Aggressive angiomyxoma: a clinicopathological and immunohistochemical study of 11 cases with long-term follow-up.

AIMS: To report the clinicopathological and immunohistochemical features and longer term biological behaviour of aggressive angiomyxoma, an uncommon mesenchymal neoplasm occurring predominantly in the pelvi-perineal region of adults. Using immunohistochemistry, possible overexpression of CDK4 and MDM2 was analysed, which might point to (cyto)genetic alteration(s) in chromosome region 12q13-15, an area reported to be altered in this tumour entity. METHODS AND RESULTS: Cases (n=11) of aggressive angiomyxoma were retrieved from the consultation files of the Comprehensive Cancer Centre of the Middle Netherlands (IKMN) panel for soft tissue tumours. Clinical and follow-up information were obtained, and immunohistochemical analysis was performed using antibodies directed against vimentin, cytokeratin AE1/AE3, desmin, alpha-smooth-muscle actin, CD34, S-100 protein, oestrogen receptors, CDK4 and MDM2. Five patients were female (age range 24-47 years; median 39 years), and six patients were male (age range 36-69 years; median 44.5 years). Of 11 cases, 10 arose in the pelvi-perineal area and 1 arose in the abdominal cavity in close relation to the bladder. Morphology was consistent with previous reports of this entity. Immunohistochemically, 8 of 11 cases were desmin positive (5 of 5 positive in females; 3 of 6 positive in males), 6 of 11 cases were positive for alpha-smooth-muscle actin, 5 of 11 cases were CD34 positive, 11 of 11 cases, irrespective of gender, were positive for oestrogen receptors and 3 of 11 cases were positive for cytokeratin AE1/AE3. Strong, diffuse nuclear positivity for CDK4 expression was present in all 6 cases tested, while only 1 of 11 cases tested for MDM2 showed weak focal positivity. Clinical follow-up in all cases (range 1-216 months; median 72 months) showed one local recurrence (9%) after 36 months. No metastases or tumour-related deaths were noted. CONCLUSIONS: The sex distribution of cases reported in this study was roughly equal, in contrast to previous reports emphasising the predominance of this tumour in females. Our study confirms the local aggressive nature of aggressive angiomyxoma, although our local recurrence rate is lower than previous reports (9% versus 36-72%); no metastases and/or disease-related patient deaths were documented. All cases arising in females were positive for desmin, while three of the six cases arising in males were negative for desmin, supporting previous findings and indicating that the lesion may be somewhat different in males. The strong diffuse positivity for CDK4 in all six cases tested goes some way in implicating CDK4, either directly or indirectly, in tumourigenesis. The negative immunostaining for MDM2 would argue against functional amplification of this gene.

Classification and grading of soft-tissue sarcomas.

This article provides an overview of the classification of soft-tissue sarcomas as influenced by modern histopathologic techniques. The overview is followed by a practical grading system for these tumors based on a study of 282 eligible patients. The primary tumors of this trial were adequately treated. The quantitative data (mitotic count and size of the tumor) were based on the results of the statistical analysis.

[Consensus soft tissue tumors. Dutch Workgroup Soft-Tissue Tumors]. / Consensus weke-delentumoren. Nederlandse Werkgroep Weke-delentumoren.

Soft-tissue sarcomas constitute a rare group of malignant tumours with histopathological features of connective, muscular, fatty or peripheral nervous tissue. The prognosis at manifestation depends on only two factors: the spread, both local and remote, and the biological behaviour of the tumour. The latter factor cannot be influenced but the former can: by inexpert manipulation. Consequently, tumours suspected of being soft-tissue sarcomas require multidisciplinary management from the beginning, with the team members familiar with each other's diagnostic and therapeutic skills. Imaging diagnostic methods should precede invasive methods for collection of material for pathological examination. The number of mitotic figures observed at microscopical examination of the tissue is an important prognostic feature. Surgical resection is the treatment of first choice. Radiotherapy is indicated in grade 3 tumours, after recurrence surgery, and when radical resection would involve too much mutilation. Chemotherapy is only given in the context of clinical trials. Surgical treatment of lung metastases may be indicated in selected patients. Regional isolated perfusion with tumour necrosis factor may be an alternative for limb amputation.

Detection of calcitonin and calcitonin gene-related peptide mRNA in human medullary thyroid carcinoma. A retrospective study.

In situ hybridization finds many applications in modern pathology. In many cases, special attention is paid to the processing of the tissues prior to in situ hybridization. In order to investigate the value of RNA in situ hybridization (RISH) in retrospective studies, we performed RISH for calcitonin and calcitonin gene-related peptide (CGRP)-I and -II mRNA in eight medullary thyroid carcinomas processed in 1981-1983. RISH was successful with radioactive calcitonin and CGRP-I probes. With biotinylated probes, only calcitonin-specific probes gave adequate results. The concentrations of CGRP mRNA were probably too low to be detected by non-radioactive RISH. The results of RISH were correlated with the immunohistochemical localization of the polypeptides. The results matched in all cases except one, where hybridization for calcitonin mRNA was found, but no immunoreactive calcitonin polypeptide. We conclude that RISH can be successfully used for retrospective analysis, even after long storage of tissue embedded in paraffin.

Grading of soft tissue sarcomas: experience of the EORTC Soft Tissue and Bone Sarcoma Group.

A practical grading system for soft tissue sarcomas was developed, based on 282 eligible patients entered in an EORTC adjuvant clinical trial. The primary tumours in this trial had to be adequately treated. Histopathological parameters, which appeared significant in two preceding studies, were tested. These parameters were differentiation of the tumour, presence and amount of necrosis, the presence and amount of myxoid areas and the number of mitoses. In addition, the size of the tumour was also analysed. The quantitative data (mitotic count and size of the tumour) were not a priori grouped, but were divided into categories based on the results of the statistical analysis. Based on a multivariate analysis only mitotic count, the presence or absence of necrosis and the size of the tumour were significantly correlated with the duration of survival or the time to distant metastases. Of these parameters, the mitotic count was the most important.

No constant relationship between islet amyloid polypeptide (IAPP) and insulin expression in insulinomas.

The relationship between insulin and islet amyloid polypeptide (IAPP) expression was studied at the level of RNA and peptide in 3 patients operated on for insulinoma. Two patients had a solitary tumour; one patient suffered from the MEN-I syndrome. In the 3 tumours, as in normal pancreas, the same 2 IAPP-specific mRNA species, approximately 1600 and 2100 nucleotides, respectively, were detected. In 2 patients, the IAPP mRNA concentration of the tumour was lower than the insulin mRNA concentration; in the third patient, however, the specific IAPP hybridization signal was at least equal to that for insulin. Amyloid deposits were found in one solitary tumour and in the tumour from the MEN-I patient, both staining strongly positive with anti-IAPP antibodies; cytoplasmatic IAPP was weak. In conclusion, at least in some insulinomas, no constant relationship exists between insulin and IAPP expression.

Expression of the first calcitonin/CGRP gene in spontaneous and transplanted rat medullary thyroid carcinoma: a comparison of dot-blot analysis, in situ hybridization, and immunohistochemistry.

Calcitonin (CT) and calcitonin gene-related peptide (CGRP) are encoded by a single gene, the CALC-I gene. They are expressed in the thyroid and in the nervous system by alternative splicing of the pre-messenger RNA derived from the CALC-I gene. In medullary thyroid carcinoma (MTC), a malignancy derived from the calcitonin-producing C-cells in the thyroid, production of calcitonin and CGRP is a common feature. We investigated the CT and CGRP production of four spontaneous MTCs transplanted three to four times and 14 MTC lines transplanted for several years in WAG/Rij rats, a strain with hereditary MTC. The expression of CT and CGRP in the spontaneous and in the transplanted tumors was studied by means of RNA in situ hybridization (RISH), dot-blot analysis, and immunohistochemistry. A down-regulation of CT production in transplanted compared with spontaneous tumors was observed, but an inverse relation between CT and CGRP mRNA content in both spontaneous and transplanted tumors was not observed. In this study, RISH proved to be as sensitive as dot-blot analysis to detect gene expression in tissue samples. The different approaches of analyzing the gene expression in tissue samples (the cellular localization of gene expression by ISH vs the analysis of an extract of a total tissue sample with dot-blot analysis) showed that each technique is equal in value and that they are complementary to each other.

Malignant fibrous histiocytomas in dogs and cats: an immunohistochemical study.

Immunohistochemical staining was performed on seven canine and 10 feline soft tissue tumours histologically diagnosed as malignant fibrous histiocytomas (MFHs) or MFH-like tumours, and eight other histologically specified tumours (non-MFH). This was done to determine if commercially available antibodies that are used routinely in human diagnostic pathology for MFHs would express the same immunohistochemical patterns in canine and feline MFHs and MFH-like tumours. The antibodies were directed against human alpha 1-anti-trypsin (AT), human alpha 1-anti-chymotrypsin (ACT), human lysozyme, bovine S-100 protein and human desmin. AT did not show any immunoreactivity in the tissues investigated. Except for one MFH, all canine MFHs and other soft tissue tumours with a 'histiocytic' character stained for lysozyme and not for S-100. Six out of seven canine MFHs and MFH-like tumours stained positive for desmin as did most non-MFH sarcomas. Most of the canine and feline MFHs and MFH-like tumours were positive for ACT. These findings for ACT staining in canine and feline MFHs and MFH-like tumours are in agreement with the findings in human MFHs. The immunohistochemical results of canine MFHs and MFH-like tumours were different from those in cats. Feline MFHs differed from canine MFHs for both lysozyme and desmin staining.

Islet amyloid polypeptide (IAPP) is synthesized in the islets of Langerhans. Detection of IAPP polypeptide and IAPP mRNA by combined in situ hybridization and immunohistochemistry in rat pancreas.

We investigated the localization of IAPP mRNA by means of in situ hybridization in tissue sections of rat pancreas. A 35S-labeled, IAPP-specific DNA probe--hybridized specifically in the islets of Langerhans. This localization was confirmed by immunohistochemical localization of insulin and IAPP polypeptides on adjacent tissue sections. Moreover, combined in situ hybridization of IAPP mRNA and immunohistochemistry of insulin and IAPP polypeptide on the same section, using insulin as specific marker shows the presence of IAPP mRNA in the islets of Langerhans.

Cellular expression of K-type pyruvate kinase in normal and neoplastic human tissues.

The cellular expression of K-type pyruvate kinase was studied immunohistochemically in several normal and neoplastic tissues of human origin. The authors used the monoclonal antibody, designated as ES1, which was raised against human K-type pyruvate kinase. In contrast to the normal counterparts, a strong immunoreactivity was found in a rhabdomyosarcoma (n = 1), in a carcinoma of the pancreas (n = 1), and in neurofibromas (n = 2). Furthermore, the staining in leiomyosarcomas (n = 2) was shown to be more intense when compared with both normal smooth muscle cells and leiomyomas (n = 2). These findings show that knowledge about the cellular expression of the K-type pyruvate kinase identifies cell types for which its expression serves as oncodevelopmental marker. In addition, these immunohistochemical studies give information whether shifts toward K-type containing isozymes of pyruvate kinase, which are determined by electrophoresis in whole cytosolic extracts of various tumors, are due to an altered gene expression or due to proliferation of cells which normally express already the K-type pyruvate kinase. The first possibility probably occurs in rhabdomyosarcomas. The latter possibility seems to be valid for astrocytomas because astrocytes express the K-type pyruvate kinase in normal brain.

Two cell lines with epithelial cell-like characteristics established from malignant fibrous histiocytomas.

Two malignant fibrous histiocytoma (MFH) cell lines were established: one from a storiform-pleomorph subtype and the other from a myxoid one (codes, MFH-3 and MFH-4). Light microscopic examination revealed large rounded cells, growing mostly separately, in both cell lines. Their ultrastructure was different in various aspects. The MFH-3 cells showed abundant lysosomal activity, a well-developed Golgi apparatus, and a few desmosome-like cell contacts. The MFH-4 cells had a well-developed rough endoplasmic reticulum, delicate bundles of tonofilaments, the formation of pseudoacini, and the presence of small completely developed desmosomes. Based on immunostaining and immunoblotting assays of cultured cells, both cell lines expressed immunoreactivity for vimentin; cytokeratins 7, 8, and 18; desmin; and laminin, but they lacked reactivity for cytokeratins 10 and 19, neurofilament, alpha-smooth muscle actin, S-100 protein, collagen type IV, carcinoembryonic antigen, and antigens specific for macrophages. Fibronectin and, to a variable extent, glial fibrillary acid protein and epithelial membrane antigen (EMA) were detectable in MFH-3 cells only. Furthermore, a 60-kilodalton band was present in both cell lines which was reactive for cytokeratins 8 and 18. The MFH-3 cells had the capacity to grow as xenografts with a carcinoma-like pattern. The cells retained their immunoreactivity for vimentin and cytokeratin 8 and showed the presence of desmosomes. Several of these immunophenotypic features also were noticed in established sarcoma cell lines and in short-term cultures of fibroblasts, smooth muscle cells, and endothelial cells. However, experimental data on the two MFH cell lines show that the MFH cell line may express some immunophenotypic and ultrastructural features considered to be specific for epithelial cells. The MFH cells may originate from multipotential mesenchymal cells with a capacity to differentiate to fibroblast-like cells, and less frequently, to epithelial cells, smooth muscle cells, and Schwannian cells. Such a differentiation became evident when these cells were adapted to culture conditions or grew in nude mice.

Pyruvate kinase activity and isozyme composition in normal fibrous tissue and fibroblastic proliferations.

Pyruvate kinase (PK) was studied in 57 fibroblastic and fibrohistiocytic proliferations and normal fibrous tissue (n = 10). The specific activity was significantly increased in malignant tumors (1.67 +/- 0.25) compared with normal tissue (0.26 +/- 0.04; P less than 0.001) and benign proliferations (0.52 +/- 0.05; P less than 0.005). Although an overlap exists between aggressive fibromatosis and the benign group, high values of PK activity are indicative of Grade 2 and 3 malignancy. Significant shifts in isozyme pattern, favoring the expression of K-type subunits were found in tumors with a metastasizing potential and aggressive fibromatosis. These changes in the isozyme pattern of PK in aggressive fibromatosis may act as another argument to place them in the category of malignant fibroblastic tumors.

Hybridohistochemical demonstration of alternative splicing of the CALC-I gene.

Precursor RNA is processed to mature mRNA by excision of the noncoding introns. This process is called splicing and usually is studied by in vitro transcription experiments and by Northern blotting. The pre-mRNA of the first calcitonin gene (CALC-I) can be spliced alternatively into two different mRNAs encoding either for calcitonin or for calcitonin gene-related peptide (alpha-CGRP). The expression of CALC-I was studied by in situ hybridization in the nervous system of the rat with calcitonin-specific and CGRP-specific probes. We found evidence for alternative splicing of the pre-mRNA of CALC-I in the motor neurons of the spinal cord. alpha-CGRP mRNA was found in the nuclei and the cytoplasm of the spinal motor neurons, whereas hybridization with a calcitonin-specific probe was located strictly in the nuclei. Because no calcitonin RNA and protein was detected in the cytoplasm of the cells, we concluded that the hybridization obtained in the nucleus was the pre-mRNA encoding for alpha-CGRP.

Distribution of actin isoforms in sarcomas: an immunohistochemical study.

The actin immunophenotype of eight benign mesenchymal tumors, 14 nonsarcomatoid tumors, and 46 sarcomatoid tumors was studied, using monoclonal antibodies (MoAb) specific for alpha-smooth muscle actin (clone 1A4), alpha- and gamma-smooth muscle actin (designated CGA7), and muscle actin (designated HHF35) on frozen sections. Tumor cells of nonsarcomatoid tissues were not reactive, but all leiomyomas and five of the seven leiomyosarcomas reacted with the three MoAbs. One leiomyosarcoma was immunoreactive for the MoAb 1A4 only. One of the six malignant schwannomas showed staining for muscle actin (HHF35). The 22 malignant fibrous histocytomas (MFH) expressed these actin isoforms in various degrees. One case immunoreacted with all three MoAbs, three reacted with 1A4 only, seven reacted with CGA7 and HHF35, and two reacted with HHF35 only. Nine MFHs were not immunoreactive for any of the MoAbs specific for (smooth) muscle and actin. In addition, the expression of desmin and collagen type IV was investigated for the group of leiomyosarcomas and MFHs. Desmin was found in five leiomyosarcomas and in two MFHs. Collagen type IV was seen in all leiomyosarcomas, and was seen weakly in a few small areas in four MFHs. When we take into account the expression of all markers tested [( smooth] muscle actin, desmin, and collagen type IV), then six of the 22 MFHs were unreactive for all these markers. Five of these six tumors were located intramuscularly, whereas only half of the total number of MFH cases had an intramuscular location. The fact that 15 of 22 MFHs displayed one or more markers linked with (smooth) muscle differentiation suggests that some of the MFHs may be classified as poorly differentiated leiomyosarcomas, and that MFH is not a unique entity.

Insulin-like growth factor gene expression in human smooth muscle tumors.

A role for insulin-like growth factors (IGF) in autocrine or paracrine growth stimulation of tumor cells has been proposed for tumors of different origins. We have studied IGF gene expression in human uterus smooth muscle (myometrium) and in a panel of benign (leiomyoma) and malignant (leiomyosarcoma) smooth muscle tumors. Using RNA transfer blot analysis we could demonstrate that in smooth muscle tissue and tumors IGF genes are differentially expressed. The mRNA species detected had the same size as reported for IGF mRNAs from other tissues. However, the abundance of the IGF gene transcripts varied from tissue to tissue. The amounts of IGF mRNAs detected in smooth muscle tumors were compared to the levels found in normal smooth muscle. The IGF-I gene was expressed at high levels in normal myometrium and in leiomyomas but appears to be repressed in leiomyosarcomas. Also the IGF-I peptide was detected in myometrium and in leiomyomas, but in leiomyosarcomas the level was substantially lower. The IGF-II gene was expressed at low levels in normal myometrium and leiomyomas but is activated in leiomyosarcomas. With increasing malignancy from the two major IGF-II mRNA species, 6.0 and 4.8 kilobases, in particular the 6.0-kilobase mRNA is produced at higher levels. In conclusion, these data suggest that for IGF-I a role in tumor cell growth is not likely, but probably IGF-II is involved in malignant smooth muscle tumor growth progression.

Expression of insulin-like growth factor 1 in sarcomas.

The expression of insulin-like growth factor-1 (IGF-1) was studied in normal tissues, in eight benign lesions and in 50 sarcomas. In palmar fibromatosis the spindle cells in cell-dense areas exhibited a strong immunoreactivity. IGF-1 was variably found in leiomyosarcomas (7/8), malignant schwannomas (7/9), synovial sarcomas (2/3), liposarcomas (3/6), fibrosarcomas (1/3), malignant fibrous histiocytomas (10/18) and in one angiosarcoma. Two rhabdomyosarcomas failed to express IGF-1 and only the spindle cell component of synovial sarcomas was positive. Immunoreactivity for IGF-1 in 10 malignant filrous histiocytomas (MFH) appeared to be related to co-expression of smooth muscle actin. These findings imply that MFHs can be subdivided into a group of tumours which are devoid of morphological and immunophenotypic evidence of differentiation and a group which manifest immunophenotypic differentiation.

The relevance of the DNA index and proliferation rate in the grading of benign and malignant soft tissue tumors.

The relevance of the DNA index (DI) to malignancy grading and the relationships between mitotic score, Ki-67 score, and the proportion of cells in the G1-phase (G1PF) of the cell cycle, as proliferative indicators, were investigated in benign (n = 8) and malignant (n = 46) soft tissue sarcomas. Although DI was not found to be related to the histology of the sarcomas, it was associated with malignancy grade, i.e., 14%, 25%, 42%, and 86% of benign, Grade 1, Grade 2, and Grade 3, respectively had a DI greater than 1. The relevance of aneuploidy in benign tumors is discussed and the literature reviewed. A subgroup of the malignant tumors (n = 13) had both a low mitotic score and a low Ki-67 value. However, a distinct correlation between Ki-67 and the mitotic score could not be shown. For malignant tumours G1PF was related to DI, i.e., low G1PF occurred in tumors with diploid DNA content. Low Ki-67 scores were observed in 59% of diploid tumors and in 20% of aneuploid tumors. However, it appeared that G1PF and Ki-67 were not correlated. In conclusion, (1) benign and Grade 1 tumors were predominantly diploid with high G1PF and low Ki-67 values, (2) most of Grade 3 tumors were aneuploid (86%) with low G1PF and high Ki-67 values, and (3) the group of Grade 2 tumors could be divided into two subgroups either with the characteristics displayed by benign and Grade 1 tumors (DI = 1, low Ki-67 scores, and high G1PF) or with the characteristics exhibited by Grade 3 tumors (DI greater than 1, high Ki-67 scores, and low G1PF). Hence, supplementary to the grading of soft tissue tumors, DI, G1PF, and Ki-67 score could be useful as prognostic parameters in soft tissue tumors.

Detection of calcitonin-encoding mRNA by radioactive and non-radioactive in situ hybridization: improved colorimetric detection and cellular localization of mRNA in thyroid sections.

The localization of mRNA encoding calcitonin was studied by in situ hybridization using 35S-labeled RNA probes and biotin-labeled DNA probes. Radiolabeled probes were detected by autoradiography and biotin-labeled probes by streptavidin-biotin-peroxidase. To intensify the colorimetric signal, the indirect avidin-biotin complex (ABC) method was performed. However, the results were often variable. To improve the sensitivity, the peroxidase reaction signal was enhanced with a gold-silver deposit intensification reaction. To shorten the incubation times and to enhance the colorimetric reaction, several reaction steps were performed in a microwave oven. The localization of calcitonin mRNA in thyroid tissue, as detected with in situ hybridization, was confirmed by immunohistochemical localization of the calcitonin polypeptide. The results of in situ hybridization using biotinylated probes were compared to in situ hybridization using radioactive probes. Our data show that the results of in situ hybridization applied on frozen and paraffin-embedded sections using biotinylated DNA probes, detected with an indirect streptavidin-biotin-peroxidase reaction and intensified by silver-gold enhancement, were comparable to those obtained with radioactive probes. The localization of calcitonin encoding mRNA was in agreement with the localization of the calcitonin polypeptide.

Activity of glycolytic enzymes and glucose-6-phosphate dehydrogenase in lipoblastic and neurogenic proliferations.

The alterations in specific activity and/or isozyme pattern of hexokinase, phosphofructokinase, aldolase, enolase, pyruvate kinase and glucose-6-phosphate dehydrogenase were studied in the tissue specimens of 26 patients with lipoblastic tumors and 28 patients with tumors of neurogenic origin. Although the biochemical data demonstrated that the activities of most enzymes studied were elevated in the specimens of the malignant tumors, only the differences in activity of hexokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase measured between benign and malignant neurogenic tumors were significant. In malignant tumors, especially those of neurogenic origin, the isozyme pattern of pyruvate kinase showed a shift towards K-type subunits.