Tumors of the pancreas with osteoclast-like and pleomorphic giant cells: an immunohistochemical and ploidy study.

BACKGROUND: Tumors of the pancreas with osteoclast-like giant cells are of uncertain histogenesis and aggressiveness. Their relationship, if any, to undifferentiated (anaplastic) carcinomas of the pancreas with pleomorphic giant cells is also not clear. METHODS: Eleven tumors with osteoclast-like giant cells were studied by immunohistochemistry for epithelial and mesenchymal markers, as well as for a proliferation marker (Ki67) and p53 protein expression. Cytometric image analysis for nuclear DNA content was also performed. K-ras mutations were investigated by DNA sequence analysis. RESULTS: Neoplastic, predominantly spindle-shaped cells and osteoclast-like giant cells were positive for mesenchymal markers CD68, LCA, and A1ACT. These spindle-shaped cells were also positive for human muscle actin. Spindle-shaped cells of seven tumors were also positive for epithelial markers carcinoembryonic antigen, epithelial membrane antigen, or keratin. Nine tumors contained a variable number of pleomorphic giant cells in addition to osteoclast-like giant cells. Pleomorphic giant cells were much less positive for mesenchymal markers than were osteoclast-like giant cells, but they were positive for some epithelial markers. A high percentage of spindle-shaped and pleomorphic giant cells were positive for Ki67. Diploid and aneuploid populations were present in varying proportions in both spindle cells and pleomorphic giant cells. The nuclei of osteoclast-like giant cells, however, were diploid and not proliferating. Spindle-shaped and pleomorphic giant cells were positive for p53 protein in 5 of 10 cases. Five of six tumors studied were positive for K-ras mutations. CONCLUSION: The distinction between tumors with osteoclast-like giant cells and undifferentiated carcinomas with pleomorphic giant cells is often not clear-cut. Both types of tumors have mesenchymal and epithelial characteristics in varying proportions and may arise from an undifferentiated pancreatic stem cell. Long-term survival of two patients suggests that some tumors with osteoclast-like giant cells may have a better prognosis than the usual pancreatic ductal adenocarcinoma.

Telomere-telomere interactions and candidate telomere binding protein(s) in mammalian sperm cells.

We have used fluorescent in situ hybridization to localize telomeres within the nuclei of sperm from six mammals (human, rat, mouse, stallion, boar, and bull). In minimally swollen sperm of mouse and rat, most of the telomeres are clustered within a limited area in the posterior part of nuclei. In sperm of other species, telomeres associate into tetrameres and dimers. On swelling of sperm cells with heparin/dithiotriethol, telomere associations disperse, and hybridization signals become smaller in size and their numbers approach or correspond to the number of chromosome ends in a haploid genome. Quantitation of telomere loci indicates that dimeric associations are prominent features of mammalian sperm nuclear architecture. Higher order telomere-telomere interactions and organization develop during meiotic stages of human spermatogenesis. At this stage, telomeres also become associated with the nuclear membrane. In an attempt to elucidate the molecular mechanisms underlying telomere interactions in sperm, we have identified a novel protein activity that binds to the double-stranded telomeric repeat (TTAGGG)n. Sperm telomere binding protein(s) (STBP) was extracted from human and bull sperm by 0.5 M NaCl. STBP does not bind single-stranded telomeric DNA and is highly specific for single base substitutions in a duplex DNA sequence. Depending on the conditions of binding, we observed the formation of several nucleoprotein complexes. We have shown that there is a transition between complexes, which indicates that the slower migrating complex is a multimer of the higher mobility one. We propose that STBP participates in association between the telomere domains which were microscopically observed in mammalian spermatozoa.

A brief staurosporine treatment of mitotic cells triggers premature exit from mitosis and polyploid cell formation.

At any point during the progression of many tumor types, cells can develop a hyperploid DNA content. Hyperploid tumors are significant more aggressive, with a higher growth rate and a poor patient prognosis. Yeast genetics have implicated three important genes involved in DNA ploidy changes: cdc2, cyclin b, and a specific inhibitor of the p34(cdc2)/cyclin B kinase, rum1. Mutations in these genes uncoupled the dependence mitosis on DNA replication in the fission yeast, Saccharomyces pombe. It was proposed that the inactivation of the mitotic kinase complex, p34(cdc2)/cyclin B, induces a G(1), state wherein the cells re-replicate their DNA without an intervening mitosis. We show in this report that treatment of only M phase-arrested mouse cells, with the protein kinase inhibitor staurosporine, induced polyploidy. Nocodazole-arrested metaphase FT210 cells were pulsed with 100 ng/ml of staurosporine for 1 h. This 1-h treatment results in the inhibition of the mitotic p34(cdc2) kinase. The inhibition of the mitotic kinases leads to a reduction in the histone H1 and H3 mitotic-associated phosphorylations, chromosome decondensation and nuclear membrane reformation. When released into normal growth medium, these cells are reset to a G(1)state, re-replicate their DNA without completing mitosis, and become octaploid.

The influence of volumetric tumor doubling time, DNA ploidy, and histologic grade on the survival of patients with intracranial astrocytomas.

PURPOSE: To improve the prediction of individual survival in patients with intracranial astrocytomas through the analysis of volumetric tumor doubling time (VDt) and DNA ploidy. METHODS: A pilot study was retrospectively conducted on a group of 25 patients with intracranial astrocytomas in whom recurrent and/or progressive disease was observed on serial contrast-enhanced CT or MR examinations. VDt was computed using two or more data points from a semilogarithmic plot of tumor volume versus time. Size-adjusted survival was calculated using a method based on VDt and initial tumor volume to decrease the lead time bias attributable to differing tumor sizes at presentation. RESULTS: Slower VDt was associated with significantly longer survival and size-adjusted survival as determined by a univariate Cox proportional hazard analysis. Aneuploidy was a significant indicator of poor survival. Aneuploid and multiclonal astrocytomas had poor size-adjusted survivals compared with diploid astrocytomas. Grade IV astrocytomas had significantly poorer survival and size-adjusted survival compared with lower grades (I to III), which individually were not significantly correlated. However, grade IV histology was not a significant independent predictor of size-adjusted survival in a multivariate Cox model, whereas VDt and DNA ploidy remained significant. VDt also had a significant direct linear correlation to survival and size-adjusted survival. CONCLUSIONS: VDt and DNA ploidy were more sensitive than histologic grading as indicators of individual survival. Initial tumor size needs to be considered when staging and assessing survival in patients with intracranial astrocytomas.

Alu repeated DNAs are differentially methylated in primate germ cells.

A significant fraction of Alu repeats in human sperm DNA, previously found to be unmethylated, is nearly completely methylated in DNA from many somatic tissues. A similar fraction of unmethylated Alus is observed here in sperm DNA from rhesus monkey. However, Alus are almost completely methylated at the restriction sites tested in monkey follicular oocyte DNA. The Alu methylation patterns in mature male and female monkey germ cells are consistent with Alu methylation in human germ cell tumors. Alu sequences are hypomethylated in seminoma DNAs and more methylated in a human ovarian dysgerminoma. These results contrast with methylation patterns reported for germ cell single-copy, CpG island, satellite, and L1 sequences. The function of Alu repeats is not known, but differential methylation of Alu repeats in the male and female germ lines suggests that they may serve as markers for genomic imprinting or in maintaining differences in male and female meiosis.

In favour of an oncofoetal concept of bronchogenic carcinoma development.

Our recent studies in a heterotopic model of non-small cell lung cancer in dogs (subcutaneous bronchial autografts treated with 3-methylcholanthrene) have provided evidence that alveolar type II cells may newly arise during initial phases of bronchial carcino-genesis. In the light of these novel findings, which are in agreement with our observations in human non-small cell lung cancer, and in view of present insights into embryonic lung differentiation, we discuss evidence that favours a new, oncofoetal concept of bronchogenic carcinoma development. According to this concept, the primary cells of origin for these tumors are undifferentiated primordial-like cells that derive from bronchial epithelial cells present in major bronchi or their divisions by retrodifferentiation. Such primordial-like cells of origin undergo novel differentiation into the potential (alveolar, bronchial or primordial) tumor stem cells, which occupy the dividing cellular layers of the (pre)neoplastic lesions and constitute the actively dividing and invading part of the neoplasm. Examples of tumors that may originate from alveolar tumor stem cells are carcinomas of the bronchiolo-alveolar, papillary, acinar, and adenoid-cystic types. Squamous cell carcinomas could possibly belong to this group as well, but much more evidence is required to reach conclusions regarding this type of cancer. We suggest that epithelial retrodifferentiation followed by novel differentiation (oncofoetal mechanism) is fundamental in bronchial carcinogenesis.

DNA content of head and neck squamous carcinoma by flow and image cytometry.

OBJECTIVE: To compare the measurement of quantitative DNA in squamous cell carcinoma of the head and neck by flow cytometry and image cytometry. DESIGN: Comparison of image cytometry to the more commonly used flow cytometry using paraffin-embedded tissues. SETTING: University of California, Davis Medical Center, Sacramento. A 472-bed university teaching hospital. PATIENTS: Records of 26 patients with squamous cell carcinoma of the tongue, base of tongue, and larynx were obtained from the case files of an otolaryngologist-head and neck surgeon. They were reviewed for staging and follow-up. RESULTS: We demonstrated a 96% concordance rate between the methods. A solitary discrepant case was aneuploid by image cytometry and diploid by flow cytometry. The specimen involved tumor infiltrated by lymphocytes that may have masked the aneuploid population to measurement by flow cytometry. Quantitative DNA analysis correlated moderately well with tumor grade, tumor stage, and patient outcome with a minimum of 6 years of follow-up. All patients with diploid tumors were long-term survivors. CONCLUSIONS: Both methods provide accurate quantitative DNA analyses in squamous cell carcinoma of the head and neck. The methods are highly correlative and yield similar predictive data regarding tumor behavior and prognosis.

A step section method for full histopathological assessment of carcinogen-affected target tissue during respiratory carcinogenesis.

Studies of carcinogenesis that are not limited to overt neoplasms but also involve evaluations of preneoplastic stages require histopathological assessment of the entire carcinogen-affected tissue so that the true nature and sequence of the progressive process can be determined. The customary serial sectioning approach achieves this goal, but at an inordinate logistic cost. In studies of hamster bronchial carcinogenesis, a step section method was compared to a quasi-random approach and to the customary serial section method. The step section method achieved the same diagnostic completeness as serial sectioning, but at a two orders of magnitude reduction in costs.

Regression of bronchial epithelial cancer in hamsters.

For bronchogenic carcinoma, if and when the sequential process of carcinogenesis is reversible is fundamental to chemoprevention research. In our hamster model, focally originating non-small cell lung carcinoma (NSCLC) develops via a reproducible sequential process of carcinogenesis by 180 days after endobronchial sustained-release implants (SRIs) of 10% benzo(a)pyrene. In this study, 114 hamsters received removable 10% benzo(a)pyrene SRIs. Short-term controls were sacrificed in 3 groups at 50, 65, and 80 days after SRI placement. Three experimental groups had SRIs removed at 50, 65, and 80 days after placement, and sacrifice was delayed until 100 to 180 days later. Long-term controls retained SRIs until sacrifice at 180 or 240 days after SRI placement. All long-term controls had NSCLC. Preneoplastic change was more common in 50- and 65-day controls, as compared with hamsters with equal duration of SRI exposure whose sacrifice was delayed until 100 to 180 days after SRI removal (p < 0.05). The 56% incidence of early NSCLC in hamsters sacrificed after 80 days of SRI exposure decreased to 5% in hamsters that had delayed sacrifice after SRI removal after 80 days of exposure. At the 10% benzo(a)pyrene dose used, hamster bronchial epithelium requires more than 80 days of continuous exposure to become irreversibly committed to NSCLC uniformly. Microinvasive NSCLC in hamsters often regresses, and it is not necessarily a precursor of overt invasive cancer. The removable SRI model provides new opportunities to evaluate chemoprevention of NSCLC and the related molecular-genetic control mechanisms.

A light microscope study of linker histone distribution in rat metaphase chromosomes and interphase nuclei.

Several subtypes of the linker histone H1 are present in normal rat kidney epithelial cells (NRK-52E). Although H1 is essential in nucleosome and chromatin packaging or condensation, the unique functions of these very basic proteins are largely unknown. There has been much speculation on the role of each H1 variant on developmentally regulated or tissue specific gene expression. We have examined the global distribution of several H1 subtypes on metaphase chromosomes in an attempt to uncover large-scale differences in chromatin condensation. Polyclonal antibodies raised against HPLC-purified rat H1 subtypes revealed a pattern much like G or Q bands for all H1 variants tested on chromosomes harvested with either aqueous or organic spreading methods. H1(0), a less abundant form of H1, may be associated with terminally differentiated or senescent cells. In cultures treated to induce higher levels of H1(0) there were no visible differences at the light microscope level in the antibody banding pattern between induced and noninduced cells. The distributions of H1 subtypes on chromosomes may be visible in different tissues when viewed at higher magnifications. While chromosome patterns were consistent with the antibodies tested, the interphase nuclei displayed clear differences. An epitope specific for anti-H1A antibody is present in the nuclear envelope and is possibly used for chromosomal location or anchorage. Anti-H1B antibody did not specifically label the nuclear envelope, nor did anti-H1(0) antibody. Highly concentrated regions of H1(0) surround the nucleoli, possibly indicating a cluster of genes that are poised for transcription.

Expression of alveolar type II cell markers in acinar adenocarcinomas and adenoid cystic carcinomas arising from segmental bronchi. A study in a heterotopic bronchogenic carcinoma model in dogs.

The type II alveolar epithelial cell is one of two pluripotential stem cell phenotypes in normal mammalian lung morphogenesis; cells manifesting this phenotype have been found to constitute bronchioloalveolar regions of canine adenocarcinomas. We now studied type II cell expression in canine acinar adenocarcinomas and adenoid cystic (bronchial gland) carcinomas, using the same bronchogenic carcinoma model (subcutaneous bronchial autografts treated with 3-methylcholanthrene). Distinctive features of type II cells are the approximately cuboid cell shape, large and roundish nucleus, immunofluorescent staining of the cytoplasm for the surfactant protein SP-A, and presence of multilamellar bodies or their precursory forms. Cells with these type II cell characteristics were found in the basal epithelial layer of all tumor lesions and in upper layers as far as the lumen, singly or in clusters; they were also found in early invasive carcinomatous lesions but not in bronchial glands or bronchial epithelium before carcinogen exposure. Immunoblots of tumor homogenates showed reactive proteins within size classes of SP-A (28 to 36 kd) or its dimeric form (56 to 72 kd). These findings and those previously reported are consistent with the concept that chemical carcinogenesis in the adult bronchial epithelium may lead to type II cell carcinomas of varying glandular (acinar, adenoidcystic or bronchioloalveolar) growth patterns.

Heteroduplex formation: a potential source of genotyping error from PCR products.

The formation of heteroduplexes from polymerase chain reaction (PCR) products has recently become a diagnostic tool that is routinely used for the prenatal detection of small deletions or insertions in a number of disease-causing alleles. We present evidence illustrating that heterozygous PCR products can manifest 'invisible' heteroduplexes that can ultimately lead to genotyping errors. Justifications for these 'invisible' heteroduplexes and requisite parameters to optimize their detection are presented.

Prenatal diagnosis by enzymatic amplification and restriction endonuclease digestion for detection of haemoglobins A, S and C.

The detection of the single base pair mutations at codon 6 of the beta-globin gene is important for the prenatal diagnosis of sickle-cell anaemia and SC disease. A novel procedure has been designed to create a restriction site at both the beta A and beta C alleles to facilitate the discrimination of haemoglobins A, S and C. The general principle of this procedure is to enzymatically amplify genomic DNA using a modified primer containing an altered 3'-terminal nucleotide to create these restriction sites. After this modified primer has been efficiently incorporated into amplified DNA, the PCR products are digested with the restriction enzymes Ava I and Sty I. Ava I recognizes a site in amplified DNA containing a beta A allele, and Sty I recognizes a site in DNA containing a beta C allele. Since the beta A and beta C alleles can be distinguished directly by the presence of a restriction site, the beta S allele can be identified indirectly. All three beta-globin alleles are easily distinguished by size and pattern of electrophoresed fragments on agarose gels. This procedure is specific and sensitive, thus permitting rapid, economical diagnosis of sickle-cell anaemia and SC disease.

The cytology of pediatric masses: a differential diagnostic approach.

In the United States, fine-needle aspiration biopsy (FNAB) and other cytodiagnostic methods have been underutilized in the evaluation of masses in the pediatric age group. Cytopathologists and cytotechnologists are therefore relatively unfamiliar with the cellular features of lesions that occur in children. On the basis of the cytologic findings from 64 pediatric cases, including 56 FNABs and 8 intra-operative imprints, a differential diagnostic approach to lesions in this age group is presented. The majority of cases can be placed into 1 of 5 cytomorphologic categories: (1) round-cell pattern, (2) mixed inflammatory pattern, (3) spindle-cell pattern, (4) epithelial pattern, and (5) cystic pattern. Once a cytomorphologic category is determined, evaluation for unique cellular features, special studies, and clinical correlation allows a specific diagnosis to be made in most cases. Pitfalls in pediatric cytopathology are illustrated by discussion of the following cases: a renal Burkitt's lymphoma mimicking a Wilms' tumor, a traumatic neuroma masquerading as a recurrent malignant schwannoma, Langerhans-cell histiocytosis resembling granulomatous inflammation, and a cystic granuloma that mimicked a branchial cleft cyst. Consideration of these problems and use of the recommended diagnostic approach will aid in interpretation in this difficult area.

Metastases from fresh human non-small cell lung cancers propagated in nude mice.

Specimens from 69 freshly resected human non-small cell lung cancers (NSCLC) were transplanted into nude mice. Twelve mice died before the transplants were evaluable. There were 4 takes of 12 evaluable transplants into untreated athymic nude mice and 24 takes of 45 evaluable transplants into nude mice with decreased natural killer (NK) cell activity. Fourteen tumor lines were propagated into 2 or more successive transplant generations. Distant metastases occurred from the mid-dorsal transplant site in 8 of 81 (9.88%) recipients of 4 of those tumor lines, after 3-9 consecutive tumor growth cycles. Xenotransplantation of freshly resected human NSCLC provides a model with potential for serial assessment of cellular changes related to metastatic capability.

Metastatic behaviour of canine lung carcinoma in autochthonous and xenotransplant hosts.

Specimens from 24 chemically induced canine non-small cell lung cancers (NSCLC) were xenotransplanted into nude mice. Twenty-one tumour lines were established in serial transplantation; four were from NSCLC that arose from orthotopically induced NSCLC in four dogs, and 17 were from NSCLC that arose heterotopically in 15 subcutaneous bronchial autografts (SBA) in seven dogs. Distant metastases developed in recipients of two orthotopic NSCLC after three and eight consecutive tumour growth cycles; no metastases have occurred after three and six growth cycles of two other orthotopic tumour lines. Recipients of eight heterotopic tumour lines developed metastases after 3-9 consecutive tumour growth cycles, while no metastases have occurred after 4-11 growth cycles in recipients of nine other heterotopic tumour lines. In three instances, both metastasizing and non-metastasizing tumour lines resulted from NSCLC that arose in different SBAs in the same dog. These findings indicate that, in the canine SBA bronchogenic cancer model as expanded by tumour xenotransplantation, those molecular events involved in both the initiation and the full progression of a single cancer may be investigated serially and concomitantly.

Lung cancer model for study of the metastatic process.

In our hamster model of focal, chemically induced nonsmall cell lung cancer (NSCLC), we studied metastases in autochthonous hamster hosts (n = 300) and in syngeneic hamster and nude mice recipients (n = 230) of serial tumor transplants. Metastases in autochthonous hosts and transplant recipients occurred in regional lymph nodes, liver, and adrenals. In autochthonous host hamsters no metastases were noted from microinvasive (n = 112) or visible cancer less than 3.0 mm in diameter (n = 66); the incidence of metastasis was 8.2% (4/49) from 3- to 10-mm cancers and 22% (16/73) from cancers 10 mm in diameter or larger (p less than 0.05). Serial transplants were used to evaluate the metastatic propensity of 20 primary and six metastatic NSCLCs. Six primary NSCLCs that metastasized in the autochthonous host and six metastatic NSCLCs all metastasized promptly in recipients. This expression of metastatic potential was significantly different (p less than 0.05) from 14 primary cancers without autochthonous host metastases. Eight of the 14 caused no metastases in recipients, even after 5 to 11 tumor growth cycles; metastases occurred from the other six primary NSCLC after 3 to 12 tumor growth cycles in transplant recipients. Primary hamster NSCLCs metastasize in the autochthonous host with a frequency and a distribution pattern similar to human NSCLCs. A new model to study serially the cellular changes that govern the process of metastasis in NSCLC has been developed.

Fine needle aspiration biopsy of superficial masses in children.

Fine needle aspiration biopsy (FNAB) is an underused diagnostic procedure in children, particularly in the evaluation of superficial masses. A total of 54 FNABs of superficial masses were performed in children aged 1 month to 15 years. Adequate material for diagnosis was obtained in 50 attempts. The cytologic diagnosis increased clinical understanding and provided a guide for treatment in 46 of the 50 cases. The cytologic diagnosis was confirmed in 15 of 19 patients who underwent an operation. Surgical intervention was obviated in 31 patients. There was one false-positive diagnosis of cancer. We describe the role of FNAB in children and its technique, accuracy, and diagnostic problems.