Raymond L. Teplitz

Correlation of Ki-67, p53, and Adnab-9 immunohistochemical staining and ploidy with clinical and histopathologic features of severely dysplastic colorectal adenomas.

Variations of Ki-67, p53, and Adnab-9 monoclonal antibody reactions in colonic adenomas may be associated with colonic cancer risk. We studied the predictive value of these markers for adverse behavior in severely dysplastic colorectal adenomas, such as an associated carcinoma, multiplicity of adenomas, and subsequent development of adenomas. For this purpose we compared the clinical, gross, and histologic characteristics of highly dysplastic index polyps in 42 patients with Ki 67, p53, and Adnab-9 immunostaining and other molecular markers. Polyps were removed endoscopically, and severely dysplastic polyps were stained immunohistochemically with Ki-67, Adnab-9, and p53 protein by the avidin biotin conjugate (ABC) technique. Quantitative DNA (QDNA) was analyzed by computer-assisted image analysis. Ki-67 immunohistochemistry showed reversal of normal distribution of nuclear staining from the normal basal position to the upper third of the colonic crypts. This abnormality of immunostaining in dysplastic adenomas was the earliest detected by the panel we used. A statistically significant correlation was seen between invasiveness of carcinoma in the index polyp and polyp size (P = 0.003), sessile morphology (P = 0.037), and villous or tubulovillous histology (P = 0.019). In the index adenoma, p53 positivity was correlated with multiplicity at initial examination (P = 0.053), villous histology (P = 0.053), invasiveness of carcinoma (P < 0.003), and recurrence of colorectal adenomas (P = 0.025). Although p53 positivity and aneuploidy were correlated with invasiveness of carcinoma in the index polyp (P = 0.025), Adnab-9 positivity was not. However, Adnab-9 positivity in the index polyp was associated with multiplicity of adenomas (P = 0.04) as well as recurrence of adenomas (P < 0.024). In conclusion, in addition to the morphologic and histologic markers already known, Ki-67, Adnab-9 antibody, and p53 protein may be prognostic indicators useful in follow-up of patients with severely dysplastic colorectal adenomas. Adnab-9 antibody may identify a field defect in above-average-risk adenoma-bearing patients.

Activity banding of human chromosomes as shown by histone acetylation

The expression of genes in mammalian cells depends on many factors including position in the cell cycle, stage of differentiation, age, and environmental influences. As different groups of genes are expressed, their packaging within chromatin changes and may be detected at the chromsomal level. The organization of DNA within a chromosome is determined to a large extent by the positively charged, highly conserved histones. Histone subtypes and the reversible chemical modifications of histones have been associated with gene activity. Active or potentially active genes have been associated with hyperacetylated histones and inactive genes with nonacetylated histones. Sodium butyrate increases the acetylation levels of histones in cell cultures and acts as both an inducer of gene activity and as a cell-cycle block. We describe a method to label the interphase distribution of DNA associated with various histone acetylation stages on chromosomes. Nucleosomes from untreated and butyrate-treated HeLa cells were fractionated by their acetylation level and the associated DNA labeled, and hybridized to normal human chromosomes. In the sodium butyrate-treated cells the resulting banding patterns of the high- and low-acetylated fractions were strikingly different. DNA from low-acetylated chromatin labeled several pericentric regions, whereas hybridization with DNA from highly acetylated chromatin resulted in a pattern similar to inverse G-bands on many chromsomes. The results from noninduced cells at both high and low acetylation levels were noticeably different from their induced counterparts. The capture and hybridization of DNA from interphase chromatin at different acetylation states provides a “snap-shot” of the distribution of gene activity on chromosomes at the time of cell harvest.

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.

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.

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.

Sequential Flow Cytometry and Single Gene Analysis by Enzymatic Amplification and Allele Specific Oligonucleotide Hybridization of Urothelial Cells

In this report, we show that fresh urothelial cells first subjected to propidium iodide staining and flow cytometric quantitative DNA analysis can then be used for DNA analysis using the PCR and hybridization with oligonucleotide probes for the detection of specific B-globin alleles

MEASUREMENT OF TOTAL DNA TO ASSESS LUNG CANCERS

an DNA ploidy alone be used to discriminate beC tween metastases and second primary neoplasms? Ichinose and associates [l] suggest that difference in DNA ploidy as measured by flow cytometry can be used as a criterion by which to distinguish between second primary lung adenosquamous carcinomas and metastases from a primary neoplasm. This contention is an example of a general problem, and therefore it deserves close By their own criteria, Ichinose and associates state that primary adenosquamous carcinoma of the lung occurs at a low frequency (2%). Because this is such an uncommon tumor, on statistical grounds alone, one must question the frequency with which a second primary adenosquamous cancer of the lung could be expected to occur. This consideration alone may make the results of their study questionable. The weakness of flow cytometry as a method for detecting low-frequency aneuploid clones was not addressed by Ichinose and associates. In alleged second primary tumors, in which the ploidy of the second clone appears to differ from the ploidy in the initial primary tumor, it is possible that the second clone was present in scrutiny.