DNA-flow cytometry, DNA-image cytometry and proliferation index (MIB-5) in irradiated rat salivary glands and salivary gland tumors.

AIM: Salivary glands (SG) can become atrophic following radiation exposure. Malignant transformation of SG in a radiation field is another known sequela of patients who have been treated by radiotherapy for a malignant tumor in the head and neck region. The aim of this study was to investigate cytogenetic alterations and to determine the proliferation index (PI) of SG of rats subjected to various total dosages of fractionated X-rays. MATERIALS AND METHODS: We investigated rat SG, subjected to 20, 40, or 60 Gy exposure by X-rays to the left neck and skull base. Non-irradiated rats served as a control group. Tumors originating from the SG were histologically-diagnosed following the descriptions for human SG tumors. The MIB-5 antibody was used to determine the PI. The ploidy was determined by flow and image cytometry (FCM, ICM). RESULTS: We consistently recorded diploid histograms in the FCM in irradiated glands. ICM revealed aneuploid histograms in 6/22 tumors, 3 of them were Auer Type III or IV. The PI showed a dose- and time-dependent course, indicative of variable regeneration properties of the parenchyma. Statistically significant differences were found for the PI within the irradiation groups and comparing irradiated SG and tumors. CONCLUSION: Irradiation of rat SG can cause almost complete loss of function. On the other hand, the PI remained in animals subjected to 40 Gy and investigated 1 year after completion of radiation at a level up to 10-fold higher than in untreated controls. The PI in carcinoma is higher in this species than in irradiated SG. Constantly elevated PI could support the development of cancer in SG.

Combined analysis of tumor growth pattern and expression of endogenous lectins as a prognostic tool in primary testicular cancer and its lung metastases.

The aim of this study was to glyco- and immunohistochemically analyze expression of distinct growth/adhesion-related markers of primary testicular carcinomas and their lung metastases in relation to the risk of developing lung metastases and survival of patients, and to correlate immunohistochemical staining profile and syntactic structure analysis in order to delineate new prognostic parameters for this tumor type. Clinical features of 50 patients with primary testicular carcinomas and their corresponding lung metastases were evaluated and compared to those of a control cohort of 25 cases. The set of eight probes including labeled galectins-1 and -3, specific non-cross-reactive antibodies against galectins-1, -3, and -8 as well as anti-Ki-67, anti-bcl-2, and anti-p53 was applied to formalin-fixed, paraffin-embedded tumor sections of both primary and metastatic lesions. Syntactic structure analysis computed staining intensities and structural features of the tumor cells. These parameters were set into relation separately and in combination to clinical data including tumor stages, smoking habits, applied cytostatic therapy, disease-free interval, and survival. The risk of testis cancer patients to develop lung metastases depends in descending order on the tumor cell type (non-seminoma versus seminoma), tumor cell heterogeneity (mixed versus monomorphous cell type), age of patients, and pT stage. The extent of differential expression of galectin-related features between primary and secondary lesions was pronounced. Prognostic correlations for distinct galectin-related features were delineated in combination with data from syntactic structure analysis, for example cluster radius of galectin-3-positive tumor cells and post-surgical and total survival. Lengths of disease-free interval and total survival of patients were also correlated to characteristics obtained by syntactic structure analysis and their combination with galectin data in the first place, then to smoking habits, percentage of proliferating cells in the primary and secondary tumors, and finally to expression of certain galectins and of p53. Patients with non-seminoma testicular cancer should be thoroughly controlled for lung metastases. Regarding marker selection, our study underscores that further investigation of the growth-regulatory network of galectins is clearly warranted.

Comparison of in-situ hybridization, direct and indirect in-situ PCR as well as tyramide signal amplification for the detection of HPV.

One hundred paraffin-embedded cervical biopsy specimens were tested for the presence of human papilloma virus (HPV) by in situ hybridization (ISH), and by direct and indirect in situ PCR (IS-PCR) in order to evaluate the efficiency of the different in situ methods in detecting HPV infection. ISH was performed using either commercial DNA probes or a cocktail of 5'-digoxigenin labeled oligoprimers. The same were used for ISH during indirect IS-PCR. To enhance the sensitivity of ISH several polymers, i.e., polyvinyl alcohol (PVA), polyethylene glycol, and polyvinylpyrrolidone were added to the alkaline phosphatase nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) reaction. Furthermore, tyramide signal amplification (TSA) was tried for signal amplification. Those samples treated with PVA during the NBT/BCIP reaction did not show any signal amplification whereas those treated with TSA exhibited a dramatic increase in sensitivity with usually acceptable signal to noise ratios. Our results show that, regarding sensitivity, ISH with subsequent signal amplification by TSA can be used as an almost equivalent alternative to the more cumbersome IS-PCR on routinely processed tissue specimens. When considering reproducibility, it is superior to IS-PCR.

Karyotype evolution in a case of uterine angioleiomyoma.

Clonal karyotypic alterations of a uterine angioleiomyoma of a 41-year-old woman are reported. Cytogenetically a stemline of the tumor and two related subclones with additional abnormalities due to karyotypic evolution were identified: 46,X,t(X;11)(p11.4;p15)/46, idem,inv(2)(p15q13)/46, idem,inv(2)(p15q13),t(5;20)(q13;q13.2). None of the aberrations observed in the present case has been reported in uterine smooth muscle tumors before.

A third case of a low-grade endometrial stromal sarcoma with a t(7;17)(p14 approximately 21;q11.2 approximately 21).

Cytogenetic analysis of a low-grade endometrial stromal sarcoma of the uterus in a 52-year-old woman revealed the karyotype 46,XX,t(7;17)(p14 approximately 21;q11.2 approximately 21),der(7)t(7;16)(p14-15;q22)t(7;9) (q22;q22), der(9)t(7;9)(q22;q22),del(16)(q22). The t(7;17) was identical to an aberration observed in two other cases of endometrial stromal sarcomas, thus confirming the idea that it constitutes a non-random aberration for this type of tumor.

[Genetic studies of differential fatty tissue tumor diagnosis]. / Genetische Untersuchungen zur Differentialdiagnose von Fettgewebstumoren.

Adipose tissue tumors are often characterized by typical or even specific chromosomal alterations. In some of the cases the molecular background of these microscopically visible alterations was already elucidated. In myxoid liposarcomas the translocation t(12;16) creates a fusion gene between the CHOP gene and the FUS gene and in lipomas the HMGI-C gene becomes rearranged by structural aberrations involving chromosomal region 12q14-15. Based on examples of a lipoma, a well-differentiated liposarcoma, a myxoid liposarcoma, and an aggressive angiomyxoma it is demonstrated in the present paper how cytogenetic investigation can be used as an additional tool for an improved diagnosis of adipose tissue tumors. Furthermore, the detection of molecular mechanisms underlying the visible cytogenetic alterations will certainly significantly increase our knowledge about the pathogenesis of these diseases.

[Merkel cell tumor or neuroendocrine skin carcinoma]. / Merkel-Zell-Tumor oder neuroendokrines Hautkarzinom.

Merkel cell carcinoma is a rare malignant tumor of the skin with predominance in older patients; 78.6% of patients are older than 59 years. Female and male patients are equally involved in the age group below 60 years. After 60 years, Merkel cell carcinomas are more often observed in female patients. The tumor is most often located in the head and neck region (50.8%) or the extremities (33.7%). The average size is 29 mm at presentation. Clinically, only a presumptive diagnosis of Merkel cell carcinoma can be established. The definite diagnosis is made by histological, especially immunohistological methods (detection of intermediate filaments and neuroendocrine markers). The therapy of choice is local excision. Secondary therapy may be a combination of operation and radiation or chemotherapy. Since this combination may reduce the risk of recurrences it should be applied for patients with poor prognostic features. Especially in young patients, additional lymphadenectomy should be discussed. Clinical control is necessary. Distant metastases should be treated by chemotherapy. Bad prognostic features are: lymph node metastasis, size larger than 2 cm, male sex.

A hamartoma of the breast with an aberration of 12q mapped to the MAR region by fluorescence in situ hybridization.

Cytogenetic studies of a breast adenolipoma (hamartoma) of a 58-year-old patient revealed a karyotype 46,XX,add(4)(?),add(6)(q?),der(7)t(7;12)(q11.1 or q11.2;q11 or q12),der(12). To our knowledge, this is the second report of an aberration involving 12q12-15 in a hamartoma of the breast. By FISH studies, we found this chromosome 12 translocation breakpoint to be mapping within the MAR (Multiple Aberration Region). MAR is known to be a major cluster region of chromosome 12 breakpoints of benign solid tumors such as uterine leiomyoma, lipoma, and pleomorphic salivary gland adenomas, therefore raising the possibility that the same gene is involved in hamartoma of the breast as in these three benign solid tumors.

Cytogenic and molecular analysis of an aggressive angiomyxoma.

Aggressive angiomyxoma is a rare mesenchymal tumor occurring mainly in the vulvar region extending into the paravaginal and perirectal region. Histologically, this tumor is rich in vascular structures and in collagen fibers and is of myxoid appearance. Cytogenetic and molecular analysis was performed on a case of an aggressive angiomyxoma and revealed clonal karyotypic abnormalities. All 50 metaphases analyzed showed a translocation involving the chromosomal region 12q14-15. Chromosomal aberrations involving the breakpoint region 12q14-15 are frequently seen in a variety of other mesenchymal tumors as uterine leiomyomas, lipomas, hamartomas of the lung, liposarcomas, or hemangiopericytomas. Therefore, this breakpoint region seems to be the most frequent chromosomal abnormality associated with the initiation of human mesenchymal neoplasms. To narrow down the breakpoint region on a molecular level in the cells of the angiomyxoma we performed FISH analysis with different cosmid clones originating from a yeast artificial chromosome and cosmid contig overspanning parts of the region 12q14-15. We were able to narrow down the region to approximately 70-80 kb belonging to an area designated a multiple aberration region, because it also includes the breakpoints of leiomyomas, lipomas, and pleomorphic adenomas with 12q13-15 abnormalities. Our molecular and cytogenic data suggest that angiomyxomas or at least a subset of them molecularly belong to the benign group of mesenchymal tumors showing multiple aberration region involvement.

Trisomy 8 and 18 as frequent clonal and single-cell aberrations in 185 primary breast carcinomas.

For cytogenetic investigations short-term cultures of 185 breast carcinomas (135 invasive ductal, 21 invasive lobular, 12 invasive ductal with intraductal components, seven heterogeneous, six intraductal, four invasive ductal and lobular) were prepared. Cytogenetic examinations revealed clonal abnormalities in 39 cases with a predominance of simple numerical chromosome changes, i.e., trisomies of chromosomes 7, 8, and 18. One hundred forty-six tumors did not show clonal abnormalities, but single-cell aberrations other than monosomies occurred in 79 of these tumors. Compared to cells of epithelial hyperplasia of the breast, amniotic fluid cells, and cells from pleomorphic adenomas cultivated using the same medium, the frequency of single-cell trisomies was significantly higher. Trisomy 8 was not only found as a clonal aberration in 10 cases but was also the most frequent non-clonal aberration. Trisomy 7 and 18 were also frequent clonal as well as non-clonal cytogenetic deviations.

Fibroadenoma of the breast showing a translocation (6;14), a ring chromosome and two markers involving parts of chromosome 11.

The cytogenetic findings of a recurrent fibroadenoma of a 25-year-old woman are reported. Of 58 metaphases karyotyped after G-banding, 27 showed an apparently normal karyotype and 31 the karyotype 48,XX,del(6)(q21),r(11)(?) + der(11)x2,der(14)t(6;14)(q21;q32). By fluorescence in situ hybridization studies using a chromosome 11 specific painting probe, we were able to show that the two marker chromosomes and the ring contained chromosome 11 DNA.

[Metastasis surgery in granulosa cell tumor--determining indications and tumor after care]. / Metastasenchirurgie beim Granulosazelltumor--Erörterungen zur Indikationsstellung und Tumornachsorge.

The granulosa cell tumour is a rare and malignant neoplasia of the ovary. As it has a low grade of malignancy a long course of the disease is observed. Following surgical resection, life-long tumour follow-up is recommended. Based on a case report and a review of the literature, the possible sites of metastases are demonstrated. This paper presents the latest therapeutic concepts and discusses the usefulness of metastasis resection.

A recurrent marker chromosome involving chromosome 1 in two mammary tumors of the dog.

An apparently identical marker chromosome resulting from a chromosome 1. translocation was found in the mammary carcinomas of two bitches. Although these karyotypic aberrations were the sole clonal aberrations detected, it was not possible to unambiguously identify the material translocated to the chromosome 1 in either animal. Our observations, however, represent the first report of a recurring marker chromosome in mammary tumors of the dog and suggest that these tumors may become an interesting model for human breast cancer.

Mucoepidermoid tumor of the parotid gland showing a translocation (3;8)(p21;q12) and a deletion (5)(q22) as sole chromosome abnormalities.

The cytogenetic findings in a mucoepidermoid tumor of the parotid gland are described. In addition to a t(3;8)(p21;q12), all cells analyzed showed a deletion of part of the long arm of chromosome 5. Although the typical translocation of benign pleomorphic adenomas was found, histologic examination did not show remnants of a pleomorphic adenoma. Nevertheless, we assumed that the malignant tumor most likely arose in a preexisting pleomorphic adenoma. As a mechanism for malignant transformation, the loss of a suppressor gene located on the long arm of chromosome 5 is discussed.

Prognostic factors for adenoid cystic carcinoma of the head and neck: a retrospective evaluation of 96 cases.

Cases of adenoid cystic carcinomas of the salivary (n = 90) and lacrimal glands (n = 6) from the years 1965-1980 were evaluated retrospectively with regard to clinical, epidemiologic and histomorphologic parameters, and in 52 cases, nuclear DNA content was assessed using a single cell scanning cytophotometry procedure in order to determine prognostic factors. Clinical courses were poor with a high incidence of recurrences, hematogenous metastases and deaths from tumor. Histology was related to prognosis, glandular tumors showing a better prognosis than solid ones. Tumor size greater than 4 cm was correlated with an unfavorable clinical course in all cases. Cytophotometry yielded various types of histograms (7 diploid, 10 proliferative, 14 triploid, 19 atypical, 2 tetraploid). Significant correlations were found as to the time of survival, tumors with diploid histograms showing the longest intervals and those with atypical ones the shortest. Although the prognosis of adenoid cystic carcinoma remains poor, cytophotometry can offer additional prognostic information in the individual case.

Radioprotection of minipig salivary glands by orciprenaline-carbachol. An ultrastructural and semiquantitative light microscopic study.

Parotid and submandibular glands of miniature pigs were exposed to 36 Gy X-irradiation given as 6 x 6 Gy in 3 weeks. Half of the animals received orciprenaline and carbachol before each dose. The effects were analysed 6 months later by light and electron microscopy. Ultrastructural examination showed less change in the pretreated glands. Semi-quantitative light microscopic data confirmed the significance of the differences. Acinar cells of both glands were significantly more numerous (P less than 0.01) and the cells were better preserved (P less than 0.01) in the pretreated group. The effect was more pronounced in the parotid gland, which appeared almost normal. Intercalated ducts of the parotid glands (P less than 0.01) and striated ducts of the submandibular glands (P less than 0.05) showed less change in pretreated animals. The findings confirm the radioprotective effect of pharmacologically induced degranulation of acinar cells. The biological effects of the radiation schedule (cumulative radiation effect value 18.76) as well as the dosage of orciprenaline and carbachol are within the normal range of medical treatment. Similar results may be expected from future studies in man.

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

Acinic cell carcinomas of the Salivary Gland Registry, Institute of Pathology, University of Hamburg, West Germany, from 1965 to 1980 (n = 55) were evaluated retrospectively with respect to histologic, cytophotometric, and clinical data. The majority of the tumors (92.8%) were located in the parotid gland. Two thirds of the patients were female; one third were male. Mean age at primary diagnosis was 55.4 years. The tumors were graded into highly differentiated (76%) or less differentiated forms (24%) according to classic histologic and cytologic criteria. The clinical course was characterized by no recurrence in 15 cases; in 17 cases, recurrences developed, and 12 patients died of their tumor, some as late as 240 months after primary diagnosis. Differentiation showed a weak correlation with the clinical course. In 35 cases, nuclear DNA content of tumor cells was assessed cytochemically. The tumors were "diploid" or "near-diploid" in 34 cases; DNA content showed no correlation to the clinical course. As a result of long-term follow-up, it becomes evident that acinic cell carcinoma is prone to develop recurrences and metastases. Complete tumor removal during the primary operation seems to be important for controlling the disease inasmuch as the ostensible prognostic predictors evaluated here proved to be unreliable.