneuploidy in 7 patients, whereas microspectrophotometry showed diploidy in 5 tumours, tetraploidy in 3 and aneuploidy in 1. Methodological limitations may explain the discrepancy in the results obtained by the two methods. However, both the flow cytometry and the microspectrophotometry method show the presence of aneuploid DNA patterns in GH-producing pituitary adenomas despite their benign growth characteristics and the clinically benign course of the disease. This comparative study with two methods measuring DNA content, shows that depending on the criteria used for diploidy-aneuploidy, the frequency of aneuploidy will vary. In this material of 26 GH-producing adenomas, 46% were aneuploid according to flow cytometry and 23% according to microspectrophotometric. However, no correlation to tumour size or GH levels was found with either method when patients with aneuploid and diploid tumours were compared. Therefore, no clinical significance can so far be drawn from these results.

Cytophotometry.

Basic differences and similarities of flow and static cytophotometric instruments are viewed with regard to technical peculiarities and applicability in the fields of biology and tumor pathology. In flow systems, precision of the information concerning quantities of constituents is high, whereas precision of information concerning morphologic parameters is low. In static systems, morphologic identification by a trained operator can be used advantageously to classify any given cell interactively, thus replacing a large number of flow parameters. This is why the vast majority of automated static machines used in pathology are still more or less semiautomatic or interactive. Carefully performed specimen-adapted cell preparation procedures in which each step is strictly supervised, highly standardized staining methods, and internal controls are prerequisites in order to obtain reliable cytochemical results. In a number of human tumors with well-controlled cytopathologic and/or histopathologic and clinical data, quantitative cytochemical analysis has been demonstrated to provide diagnostic and prognostic information complementary to that obtained by conventional clinical and morphologic methods.

Intratumoral variations in DNA distribution patterns in mammary adenocarcinomas.

Correlated flow-cytometric (FCM) and microspectrophotometric (MSP) techniques were applied to investigating whether intratumoral variations in the DNA distribution patterns of 21 primary mammary adenocarcinomas can occur. Although neoplastic cell populations with both diploid and tetraploid (i.e., euploid) distribution patterns could be found in varying proportions in some of the tumors, there was no evidence in any tumor nodule for the presence of euploid populations in one part and aneuploid populations in another. This statement was based on the results of the MSP technique, where the assessments were made on cytodiagnostically identified neoplastic cells. Also, when applying the FCM technique the statement was found to be essentially valid; only one of the tumor nodules showed a DNA distribution pattern that, by means of the criteria used in this procedure, was defined as being both euploid and aneuploid. Here, however, the technique consists of assessments made on a great number of microscopically non-identified cells. It was concluded that when conflicting reports are given from different laboratories on the prognostic value of the cytochemically assessed DNA distribution patterns in breast carcinomas, they are not likely to be attributed to intratumoral DNA heterogeneity but, rather, to differences in the methods used and in the criteria applied for the so-called ploidy assessments.

Epithelial-myoepithelial duct carcinoma of salivary glands: a follow-up and cytophotometric study of 21 cases.

The Salivary Gland Registry provided 21 cases of epithelial-myoepithelial duct carcinoma of salivary glands from 1965-1980 which were evaluated retrospectively for clinical follow-up and cytophotometric data; 81% were localized in the major, 19% in the minor salivary glands. The male:female ratio was 1:1.1, 10 patients (47.6%) being men and 11 (52.4%) women. The youngest patient was 27, the oldest 91 y old. The mean age was 59.3 y (overall), 57.9 y (women) and 61.0 (men). The clinical course was characterized by lymph node metastases present at initial diagnosis and local recurrences in 23.5%. No patient died of the tumor. In 12 cases, cytochemical assessment of nuclear DNA by means of single cell scanning cytophotometry yielded diploid histograms. According to clinical and cytophotometrical data, epithelial-myoepithelial duct carcinoma of salivary glands can be regarded as a proper tumor entity of low grade malignancy.

Nuclear DNA measurements in follicular thyroid adenomas.

The nuclear DNA content was measured retrospectively in histologically confirmed follicular adenomas in 20 patients, and prospectively in 25 patients. In the retrospective group two of the adenomas, and in the prospective group three of the adenomas were classified as atypical due to high cellularity and nuclear atypia, but with no sign of micro-invasive growth. In the group of patients studied retrospectively, the two patients with atypical adenoma had an aneuploid DNA pattern. One of these died 11 years after diagnosis with generalized disease. The other patient in this group with an aneuploid atypical adenoma is alive, as are all the remaining 18 patients with diploid adenomas. In the prospective group there were five aneuploid tumours. The atypical adenomas were aneuploid in two cases and polyploid in one case. No patient in the prospective group has shown any evidence of recurrence so far. Although DNA measurements are of value in distinguishing between low and high malignant potential in invasive tumors, the prognostic information if no invasion is found is uncertain. The importance of examining an adequate number of tissue blocks from the capsular region is emphasized.

Cytochemical assessments of the nuclear DNA distribution pattern by means of image and flow cytometry in thyroid neoplasms and in non-neoplastic thyroid lesions. A comparative methodological study.

The two most prevalent techniques for cytochemical DNA assessment of the nuclear DNA distribution pattern in neoplastic cells are image cytometry (ICM) and flow cytometry (FCM). The aim of the present study was to compare the results of nuclear DNA assessments, obtained by means of these two methods, in fresh surgical biopsy specimens from the thyroid gland, both in neoplasms and in nonneoplastic lesions. Material for DNA analysis was taken preoperatively by fine needle aspiration (FNA) biopsy from 13 papillary thyroid carcinomas and analyzed by the two methods. Surgical specimens were taken from 48 papillary thyroid carcinomas (one of which showed low differentiated papillary and anaplastic giant cell formations at autopsy), 2 follicular carcinomas, 66 follicular adenomas, 7 medullary carcinomas as well as from the nodules of 17 non-toxic colloid goitres and 19 specimens from the diffusely hyperplastic thyroid parenchyma in patients with hyperthyroidism. For the ICM assessments, FNA biopsies or imprints were made from the macroscopically identified fresh thyroid biopsy specimens; for the FCM assessments FNA specimens from the same region were used. In 155 out of the 159 specimens the results obtained by means of the two methods were the same. The DNA distribution pattern in 106 of the neoplasms and in all the 36 non-neoplastic lesions were of the "euploid" type (i.e. "diploid" or diploid/tetraploid"), whereas that of 17 neoplasms were of the "aneuploid" type. Fifteen of the histopathologically benign follicular adenomas showed a cytochemical DNA distribution pattern that by means of FCM was of the "aneuploid" type.(ABSTRACT TRUNCATED AT 250 WORDS)

Mucoepidermoid tumors of the salivary glands. Correlation of cytophotometrical data and prognosis.

The mucoepidermoid tumors of the Salivary Gland Registry, Institute of Pathology, University of Hamburg, Western Germany, were evaluated retrospectively with regard to epidemiologic data, clinical follow-up, and cytophotometric data. Clinical data were obtained in 71 cases. Tissue from 46 cases was studied by single cell scanning cytophotometric analysis. Two thirds of the tumors were located in the major salivary glands, the parotid being the most common site, one third occurred in the minor salivary glands. The age range was from 6 to 81 years; peaks were observed in the fourth and seventh decades; the sex distribution was almost equal. By means of a single cell scanning cytophotometric device, a division into "diploid" and "atypical" patterns was possible. The clinical course was well correlated with these two groups, the atypical group showing generally an unfavorable course. Especially in poorly differentiated tumors, selection of clinically aggressive tumors was possible by their atypical DNA distribution pattern. Consequently, single cell DNA assessment can be a useful supplementary tool in the clinicopathologic and prognostic evaluation of mucoepidermoid tumors of the salivary glands.

Prognosis of salivary adenocarcinomas. A retrospective study of 52 cases with special regard to cytochemically assessed nuclear DNA content.

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.

Tumour pathology of the neuron-paraneuron system and its evolutionary background, with special attention to neuro-endocrine differentiation in prostate and mammary carcinomas.

From results of phylogenetical investigations of the neuron-paraneuron or neuroendocrine system it is known that cells producing hormonal peptides appear first in the nervous system, then also as disseminated cells of an open or closed type in the mucosa of the alimentary tract, and, lastly, as the parenchyma of the classical endocrine glands. In all these three parts of the neuroendocrine system the parenchymal cells can undergo neoplastic transformation, expressing "eutopic" and/or "ectopic" peptide hormones and/or biogenic amines. The neuro-paraneuronal tumours arising in the central or peripheral nervous system in man are rare. In contrast, they are more common when they originate from the disseminated neuroendocrine cells in the gut mucosa, and when they arise in the endocrine glands. Here, a broad spectrum of histopathologically benign or malignant neoplasms, hyperplastic nodules, and other tumour-like lesions occur, some of which are associated with well-known clinicopathological entities. Typical examples are the gastrointestinal carcinoids, pancreatic insulomas, pituitary adenomas, medullary thyroid carcinomas, and phaeochromocytomas. The fact that neoplastically transformed cells of some traditionally non-endocrine tumours, e.g., mammary and prostatic carcinomas, can undergo neuroendocrine differentiation has recently become well established. The "malignancy potential" of neuroendocrine carcinomas and that of the paraneuronally differentiated cells of prostatic and mammary carcinomas have both been studied by means of cytochemical DNA assessments of the nuclei of the tumour cells, applying a recently developed deparaffinisation-disintegration procedure using archival material. So far, it seems as if about 1/4 of common gut carcinoids show a DNA distribution pattern of the "aneuploid" type, whereas the other 3/4 are "euploid" i.e., show a "diploid" or "diploid/tetraploid" DNA distribution pattern. The cell nuclei in areas in prostatic and mammary carcinomas with high amounts of paraneuronally differentiated cells displayed to a greater extent "euploid" DNA patterns than those in areas of the same carcinomas without such paraneuronal cells. Thus, the "malignancy potential" of the neoplasms of neuro-paraneuronal cells seems to be rather low.

The relationship between nuclear DNA content in salivary gland tumors and prognosis. Comparison of mucoepidermoid tumors and acinic cell tumors.

Differences in prognosis between salivary gland mucoepidermoid tumors and acinic cell tumors were compared by means of conventional histopathological grading and nuclear DNA content which was assessed cytochemically by a scanning cytophotometric procedure. The mucoepidermoid tumors were found to show a stronger correlation between histopathological grading and prognosis than did the acinic cell tumors. By using DNA quantification, valuable additional information could be obtained for predicting the behavior of the mucoepidermoid tumors, whereas there was no correlation between DNA content and prognosis for the acinic cell tumors. Regarding the relatively "benign" clinical course of most mucoepidermoid tumors, the term "tumor"--as proposed by the World Health Organization's classification--seems appropriate. In contrast, the more severe clinical courses of the acinic cell tumors justify the use of the term "carcinoma" instead.

DNA-determination in the clinical management of patients with meningioma or haemangioblastoma.

The histopathology and clinical course of 15 benign (grade I) and nine anaplastic (grade II-III) meningiomas was reviewed and compared with the nuclear DNA distribution patterns of the tumour cells as determined by Feulgen staining with ensuing image and flow cytometry. In addition, eight haemangioblastomas were studied. One benign meningioma, three anaplastic meningiomas and two haemangioblastomas were aneuploid. The outcome could be well predicted from standard histopathological criteria and a critical evaluation of the extent of removal at operation. The determination of nuclear DNA contents did not add further information useful in the management of these meningioma patients. Histopathologically aggressive or frankly anaplastic tumours had a bad outcome. The clinical results underline the importance of aggressive treatment of anaplastic meningiomas.

Primary structures of somatostatins from the islet organ of the hagfish suggest an anomalous pathway of posttranslational processing of prosomatostatin-1.

The cyclostomes represent the first class of vertebrate in evolution to develop an endocrine pancreas. Two peptides with somatostatin-like immunoreactivity were isolated from the islet organ of one such cyclostome, the Atlantic hagfish (Myxine glutinosa). The primary structure of the more abundant peptide was established as: Ala-Val-Glu-Arg-Pro5-Arg-Gln-Asp-Gly-Gln10-Val-His-Glu-Pro- Pro15-Gly-Arg-Glu-Arg-Lys20-Ala-Gly-Cys-Lys-Asn25-Phe- Phe-Trp-Lys-Thr30-Phe-Thr-Ser-Cys. The second peptide, comprising 27% of the total immunoreactivity in the islet extract, was identical to mammalian somatostatin-14. The pathway of posttranslational processing of prosomatostatin in the hagfish islet differs markedly from the pathway in the higher vertebrates. In the mammalian pancreas, prosomatostatin is cleaved at the site of the single arginyl residue (corresponding to position 6 in hagfish somatostatin-34) and at the arginine-lysine site (corresponding to positions 19 and 20 in the hagfish peptide) to generate somatostatin-14 and somatostatin-28(1-12)-peptide. In the hagfish islet, Arg6 is not used as a cleavage site and cleavage at Arg19-Lys20 represents only a minor pathway of processing. The data provide further evidence of the strong evolutionary pressure to conserve the complete amino acid sequence of somatostatin-14.

Neuroendocrine tumors of the gastrointestinal tract.

Neoplastic proliferations of neuroendocrine cells (NE) may occur throughout the entire GI tract but affect particularly appendix and ileum ("midgut carcinoids"), rectum ("hindgut carcinoids"), as well as stomach and the duodenum ("foregut carcinoids"). Only more exceptionally, they arise in the esophagus, jejunum and colon. The NE tumors encompass a heterogeneous gross and microscopic structural spectrum, ranging from inconspicuous microproliferations ("mucous membrane nevi") to bulky tumor masses. Their growth patterns are usually characteristic and easily recognized. In doubtful cases their NE differentiation becomes established by a characteristic silver affinity, by the ultrastructurally observed presence of characteristic "endocrine" secretion granules, and by immunohistochemically detectable occurrence of "pan-NE markers" (neuron-specific enolase, chromogranins, and synaptophysin), biogenic amines (mainly serotonin), and neurohormonal peptides. Foregut carcinoids usually contain serotonin, gastrin, and somatostatin, midgut carcinoids often only serotonin and tachykinins, whereas the hindgut carcinoids as a rule are multihormonal with a wide spectrum of hormonal peptides, including even insulin. Most GI NE tumors are found in the appendix (50%) and the ileum (30%). Practically all (98%) of the appendiceal NE tumors are benign. They have recently been proposed as arising from apparently Schwann-cell-related NE cells in the submucosa, whereas the ileal--and probably also all the other non-appendiceal NE tumors--are derived from the totipotential cells in epithelial crypts of the mucosa. Among the ileal NE neoplasms a large number can metastasize and result in a fatal outcome. The ability to metastasize is related to the size and to the multiplicity of the primary tumors at the time of initial diagnosis and, to some extent, to their histopathologic growth pattern. Now, some relationship between the prognosis and the cytochemically assessed nuclear DNA content of the NE tumor cells has also been established; not less than about 1/4 to 1/3 seem to be aneuploid. Almost 90% of the rectal carcinoids are benign. Exceptionally, a highly malignant NE neoplasms can arise from the colon/rectum--as well as from the esophagus--composed of NE cells of small and intermediate size. The NE tumors of the stomach are often composed of ECL (enterochromaffin-cell-like) cells; such ECL cell carcinoids are related to atrophic gastritis with pernicious anemia; experimentally, they can be induced by hypergastrinemia in rats. Duodenal carcinoids often contain psammoma bodies and can be associated with neurofibromatosis.

Diagnostic and prognostic impact of cytochemically assessed nuclear DNA contents in human adenocarcinomas of the mammary gland.

Cytochemical assessments of the nuclear DNA contents in carcinomas of the breast can be used for both prognostic and diagnostic purposes. Two main techniques are currently being used, viz. flow cytometry (FCM) and microspectrophotometry (MSP). An account of their advantages and disadvantages is given. In addition, an old, rather crude, cytophotometric technique can be used for histopathological sections of paraffin-embedded specimens. The principal sampling procedures are fine-needle aspiration biopsy and the so-called imprint technique, where the specimens are made from the cut surface of the freshly excised operation specimen. Paraffin-embedded histopathological material can also be used, applying a newly developed MSP procedure, where isolated nuclei from deparaffinized/disintegrated specimens are analyzed. Most important is that intact cell nuclei, representative for the whole tumour nodule, can be obtained. The simultaneous use of FCM and MSP is also of utmost importance for the reliability in the interpretation of the results. Then, a kind of "DNA malignancy grading" is obtained that in several investigations has proven itself to be an excellent prognosticating tool that can be used for making an adequate choice of therapy for the individual patient. The diagnostic value of the results of the cytochemically assessed nuclear DNA distribution patterns is not so high as the prognostic one. Tumours with a diploid type of nuclear DNA content can be found both among benign and malignant neoplasms. However, a neoplasm with an aneuploid DNA distribution pattern can almost certainly be considered highly malignant.