The Value of Cytometric DNA Analysis as a Prognostic Tool in Neuroendocrine Neoplastic Diseases

Path. Res. Pract. 191,281-303 (1995)

Original Papers

The Value of Cytometric DNA Analysis as a Prognostic Tool in Neuroendocrine Neoplastic Diseases U. G. Falkmer and S. Falkmer Tumour Pathology Unit, Department of Oncology and Pathology, Karolinska Institute and Hospital, Stockholm, Sweden

SUMMARY In several traditionally non-endocrine, common, human, neoplastic diseases, it has become well established during the last few years, that cytometric analyses of the DNA distribution pattern of the nuclei of tumour cells can be an excellent supplement to the conventional prognostic tools, (such as clinical staging and histopathologic malignancy assessments). When analogous studies of the value of DNA analysis by means of flow cytometry and/or image cytometry are made in neuroendocrine (NE) neoplastic diseases, the ensuing results often become rather disappointing. Thus, clear-cut aneuploid DNA histograms can be found in the neoplastic cell nuclei of clinically and histopathologically completely benign NE adenomas (and even hyperplastic nodules). In contrast, highly aggressive NE carcinomas not seldom reveal themselves to be composed of tumour cells with nuclei, displaying an euploid, i.e. normal, DNA pattern. Statements of this kind have been based on the results of comprehensive investigations in several laboratories, analysing such NE tumours as insulomas/insular carcinomas, bronchial/gastrointestinal carcinoids, phaeochromocytomas, paragangliomas, neuroblastomas, adenomas of the anterior pituitary gland, parathyroid adenomas, medullary carcinoma of the thyroid and Merkel-cell tumours of the skin. Thus, the prognostic value of the cytometric DNA ploidy pattern of the nuclei of neoplastic parenchymal cells is definitely lower in NE tumours than in most of the traditionally non-endocrine carcinomas and sarcomas Data from published and unpublished series of these kinds of NE tumours, and those of prostatic and breast carcinomas with NE differentiation, are given. By means of a new, consecutive double staining technique, it was shown that in idiopathic nesidioblastosis, the hyperinsulinism is caused by beta cells with a nuclear DNA ploidy pattern of euploid type. By the same technique, it can be shown that in the pathogenesis of the hypergastrinaemia-induced ECL-cell carcinoids of the stomach, a switch from an euploid to an aneuploid nuclear DNA distribution pattern occurs in the ECL-cells when they pass from a state of hyperplasia to that of a genuine neoplasia. In neuroblastomas, a triploid (i.e. aneuploid) DNA pattern is part of an algorithm capable of predicting a 96% survival rate, whereas a diploid/tetraploid (i.e. euploid) DNA pattern predicts a 0% survival.

Paper held at Meeting of European School of Pathology in Torino in 1992 on the subject of "Endocrine Pathology". © 1995 by Gustav Fischer Verlag, Stuttgart

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Introduction Many neuroendocrine (NE) neoplastic diseases are notorious for the difficulties encountered in assessing their prognosis for the individual patient. This statement is particularly valid for such well-known NE tumours as pancreatic endocrine tumours (insulomas)38,64, carcinoids of the gur70' 108, 109 and medullary carcinomas of the thyroid 45 , 72, 83. To a great extent, the statement also holds true for phaeochromocytomas and paragangliomas 30,114. Even in adenomas of the anterior pituitary gland, there is not always a good conformity between the histopathological malignancy assessments and their clinical and radiologic diagnostic data 53,68. Although, admittedly, conventional clinical staging of the extent of the neoplastic disease, the size of the primary tumour, its histopathological degree of invasive growth pattern and its cytological characteristics (including incidence of mitotic figures, degree of anaplasia, etc.), can, in most cases, give a fairly adequate assessment of the prognosis and subsequent course of the NE neoplastic disease 6o , there are, nevertheless, surprisingly often clear-cut failures in these respects. For this reason, supplementary prognostic tools have long been sought in NE neoplastic diseases by means of which some cell-biological features of tumour cells could be found, offering additional pieces of information in regard to the proliferative capacity of the neoplasm and its aggressiveness, particularly its tendency to dissemination 7, 119. In traditionally non-NE carcinomas and sarcomas, one such tool has, during the last decade, become of great value; namely cytometric DNA analysis of the nuclei of the neoplastic cells 5,31. Here, two techniques predominate: flow cytometry (FCM)98 and image cytometry (ICMf8. Several handbooks and monographs have documented the value of both techniques5, 6, 31, 32, 78, 97, 98. As a crude rule of thumb, it can be said that neoplastic diseases in which the FCMlICM nuclear DNA distribution pattern of the tumour cells is found to be of aneuploid type, run a rapidly progressive clinical course with extensive metastatic spread, early cachexia and death of the patient, whereas those with an euploid (diploid or tetraploid) (i.e. normal) DNA pattern of the neoplastic cell nuclei are less aggressive and can even be completely benign5, 6, 31, 78, 97. In most kinds of NE tumours, both FCM and ICM DNA analyses have also been made. As accounted for below, the results in regard to the value of these techniques as a prognostic tool in NE neoplastic diseases, have sometimes been promising but often rather disappointing; obviously, great variations exist between different tumour entities and the techniques applied. Clinically and histopathologically completely benign NE adenomas can be found to be equipped with neoplastic parenchymal cells displaying nuclei with a highly aneuploid DNA distribution pattern. Conversely, aggressive NE carcinomas can show an FCMlICM nuclear DNA ploidy pattern of euploid type.

The aim of the present work is to give a critical review of the experiences obtained in our and other endocrine pathology laboratories by means of FCM and ICM DNA analyses performed in some major entities of NE neoplastic diseases. Both published and unpublished data will be included. Some speculations will also be included about the possibilities that technical sources of errors might explain the so-called paradoxial DNA aneuploidy in NE adenomas.

Results

Pancreatic NE Tumours (Insulomas; Islet-Cell Adenomas/Carcinomas) and Nesidioblastosis Insulomas: The endocrine tumours of the pancreas the insulomas or islet-cell adenomaslcarcinomas - form that group of NE tumours in which the above-mentioned "paradoxial DNA aneuploidy" is most conspicuous 4. Here, in a recent report from our laboratory2, the data from the pertinent literature were briefly reviewed. We also added our own results of combined FCM and ICM DNA analyses, consisting of 18 cases. We could confirm several previous statements, for instance that a fairly high incidence of clear-cut DNA aneuploidy - both by means of FCM and ICM - is characteristic for the neoplastic cell nuclei of insulinproducing insulomas ("insulinomas"), although there are reports stating that the majority of them display low proliferative activity38, 52, 64, 65. As a matter of fact, all 8 cases of this kind in our material were found to be equipped with tumour cell nuclei displaying an undisputable DNA aneuploidy, either by means of FCM, ICM or both (Fig. 1), despite the fact that all 8 tumours showed the typical appearance of a small, well encapsulated, solitary NE nodule (Fig. 1), histopathologically composed of highly differentiated, insulin immunoreactive, beta cells without any signs of cellular or nuclear atypia and practically lacking mitotic figures. The characteristic amyloid (Fig. 1) was often present together with immunoreactivity of the beta cells for lAPP (islet amyploid poly-peptide)64. None of the 8 patients with clinically and histopathologically benign insulin-producing insulomas with a nuclear DNA ploidy pattern of aneuploid type showed any evidence of recurrence or disseminated NE neoplastic disease. No intratumoral heterogeneity31,32 was found with regard to the FCMlICM DNA ploidy pattern. Another source of error, namely the appearance of artificial "aneuploid" peaks in the FCMlICH DNA histograms evoked by tissue autolysis 3, could be excluded. As described below, the operation specimens were immediately taken care of, and surgical ischemia of the NE tumour during operation was consistently avoided. Such a high incidence of DNA aneuploidy is usually not met in most human carcinomas or malignant lymphomas 2. It should also be pointed out, however, that chemotherapy can evoke a switch from euploidy to aneuploidy23, and that highly differen-

DNA Analysis in Neuroendocrine Tumours· 283

In the clinically and histopathologically malignant insulomas (islet-cell carcinomas), both those producing peptide hormones and those hormonally inactive, the FCMlICM DNA ploidy data were found to be more variable. There was also some discrepancy between the FCM and ICM data 2 and a great range of proliferative activity when measured immunohistochemically by means of the nuclear Ki67 proliferation antigen 52 • Nevertheless, an FCMIICM DNA analysis of the nuclear DNA distribution pattern of the neoplastic parenchymal cells was found to be of some value as a prognostic tooF, 65. When a DNA index (DI) (i.e. the position of the main peak of the DNA histogram on the abscissa when the position of the diploid ("2c") peak of the standard cells is designed as "1.0", and that of a tetraploid ("4c") peak is given the value "2.0") is calculated for these tumours, it was found that the DIs obtained from the FCM data correlated fairly well (p = 0.01) with the final outcome of the neoplastic disease2 • This observation was particularly evident in regard to the predominantly glucagon-producing insulomas and the hormonally inactive ones 65 •

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Fig. 1. Low-power photomicrograph (top) of a small, well encapsulated, benign, insulin-producing islet-cell adenoma with amyloid (middle) and lAPP immunoreactivity, and its ICM DNA histogram (bottom) of the neoplastic cell nuclei. They show a clear-cut aneuploid DNA distribution pattern. The main cell population shows a DNA Index of 1.5 or 3.0c. This is a typical example of the so-called paradoxial aneuploidy of histopathologically and clinically completely benign NE tumours (van Gieson stain; x 7 and x 350).

tiated, insulin-secreting islet-cell adenomas seldom conform to the DNA cytometric rule that "benign tumours are euploid 38 , 52, 64, 65".

Nesidioblastosis: With this background, we thought is would be of particular interest to perform an analogous DNA cytometric investigation of the nuclei of insulin-immunoreactive parenchymal cells in the pancreas of infants with persistent neonatal hypoglycaemia, with hyperinsulinism and idiopathic nesidioblastosis (PNHHN)38, 65. The "nesidioblastotic" insulin-producing beta cells are generally considered to be hyperplastic and not genuinely neoplastic 67, at least not in the idiopathic form of nesidioblastosis 38 , 65. By means of ICM, the nuclear DNA distribution pattern was analyzed of hyperplastic beta cells in subtotal resection specimens from the pancreas of 6 infants with the classical PNHHN picture29 • The technique we used 33 was the one described below for the analogous analyses of ECL (EnteroChromaffin-Like) cells of the gastric corpus mucosa. The images and XIY-positions of the nuclei of the insulin cells in the cut sections of the formalin-fixed, paraffin-embedded specimens, were recorded and stored by a computer. After de staining with methanol and restaining according to the Feulgen procedure, the insulin-cell nuclei were retrieved and their ICM DNA ploidy pattern analyzed. The ensuing DNA histograms were compared with those derived from analogous analyses of the genuinely neoplastic beta cell nuclei from one pancreatic gland in which the hyperinsulinism was caused by an insulin-producing adenoma. All the DNA histograms from the beta cell nuclei of the 6 non-neoplastic "nesidioblastotic" pancreatic glands showed distinctly diploid or tetraploid (normal) DNA distribution patterns (Fig. 2). In contrast, the case with neoplastic beta cells displayed a nuclear DNA ploidy pattern with signs of high proliferative activity, though still essentially diploid (Fig. 2). These observations confirm the above-mentioned working hypothesis that idiopathic PNHHN is a non-neoplastic disease.

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Fig. 2. Two medium-power and one high-power photomicrographs of the pancreas of an infant (out of six), suffering from PNHHN, showing the characteristic features of idiopathic nesidioblastosis with its disseminated insulin-producing islet parenchymal cells (bright in the middle photomicrograph), often showing some large nuclei (arrows; right). The ICM DNA histogram obtained by means of the double-consecutive staining technique of the nuclei of these insulin cells is shown in the lower left part of the panel. There is a distinctly diploid DNA distribution pattern. The main peak is somewhat broader than when only the Feulgen staining is used, e.g. in the DNA histogram shown in Fig. 1. For comparison, the ICM DNA histogram of the nuclei of an insulin-producing, benign islet-cell adenoma in an adult patient with hyperinsulinism is shown in the lower part of the panel. Here, several so-called scattered cells outside the "diploid" peak appear in the DNA histogram, indicating that the neoplastic insulin cells have a slightly higher proliferative potential than the only hyperplastic beta cells in the pancreatic islets of the six patients with PNHHN (H & E stain; x 150; insulin immunofluorescence; x 300; and H & E stain; x 450).

Carcinoid Tumours It is well-known that NE tumours of the carcinoid type can appear in a multitude of locations in the human body96, 121, and that their degree of aggressiveness can vary with the anatomic site of their primary lesion 7o , 108, 109. Thus, the most common carcinoid tu-

mours, namely the classical appendiceal carcinoids 8o, 81, 82, are almost completely innocuous and usually discovered only by incidence. Likewise, almost 90% of the carcinoids in the rectum, another common site 59 , never display metastatic spread or any kind of endocrine syndrome 14 , 116. Also, the so-called EeL cell carcinoids of the stomach seldom give rise to metastatic

DNA Analysis in Neuroendocrine Tumours· 285

lesions 10, 110. In contrast, the second most common carcinoids, namely those arising in the ileum, are notorious for their inherent ability to metastasize and, usually44 when the liver is involved in the dissemination of the neoplastic disease, give rise to the classical carcinoid syndrome 14, 70, lOB, 109. In other anatomic locations, in particular the bronchi B4, the incidence of metastatic dissemination is highly variable 96 , 121. With this information, it is clear that the need for DNA cytometric analysis of the neoplastic cell nuclei is most urgent in ileal ("mid-gut") and bronchial carcinoids. It is also these kinds of carcinoids which have been most studied. One draw-back with the ileal and bronchial carcinoids is the one trait they have in common with practically all other human neoplasms, benign as well as malignant; namely that their etiology and pathogenesis are essentially unknown, although a pathogenetic step is, obviously, their tendency to be associated with hyperplastic proliferations of their cells of origin 1B, 19.

lesions in the regional lymph nodes or in the liver practically never appear in the gastrin-induced carcinoids 10, 110, 113. There are, however, other rare, gastric carcinoids which are obviously not associated with hypergastrinaemia; here, metastases may appearl0. Multiple primary lesions occur in the stomach mucosa and can resolve spontaneously47, 90, 110.

ECL Cell Carcinoids: Among the gastrointestinal carcinoids there is, however, one recently discovered exception to the statement made above; the considerably less common ECL carcinoid of the stomach mucosa. Here, the etiology has been proven to be hypergastrinaemia 19,79, lOB and the stepwise pathogenesis has been experimentally established in detail 49, 51, 79, 107. The first effect of the hypergastrinaemia - experimentally induced via proton-pump inhibitor drugs 49 ,51 partial corpectomy79 or evoked (clinically) via atrophic gastritis50, lOB, 110, with or without concomitant pernicious anaemia - is a diffuse hyperplasia of the EeL cells lB , 19. The next step is a linear hyperplasia. The first sign of a developing tumour is when the linear hyperplasia becomes nodular. These lesions are still, however, reversible and non-neoplastic 49 . The "point of no return" is reached when the nodular hyperplasia switches over to an intramucosal microcarcinoid. An invasively growing ECL cell carcinoid is the neoplastic end stage. As mentioned above, metastatic

Fig. 3. High-power photomicrograph of an EeL cell carcinoid of the stomach, illustrating the so-called consecutive double staining technique we elaborated in our laboratory for making reM DNA analyses of the nuclei of immunohistochemically and histopathologically identified NE parenchymal cells in the gastrointestinal mucosa and the pancreatic islet parenchyma. The small inset (top right corner) shows some chromogranin-immunoreactive EeL cells (black; arrow heads) of the carcinoid. The main photomicrograph gives a magnified picture of the Feulgen-stained nuclei of exactly the same cells (arrow), retrieved after the destaining-restaining procedure described in the text. In the middle part of Fig. 3 the IeM DNA histogram of the nuclei of neoplastic EeL-cells is given, showing that they display an aneuploid DNA distribution pattern. For comparison, the corresponding nuclear DNA distribution pattern of some EeL cells is shown in the lower-most part of Fig. 3, in which the EeL cells are just hyperplastic and not genuinely neoplastic. Here, the nuclei still display a normal, diploid, DNA distribution pattern.

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In order to see whether or not this stepwise evolution of an ECL cell carcinoid has some corresponding alterations in the cytometric DNA histograms from the nuclei of the hyperplastic/neoplastic ECL celis, a new method, devised in our laboratory33, was tried. An image analysis system equipped with a CCD-camera of high resolution and a microscope with an automatic object table device (Merzhauser) was used. For the visualization of the immunoreactive cells, conventional 4-llm-thick, paraffin sections of formalin-fixed specimens were assessed, using the Avidin-Biotin-Complex technique with alkaline phosphatase as enzyme and Fast Red as chromogene. The antisera used (both polyclonal and monoclonal) were raised against the two common "NE markers" chromogranin NB and neuron-specific enolase, as well as against various peptide hormones, serotonin and histamine, including activation of histidine decarboxylase 42,111. The nuclei of the immunoreactive cells and their images were recognized by the pathologist and their X- and Y-coordinateaxis-positions were stored in the computer. The chromogene was then destained with methanol and the sections were restained according to the Feulgen procedure. The stored images of the nuclei of the immunoreactive cells and their X- and Y-axis-positions were recalled by means of the programme mode (ACAS-System, Bargteheide, Hamburg) controlling the automatic object table device. After this re-identification of the immunoreactive celis, their cytometric nuclear DNA content could be assessed according to our routine ICM procedure. As shown in Fig. 3, the histopathological switch from a state of hyperplasia to that of real neoplasia of the ECL cells was found to be accompanied by a change in their ICM DNA ploidy pattern. In this particular case, the slight DNA aneuploidy of the nuclei of the ECL cell carcinoid cells should be looked upon as an additional example of the "paradoxial aneuploidy of NE tumours," rather than a sign of highly malignant neoplastic features. As mentioned above, these gastrininduced ECL cell carcinoids show no aggressive features, clinically or experimentally.

Ileal Carcinoids: Whether or not this new consecutive-double staining technique offers improvements in using DNA cytometry as a supplementary prognostic tool in ileal carcinoids, will be the subject of forthcoming investigations. Preliminary observations, illustrated in Fig. 4, show how a "tetraploid" carcinoid cell population in an otherwise seemingly homogenously "diploid" ileal carcinoid can be detected in this way. Perhaps this technique also gives some insight into the "paradoxial aneuploidy of NE tumours". It is not unusual for subpopulations to be discovered immunohistochemically even in the most homogenously appearing insular carcinoids52 . Maybe such subpopulations have a decisive importance for the subsequent clinical course of the neoplastic disease. Regarding ileal carcinoids, we are presently engaged in extending our previous studies 14, 17, 28 concerning the

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Fig. 4. Differences between the image cytometric DNA ploidy patterns of the nuclei of the neoplastic parenchymal cells of an ileal carcinoid, when the assessments were haphazardly made (upper DNA histogram) over large areas of the tumour, and when the cytometric measurements were performed on immunocytochemically identified neoplastic cells (lower DNA histogram). validity of using the ICM DNA ploidy pattern as a prognostic tool, supplementary to fundamental clinical and histopathological data 60 • The clinical course of 37 patients with ileal carcinoids was followed up to 20 years after operation; correlations were made between data from the histopathological/immunohistochemical examinations and with from a conventional ICM DNA analysis of neoplastic cell nuclei, irrespective of their production of peptide hormones and/or serotonin34 . The results are shown in Fig. 5. Our preceding pilot study28 was based on the ICM data obtained from a series of 8 patients suffering from metastasizing ileal carcinoids. It aimed at investigating if any differences existed in the DNA ploidy pattern between those carcinoids in which the neoplastic disease ran a rather "benign" course, with survival times of the

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Fig. 5. Survival times of patients with ileal ("midgut") carcinoids, related to the reM DNA ploidy patterns of the nuclei of the neoplastic parenchymal cells. Deaths from intercurrent diseases have been recorded in the diagrams as deaths from the neoplastic disease, provided widespread dissemination of the carcinoid was found at autopsy. Obviously, there is - in regards to the course of the neoplastic disease and the length of survival - no clear-cut difference between patients suffering from carcinoids with a nuclear DNA ploidy pattern of euploid (diploid) type (n = 29) and those affected by carcinoids equipped with neoplastic parenchymal cells showing a nuclear DNA distribution pattern of aneuploid type (n = 8). For comparison, the clinical staging is also included (lower diagram).

patients exceeding 5 years, and those in which the patients died from a rapidly progressive disease. Using a rather blunt ICM technique, some minor differences could actually be found 28 . Now, with this more variegated and numerous patient material at hand, it is clear that - even by means of the present, considerably more reliable, ICM method 31 - the value of using the ICM DNA analysis data as a supplementary prognostic cytochemical tool is of little or no value (Fig. 5). The overwhelming majority (about 80%) of the carcinoids - irrespective of whether they occurred in patients with a progressive, rapidly fatal, neoplastic disease or not - were found to be composed of neoplastic parenchy-

mal cells whose nuclei displayed an euploid (essentially diploid) DNA distribution pattern 34 . The results were the same whether the patients had been treated with chemotherapy or not23 • Our findings actually conform with the results of similar investigations performed in several other laboratories, using ICM or FCM DNA analyses in their malignancy assessment routines 91 , 117 and/or immunohistochemical techniques to measure the proliferative activity of the carcinoid neoplastic cells 52 . Thus, clearly aneuploid DNA distribution patterns turned out to be rare (about 20%), even in those cell nuclei belonging to the most aggressive carcinoids. It should be emphasized here, however, that in other primary sites of the carcinoids, e.g. rectal carcinoids, a better correlation was obtained between the DNA cytometry data and the prediction of the malignant potential of the tumour cells together with that of the subsequent course of the disease 15 ,116. A "near-hypertriploid" DNA distribution pattern indicated a rapidly progressive, fatal, rectal carcinoid neoplastic disease15.

Bronchial Carcinoids: The NE tumours of the bronchi are looked upon nowadays as a broad spectrum of neoplasms, extending from a highly differentiated typical bronchial carcinoid via the so-called atypical carcinoids and well-differentiated NE carcinomas, to the poorly differentiated intermediate- and small-cell bronchial carcinomas of the classical oat-cell type 48 , 58, 69. In regard to latter three types, the overwhelming majority are reported to be composed of neoplastic cells with a cytometric nuclear DNA distribution pattern of aneuploid type 58 . Although there is a greater variation in the corresponding results between different laboratories concerning genuine carcinoids, there are DNA cytometric observations that indicate a significant increase in the DNA content from typical carcinoids via atypical ones to small-cell NE carcinomas 69 . Aneuploidy was seen in about 80% of the atypical carcinoids but only 20% in the typical 84 . When, however, attempts were made to assess the malignant potential of the typical and atypical carcinoids, the DNA cytometry data were often found to be of limited value 120 . We can confirm observations of this kind in an unpublished study from our laboratoryl00 on 29 patients; 24 of them suffered from bronchial carcinoids histopathologically classified as "typical"; 5 patients, "atypical". By means of the old Adams ICM technique, the nuclear DNA distribution patterns were found to be of diploid type in all 3 of the 24 "typical" carcinoids, but only in one of the 5 "atypical." Clear-cut aneuploid nuclear DNA distribution patterns were not observed. Two patients died in less than one year from their neoplastic disease; their tumours were large (> 3.5 em), histopathologically "atypical" or "fascicular", and the neoplastic cell nuclei displayed a DNA ploidy pattern of "non-diploid" type. Otherwise, there was no clear-cut correlation between the size of the tumour, the histopathological type of growth pattern, the cyto-

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metric DNA analysis data, and the course of the neoplastic disease (Fig. 6). Thus, it can be concluded that bronchial carcinoids, irrespective of size and growth pattern, are usually equipped with neoplastic cells whose nuclei are cytometrically DNA diploid. No reliable correlation seems to exist between the DNA ploidy pattern and the course of the neoplastic disease 12o • Phaeochromocytomas and Paragangliomas

The value of data derived from FCM and/or ICM DNA analyses of nuclei of neoplastic parenchymal cells of phaeochromocytomas (Fig. 7A) and paragangliomas (Fig. 7B) has recently been accounted for in a review from our laboratories 3o in a monograph on the diagnostic histopathology of the NE tumours. In addition, an original report was given a couple of years ago on the possibility of combining immunohistochemical peptide hormone data with data from ICM DNA analyses of nuclei from neoplastic chief cells of extraadrenal paragangliomas in the malignancy assessment of NE tumours 61 • Results of analogous investigations performed in other laboratories with NE derived from

the parenchymal cells of the unitary system of paraganglia, have shown that discrepancies exist in regards to the value of the DNA ploidy data as a supplementary prognostic tool, both in phaeochromocytomas and paragangliomas 3 ?, 62, 85, 94. This is regretting since the practising histopathologist in his daily diagnostic work is not so much concerned with making the correct diagnosis of this tumour group, as in trying to assess the degree of malignancy of each individual neoplasm. Here, the phaeochromocytoma/paraganglioma group of NE tumours provide an additional example of how blunt our tools are for predicting the subsequent course of the neoplastic disease by means of cell-biological features of tumour cell nuclei and organelles. "Paradoxial aneuploidy" can also appear in this major group of NE neoplasms 3o,61. Phaeochromocytoma: Concerning phaeochromocytomas, Dr. Hansson and Jansson at the Sahlgrenska Hospital in Gothenburg have provided the material of 60 patients followed for at least 5 years after operation 43 • Both sporadic and (a few) familial (MEN-2a) cases are included. In addition to clinical staging and conventional histopathological malignancy assessments, an ICM DNA analysis has been made of the neoplastic parenchymal cells. Out of 60 intra-adrenal phaeochromocytomas from 59 patients (one patient had a bilateral tumour), 50 were classified clinicopathologically as benign (or at least "probably benign"), and 10 as malignant (or at least "possibly malignant"). As control material, 7 specimens of a histopathologically normal adrenal medulla were included in the ICM DNA analyses. The latter all turned out to be composed of parenchymal cells with a nuclear DNA distribution pattern of diploid type; thus, no tetraploid nuclei were detected. In contrast, the overwhelming majority - 28 tumours - of the benign phaeochromocytomas were found to be composed of neoplastic parenchymal cells with a tetraploid DNA distribution pattern of their nuclei; only 12 of the benign tumours were of diploid type. In 2 of them, it was even octaploid 62 • A clear-cut aneuploid DNA pattern was displayed by 8 of the benign phaeochromocytomas, i.e. an incidence of "paradoxial aneuploidy" of 16%. The main four types of ICM DNA histograms observed in our series of phaeochromocytomas are shown in Figs. 7 C-F. Of the 10 clinico-pathologically (possibly) malignant phaeochromocytomas, the diploid/tetraploid/aneuploid ICM DNA distribution patterns of the nuclei of their neoplastic parenchymal cells were 1/3/6, respectively. Thus, an euploid DNA pattern was found in 40% of these (possibly) malignant phaeochromocytomas. When survival curves of the patients with the clinically and histopathologically malignant phaeochromocytomas were made and related to the ICM DNA ploidy data, it has, until now, been found that the latter were obviously of no additional predictive value. These preliminary observations thus indicate that an ICM DNA analysis can be of some value as

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a malignancy assessment tool but not as a particularly good prognostic factor for the subsequent course of the neoplastic disease. Here, our results conform with those of some previous investigations made by means of FCM54, 105, but conflict with those obtained by means of ICM93. Thus, it can only be concluded that the value of cytometric DNA analyses in phaeochromocytomas still suffers from the same limitations as in most other NE neoplasms.

Thus, it can be concluded that DNA cytometry as a prognostic tool, supplementary to the fundamental clinical data and histopathological malignancy assessments, actually can be of some value regarding extraadrenal paragangliomas. This does not always seem to be the case in the phaeochromocytomas of this major group of NE tumours.

Paraganglioma: The situation seems to be rather similar when we are dealing with extra-adrenal paragangliomas. Admittedly, it has previously been stated that the DNA ploidy of these tumours cannot serve as a predictor for an expected growth pattern because unequivocal evidence of DNA aneuploidy was present in almost 40% of clinically and histopathologically benign tumours30, 61. These observations were, however, obtained by means of FCM, and paragangliomas definitely belong to those neoplasms in which it is important to use the ICM techniques rather than the FCM procedure alone to analyze the DNA ploidy pattern of nuclei of neoplastic parenchymal cells 61 , in particular the chief cells 37 . For that reason, we made a rather comprehensive, combined immunohistochemical and ICM DNA study of 22 paragangliomas from various anatomical locations; 5 of them were clinically and histopathologically assessed as malignant. As controls, one normal and 5 hyperplastic carotid bodies were used. All the 6 control carotid bodies were found to be equipped with parenchymal cells displaying a diploid type of nuclear DNA distribution pattern - irrespective of whether the carotid bodies were hyperplastic or normal. The nuclear DNA ploidy pattern of the genuinely neoplastic chief cells was found to be of enploid type in 17 out of the 22 paragangliomas (11 diploid, 6 tetraploid). Except for one case, all 17 paragangliomas had been clinico-pathologically classified as benign. The remaining 5 paragangliomas in which the nuclear DNA ploidy pattern of their chief cells was classified as aneuploid type, had actually been clinico-pathologically assessed as malignant - again with one exception. Thus, as in other NE tumours, cases of "paradoxial DNA aneuploidy" could also occur in benign paragangliomas, and - conversely - malignant paragangliomas could show an euploid nuclear DNA distribution pattern of its chief cells. It should be stressed, however, that these findings obviously are rather exceptional in paragangliomas, and that careful DNA analyses performed by means of ICM, usually give DNA ploidy data that can be of additional help in predicting the biological behaviour of this type of NE neoplasms. After our ICM study, some new FCM investigations have been made of paragangliomas 37, 94. This time, the results obtained turned out to conform with what we found in our ICM observations61 • In contrast, the numerous immunohistochemical data we had collected, did not permit any distinction between benign and malignant extra-adrenal paragangliomas 61 •

A most flagrant example of how a diploid cytometric nuclear DNA distribution pattern of the neoplastic parenchymal cells of an NE tumour can be compatible with - and even more or less predict - a highly aggressive and rapidly fatal neoplastic disease, is the neuroblastoma. Here, it is even so that a certain type of aneuploidy ("triploidy") is part of a set-up of particularly favourable prognostic variables which can - with practically 100% certainty - select those neuroblastoma patients who shall be ultimately cured from their neoplastic disease from those with a poor prognosis 20 , 66. It is actually difficult to find more dramatic proof of how the NE tumours violate the fundamental rule of DNA cytometry in human tumour pathology, that "euploidy" indicates low aggressiveness of the neoplasm and longevity of the cancer patient, whereas "aneuploidy" signifies "high malignancy" of the tumour and a progressive, rapidly fatal neoplastic disease. Together with Drs. P. Kogner, C. Dominici and B. Sandstedt at the Karolinska Hospital, one of us (UGF) has analyzed a fairly great number, viz. 84, of neuroblastomas during the last few years with regards to the cytometric nuclear DNA ploidy pattern of their neoplastic parenchymal cells. The data obtained have been correlated with those of a whole range of clinical and immunohistochemical variables, more or less known to be of prognostic value in neuroblastoma 2o, 66. Particular attention was paid to neuropeptide Y (NPY), the receptors for the nerve growth factor (NGF), and the expression of mRNAs of the oncogene N-myc and the protooncogen trk 20 , 66. The DNA ploidy assessments were made by means of both FCM and ICM, using i.a. imprints on fresh biopsy or operation specimens. From a total of 60 neuroblastomas, technically acceptable DNA histograms were obtained by means of both FCM and ICM. The results showed complete agreement. In 24 cases the DNA ploidy assessment could only be made by ICM (too much "background noise" in the FCM DNA histograms). Out of the 84 neuroblastomas analyzed by means of ICM, 41 (49%) were found to be composed of neoplastic parenchymal cells with a nuclear DNA distribution pattern of diploid type, 8 (9%) of tetraploid and 35 (42%) of cells with an aneuploid DNA pattern. In previously made DNA cytometric investigations of neuroblastomas 12 , 21, 74, 75, 92, no great intratumoral variations appeared in the nuclear DNA ploidy pattern of the tumour cells; we confirmed this observation.

Neuroblastoma

DNA Analysis in Neuroendocrine Tumours· 291

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At a closer scrutiny of the ICM DNA histograms of aneuploid type, it was observed that 22 of them could be classified as "triploid" or "near triploid" (Fig. 8). When the DNA ploidy data were correlated with results from investigations with other prognostic variables (see above), it was found that the patients who suffered from neuroblastomas composed of neoplastic parenchymal cells with this triploid type of nuclear DNA distribution pattern, were of particular interest. They turned out to belong to that category of patients who had low plasma levels of NPY and whose tumour cells showed no signs of N-myc amplification, but a clear-cut expression of both the trk proto-oncogene and the low-affinity NGF receptor. All of these patients survived; the neoplastic disease ran this favourable course, irrespective of other clinical, histopathological, or immunohistochemical variables 2o ,66. Conversely, those patients whose neuroblastoma parenchymal cells displayed a nuclear DNA ploidy pattern of the (normal) diploid type (Fig. 8), were found to belong to that category with high levels of NPY in the plasma, and tumour cells with signs of N-myc amplification but no expression of the mRNAs of trk or the low affinity receptor of NGF2o, 66. All these patients died from their neoplastic disease. In a patient with a neuroblastoma, the results of an ICM or FCM DNA analysis performed on a biopsy specimen or on an extirpated tumour can give the very first indication of what type of neuroblastoma the patient is suffering from. Thus, long before the data from the radio-immunoassays, the Northern and Southern blot analyses, the in-situ hybridizations and the chro-

mosomal analyses are available, the preliminary arrangements for adequate therapy can start20,66.

Pituitary Adenomas In the daily diagnostic work, the surgical pathologist is often faced with a request from the clinical colleagues not only to make a correct histopathological and immunohistochemical diagnosis but also a malignancy assessment of a pituitary adenoma based on a number of minimal fragments from the aspirated tumour, sucked out trans-sphenoidally by the otolaryngologist from the sella turcica. This is a difficult task. Some adenomas are so highly differentiated that their neoplastic parenchymal cells are almost structurally identical with the non -neoplastic 68 • An admixture of normal adenohypophyseal cells can form several of these minimal fragments. It is often not until some of the specimens turn out to emanate from the periphery of the lesion that it is possible to discern a surrounding capsule with compressed normal parenchyma, thus allowing an adenoma diagnosisS 3, 68. Although pituitary adenomas are generally looked upon as benign tumours - it can even be that some of these "adenomas" are actually just nodular hyperplasias 68 - it is well known that up to 20% of them show local invasions3 • If microinvasion of the adjacent dura is also taken into account, this frequency increases to almost 70% in microadenomas (largest diameter less than 10 mm) and close to 90 % in macroadenomas s3 • In addition, some adenomas may display considerable cellular and nuclear atypia and pleomorphism, together

292 . U. G. Falkmer and S. Falkmer

with the occurence of mitotic figures s3 ,68. Extremely aggressive tumours can penetrate the sphenoid sinus, other parana sal sinuses, and even the structures surrounding the base of the brains3 . Despite all these histopathological criteria of a malignant neoplasm, distant metastases are virtually exceptional. Therefore, the diagnosis "pituitary carcinoma" should be reserved for those extremely rare tumours in which metastatic lesions actually have been found, for instance in lymph nodes, liver, lungs and skeleton. The term "invasive pituitary adenoma" should be used for the rest S3 . Against this background, it is clear that the pituitary adenomas share the basic features of other NE tumours, namely the lack of frank diagnostic "criteria for malignancy" in them, and that the histopathological features are inadequate to predict the biological behaviour of the tumour S3 , 68. Consequently, several attempts have been made to introduce supplementary cell-biological tools in the malignancy grading of pituitary adenomas. Typical examples of so-called proliferation markers which have been tried, are the BrdU (bromodeoxyuridine) labelling index, immunostainings for various antigens, such as Ki-67 and p-105, the expression of DNA polymerase, and assessments of the number of nuclear organizer regions (AgNORs)S3, 68. So far, it seems as if the value of most of these proliferation markers has not been generally recognized. DNA cytometry has also been tried as a supplementary prognostic tool; the first attempts were made with FCM a decade ago S3 , 68. Then - and afterwards - the cells investigated were found to be equipped with nuclei displaying either an euploid (usually diploid) DNA distribution pattern or an aneuploid one S6 • This occurrence of nuclear aneuploidy could, however, not be correlated with any particular clinical features, such as age, sex, symptoms, the course of the neoplastic disease and the hormonal levels in the blood 56 • Neither were there any differences between pituitary adenomas with a nuclear DNA ploidy pattern of an aneuploid or euploid type regarding radiological data about the size and signs of invasive growth of the tumours, their histopathological growth patternS3 ,68 and immunohistochemical signs of hormone productions3 , 68. Since the cytometric DNA analyses in these earlier investigations were mainly made by means of FCM, it was considered worth while to collect a number of additional cases of pituitary adenomas of various kinds over a time period of at least 5 years, and to repeat the DNA analyses by means of not only FCM but also ICMs7. The main reason was namely that the specimens obtained at operation by the transsphenoidal approach can, to an unknown extent, consist of normal adenohypophyseal parenchyma. In the FCM procedure there is no control of what is actually analyzed 31 . By means of ICM, the possibility to identify the cells analyzed are fairly good. 95 pituitary adenomas were classified histopathologically and immunohistochemicallyS7. The adenomas were subdivided into two major groups. The first consisted of the classical chromophobe adenomas with no

- or only a faint and diffuse - immunoreactivity against a panel of antisera raised against the most prevalent peptide hormones in the pituitary, and with no elevated hormone levels in the patients' blood. The second group represented the typical acidophil or basophil adenomas with distinct immunoreactivities against growth hormone (GH), prolactin (PRL), or both concomitantly, and ACTH, respectively. The patients displayed the pertaining clinical findings. The former main group consisted of 49 cases, 39 of which could be analyzed by means of both FCM and ICM, the rest by ICM only. In all the 39 assessments, the results obtained by means of FCM and ICM agreed regarding euploidy versus aneuploidy. Out of the 49 ICM analyzed tumours, 45 were found to be equipped with neoplastic parenchymal cells displaying a nuclear DNA distribution of euploid (44 diploid; 1 tetraploid) type. Thus, the incidence of nuclear aneuploidy was about 8 % in the chromophobe adenomas. In the second main group consisting of 45 cases (9 GH, 7 PRL, 23 GH + PRL and 6 ACTH cases in the four immunoreactive groups, respectively), the concordance of the results obtained by means of FCM and ICM was almost as good as in the first group; 28 tumours could be analyzed by means of both techniques (13 by ICM only and 4 by means of FCM); in 26 the data obtained were in complete agreement; in 2, however, the DNA ploidy pattern of the nuclei of the neoplastic parenchymal cells was assessed to be of diploid type when analyzed my means of FCM but of aneuploid type by ICM. Out of 41 adenomas assessed by means of ICM, 28 were found to be composed of tumour cells whose nuclear DNA distribution pattern was of euploid (27 diploid; 1 tetraploid) type; in 13 it was assessed as being aneuploid. Thus, the incidence of nuclear aneuploidy was 32 % in the peptide hormone immunoreactive adenomas; i.e. four times higher than in their chromophobe counterparts. There was also an additional group of 6 patients with pituitary adenomas; here, there were increased concentrations of growth hormone or prolactin in the blood but no corresponding immunoreactivity in the neoplastic parenchymal cells. The incidence of DNA aneuploidy in the nuclei of these tumour cells was found (by means of ICM) to be the same as in the corresponding group with presence of immunoreactivity in the neoplastic cells, namely 2 out of 6 (one of these 2 tumours was classified as "diploid" by means of FCM). These observations are, to a major extent, derived from a recent report from our laboratories 57 • For the present review they are of particular interest in three major respects. Firstly, they show that the use of FCM instead of ICM can - as expected - give a falsely diploid DNA distribution pattern. Whether or not this is a result of an analysis of the nuclei of the normal parenchymal cells instead of those of the neoplastic ones, is difficult to decide. It is, however, a plausible explanation of this discrepancy. It should be stressed that our results show that if such a methodological pitfall in FCM exists, it is very exceptional (3 % ). Secondly, our present observations give an excellent

DNA Analysis in Neuroendocrine Tumours· 293

additional confirmation of the initial statement that pituitary adenomas definitely belong to that group of NE neoplasms displaying a clear-cut paradoxial aneuploidy. As previously found 56 , there was no relationship whatsoever, between the nuclear DNA ploidy pattern of the neoplastic parenchymal cells and the clinical, radiological, or histopathological variables. As a supplementary diagnostic or prognostic tool, DNA analyses of pituitary adenomas must, consequently, be looked upon as rather blunt. Thirdly, the present observations also confirm results, previously obtained in our own56 and in other laboratories53 , 68, that the incidence of pituitary adenomas, equipped with tumour cell nuclei displaying a DNA distribution pattern of aneuploid type, is higher (about 1 out of 3) in histopathologically highly differentiated, hormonally active neoplasms than in the chromophobe adenomas without any clinical or histopathological signs of hormone overproduction (about 1 out of 12). These findings are somewhat difficult to explain cell-biologically. If parallels are to be drawn to the conditions in another NE organ, namely the islets of Langerhans, it is so that their structural homologues to the pituitary chromophobe cells might be agranular or poorly granulated islet parenchymal cells 9,27. They are normally rare but can increase considerably in number in conditions with a need for regeneration, for instance in the islets of the rest of the pancreas after a subtotal pancreatectomy9, 2? and in the restitution phase of transient alloxan diabetes of spontaneous or hereditary diabetes mellitus 9,27. A neoplastic variant of these agranular or sparsely granulated "chromophobe" islet cells appears in poorly differentiated islet-cell carcinomas2? From this parallel, based on light-microscopical, immunohistochemical, and ultrastructural similarities only, it could be assumed that the agranular or sparsely granulated islet cells represent young, not yet fully differentiated, precursor cells 9,27, and that the pituitary chromophobes could also be "reserve" cells, ready to mature into fully granulated acido- or basophilic parenchymal cells with various kinds of peptide hormone production 53, 68. When they are neoplastically transformed, it would be no greater surprise if they displayed a higher incidence of nuclear DNA aneuploidy than that of an adenoma composed of highly differentiated, amply granulated parenchymal cells, difficult to differ histopathologically from their normal counterparts. Although this is not too seldom the case in the poorly differentiated insulin-producing islet-cell carcinomas, in comparison with the highly differentiated benign insulomas (see above), the reverse obviously holds true for the pituitary adenomas. Thus, the paradoxial data from a DNA-cytometrical point of view, is even more marked than in most other NE tumours. This implies that a solution to the paradox can, in the future, perhaps be found in the hormonally active pituitary adenomas.

Parathyroid Adenomas In general, the parathyroid glands form a contrast to the adenohypophysis in regards to the value of cytometric DNA analyses as a diagnostic and prognostic tool, supplementary to the fundamental clinical and histopathological data. Although there are reports that "paradoxial DNA aneuploidy" can occur in the parathyroid glands, not only in completely benign adenomas?? but even in simple hyperplastic nodules 115, the incidence of such findings is apparently low55 . Generally speaking, cytometric DNA analyses are often reported to be of some diagnostic and prognostic value when an assessment of the proliferative capacity of parenchymal cells of a parathyroid lesion shall be made 115 . Thus, in the extremely rare, metastasizing parathyroid carcinomas, the majority of neoplastic cells are equipped with nuclei displaying an aneuploid DNA distribution pattern 55 , 102, 103, 104. Conversely, normal and simply hyperplastic parathyroid parenchymal cells practically always harbour nuclei with a diploid pattern 115 . Here, it should be added that we have recently found that the occurrence of so-called scattered cells in the ICM DNA histograms of the nuclei of parathyroid parenchymal cells, displaying different kinds of hyperplasia, can be of diagnostic value 115 . The cytometric concept "scattered cells" is defined as those cells whose nuclei display a DNA distribution pattern, falling outside the usual diploid or tetraploid peaks in the DNA histograms, but still so evenly distributed between the GOG 1 and G2M peaks that their presence does not cause the DNA pattern to become classified as an aneuploid type 115 . By means of using BrdU incorporation in proliferating cells and the immunohistochemical technique with an antibody against BrdU (Bromodeoxy-uridine), we (together with Drs. H. Kouvidou, O. Ahrens and A. Haag) have been able to show that these "scattered cells" actually represent cells, whose nuclei are in the S-phase of the cell cycle (unpublished). As shown in Fig. 9, such cytometric evidence for cell proliferation in parathyroid parenchyma is rather conspicuous in parathyroid glands autographed in skeletal musculature but can also be fairly prominent in nodular hyperplasia. In order to get an idea of the incidence of "paradoxial DNA aneuploidy" in benign parathyroid adenomas, and - at the same time - to compare the degree of reliability of procedures in the DNA analysis of parathyroid lesions, parathyroid glands from a total number of 235 patients with primary hyperparathyroidism (HPT) were investigated. The data given here are preliminary and some of them are included in a report which is still in preparation71. In these 235 patients, HPTwas found to be caused by genuine adenomas in 216 (in each of 6 of these patients even two adenomas were detected). Thus, a total of 222 adenomas were examined histopathologically; 3 of them were excluded, mainly because they were "atypical" in some respects and, consequently, did not fulfil the histopathological crite-

294 . U. G. Falkmer and S. Falkmer

ria of a completely benign parathyroid adenoma72. Out of the remaining 219 adenomas, the overwhelming majority was found to be of the common chief-cell type, either pure or admixed with more or less distinctly differentiated oxyphil cells; only in 18 adenomas were the oxyphil cell type predominant. In 19 of the 235 patients with HPT, the disease was found to be caused by a diffuse or nodular hyperplasia of the parathyroid glands;

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from these 19 patients, 35 glands were examined histopathologically. The material available for cytometric DNA analysis from the 219 benign adenomas was sufficient enough to allow ploidy assessments by means of both FCM and ICM in 155 of them. In 5 cases neither of the two techniques gave DNA histograms that could be used for optimal ploidy assessments. The technical failures were particularly numerous when the DNA analyses were made by the FCM procedure; here, 48 adenomas had to be analyzed only by ICM. In 11 adenomas the ICM measurements either failed (2 cases) or were - for various reasons - not performed (9 cases); here, only the FCM results were available. The frequency of technical failures in the FCM procedure was thus about 22%. The corresponding figure for the ICM technique was 2. In those 155 adenomas with technically satisfactory data from both FCM and ICM assessments, complete conformity was arrived at in 144, i.e. in 93%. If differences of diploidy versus tetraploidy are disregarded both belong, after all, to the concept "euploidy"31 the conformity was as high as 99%. Only two exceptions occurred; one case was assessed as diploid by means of FCM but as aneuploid by ICM; in a second case the results obtained were just the opposite. In the 203 cases analyzed by means of ICM, the nuclear DNA distribution pattern of the neoplastic parenchymal cells was found to be of diploid type in 150, of tetraploid type in 29 and of aneuploid type in 24. Thus, the incidence of "paradoxial aneuploidy" ... Fig. 9. One low-power (top) and one high-power (middle) photomicrograph of a parathyroid gland, transplanted to the skeletal musculature of a patient with secondary HPT with relapse. In addition, examples are given of IeM DNA histograms from the nuclei of parathyroid parenchymal cells with a clear-cut diploid nuclear DNA distribution pattern in various states of hyperplasia, showing the relative incidence of so-called scattered cells. It is the small columns on the Xaxis of the histograms, located to the right of the large diploid peak which indicate the "scattered cells". The four examples of DNA histograms are also chosen from the parathyroid parenchyma of patients with secondary HPTwith relapse. In addition to those from normal parenchyma and/or diffuse hyperplasia at the initial operation, the histograms are also from nodular hyperplasia, from the parathyroid parenchyma at operation for recurrence and from a parathyroid gland autografted in skeletal musculature. The relative incidence of the "scattered cells" reflects the cells in the S-phase of the cell cycle32 , 115; thus, high percentages of "scattered cells" in the DNA histograms indicate high cell proliferation activity of the parenchyma. As shown in the four DNA histograms, the highest proliferative activities seem to occur in nodular hyperplasia (particularly at reoperation for recurrency) and in the autografted parathyroid parenchyma. It has been hypothesized that this can be an expression of the possibility that stimulatory factors in secondary, renal HPT are focused on a smaller parenchymal cell mass than that present before the primary operation l15 • As shown in the photomicrographs,

there are no signs of cellular or nuclear atypia, invasive growth or presence of mitotic figures (H & E; x 150 and x 500),

DNA Analysis in Neuroendocrine Tumours· 295

amounted to 12 % in these benign parathyroid adenomas. There was no relationship to the topography, size or cellular compositions of the adenomas. It should be added that there was no single case of nuclear DNA aneuploidy of hyperplastic chief cells in the 35 parathyroid glands from those 19 patients in whom the HPTwas caused only by a diffuse or nodular hyperplasia. In the three atypical adenomas the nuclei of the neoplastic parenchymal cells displayed an ICM DNA distribution pattern of euploid type. There were no HPT reccurrences in the patients with adenomas. When our present data are compared with those of some recent reports in this field, both conformities 77, 103 and discrepancies 55 are found. Incidences of aneuploid type of nuclear DNA distribution of the neoplastic parenchymal cells in adenomas from patients with HPT vary from 0%55 to 23 % 77,103; methodologic differences can contribute to the magnitude of these variations31, 115. Of particular interest are the observations that some intratumoral variations can occur in parathyroid adenomas with regard to the type of nuclear DNA ploidy pattern found in the tumour cells 103, 104; in the present study, such variations were observed in 2 cases out of27. They can, of course, also contribute to explain discrepancies in the results from different laboratories.

Medullary Thyroid Carcinoma About 10% of all carcinomas of the thyroid gland are of NE nature 72, 83. They consist of neoplastic parafollicular calcitonin (CT)-producing C-cells; this cell is still believed to be derived from the neural crest, thus supporting AGE Pearse's original APUD concept? Best known is the medullary thyroid carcinoma (MTC), but during this last decade the new entity of "thyroid carcinoma of intermediate type" (i.e. a carcinoma with follicular-parafollicular cell differentiation) has been discovered72, 73, 83; it should be added to the category of NE carcinomas of the thyroid gland. It is supposed to be extremely rare; data about the long-time natural course of this neoplastic disease are, for natural reasons, still rather fragmentary72, 73, 83. Far better investigated is MTC. As was the case with most other NE carcinomas, the oncologists and the pathologists are faced with the problem of having rather blunt tools at their disposal when trying to assess the degree of aggressiveness of the neoplastic disease in the individual patient making it difficult to predict its clinical behaviour and to install the adequate therapy106,112. Cause-specific mortality rates at 10 and 15 years have recently been reported at 15% and 26%, respectively72. 83, 95. A great number of prognostic variables have been tested by both univariate and multivariate analyses 95 . Risk group schemes for patients with MTC have been launched, based on disease stage (TNM classification), completeness of tumour resection and the results of amyloid staining in the carcinoma 95 . It has been claimed that a more favourable

clinical course is met with, not only in TNM grades I and II, complete tumour resection, and ample amounts of amyloid in the carcinoma, but also in patients with small tumours, high CT levels in blood and distinct CT immunoreactivity in the neoplastic Ccells 95 . The female sex and young age have also been suggested to be associated with longer disease-free survival, as well as inheritance (MTC as a component of the MEN-2a syndrome)41, 72, 83. The apparently poorer prognosis of patients with sporadic disease may, however, be partly related to their older age 41 , 72, 83. Quite recently, it has also been shown that immunostaining of more than 10% of the neoplastic parenchymal cells by a polyclonal antiserum to the N-myc gene product was that variable most strongly associated, both in univariate and multivariate analysis, with an unfavourable MTC outcome 99 . Against such a background of a great need for reliable prognostic indicators in MTC, it is also natural that the value of cytometric DNA analyses has been investigated. During the last decade, data have been made available from assessments by either FCM or ICM22, 41, 95, 101, 106, 112; results of correlated FCM/ ICM measurements in MTC seem to be lacking. Data are also missing regarding the occurrence of "paradoxial aneuploidy" in neoplastic thyroid C-cells; the only completely benign tumour-like lesions known here are the multiple hyperplastic nodules found in thyroid glands from patients with MTC as a part of the MEN-2a syndrome41, and they do not seem to have been assessed by means of ICM72, 83,106. In a retrospective study from our own laboratory a couple of years ago 106, the tumour parenchyma of 42 patients with MTC was analyzed with regard to its nuclear DNA distribution pattern. The ICM DNA assessments were made on isolated neoplastic cell nuclei of deparaffinized, disaggregated specimens of the tumour nodules 106 . The nuclear DNA ploidy pattern was found to be of euploid type in 35 cases (24 diploid, 11 tetraploid). Thus, only a minor fraction (one per six) of the MTC in that series were equipped with neoplastic parenchymal cells in which the ICM nuclear DNA ploidy pattern was of aneuploid type. With one exception, the 7 patients whose tumours belonged to this group had died from their MTC or were still alive but had metastases; the follow-up time periods varied from 1 to 19 years106. When conventional Kaplan-Meier survival curves were made, it was observed that a marked difference existed between the group of patients with tumours in which the nuclei of the neoplastic parenchymal cells displayed an euploid (diploid and! or tetraploid) DNA distribution pattern and that with tumours showing an aneuploid one (Fig. 10). Similar results, obtained by means of other DNA assessment techniques, had previously been obtained, both from our 112 and other22 , 41, 95, 101 laboratories. The fairly low incidence of patients with MTC cells displaying an aneuploid DNA ploidy pattern fits well with the fairly indolent course of the neoplastic disease in most cases of MTC72, 83. It should be pointed out, however,

296 . U. G. Falkmer and S. Falkmer Kaplan-Meier survival curve 100

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Fig. 10. Diagrams showing the cumulative survival of a patient group (n = 35) of MTC106 in which the ICM nuclear DNA histograms of the neoplastic cells were of euploid (diploid or tetraploid) type ("E" in the diagram), as well as that of MTC patients (n = 7) with such DNA histograms of aneuploid type ("A" in the diagram). The neoplastic disease was considerably more aggressive in the latter group.

that when FCM assessments of fresh operation specimens of MTC have been maden, 83, 106, the fraction of tumours with neoplastic parenchymal cells showing an aneuploid DNA distribution pattern in their nuclei has be~n reported to be greater than the incidence in our senes. These observations can be interpreted as showing that cytometric DNA analysis is a useful "malignancy-assessing" and prognostic instrument, supplementary to fundamental clinical and histopathological investigations 106 • This is, however, not the whole truth. In other laboratories where univariate and multivariate analyses of 12 tumour-pathologic variables have been made, the FCM DNA ploidy pattern failed to give any statistically significant power to predict increased mortality risk in MTC9S. On top of that, at closer scrutiny of our own published data 106 , it could be stated that no less than about half of the total number of patients whose tumours consisted of neoplastic C-cells with an ICM nuclear DNA distribution pattern of euploid type, were found to belong to the group with a progressive, fatal, neoplastic disease, or with remaining, recurring, and/or metastasizing tumour parenchyma. Moreover, the number of patients in this DNA euploid group who were alive without signs of MTC was found to be somewhat smaller than that of the patients in that DNA euploid group who had remaining, recurrent tumour parenchyma, and/or metastases 106 • Lastly, the neoplastic disease of one of the 7 patients whose MTC cells displayed a clear-cut aneuploid DNA distribution pattern, actually ran the course of a benign tumour 106 • It is thus obvious that in MTCas in other NE carcinomas - the data obtained from cytometric DNA analyses of the nuclei of the neoplastic parenchymal cells must be interpreted with great cau-

When looking for a very highly malignant, aggressive, and poorly differentiated NE carcinoma, it is natural to turn the attention to the Merkel-cell tumour of the skin39, 63, 118. In this rare entity, ultrastructural investigations have shown that the neoplastic parenchymal cells are so poorly differentiated that they contain only few neurosecretory granules 39 , 63. This fact implies that the conventional, granule-associated NE markers like argyrophilia, immunoreactivity for chromogranins and peptide hormones, usually give only faint light-microscopical reactions 118 ; cytosolic NE markers (like neuron-specific enolase) are needed to demonstrate the true NE nature of the carcinoma39 , 63. Here it could be assumed that, at last, an NE carcinoma would be found that might adhere to the rule of thumb of DNA cytometry as a diagnostic and prognostic tool that the more anaplastic the carcinoma, the more distinctly aneuploid is the cytometric nuclear DNA distribution fattern of its neoplastic parenchymal cells s, 31, 7 ,97. It is known that heterozygous allelic deletions occur of varying extent on distal chromosome 1 p in the neoplastic DNA of Merkel-cell carcinoma46 • Cell

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Fig. 11. An ICM DNA histogram, showing the nuclear ploidy pattern of the neoplastic cells of a Merkel-cell carcinoma of the skin (in a 75-year-old man with a rapidly growing lesion). It is a distinctly aneuploid pattern with a proliferation index of above 10%. This conforms with the histopathologic picture indicating a poorly differentiated, rapidly growing, highly malignant NE carcinoma. It should be emphasized, however, that Merkel-cell carcinomas with an identical clinical history and histopathological picture can display a quite normal, diploid nuclear DNA distribution pattern of neoplastic cells; 3 out of 6 Merkel-cell tumours recently studied in our laboratory showed this phenomenon.

DNA Analysis in Neuroendocrine Tumours· 297

During these last few years, we at the Karolinska Hospital secceeded in collecting - mainly through the courtesy of Drs. V. Bjarnhagen and A. Haag - a total of 6 Merkel-cell tumours of the skin in 6 patients. Their age range was 69 - 85 years of age; one female patient was exceptional in this respect - she was only 20. The sex distribution was equal. Histopathologically, immunohistochemically, and ultrastructurally, all the tumours fulfilled the diagnostic criteria of a Merkel-cell tumour. The cytometric DNA analyses of the nuclei of the neoplastic parenchymal cells were made by means of both FCM and ICM (using "imprints" made at operation). Much to our surprise, 3 of them were found to be equipped with tumour cells displaying a nuclear DNA distribution pattern of clear-cut diploid type; the rest were of aneuploid type (Fig. 11). There was good agreement between the FCM and ICM data. The SPF was less than 5% in the 3 "diploid" tumours but above 10% in the "aneuploid" ones. No patients had a progressive fatal neoplastic disease. We think that our small Merkel-cell tumour study is another good illustration of the great limitations that cytometric DNA analyses can suffer when applied to NE neoplasms.

Malignant Melanoma In this connection39 , it may be worth while to briefly mention the quite recent observations made by Dr. Bjarnhagen at the Karolinska Hospital in her comprehensive series of works about another potentially highly malignant carcinoma of the skin, malignant melanoma 8• Malignant melanomas are notorious for their variable, and often, unpredictable clinical behaviour8; features that they have in common with true NE carcinomas 39 . Dr. Bjarnhagen and her collaborators assessed the nuclear DNA distribution patterns by means of ICM in adult melanocytes in normal skins, 20 intradermal naevi, 60 junction naevi, 107 compound naevi, 61 dysplastic naevi, 17 melanomas in situ and 101 primary malignant melanomas. All the melanocytes of the normal skin and those of the intradermal naevi were found to show a normal, diploid, nuclear DNA pattern. A clear-cut aneuploid DNA pattern in the melanocyte nuclei was recorded in 96% of the malignant melanomas, in 88% of the melanomas in situ and in 34% of the dysplastic naevi. Practically all (97-98%) of the junction naevi and the compound naevi - as well as the remaining 66% of the dysplastic naevi - displayed a nuclear DNA ploidy pattern of their melanocytes of so-called intermediate type 8 • A paradoxial aneuploidy (see above) was a rare event. When, however, 24 thin metastasizing melanomas were compared with 48 nonmetastasizing control melanomas, no differences could be found concerning the ICM nuclear DNA ploidy patterns of the neoplastic melanocytes 8 • Another surprising finding was that the nuclear DNA ploidy pattern was different in the melanocytes of the primary tumour and those of the metastases in about 1/3 of malignant

melanomas 8 • It was concluded that in melanocytic lesions ICM DNA aneuploidy data indicate increased risk of malignant transformation, and that they seem to reflect different stages in the progression to a malignant melanoma, but that they have to be combined with the results of cell proliferation assessments and morphometric data in order to more precisely differentiate between the various kinds of these lesions. The epidermal melanocytes from which the malignant melanomas are supposed to originate are, admittedly, not NE cells in the strictest sense of the word 39 , but - together with the cells of the NE system - they are nowadays included in the wider concept of "Paraneurons"36. The paraneurons are also called "the sister cells of the neurons"; they are defined as cells located outside the central and peripheral nervous system with the ability to produce at least some biogenic amines and peptides characteristics for neurons and endocrine parenchymal cells 36 . Thus, is can be concluded that the limitations of cytometric DNA analyses do not seem to be restricted to tumours of the NE .system only, but obviously include also the closely related neoplasms of paraneurons.

NE Differentiation in Traditionally Non-Endocrine Carcinomas Phylogenetical Background: When the evolution of the NE system is studied from a phylogenetical point of view, it becomes obvious that the NE system consists of three major components24 , 25. Most original is the neuronal part, formed by neurons and nerve fibres in the central and peripheral nervous system. The next step in evolution is when parts of the peripheral fraction of the neuronal part develop into the second major component, namely the disseminated endocrine cells of open or closed type in the mucosa of most hollow organs or in the epithelium of ducts and glands. These cells form the famous "Helle Zellen" system of Feyrter2\ best known among these cells are the Kultschitzky cells of the gut (from which the carcinoids originate). The third major component of the NE-systern are the classical, compact endocrine glands (including the islets of Langerhans), appearing first at the evolution stage of the earliest vertebrates 25 . As shown for the islets of Langerhans, the parenchyma of these endocrine glands develops from some of the cells of closed type in the second major component of the NE system25 . It should be emphasized that in the vertebrates not only a developmental but also a functional relationship exists between the three major components of the NE system. Typical examples are such concepts as the "brain-gut axis,,24 and the "entero-insular axis"25. Exactly the same peptide hormone (e.g. somatostatin) can be produced by NE cells in each of the three major components24 . Occurrence and Nature of NE Cells in Traditionally Non-Endocrine Carcinomas: About a decade ago, Prof. F. Bosman in Rotterdam ll gave a comprehensive

298 . U. G. Falkmer and S. Falkmer

review of the occurrence and nature of NE cells in traditionally non-endocrine carcinomas; such an NE differentiation was found to appear in a great number of carcinomas, and the knowledge of it had a surprisingly long history. Considering their almost ubiquitous presence in the mucosa, glands and organs of the body, and that NE cells belong to the second major component of the NE system13,24, it is, after all, not so peculiar that NE cells can also be part of a carcinoma, arising in the non-endocrine part of the ef:ithelium of a mucosa or that of an exocrine gland ll , 3. The tendency to undergo neoplastic transformation is markedly different in the three major components of the NE system. In the first neuronal part, this tendency is low; the various kinds of genuinely NE neoplasms are few and those existing are rare, both in the central nervous system ("primitive neuroectodermal tumours") and the peripheral (ganglio-neuromas)4o. Any admixture of NE cells in common gliomas or meningionas is virtually non-existent4o . In the second and third major component of the NE system, the tendency to undergo neoplastic transformation is, however, much greater; a fact illustrated by the present issue of "Pathology Update". Notwithstanding, it is low when compared with several other cell systems in the body. The crucial question is then: when there is an admixture of NE cells in an otherwise commonplace adenocarcinoma, shall these NE cells be looked upon as genuinely neoplastic, or as normal or hyperplastic NE cells belonging to the second major component of the NE system at the site of origin of the carcinoma trapped by it, and perhaps initiated to proliferate through the action of autocrine growth factors from the neoplastic parenchymal cells 1, 11, 13,26? One answer to that question comes from a study of metastatic lesions from such a carcinoma, located in those areas of the body where normally no NE cells are present, such as the lymph nodes or the skeleton; NE cells are there, as well 1,26. Thus, carcinomas with foci of NE cells consist of at least two phenotypes of neoplastic parenchymal cells; one of which is NE26. Then, the next important question is: is the NE phenotype more aggressive than the non-NE? Here, cytometric DNA analyses have been used in order to try to elucidate the problem26 .

DNA Cytometry of the NE Phenotype in Prostatic and Mammary Carcinomas: In the two most common

carcinomas in our country, namely adenocarcinomas of the prostate in the male 1 and of the mammary gland 86 in the female, NE differentiation is a common event l6, 26. In the prostate carcinomas, it is even so that by means of a meticulous search, almost everyone harbours at least some foci of NE cells l . Particularly numerous are the NE cells in the neoplastic parenchyma of poorly differentiated adenocarcinomas 1• In mammary carcinomas, the relative incidence of tumours with an admixture of a NE phenotype is lower86. Depending on the criteria used for identification of NE cells, the incidence figures vary from about

30% to 5 _10%86. Among the different histopathological subtypes, there are also great variations in incidence; the lobular carcinomas appear to be the type of breast cancer that is most prone to display NE differentiation 26 ,87. A comprehensive immunohistochemical, and ultrastructural characterization of the NE phenotypes has been made by Dr. P. A. Abrahamsson and his collaborators in Malmo, regarding the prostatic carcinoma, and by Prof. J. M. Nesland and his collaborators in Oslo, concerning breast cancers. The characterization encompasses i.a. such features as the production of various peptide hormones and other regulatory peptides, and a biochemical analysis of the prevalent peptide produced by the NE cells of the prostate26 . Efforts have also been made to assess the cytometric DNA ploidy pattern of the NE phenotype. In the prostatic carcinomas we have profited from the fact that a focal arrangement often occurs in the distribution of the NE phenotype 1, 26. When large enough (as assessed from sections stained with the Grimelius silver nitrate procedure), such NE foci could be cut out from thick sections of the paraffin block, and subjected to a deparaffinization -disaggregation-cytospin -Feulgen technique by means of which an ICM assessment could be made of nuclei of the NE phenotype from the neoplastic parenchymal cells26 . When analogous investigations were made of those areas of the prostatic carcinoma where NE cells were few or absent, it seemed plausible that the NE phenotype consisted of cells with a nuclear DNA distribution pattern of predominantly diploid type 26 . Analogous investigations of the DNA ploidy pattern of the NE phenotype in mammary carcinomas are still not finished 26, 35. There are, however, data available from FCM assessments of carcinomas with and without the NE phenotype, performed by Prof. Nesland and his colleagues in Os1088. The results indicate that the NE-cell-containing carcinomas show a higher incidence of neoplastic parenchymal cells in which the nuclear DNA distribution pattern is of aneuploid type, than the non-NE cancers88. In contrast, with an ICM technique, some observations were made indicating that prostatic and mammary carcinomas with high amounts of NE-differentiated cells, display euploid DNA nuclear ploidy patterns to a greater extent than those in areas of the same carcinomas without such NE-cells 26 . Together with Prof. Nesland et aI., we are now trying to supplement their mammary carcinoma FCM studies by new ICM investigations 35 , using the same paraffin blocks and applying the new consecutive-double staining technique (see above). So far, the results seem to support the preliminary observations26 .

Significance of the NE Differentiation: There is some controversy in the literature regarding the influence of the NE phenotype on the prognosis of the neoplastic disease 1 . The NE cells in prostatic carcinomas have been looked upon as a "marker of benignity" and as a feature associated with particularly aggressive vari-

DNA Analysis in Neuroendocrine Tumours· 299

ants of the neoplasm 26 • Diverging opinions have also been launched concerning the significance of the NE differentiation in mammary carcinomas26 , as well as in several of the other traditionally non-endocrine cancers that can display this phenomenon. It may be that the use of our new consecutive-double-staining technique, by means of which immunohistochemically identified NE cells can be analyzed with regard to their ICM DNA ploidy pattern, can bring some new, fundamental data about the true nature of these NE cells, helping to solve these controversies. Comments The term "DNA aneuploid" is used to describe a cell sample containing a stem line with abnormal DNA contentS, 31. Abnormalities in the karyotype or cellular DNA content are said to be a typical finding in a variety of malignant neoplastic diseases, and it has even been claimed to be the most common specific cell marker in cancers, 6, 31, 32, 78, 97. DNA aneuploidy, as detected by means of FCM and/or ICM, does not, however, detect primary chromosomal aberrations, such as structural abnormalities and specific trans locations at or near cellular oncogens, which have occurred during the neoplastic transformation; both FCM and ICM represent techniques which are far too crudes, 6, 31, 78, 97. Instead, FCMlICM DNA aneuploidy corresponds only to the secondary chromosomal aberrations that may appear in the nuclei of neoplastic parenchymal cells s, 6, 31, 78, 97. Then, the crucial question arises: are such secondary chromosomal aberrations actually all that "specific for cancer"? From what has been accounted for in the preceding sections of this report and from the results of a multitude of similar investigations in a great number of laboratories all over the world, it is obvious that great caution should be exercised in interpreting cytometrically detected nuclear changes in the DNA distribution pattern as indicating transformation of a normal parenchymal stem cell line into a neoplastic one, leading to the formation of a malignant tumours, 31, 78, 97. Particularly when dealing with hyperplastic and neoplastic lesions of the NE (and paraneuron?) system, FCM or ICM DNA aneuploidy might simply be the result of a more or less normal polyploidization processes for which most NE cells are notorious 62 • Acknowledgements This work was supported by grants from the Swedish Medical Research Council (Project No. 102), the Swedish Cancer Society (Project No. 2841), the Cancer Society of Stockholm, the King Gustaf V Jubilee Fond, and the Astra-Hassle Research Fonds, as well as the Research Fonds of the Faculty of Medicine of the Karolinska Institute, Stockholm.

Material and Methods Practically all the specimens investigated at the Karolinska Hospital consisted of freshly excised tumours, immediately taken care of in a specially designed histopathology laboratory located next to the operation theater. After measuring and weighing the tumours, imprints from the cut surface were made and samples were set apart for other special techniques such as hormonal assays and/or implantations in nude-athymic mice. The rest was cut in thin slices and fixed by immersion in neutral, buffered, 10% formalin for subsequent dehydration and paraffin embedding. Cut, deparaffinized sections, 4 - 5 Jlm thick, were used for routine stains (H & E; van Gieson's stain) and for immunohistochemical demonstration of "NE markers" regulatory peptides, such as peptide hormones, as well as biogenic amines including histamine 42 ,111. The immunohistochemical techniques and the antisera used were of the conventional type; details of which have been given in some recent reports45, 61. Exceptionally small, thin specimens were fixed in glutaraldehyde for subsequent examination by means of transmission electron microscopy and ultrastructural immunocytochemical investigations 76 • For the cytometric DNA analyses, both the FCM and the ICM techniques were used, most often in parallel. Details about the methods and instruments used, as well as all the inherent sources of errors, are given in a recent monograph31 as well as in some subsequent reports 2, 32, 61, 115. Most of the FCM assays were made on deep-frozen specimens, thawed in time for the analysis. In some NE tumour entities, the only tumour material available were paraffin blocks of conventionally formalin-fixed operation (or autopsy) specimens from other hospitals. Cut sections of these blocks were used for the same type of investigations as those from the operation theater laboratory at the Karolinska Hospital. Particular care was always exercised to check that only histopathologically unquestionably neoplastic cells were analyzed by means of ICM. The clinical data of the patients have been given in the original reports of each NE tumour entity.

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115 Tominaga Y, Grimelius L, Falkmer UG, Johansson H and Falkmer S (1991) DNA ploidy pattern of parathyroid parenchymal cells in renal secondary hyperparathyroidism with relapse. Analyt Cell Path 3: 325 -333 116 Tsioulias G, Muto T, Kubota Y, Masaki T, Suzuki K and Akasu T (1991) DNA ploidy pattern in rectal carcinoid tumors. Dis Colon Rectum 34: 31-36 117 Tsushima K, Nagorney DM, Weiland LH and Lieber MM (1989) The relationship of flow cytometric DNA analysis and clinicopathology in small-intestinal carcinoids. Surgery 105: 366-373 118 Visscher D, Cooper PH, Zarbo RJ and Crissman JD (1989) Cutaneous neuroendocrine (Merkel cell) carcinoma: An immunophenotypic, clinicopathologic, and flow cytometric study. Mod Path 2: 331-338 119 Wolfe HJ (1991) Endocrine pathology: Past, present, and future. In: Kovacs K and Asa S (Eds) Functional Endocrine Pathology. Boston, Blackwell, pp. 3-16 120 Yousem SA and Taylor SR (1990) Typical and atypical carcinoid tumors of lung: A clinicopathologic and DNA analysis of 20 tumors. Mod Path 3: 502-507 121 Zavala-Pompa A, Ro JY, Naggar el-, A, Ordonez NG, Amin MB, Pierce PD and Ayala AG (1993) Primary carcinoid tumor of testis. Immunohistochemical, ultrastructural and DNA flow cytometric study of three cases with a review of the literature. Cancer 72: 1726 -1732

Key words: Islet-cell tumours - Nesidioblastosis - Carcinoid tumours - Phaeochromocytomas - Paragangliomas - Neuroblastomas - Pituitary adenomas - Parathyroid adenomas - Medullary thyroid carcinomaMerkel-cell tumours of the skin - Malignant melanoma - Neuroendocrine differentiation in mammary and prostatic carcinomas Prof. Sture Falkmer, Tumour Pathology Unit, Karolinska Hospital, S-17176 Stockholm, Sweden, Tel. + 46 87294353 (office), + 4617562158 (home), Fax. No. + 46 8 321047 (office), + 4617562480 (home)