Ursula G. Falkmer

Expression but incomplete maturation of progastrin in colorectal carcinomas.

BACKGROUND To evaluate the hypothesis that gastrin is a local growth factor in colonic carcinomas, the expression of gastrin messenger RNA (mRNA) and peptides were examined in five human colon carcinoma cell lines, 12 solid colon carcinomas, and normal colonic tissue. METHODS Northern analysis, reverse-transcription PCR, and a library of sequence-specific radioimmunoassays were the principal methods. RESULTS Cell lines, tumors, and normal tissue all expressed a gastrin mRNA of 0.7 kilobases, and all cell lines contained incompletely processed progastrin (range, 17-54 fmol/10(6) cells). Two cell lines secreted progastrin into the media (LoVo, 25 +/- 3 pmol/L; HCT116; 12 +/- 2 pmol/L). Normal colonic tissue and all the solid tumors also contained progastrin, the concentration being higher in tumors (range, 0.4-2 pmol/g) than in normal tissue (range, 0.1-0.2 pmol/g). Only one tumor contained carboxyamidated gastrins. CONCLUSIONS Normal and neoplastic colonic mucosa both express the gastrin gene, but the posttranslational phase of expression is attenuated. The incomplete processing and low level of expression suggest that autocrine gastrin secretion has only minor significance for normal adult and most neoplastic colonic tissue.

Proliferative myositis and fasciitis

Two cases of proliferative myositis, four cases of proliferative fasciitis and one mixed form of proliferative myositis and fasciitis have been analyzed in terms of cell differentiation and DNA content. Light microscopically, the lesions were characterized by a mixture of proliferating spindle‐shaped cells and uni‐, bi‐ or occasionally multinucleated ganglion cell‐like cells. The spindle cells showed ultrastructural features and immunohistochemical properties, including an immunoreactivity for smooth muscle‐specific actin, indicative of a myofibroblastic differentiation. The ganglion cell‐like cells displayed some resemblance to active osteoblasts ultrastructurally and differed immunohistochemically from the spindle cells by being non‐immunoreactive for smooth muscle‐specific actin. None of the two cell types showed immunoreactivity for desmin, myoglobin or factor VIII RAG. It is suggested that the two cell types represent different lines of cell differentiation. The cytologic features in smears, as seen in two cases of proliferative fasciitis and one case of proliferative myositis, are considered to be characteristic of these lesions and to permit the diagnosis to be made by fine‐needle aspiration. In two of the cases, the lesion was diagnosed only cytologically and thereafter disappeared spontaneously within a month. Cytometric DNA measurements, using two different image analysis systems on Feulgen‐stained sections and smears, revealed a “diploid” spindle‐shaped cell population with a variable proportion of cells with scattered DNA values. The ganglion cell‐like cells differed from the spindle cells by having a broad DNA peak in the diploid region and additional peaks in the tetraploid region, as well as a higher proportion of cells with scattered DNA values compared with those of the spindle‐shaped cells. The results of the quantitative DNA analysis are well in keeping with the benign and proliferative nature of these lesions. However, with the technique used here, quantitative DNA analysis, does not distinguish these pseudosarcomatous fibrous lesions from diploid and tetraploid soft tissue sarcomas.

Expression of the c-erbB-2 proto-oncogene product and nuclear DNA content in benign and malignant human breast parenchyma

The expression of the c-erbB-2 proto-oncogene product was investigated immunohistochemically in 474 formalin-fixed and paraffin-embedded human breast tissue samples. The series included 32 benign and 26 hyperplastic lesions, 32 carcinomas in situ and 384 invasive breast carcinomas, 107 of which were less than 1 cm in diameter. Cytometric DNA assessments were performed on histopathologically or cytodiagnostically identified cell nuclei, using image analysis. C-erbB-2 immunoreactivity was not seen in normal parenchyma or in benign and hyperplastic lesions. Mammary carcinomas in situ were more frequently immunoreactive (59%) than invasive neoplasms (23%). Invasive tumours more than 1 cm in diameter immunoreacted more often (26%) than small invasive carcinomas (16%). C-erbB-2 expression in regional lymph node metastases was the same as in the corresponding primary tumours. Significant differences were observed between the c-erbB-2 expression in DNA diploid and aneuploid lesions; for carcinomas in situ the figures were 40% and 72%, respectively. Invasive carcinomas of DNA diploid type rarely showed c-erb-B-2 expression, irrespective of tumour size and nodal status (7–11%). DNA aneuploid tumours were more frequently immunoreactive with increasing levels during progression (32–41%). Our data indicate that genetically stable invasive mammary tumours seem rarely to express the c-erbB-2 protein, even during progression, whereas genetically unstable invasive neoplasms frequently show c-erbB-2 immunoreactivity which increases during tumour progression.

Different calculation methods for flow cytometric S‐phase fraction: Prognostic implications in breast cancer?

S-phase fraction (SPF), estimated in the flow cytometric DNA histogram, is a prognostic factor in breast cancer. There are, however, some inherent difficulties in the estimation of SPF, such as the influence of debris, aggregates, and normal cells. Most of the available SPF calculation principles try to consider these difficulties, but so far no consensus has been reached with regard to which principle is to be recommended. The aim of the present study was to investigate the prognostic impact of SPF when estimated with different calculation methods in frozen breast cancer samples from 350 patients. Two nonparametric (Rman, Rmin/both rectangle) and three parametric (ACAS/DNA-base, ModFit, and MultiCycle) calculation methods, with and without correction for debris and aggregates, were used. The mean values for SPF varied from 4.3% (ACAS/DNA-base with correction for debris and aggregates) to 9.4% (MultiCycle without any correction for background). The pairwise correlation between methods varied considerably (R = 0.72-0.98). After categorization of SPF values into low SPF (lower two tertiles) and high SPF (upper tertile), all methods yielded statistically significant Pvalues for recurrence-free survival (median follow-up time 67 months), both univariately (0.0004-< 0.0001) and multivariately (0.048-0.0004), after adjusting for nodal status, tumor size, and estrogen receptor status. SPF with background correction did not yield lower P values than SPF without. Regardless of which method was used, SPF showed similar correlations with lymph node involvement, tumor size, and estrogen receptor content. In conclusion, as the mean value of SPF for different calculation methods varies, each laboratory must be restricted to use only one method. Background correction does not seem to improve the prognostic impact of SPF in DNA histograms. Based on the experiences obtained in the present study, S-phase calculation methods without background correction may therefore be the most suitable for routine evaluation of DNA histograms of fresh frozen breast cancer material (ModFit, MultiCycle, and Rman [the latter only for experienced operators]). The nonparametric Rmin, with an automatic setting of the region used for SPF calculation, may be an alternative, but suffers from the disadvantage of not being commercially available yet.

A dna cytometric proliferation index improves the value of the dna ploidy pattern as a prognosticating tool in patients with carcinoma of the prostate

OBJECTIVES A still controversial issue is whether the results of a cytometric assessment of the DNA distribution pattern of the nuclei of the neoplastic parenchymal cells of a prostatic adenocarcinoma has additional prognostic value to that of the stage and grade of the disease. To increase the accuracy of the DNA ploidy assessments. Image cytometry (ICM) has been used and combined with the determination of an ICM proliferation index (PI) to increase its value as an additional prognosticating tool. METHODS We investigated 96 patients, followed up since diagnosis in 1980/1981 until death or, in 11 surviving patients, for an average of 14.5 years. Survival analysis was made by the conventional Kaplan-Meier method. Fine-needle aspiration biopsy was used as the major diagnostic tool. The neoplastic cell nuclei were classified as ICM DNA diploid, tetraploid, or aneuploid by means of the ploidy-establishing peak in the ICM DNA histograms, as well as the fraction of tumor cells in the S-phase. Scattered cells to the right of the ploidy-establishing peak, the S-phase fraction, and those in the G2M area of the ICM DNA histograms were counted as percent of the total number of tumor cells; this percentage was defined as the PI. Arbitrarily, tumors with a PI less than 5% were classified as having a low proliferation rate, those with a PI greater than 10% were considered highly proliferating, and those with a PI between 5% and 10% as carcinomas with an intermediate proliferation potency. RESULTS By univariate analyses, clinical stage, cytodiagnostic grade, cytometric DNA ploidy pattern and PI all had significant prognostic value. By multivariate analyses, the PI was found to add prognostic information to that of the ICM DNA ploidy pattern variable, giving it an increase in its statistical P value from 0.002 to 0.0005. As a consequence, the combination of these two variables was found to give rise to three new patient groups with regards to their prognosis: DNA group I had tumors with a diploid ICM DNA pattern with a low PI; DNA group II had tumors with a diploid or tetraploid ICM DNA tumor cell nuclei pattern with an intermediate PI; and DNA group III had a diploid or tetraploid ICM DNA pattern with high PI and all tumors with an aneuploid pattern. By multivariate analysis, including tumor grade and clinical stage, these new DNA groups (P = 0.0004) and M stage disease (P = 0.0006) were the only significant prognostic variables. CONCLUSIONS A DNA cytometric PI improves the prognosticating value of DNA ploidy. Patients with prostatic adenocarcinomas, classified as DNA group I, have a low risk of death from their neoplastic disease with deferred or hormonal treatment only.

High‐molecular weight IGF‐2 expression in a haemangiopericytoma associated with hypoglycaemia

Spontaneous hypoglycaemia is usually caused by an insulin‐producing islet‐cell tumour of the pancreas. Rarely, it can be caused by non‐islet cell tumours. Most of the tumours are of mesenchymal type, large, and slowly growing. One representative is haemangiopericytoma (HAP). The present report describes a case of a large recurrent retroperitoneal HAP associated with severe hypoglycaemia. Blood serum insulin and proinsulin concentrations were low. By means of acid‐gel chromatography and dot‐blot techniques, an increased amount of a high‐molecular‐weight IGF‐2 peptide was found. By using antigen retrieval procedures, IGF‐2‐immunoreactive tumour cells were found in specimens of the recent tumour recurrence ‐ but not in the original. When the in situ hybridization technique was used it could be shown that IGF‐2 mRNA labelling had already occurred in the original tumour specimen, 11 years before the onset of hypoglycaemic symptoms. These observations confirm the hypothesized hypoglycaemic effects of high‐molecular‐weight (HMW) IGF‐2, but also point to the presence of a prolonged compensation of this effect. A literature review, based on 17 similar cases of haemangiopericytoma with hypoglycaemia, is presented. Our observation and findings in the literature review support the idea that non‐islet‐cell tumour hypoglycaemia is caused by an overproduction of a HMW IGF‐2 peptide. The insulin‐like effect is mediated via non‐specific binding to the insulin receptors. To anticipate patients at risk of developing this kind of hypoglycaemia, the histopathological investigation should include not only immunohistochemical analyses of the presence of IGF‐2 peptide, but also in situ hybridization of the IGF‐2 mRNA expression.

The value of cytometric DNA analysis as a prognostic tool in neuroendocrine neoplastic diseases.

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.

Paragangliomas: neuroendocrine features and cytometric DNA distribution patterns A clinico-pathological study of 22 cases*

Paragangliomas from 22 patients with extraadrenal tumours of this type were studied. Neuroendocrine features were examined using immunohistochemical techniques. Twenty-two antisera raised against neuroendocrine “markers”, regulatory peptides, serotonin and intermediate filament proteins were studied in this group and cytometric DNA assessments were made by means of image cytometry. One normal and 5 hyperplastic carotid bodies were used as controls in the DNA cytometric investigations. Clinical and/or histopathological evidence of “malignancy” was present in 5 cases. The tumour cells showed heterogeneity with regard to their expression of different peptides, and the immunohistochemical analyses did not permit differentiation between benign and malignant paragangliomas. An euploid nuclear DNA distribution pattern was found in all controls and in 17 of the tumours; all except 1 were clinico-pathologically benign. An aneuploid DNA pattern was observed in 5 of the cases and some malignant features were present in 4 of these cases. DNA data may give further information apart from that obtained from the histopathological findings which may be of value in predicting the biological behaviour of this tumour type.

Flow and image cytometric study of pancreatic neuroendocrine tumours: frequent DNA aneuploidy and an association with the clinical outcome.

Eighteen pancreatic neuroendocrine (NE) tumours were analysed for nuclear DNA content by image cytometry (ICM) and flow cytometry (FCM). The DNA indices (DIs) obtained by ICM were somewhat higher than those obtained by FCM, but a major disagreement was present only in 1 case. Thirteen patients had been followed up at least for 6 years after the diagnosis or until death. At 6 years of follow-up all 4 patients with a tumour with a DI≥1.8 by ICM had died from their NE tumour or had metastatic disease, whereas all 9 patients with a smaller DI had no evidence of the disease (P=0.001). The DIs calculated from the FCM data also correlated well with the final outcome (P=0.01). A high incidence of DNA aneuploidy was found by both methods in histologically and clinically benign NE tumours; 12 (67%) were DNA aneuploid by FCM and 16 (89%) by ICM. It is concluded that pancreatic NE tumours are frequently DNA aneuploid, and both cytometric DNA methods give prognostic information in these tumours. The presence of DNA aneuploidy should not be considered as a sign of malignant behaviour in pancreatic NE tumours, whereas a large DI is associated with poor prognosis.

Disease-free Survival of Node-positive Breast Cancer Patients: Improved Prognostication by Cytometrical Parameters

Feulgen stained cytologic samples from 225 node-positive breast cancers were investigated by means of an image analysis system. From each tumor sample, 100 cells were scanned and several DNA, morphometrical and textural parameters were evaluated. The meaning of the cytometric parameters for prediction of distant metastases within five years was investigated by the stepwise Cox regression analysis. Most of the investigated DNA- and morphometrical parameters, as well as one textural feature, showed a significant univariate correlation with the clinical course. In the multivariate approach, the lymph node status (pN) was the strongest prognostic factor, followed by the histogram type, the tumor size (pT) and a textural parameter (heterochromatin area). By the linear combination of these selected variables a multivariate prognostic factor was calculated for each individual patient. Using this factor, the patients could be splitted into four groups according to their risk for distant metastases. For this, the continuous range of the multivariate factor was subdivided so that about 35% of the patients were in the middle groups and about 15% of the patients in each of the border groups with highest and lowest factors, respectively. Thus a low risk group (lowest factors) of node-positive patients could be identified with a 5-year distant recurrence-rate of only 6.5%, as well as a group of patients with a considerably worse prognosis (highest factors) and a distant recurrence-rate of 67%. Therefore, DNA, morphometrical and textural parameters can provide powerful prognostic information in node-positive breast carcinomas. Using the multivariate combination of clinical and relevant cytometrical parameters may allow a more appropriate selection of patients for adjuvant therapy.