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The occurrence, location and intensity of the basement membrane (BM) components collagen IV and laminin in benign and malignant pigment cell tumors was studied by immunohistochemical methods. The results seemed to establish the following findings: junctional nevi display varying continuity of BM; nevus cells in the dermis display more continuous and thicker BM superficially (associated with epithelial type nevus cells); superficial spreading melanoma displays discontinuity of BM. and nodular melanoma and metastatic melanoma display variable BM around tumor aggregates. The variable expression of BM components in this study showed an apparent relationship to tumor cell type and laminin and collagen IV production, partly related to clinical behaviour. [ABSTRACT FROM AUTHOR]

Hyperparathyroidism may result from parathyroid hyperplasia or adenoma, or rarely from parathyroid carcinoma. Pericentromeric inversion of chromosome 11 that results in activation of the P:RAD1/cyclin D1 gene and tumor suppressor gene loss have been described as genetic abnormalities in the evolution of parathyroid neoplasms. We studied tissue samples taken from primary parathyroid hyperplasia, parathyroid adenoma, and histologically normal parathyroid tissue by comparative genomic hybridization, fluorescent in situ hybridization, and immunohistochemistry for cyclin D1. DNA copy number changes were infrequent in primary hyperplasia (4 of 24, 17%), but common in adenomas (10 of 16, 63%; P: = 0.0059). The most common change was deletion of the entire chromosome 11 or a part of it, with a minimal common region at 11q23. This change was present in five (31%) adenomas and two (8%) primary hyperplasias. Fluorescent in situ hybridization confirmed the presence of both MEN1 alleles located at 11q13 despite deletion of 11q23 in all three cases studied. Cyclin D1 was overexpressed in six (40%) of the 15 adenomas studied, whereas none of the 27 hyperplasias (P: = 0.0010) nor the five histologically normal tissue samples overexpressed cyclin D1. Either DNA copy number loss or cyclin D1 overexpression was present in 13 (81%) of the 16 adenomas. We conclude that DNA copy number loss and cyclin D1 overexpression are common in parathyroid adenomas. The region 11q23 is frequently lost in parathyroid adenomas and occasionally in parathyroid hyperplasias, and this suggests the possibility that a tumor suppressor gene that is important in their pathogenesis is present on 11q23.

This review summarizes reports of recurrent DNA sequence copy number losses in human neoplasms detected by comparative genomic hybridization. Recurrent losses that affect each of the chromosome arms in 73 tumor types are tabulated from 169 reports. The tables are available online at http://www.amjpathol.org and http://www. helsinki.fi/ approximately lglvwww/CMG.html. The genes relevant to the lost regions are discussed for each of the chromosomes. The review is supplemented also by a list of known and putative tumor suppressor genes and DNA repair genes (see Table 1, online). Losses are found in all chromosome arms, but they seem to be relatively rare at 1q, 2p, 3q, 5p, 6p, 7p, 7q, 8q, 12p, and 20q. Losses and their minimal common overlapping areas that were present in a great proportion of the 73 tumor entities reported in Table 2 (see online) are (in descending order of frequency): 9p23-p24 (48%), 13q21 (47%), 6q16 (44%), 6q26-q27 (44%), 8p23 (37%), 18q22-q23 (37%), 17p12-p13 (34%), 1p36.1 (34%), 11q23 (33%), 1p22 (32%), 4q32-qter (31%), 14q22-q23 (25%), 10q23 (25%), 10q25-qter (25%),15q21 (23%), 16q22 (23%), 5q21 (23%), 3p12-p14 (22%), 22q12 (22%), Xp21 (21%), Xq21 (21%), and 10p12 (20%). The frequency of losses at chromosomes 7 and 20 was less than 10% in all tumors. The chromosomal regions in which the most frequent losses are found implicate locations of essential tumor suppressor genes and DNA repair genes that may be involved in the pathogenesis of several tumor types.

The genetic changes leading to thyroid cancer are poorly characterized. We studied DNA copy number changes by comparative genomic hybridization (CGH) in 69 primary thyroid carcinomas. In papillary carcinoma, DNA copy number changes were rare (3 of 26, 12%). The changes were all gains, and they were associated with old age (P = 0.01) and the presence of cervical lymph node metastases at presentation (P = 0.08). DNA copy number changes were much more frequent in follicular carcinoma (16 of 20, 80%) than in papillary carcinoma (P < 0.0001), and follicular carcinomas had more often deletions (13/20 versus 0/26, P < 0.0001). Loss of chromosome 22 was common in follicular carcinoma (n = 7, 35%), it was more often seen in widely invasive than in minimally invasive follicular carcinoma (54% versus 0%, P = 0.04), and it was associated with old age at presentation (P = 0.01). In three of the four patients with follicular carcinoma who died of cancer, the tumor had loss of chromosome 22. DNA copy number changes were found in 5 (50%) of the 10 medullary carcinomas studied. Four of these five carcinomas had deletions, and in two of them there was deletion of chromosome 22. Eleven (85%) of the thirteen anaplastic carcinomas investigated had DNA copy number changes, of which five had deletions, and one had deletion of chromosome 22. The most common gains in anaplastic carcinoma were in chromosomes 7p (p22-pter, 31%), 8q (q22-qter, 23%), and 9q (q34-qter, 23%). We conclude that DNA copy number changes are frequent in follicular, medullary, and anaplastic thyroid carcinoma but rare in papillary carcinoma when studied by CGH. Loss of chromosome 22 is particularly common in follicular carcinoma, and it is associated with the widely invasive type.

This review summarizes reports of recurrent DNA sequence copy number amplifications in human neoplasms detected by comparative genomic hybridization. Some of the chromosomal areas with recurrent DNA copy number amplifications (amplicons) of 1p22-p31, 1p32-p36, 1q, 2p13-p16, 2p23-p25, 2q31-q33, 3q, 5p, 6p12-pter, 7p12-p13, 7q11.2, 7q21-q22, 8p11-p12, 8q, 11q13-q14, 12p, 12q13-q21, 13q14, 13q22-qter, 14q13-q21, 15q24-qter, 17p11.2-p12, 17q12-q21, 17q22-qter, 18q, 19p13.2-pter, 19cen-q13.3, 20p11.2-p12, 20q, Xp11.2-p21, and Xp11-q13 and genes therein are presented in more detail. The paper with more than 150 references and two tables can be accessed from our web site http://www.helsinki.fi/lglvwww/CMG.html. The data will be updated biannually until the year 2001.

The genetic changes underlying the development of resistance to the platinum-containing drugs are poorly defined. We analyzed six resistant cell lines using comparative genomic hybridization (CGH) in order to screen and identify possible genetic changes in common. We compared parental 2008 and A2780 human ovarian cancer cells to sublines selected for resistance to cisplatin (DDP) (2008/C8, 2008/C13*5.25, 2008/A, A2780/CP); we also compared 2008 cells to sublines selected for resistance to antimonite (2008/H) and arsenite (2008/I) which demonstrated cross-resistance to DDP. DNA samples from the resistant cell lines were hybridized against DNA from the parental cells rather than from normal human cells to permit detection of only those changes associated with the development of resistance. The DNA sequence copy number changes were surprisingly numerous in the DDP, antimony, and arsenite-resistant variants of the 2008 cell line and most of the chromosomes were affected. On the other hand, in the A2780/CP subline only a few changes were found and these were limited to just four chromosomes. The most common findings among the DDP-resistant cell lines were gains of material from chromosomes or chromosome arms 2q (5 out of 6 lines), 4 (4/6), 6q (5/6), and 8q (4/6). Deletions were observed on chromosomes or chromosome arms 2p (4/6), X (4/6), 7p (5/6), 11p (4/6), and 13 (4/6). The most frequently involved chromosomal regions, affected in the majority of cell lines, were: gain of 2q14.1-q33, 4p15.2-p13, 4q22-q25, 4q31.1-q34, 6q13-q16, 8q12-q21.2, and loss of Xp22.2-q21, 7p21-p14, 11cen-p14 in sublines of 2008, and loss of 2pter-p22 and 13q21 in sublines of 2008 and A2780. The results suggest that the acquired resistance and cross-resistance to DDP in these cell lines was associated with substantial genomic instability, quite unlike the changes observed in association with the development of resistance to drugs participating in the multidrug resistance phenotype.

Thymidylate synthase (TS) provides the only de novo source of thymidylate for DNA synthesis and is a key target for cancer chemotherapeutic agents. We investigated the TS gene expression by semiquantitative reverse-transcriptase polymerase chain reaction in metastatic melanoma and compared the results with those from control tissues. The relative TS/beta-actin level ratios were 0.5, 0.9, 0.3, 0.4, and 0.5 (mean 0.5) in skin, lymph node, thyroid, muscle, and spleen, respectively. In metastatic melanoma samples, the ratios varied from 0.9 to 2.7 (mean 2.0). The differences of expression levels between these two groups of samples were statistically highly significant (p = 0.0000713). A similar statistical significance (p = 0.0002) was observed between patients achieving a complete response and patients who had progressive disease despite immunochemotherapy. There was no clear relationship between a high TS/ beta-actin ratio and the S phase fraction, as all melanomas had a high S phase fraction.

Bax is a homologue of Bcl-2 that promotes apoptosis. Bax protein levels were assessed by immunohistochemical methods in primary tumors derived from 119 women with metastatic breast cancer. These patients had received combination chemotherapy either with a once a month dosage schedule or in 4 weekly divided doses. The BAX immunostaining results were retrospectively compared with overall survival, time to tumor progression (TTP), and response, as well as several laboratory markers. Normal breast epithelium and in situ carcinomas immunostained positively for Bax. Marked reductions in Bax immunostaining were observed in 40 (34%) of 119 evaluable tumors. Reduced Bax correlated with shorter overall survival (median, 8.1 versus 15.7 months; P = 0.04), faster TTP (median, 2.0 versus 6.3 months; P = 0.009), and failure to respond (complete response, partial responses; 6% versus 42%, P = 0.01) in the subgroup of patients who received divided dose therapy. Reduced Bax immunostaining was not significant in the monthly dose group. When the two groups were combined, however, reduced Bax was significantly correlated in univariate analysis with failure to respond (21 versus 43% achieving complete response or partial response; P = 0.02), faster TTP (median, 3.7 versus 9.0 months; P = 0.02), and shorter survival (median, 10.7 versus 17.1 months; P = 0.04). Bax immunostaining was not significantly correlated with tumor histology, S-phase fraction, aneuploidy, p53 HER2, or cathepsin D, but was positively associated with Bcl-2 (P = 0.005). In multivariate analysis (Bax, tumor grade, and treatment group), reduced Bax was strongly associated with faster TTP (P approximately equal to 0.009) and shorter survival (P approximately equal to 0.001). Although highly preliminary, the finding suggest that loss of Bax immunostaining represents a novel prognostic indicator of poor response to chemotherapy and shorter survival in women with metastatic breast cancer, and raise the possibility that the subgroup of women with Bax-negative tumors may benefit from more aggressive therapy.

Chemosensitivity was analyzed by mouse subrenal capsule assay (SRCA) in 103 human melanoma metastases 79 of which were also analyzed by DNA flow cytometry. The most effective combination was dacarbazine, vincristine, and carmustine (DOB), followed by cisplatin plus etoposide (Plat-VP16). By the original criteria of the assay, 35% of tumors were sensitive to DOB treatment while 15% responded to Plat-VP16. Chemosensitivity of DNA diploid and aneuploid tumors showed no significant difference; 35% of the aneuploid tumors were sensitive to DOB and 19% to Plat-VP16, whereas 41% and 13% respectively of the diploid tumors were sensitive. An association between DNA index and chemosensitivity was observed. Growth of the implant was observed in 44% of tumors with a DNA index above 1.5 receiving DOB treatment, whereas only 12% of tumors with an aneuploid DNA index < or = 1.5 grew. No significant difference was observed in the SPF of chemotherapy sensitive and insensitive tumors, though a tendency to lower SPF was observed in sensitive tumors.

Specimens from freshly biopsied human mammary and ovarian tumours and DMBA-induced rat mammary tumours were transplanted under the renal capsule of normal, immunocompetent mice and rats. The growth of the tumour and the effects of chemotherapy to the host animals were evaluated by measuring the growth of the implanted tumour specimens. Morphological analysis of the tumours showed preserved histological structures and cellular function, as evidenced by the basement membrane component production seen using immunohistochemical stains and by the surface specialized structures, cell-to-cell contact and secretory activity observed in ultrastructural studies. Treatments with different types of drugs yielded tangible results within 6 days of transplantation, showing lymphoid infiltration and scar formation, but preserved vascular structures. The results suggest that tumours subjected to subrenal capsule assay retain their structure and function when transplanted, thus enabling the evaluation of their sensitivity to therapeutic manipulation.

Basement membrane changes in the epidermis and hair follicle apparatus resulting from topical 9,10-dimethylbenzanthracene applications were studied in mice, rats, and hamsters by light and electron microscopy and using antibodies to human collagen type IV and laminin. The basement membrane was distinct in epidermal hyperplasia, dysplasia, and papillomas, as well as around most of the keratoacanthomas and squamous cell carcinomas, which showed basement membrane irregularities, thickening, and reduplication in some areas. The invading edges of the squamous cell carcinomas with inflammatory infiltrates were devoid of laminin and collagen. Collagen IV and laminin-positive structures were observed around preserved follicular structures in rat: hair nevi and hair-follicle nevi, but partly absent around trichoepitheliomas and trichofolliculomas. Basal cell tumors were usually surrounded by a distinct basement membrane, which was lacking around some tumor cells.

17 beta-Estradiol-6-carboxymethyloxime was covalently linked to horseradish peroxidase for the cytochemical demonstration of estrogen binding sites in breast cancer tissue. The affinity of the 17 beta-estradiol-horseradish peroxidase (E2-HRP) conjugate for the estrogen receptor in a human myometrial cytosol preparation was reduced by a factor of about 16 relative to that of 17 beta-estradiol. The estradiol concentration of the E2-HRP conjugate used in the incubations was in the range 2 X 10(-9) -1 X 10(-7) mol/l which, when the reduced affinity of the conjugate is taken into account, corresponds to 1 X 10(-10) -7 X 10(-9) mol/l unbound estradiol. The cytochemical reaction was carried out on cytofuge preparations of cell suspensions of breast cancer tissue. The intensity of the cytochemical reaction was microscopically evaluated by scoring. The results were analyzed in a plot allowing the calculation of an apparent score-max and an apparent Kd value. The reaction intensity was reduced to 20-25% of the control level by a 20-fold excess of 17 beta-estradiol. The cytochemical results correlated positively with the content of estrogen receptors in the cytosol as measured by a validated radioligand method.

The alpha- and beta-subunits of spectrin are made of repeated homologous units of 106 residues. In the recently reported partial sequence of the chicken non-muscle alpha-actinin, a repetitive sequence homologous to the internal repeat in spectrin occurs several times. Both spectrin and alpha-actinin are components of the cytoskeletal network, the integrity of which is based on multiple and complex interactions. We suggest that the shared domain structure indicates common structural principles or interactions of spectrin and alpha-actinin and reflects their common evolution.

Immunological screening of a chicken gizzard cDNA expression library was used to isolate two clones encoding a part of the non-erythroid spectrin-like protein. Clones were identified by immunoblotting of the polypeptides synthesized in Escherichia coli cells transformed with cDNA cloned in the pUC8 plasmid vector using polyclonal rabbit antibodies raised against bovine non-erythroid spectrin. The sequence of an approximately 1.5-kb cDNA insert of one clone was determined. Analysis of the predicted amino acid sequence reveals that, despite differences in immunological cross-reactivity and peptide maps, the chicken non-erythroid and the human erythrocyte spectrins are highly homologous proteins. Like the human erythrocyte spectrin, the chicken smooth muscle spectrin appears also to be constructed from repeated, homologous structures of 106 amino acid residues. This is probably a universal structure motif of spectrins.

The occurrence and intensity of staining for specific antibodies against the aminoterminal propeptide of type III procollagen (PIIIP), which is indicative of the synthesis and the degradation of that collagen type, was studied in sections from normal and alcoholic livers and compared with serum PIIIP levels, serum antipyrine clearance, fibronectin distribution and morphology as revealed by conventional stains and electronmicroscopy. Positive staining for PIIIP and fibronectin was observed in the perisinusoidal space of the normal liver and in portal tracts. In alcohol-induced fatty liver positive staining increased around the central veins, in alcoholic hepatitis increased staining reaction was seen to a limited extent in areas of cell injury. Extensive reticulin and PIIIP-positive areas were found in the periportal interstitium of the cirrhotic livers and in large fibrotic areas extending into the surrounding parenchyma in cases of active disease. The results show a distinct relationship between collagen type III metabolism, morphologically detectable hepatic injury and liver cell function tests, with tissue deposition occurring later in the disease process than biochemically detectable serum collagen levels and signs of altered liver cell function.

Changes in basement membrane (BM) structure were studied in functioning and hyperplastic endometrium, in adenocarcinomas with various degrees of differentiation and in progesterone-treated adenocarcinomas using electron microscopy and immunohistochemical staining with antibodies against human type IV collagen and laminin. These BM components were distinctly visualized as narrow, continuous bands beneath the epithelium and around the endometrial glands in functioning, atrophic and hyperplastic endometrium. In well-differentiated endometrial carcinomas there was mostly a continuous BM, though occasional disruptions were seen. The undifferentiated tumors, on the other hand, were characterized by the absence of a continuous BM structure, although irregular patches of BM material were found within the neoplasm. Hormonal treatment caused the reappearance of the BM structures. According to these results, the visualization of the BMs in the endometrium not only increases our understanding of tumor behavior, but can also be used as an aid for the classification and treatment of endometrial neoplasms.

Solid cell nests of the thyroid display histological features that resemble squamous epithelium. In the present study we investigated whether these ultimobranchial nests express a renal tubular cell cytokeratin which is found to be widely distributed in epithelia but does not appear in squamous tissue. All 17 solid cell nests studied, in contrast to squamous epithelium lining an intrathyroid thyroglossal cyst, stained positively for this intermediate filament. These findings, together with the gland-like features shown ultrastructurally by these ultimobranchial remnants, further support the view that solid cell nests of the thyroid should not be regarded as a kind of intrathyroid squamous inclusion. In addition, they were electron-microscopically comparable to mammalian ultimobranchial tissue.

A monoclonal antibody (Mab) against a synthetic peptide, SEDYGKDL, corresponding to one conserved sequence in the chicken alpha-fodrin repeats reacts in immunoblotting with avian alpha-spectrin and alpha-fodrin, both mammalian spectrins and with mammalian alpha-fodrin. This Mab also reacts with alpha-actinin in both chicken and human cells. Our results confirm the previously detected structural homology between spectrins and alpha-actinin and implicate their common evolutionary origin.

The complete nucleotide sequence coding for the chicken brain alpha-spectrin was determined. It comprises the entire coding frame, 5'- and 3'-untranslated sequences terminating in a poly(A)-tail. The deduced amino acid sequence shows that the alpha-chain contains 22 segments, 20 of which correspond to the typical 106 residue repeat of the human erythrocyte spectrin. Some segments non-homologous to the repeat structure reside in the middle and COOH-terminal regions. Sequence comparisons with other proteins show that these segments evidently harbour some structural and functional features such as: homology to alpha-actinin and dystrophin, two typical EF-hand structures (calcium-binding) and a putative calmodulin-binding site in the COOH-terminus and a sequence homologous to various src-tyrosine kinases and to phospholipase C in the middle of the molecule. Comparison of our sequence with other partial alpha-spectrin sequences shows that alpha-spectrin is well conserved in different species and that the human erythrocyte alpha-spectrin is divergent.

Basement membrane changes in human skin exposed to UV irradiation were studied by light and electron microscopy and antibodies to human collagen IV and laminin were compared to similar lesions in non-sun-exposed areas. A distinct basement membrane was seen in cases of epidermal solar keratosis, even with marked dysplasia and Bowenoid type lesions, and also around most of squamous cell carcinomas which showed basement membrane irregularities, thickening and reduplication. The invading edges of the squamous cell carcinomas with inflammatory infiltrates were devoid of laminin and collagen. Basement membrane disruption was also observed in lichen planus-type solar keratosis with severe inflammation, but with no evidence of malignancy. Alterations in basement membrane structure and location were associated with cutaneous morphological abnormalities not due to UV irradiation as such.

Specimens from 16 freshly biopsied human tumors, two mammary adenocarcinomas, ten ovarian adenocarcinomas, two squamous cell carcinomas, one malignant histiocytoma and one chondrosarcoma of the bone, two human ovarian adenocarcinomas established by transplantation into nude mice and two adenocarcinomas induced in rat mammary gland were transplanted under the renal capsule of 510 normal immunocompetent mice and 180 rats and the effects of chemotherapy were evaluated. The results showed successful transplantation of all types of tumors in both animal species. Morphological analysis revealed preserved glandular structures with surface microvilli, mucin and CEA production and partially preserved basement membranes. Treatment with cyclophosphamide, vinblastine, adriamycin and cisplatin caused cell shrinkage, degradation and partial or total disappearance of the tumor cells. Vascularization was distinct in all specimens. A cellular infiltrate was found frequently but not consistently. A common end stage was a fibrotic scar with no cellular activity, occasionally giving a misleading impression of a growing tumor on gross observation. The results were obtained rapidly and suggest that the subrenal capsule assay would be useful for evaluating the sensitivity of human tumors to therapeutic manipulation, but needs supplementary histological examination.

The location and amount of the basement membrane (BM) components collagen IV and laminin were studied in ovarian epithelial, sex cord-stromal and germ cell tumors. BM structures were found in the epithelial stromal interface of benign surface epithelial tumors and, though discontinuous, around well-differentiated tumor islets, being less well developed in invasive undifferentiated neoplasms. The stromal components in Müllerian mixed tumors had less distinct BM structures, a finding useful for the classification of these neoplasms. Thecomas and fibromas had scanty collagen IV and laminin; granulosa cell tumors contained large amounts of BM material. A fine diffuse BM-positive pattern occurred in dysgerminomas and endodermal sinus tumors; BM structures in cystic teratomas were distinct. Collagen IV and laminin were well-developed in benign and slow-growing tumors with epithelial components and in their metastases, but less distinct in stromal tumors and highly malignant undifferentiated tumors, showing the usefulness of this method for the clinical and biological classification of such tumors.