Interphase cytogenetics on paraffin sections of malignant pleural mesothelioma. A comparison to conventional karyotyping and flow cytometric studies.

We performed in situ hybridization (ISH) studies of malignant pleural mesotheliomas to detect numerical aberrations of chromosomes 1 and 7 in interphase nuclei of paraffin sections of 13 cases that had been analyzed previously by conventional karyotyping and flow cytometry. The hybridizations were performed with the biotin-labeled probes recognizing repetitive DNA sequences in the (peri)centromeric regions of chromosomes 1 (1q12) and 7(7cen). Application of histologic sections allowed us to analyze the tumor cells only. Comparison of the karyotype and ISH studies showed that the same chromosome copy numbers were detectable by both methods in 13 (chromosome 1) and in 12 (chromosome 7) cases evaluable by ISH. DNA indexes determined in the paraffin-embedded tumor material corresponded with the ISH findings. As compared with karyotype analysis, ISH showed a larger heterogeneity in chromosome copy numbers. The results can be divided into three groups: 1) Monosomy or disomy of chromosomes 1 and 7 was detected by both methods in two cases; 2) in four cases, disomy of both chromosome 1 and 7 was observed in most of the cells by ISH analysis, and karyotype analysis had shown clear polyploidization in three of these cases; 3) in seven cases, supernumerary copies of chromosomes 1 and/or 7 were present in an evident fraction (27-80%) of the cells analyzed by ISH, and karyotype analysis confirmed the aberrant copy numbers in five of these cases. On the other hand, ISH showed copy numbers not detected by karyotype analysis in six of the seven cases. Thus, by combining karyotype and interphase cytogenetic studies, complementary information about chromosomal aberrations in mesothelioma is obtained.

In situ detection of supernumerary aberrations of chromosome-specific repetitive DNA targets in interphase nuclei in human melanoma cell lines and tissue sections.

The use of non-radioactive in situ hybridization (ISH) with chromosome-specific repetitive DNA probes to study genomic changes, aneuploidy, and heterogeneity during melanocytic tumor progression, relies on its applicability to non-mitotic interphase nuclei, present in cell suspensions and tissue sections. Therefore, we studied the feasibility of detecting numerical aberrations with respect to the (peri-) centromere regions of chromosomes 1 and 7 in intact nuclei of two human melanoma cell lines with different metastatic behavior in nude mice. In addition, we used paraffin sections from xenograft lesions, obtained by inoculation of these cell lines in nude mice (subcutaneous tumors and spontaneous lung metastases). Paraffin sections from the original primary cutaneous melanoma (with a subepidermal and a dermal part) and two loco-regional metastases were also studied, one of which was the source for the cell lines. These cells and tissues represent examples of materials used in different approaches to the study of melanocytic tumor progression. Regarding the targeted sequences, ISH analysis showed that both cell lines were heterogeneous and aneuploid. The results correlated well with those obtained by ISH on metaphase spreads. Differences between the lines, which could not be detected by flow-cytometric or conventional karyotyping analysis, included data suggestive of a polyploid subpopulation and an extra copy of chromosome 7 in the metastasizing cell line. The polyploid population could be detected also in the paraffin sections of the corresponding subcutaneous xenografts and lung metastases in the mice. Both areas in the patients' primary melanoma could be evaluated separately and showed similar supernumerary aberrations of the chromosome-specific targets. These abnormalities matched those found in both metastases. Our results demonstrate that ISH can be used to visualize genomic abnormalities at the single-cell level in melanocytic nuclei in their natural context, which makes it a promising tool in the histopathology of melanocytic lesions and in the study of melanocytic tumor progression.

Interphase cytogenetics of hematological cancer: comparison of classical karyotyping and in situ hybridization using a panel of eleven chromosome specific DNA probes.

Numerical chromosome aberrations were detected in hematological cancers by nonradioactive in situ hybridization (ISH) procedures, using centromere specific probes for chromosomes 1, 7, 8, 9, 10, 11, 16, 17, 18, X, and Y. All 15 cases could be evaluated by ISH for these 11 probes. Our experiments show that in seven of these randomly selected leukemia bone marrow cell suspensions numerical aberrations for one or two chromosomes could be detected by this method. The results of ISH on interphase nuclei and in some cases on metaphase preparations were compared with karyotyping data. Seven cases of chromosomal aberrations observed with ISH (three for monosomy and four for trisomy) were confirmed by this classical cytogenetic technique, whereas in five instances an aberration was found only with ISH (twice for monosomy, twice for trisomy, and one disomy for the Y-probe). One case of a trisomy for chromosome 1 observed by ISH on interphase nuclei could be explained by a marker chromosome, a finding that was further substantiated by ISH on metaphase spreads. In this case double-target ISH on interphase cells with the probes for chromosomes 1 and 16 strongly suggested a translocation between these chromosomes. Also, in one case a marker chromosome could be characterized as a translocation between chromosomes 7 and 17. In this latter case the cytogenetic examinations revealed only monosomy for chromosomes 7 and 17 in addition to noncharacterized marker chromosomes. Our results indicate that the nonradioactive ISH procedure in combination with chromosome specific repetitive centromeric probes is a powerful tool for studying both numerical and structural chromosomal aberrations in interphase nuclei of leukemias. It may therefore become a valuable and routine diagnostic tool in addition to the existing karyotyping procedures.

Numerical chromosome 1, 7, 9, and 11 aberrations in bladder cancer detected by in situ hybridization.

Forty transitional cell carcinomas of the human urinary bladder (TCCs) were examined for numerical aberrations of chromosomes 1, 7, 9, and 11 by in situ hybridization using chromosome-specific probes. Our interphase cytogenetic study of 24 low-grade, noninvasive TCCs, which were near-diploid by flow cytometry, showed a numerical aberration for at least 1 of these chromosomes in 14 of these cases. Most strikingly, a monosomy for chromosome 9 was found in 9 of 24 low-grade TCCs. A trisomy for chromosomes 1, 7, and 11 was detected in 5, 2, and 1 case(s), respectively. In 1 case a monosomy for chromosome 1 was detected by in situ hybridization. Monosomy for chromosome 9 was the only detected numerical change in 5 low-grade TCC cases. Examination of 16 invasive TCCs showed extra copies for chromosomes 1 and 7 in 7 flow cytometrically diploid cases with numerical chromosome aberrations; also, loss of chromosome 9 was detected. In 5 invasive and 2 noninvasive aneuploid/tetraploid TCCs a profound imbalance between the different chromosomes was found. In 5 of these cases an evident underrepresentation of chromosome 9 in comparison to chromosomes 1, 7, and 11 was detected. This underrepresentation of chromosome 9 in diploid, as well as aneuploid, TCCs, and in some cases the constant ratio between this chromosome and the other chromosomes, may be explained by a process of tetraploidization. Therefore, loss of chromosome 9 may be one of the primary genetic events in TCC oncogenesis, with secondary events, such as tetraploidization, correlated to tumor progression. Our results show that in situ hybridization can be routinely used to study important cytogenetic changes which occur during the development of a malignant disease.

Detection of numerical chromosome aberrations in bladder cancer by in situ hybridization.

The nuclear DNA content of 53 transitional cell carcinomas (TCCs) of the urinary bladder, as determined by flow cytometry (FCM), was compared with chromosome ploidy as detected by nonradioactive in situ hybridization (ISH). For this purpose, probes for repetitive DNA targets in the (peri) centromeric region of chromosomes 1 and 18 were used. Hybridization results with both probes of 35 TCCs, which had a DNA index of approximately 1.0 as concluded from FCM, showed evident chromosome 1 aberrations in approximately 25% of the tumors, and in a few cases an aberration for chromosome 18 was detected. Comparison of the ISH spot numbers for both chromosomes showed in most cases a higher number for chromosome 1 than for chromosome 18. ISH on 18 cases of TCCs, which showed a single peak in FCM with a DNA-index of 1.2 to 3.2, exhibited a profound heterogeneity. In these TCCs the ratio between chromosomes 1 and 18 varied over a wide range, resulting in cases showing more hybridization signals for chromosome 1 than for chromosome 18 or the opposite. Furthermore, using ISH minor cell populations showing polyploidization and giant cells containing numerous ISH signals could occasionally be detected. Results showed that interphase cytogenetics by ISH enable a fast screening of numerical chromosome aberrations and detection of different cell populations within one tumor, which was apparently homogeneous according to FCM.

Intermediate filament expression and the progression of prostatic cancer as studied in the Dunning R-3327 rat prostatic carcinoma system.

To evaluate if there is any consistent relationship between the expression of intermediate filament proteins (IFP), particularly keratins, and the degree of malignancy of prostatic cancer cells, a series of nine Dunning rat prostatic cancer sublines that span the entire spectrum of progression of prostatic cancer were studied immunocytochemically by the use of a variety of antibodies specific for keratins, vimentin, or desmin. For the keratin studies, monoclonal antibodies with either a general reactivity to several keratins or highly specific for either luminal or basal epithelial cells of the normal rat prostate were used. By use of an antibody specific for luminal cell keratin 18, the luminal tumor cells of the well-differentiated, slow-growing H and HI-S sublines were positively stained. In most of the sublines with a more advanced state of progression (i.e., the moderately differentiated, moderately fast growing HI-M; the poorly differentiated, faster growing HI-F; and the anaplastic, very fast growing AT-1, AT-2, and MAT-Lu tumors), however, no expression of keratin specific for luminal cells was detected. In addition, several of the most advanced sublines (i.e., AT-1, AT-2, and MAT-Lu) were negative using any of the keratin antibodies. In contrast, several of the other sublines with the most advanced degree of progression (i.e., the anaplastic, very fast growing MAT-LyLu tumor derived from the AT-1 subline; and the anaplastic, very fast growing AT-3 tumor, derived from the HI-F subline), however, were positively stained with the keratin antibody specific for the luminal cells. By use of the keratin antibody specific for the basal cells of the normal rat prostate, the basal tumor cells of the well-differentiated slow-growing H and HI-S tumor were positively stained. This positive staining for basal cell keratin was also found in the HI-M and HI-F tumors, while the AT-1, AT-2, MAT-Lu, MAT-LyLu, and AT-3 were negative with this antibody. Thus, a loss in staining for basal cell keratin was consistently associated with the most advanced state of tumor progression. Vimentin-positive staining was demonstrated either alone or with keratin-positive staining in part of the epithelial cancer cells of all the sublines. An increase in the positive staining for vimentin was consistently associated with a more advanced state of tumor progression. Desmin-positive staining was found only in smooth cells present within the various tumor sublines.(ABSTRACT TRUNCATED AT 400 WORDS)

Tissue distribution of keratin 7 as monitored by a monoclonal antibody.

Monoclonal antibody (RCK 105) directed against keratin 7 was obtained after immunization of BALB/c mice with cytoskeletal preparations from T24 cells and characterized by one- (1D) and two-dimensional (2D) immunoblotting. In cultured epithelial cells, known from gel electrophoretic studies to contain keratin 7, this antibody gives a typical keratin intermediate filament staining pattern, comparable to that obtained with polyclonal rabbit antisera to skin keratins or with other monoclonal antibodies, recognizing for example keratins 5 and 8 or keratin 18. Using RCK 105, the distribution of keratin 7 throughout human epithelial tissues was examined and correlated with expression patterns of other keratins. Keratin 7 was found to occur in the columnar and glandular epithelium of the lung, cervix, breast, in bile ducts, collecting ducts in the kidney and in mesothelium, but to be absent from gastrointestinal epithelium, hepatocytes, proximal and distal tubules of the kidney and myoepithelium. Nor could it be detected in the stratified epithelia of the skin, tongue, esophagus, or cervix but strongly stained all cell layers of the urinary bladder transitional epithelium. When applied to carcinomas derived from these different tissue types it became obvious that an antibody to keratin 7 may allow an immunohistochemical distinction between certain types of adenocarcinomas.

Application of antibodies to intermediate filament proteins in simple and complex tumors of the female genital tract.

Polyclonal antibodies to cytokeratins, vimentin, and desmin and monoclonal antibodies to vimentin and to individual cytokeratin polypeptides, specific for glandular epithelia (RGE 53) or kertinizing stratified squamous epithelia (RKSE 60), have been applied in gynecological tumors with simple or complex composition. In general, tumors with simple composition showed reaction patterns fitting their known epithelial or mesenchymal nature, i.e., cytokeratin positivity in epithelial tumors only, vimentin positivity in mesenchymal tumors, and expression of desmin and vimentin in muscle cell tumors. Rather frequently, coexpression of cytokeratins and vimentin was noted in endometrial adenocarcinomas. Tumors with complex composition, such as müllerian mesodermal mixed tumors (MMMTs), that may pose considerable problems in conventional histopathology revealed various reaction patterns, with either expression of only cytokeratins or coexpression of cytokeratins and vimentin in carcinomatous areas and expression of only vimentin in sarcomatous areas. However, in addition, some MMMTs contained cells that were also positive for desmin. Intermediate filament protein immunohistochemistry appeared to be helpful in establishing a diagnosis of MMMT and in characterizing the different tumor components, which may prove to be useful in the evaluation of gynecological treatment protocols.

Co-expression of glial fibrillary acidic protein- and vimentin-type intermediate filaments in human astrocytomas.

The expression of intermediate filament (IF) proteins was studied in 71 cases of malignant human astrocytoma and in 17 cases of reactive gliosis, using immunocytochemical techniques with polyclonal and monoclonal antibodies to glial fibrillary acidic protein (GFAP) and vimentin. In all cases of astrocytoma, varying in degree of malignancy from grade I to grade IV, co-expression of GFAP and vimentin was found. No change in vimentin- or GFAP-IF expression with increasing anaplasia was seen. In addition astrocytic cells in reactive gliosis showed simultaneous expression of GFAP and vimentin. The intracellular distribution of these IF proteins differed. Vimentin was found to be located in a more juxta-nuclear position, whereas GFAP immunoreactivity showed a more intense staining of the cellular processes. Astrocytes in reactive gliosis behaved more or less like neoplastic cells. However, thin cell processes of reactive astrocytes in the cortex and superficial white matter only contained GFAP immunoreactivity. Simultaneous expression of GFAP and vimentin and their proportion in malignant and reactive glial cells are discussed in the light of earlier reports on the IF content of glial cells during development and maturation, in which vimentin precedes GFAP-expression. The existence of two separate (functional) IF systems in astroglia is suggested.

Immunohistochemical identification of Langerhans cells in normal epithelium and in epithelial lesions of the uterine cervix.

Using the double label indirect immunofluorescence technique we have studied vimentin-positive cells present in normal ecto- and endocervical epithelium, subcolumnar reserve cell hyperplasia, and squamous metaplastic and dysplastic epithelium of the uterine cervix. Monoclonal antibodies to Ia- and T6-antigens were applied in the examination of the expression of these membrane markers by such cells. Our studies reveal the presence of a relatively large number of vimentin-positive and T6-positive (Langerhans) cells in normal ectocervical stratified squamous epithelium, a small number in endocervical columnar epithelium, and a larger number in subcolumnar reserve cell hyperplasia and in immature squamous metaplasia. In this respect, mature squamous metaplastic epithelium shows a great resemblance to normal ectocervical stratified squamous epithelium. In contrast with previous reports in the literature we could only demonstrate small numbers of Langerhans cells in cases of dysplasia. The clinicopathological significance of these findings is discussed.

Expression of cytokeratins in early neoplastic epithelial lesions of the uterine cervix.

Polyclonal and monoclonal antibodies to cytokeratin polypeptides were used to study the expression of these intermediate filament proteins in normal, squamous metaplastic, and neoplastic epithelium of the uterine cervix, in order to investigate the morphogenesis of early epithelial changes preceding cervical squamous cell carcinoma. A polyclonal keratin antiserum showed a positive reaction in all different epithelial cell types of the uterine cervix. A positive reaction was also found in subcolumnar reserve cell hyperplasia, in squamous metaplastic and dysplastic cells, and in (squamous) carcinoma in situ. A monoclonal antibody specific for columnar epithelium (RGE 53) gave a positive reaction in endocervical columnar cells and in some immature metaplastic cells but was negative in subcolumnar reserve cells, squamous (metaplastic) cells, dysplastic cells, and most cases of carcinoma in situ. Another monoclonal cytokeratin antibody (RKSE 60) pointed to early keratinization in light microscopically nonkeratinizing squamous (metaplastic) and dysplastic epithelium. A possible overlap in staining patterns of RGE 53 and RKSE 60 was seen in some cases of immature metaplasia. Morphologic changes occurring in the transformation zone upon dedifferentiation are accompanied by alterations in cytokeratin expression. Similarities in cytokeratin expression were found between dysplasia and carcinoma in situ on one hand and subcolumnar reserve cell hyperplasia and squamous metaplasia on the other. This study favors an epithelial origin and a squamoid nature of subcolumnar reserve cells.

The use of antibodies to intermediate filament proteins in the differential diagnosis of lymphoma versus metastatic carcinoma.

Forty-nine cases encompassing 16 different types of malignant lymphoma were examined for their intermediate filament protein (IFP) type by indirect immunofluorescence microscopy of cryostat sections. In all cases, vimentin was shown to be the only IFP type detectable in these tumours. Lymphomas are negative for keratin and desmin, which are characteristic for benign and malignant epithelial or muscular tissues respectively. In addition, eighteen cases are described in which antibodies to intermediate filament proteins were used successfully to distinguish between lymphoma and metastatic carcinoma where differential diagnosis was difficult or impossible on the basis of routine histology.

Cytokeratin expression during neoplastic progression of human transitional cell carcinomas as detected by a monoclonal and a polyclonal antibody.

Antibodies to intermediate filament proteins were used to study different cell layers in normal human transitional epithelium, 16 human transitional cell carcinomas, and two cell lines derived from human bladder carcinomas. Conventional rabbit antisera to human skin keratins stained all layers of the transitional epithelium from bladder, ureter, and kidney. A slightly higher staining intensity was found in the basal and superficial layers as compared with the intermediate cell layers. A monoclonal antibody to cytokeratin 18 (RGE 53), however, stained only the superficial cell layer of transitional epithelium, the so-called umbrella cells. In well-differentiated (grade I) transitional cell carcinomas, RGE 53 stained only the superficial cells of papillary structures. In higher grade papillary tumors, RGE 53 also stained cells within the basal and intermediate layers, whereas in high-grade, invasive tumors almost all tumor cells were RGE 53 positive. These results show that monoclonal antibodies to cytokeratins can provide both an indication of processes involved in neoplastic progression of bladder tumors and a means of studying the molecular relationship of the tumor cells to normal cells.

Antibodies to cytokeratin and vimentin in testicular tumour diagnosis.

Thirteen primary and metastatic testicular germ cell tumours, including classical and anaplastic seminomas, and non-seminomatous testicular tumours were examined for their intermediate filament protein (IFP) types. The seminomas were shown to react with a monoclonal and a polyclonal antibody to bovine lens vimentin, while non-seminomatous germ cell tumours were strongly positive for a polyclonal and a monoclonal antibody to cytokeratin. In one case of seminoma with elevated serum levels of beta HCG and alpha FP, cytokeratin positive tumour cells were found. In the case of teratocarcinoma, several components of the tumour could be distinguished using a combination of antisera in double-label immunofluorescence microscopy. The glandular component of this tumour was positive with the polyclonal antikeratin, but also with the monoclonal cytokeratin antibody specific for glandular epithelia (RGE 53). However, the squamous component was negative with this latter antibody. Strikingly, the spindle cell component showed focal positivity for vimentin, with coexpression of cytokeratin and vimentin in some cells. Our data show that antibodies to cytokeratin and vimentin can be helpful in the diagnosis of testicular germ cell tumours, especially in the differentiation between seminomas and non-seminomatous tumours.

Immunocytochemical demonstration of intermediate filaments in a granular cell ameloblastoma.

The nature and location of intermediate filament proteins (IFP) may provide new insights into the origin and differentiation of neoplastic cells. An immunofluorescent study of these IFP in a case of a granular cell ameloblastoma revealed that all tumor cells contained the IFP keratin. Some granular cells, however, also contained the IFP vimentin, which is considered specific for mesenchymal tissues only. The implications of these observations are discussed. Study with monoclonal antibodies indicated the origin of the ameloblastoma from non-keratinized squamous epithelium. A comparison of the anti-keratin immunofluorescence pattern of the ameloblast-like cells in the present tumor with ameloblasts in the tooth germ revealed no similarities, indicating that despite some resemblance of the peripheral columnar cells to ameloblasts, these cells differ in other aspects.

Monoclonal antibody to keratin filaments, specific for glandular epithelia and their tumors. Use in surgical pathology.

A monoclonal antibody (RGE 53) against keratin intermediate filaments was prepared by fusing myeloma cells with splenic lymphocytes from mice immunized with a HeLa cell cytoskeleton preparation and human callus keratins. The antibody, selected for fibrillar staining in HeLa cells and a negative reaction on human skin, was tested on frozen sections from normal and malignant human tissues. RGE 53 specifically recognizes glandular epithelial cells from female breast, digestive, respiratory, and urogenital tracts, endocrine and exocrine tissues, and mesothelial cells. No reaction is found in stratified squamous epithelia or nonepithelial tissues. Furthermore, RGE 53 can distinguish adenocarcinomas and mesotheliomas, which stain positively in the indirect immunofluorescence technique from squamous cell carcinomas and nonepithelial tumors, which are negative for this antibody. RGE 53 may, therefore, be a useful tool for differential diagnosis in surgical pathology, especially in those cases in which anaplastic carcinomas have to be characterized.

Antibodies to intermediate filament proteins in the immunohistochemical identification of human tumours: an overview.

Intermediate-sized filament proteins (IFP) are tissue specific in that antibodies to keratin, vimentin, desmin, glial fibrillary acidic protein (GFAP) and the neurofilament proteins can distinguish between cells of epithelial and mesenchymal origin as well as of myogenic and neural origin respectively. Malignant cells retain their tissue-specific IFP, which makes it possible to use these antibodies in tumour diagnosis. Carcinomas are exclusively detected by antibodies to keratin. Monoclonal antibodies to keratin have allowed the differentiation between subgroups of epithelial tumours until now between adenocarcinomas and squamous cell carcinomas. Lymphomas, melanomas and several soft tissue tumours are distinctly recognized by antibodies to vimentin. On the other hand, rhabdomyosarcomas and leiomyosarcomas are positive for desmin, while astrocytomas give a strong reaction with GFAP antibodies. Thus, antibodies to IFP are useful tools for differential diagnosis in surgical pathology.

Mesodermal mixed tumor. Diagnosis by analysis of intermediate filament proteins.

A 58-year-old patient presented with poorly differentiated adenocarcinoma, probably primary in the ovary and, later in the course of her illness, with pure pleomorphic rhabdomyosarcoma. There was no evidence by light or electron microscopy of a mixture of these two tumor types. Further analysis by immunoperoxidase demonstrated scattered desmin-positive (muscle) cells in the adenocarcinoma portion of the tumor, establishing the diagnosis of malignant mesodermal mixed tumor.