Implications of recent epidemiologic studies for the linear nonthreshold model and radiation protection.

The recently published NCRP Commentary No. 27 evaluated the new information from epidemiologic studies as to their degree of support for applying the linear nonthreshold (LNT) model of carcinogenic effects for radiation protection purposes (NCRP 2018 Implications of Recent Epidemiologic Studies for the Linear Nonthreshold Model and Radiation Protection, Commentary No. 27 (Bethesda, MD: National Council on Radiation Protection and Measurements)). The aim was to determine whether recent epidemiologic studies of low-LET radiation, particularly those at low doses and/or low dose rates (LD/LDR), broadly support the LNT model of carcinogenic risk or, on the contrary, demonstrate sufficient evidence that the LNT model is inappropriate for the purposes of radiation protection. An updated review was needed because a considerable number of reports of radiation epidemiologic studies based on new or updated data have been published since other major reviews were conducted by national and international scientific committees. The Commentary provides a critical review of the LD/LDR studies that are most directly applicable to current occupational, environmental and medical radiation exposure circumstances. This Memorandum summarises several of the more important LD/LDR studies that incorporate radiation dose responses for solid cancer and leukemia that were reviewed in Commentary No. 27. In addition, an overview is provided of radiation studies of breast and thyroid cancers, and cancer after childhood exposures. Non-cancers are briefly touched upon such as ischemic heart disease, cataracts, and heritable genetic effects. To assess the applicability and utility of the LNT model for radiation protection, the Commentary evaluated 29 epidemiologic studies or groups of studies, primarily of total solid cancer, in terms of strengths and weaknesses in their epidemiologic methods, dosimetry approaches, and statistical modelling, and the degree to which they supported a LNT model for continued use in radiation protection. Recommendations for how to make epidemiologic radiation studies more informative are outlined. The NCRP Committee recognises that the risks from LD/LDR exposures are small and uncertain. The Committee judged that the available epidemiologic data were broadly supportive of the LNT model and that at this time no alternative dose-response relationship appears more pragmatic or prudent for radiation protection purposes.

Ultrasound-detected thyroid nodule prevalence and radiation dose from fallout.

Settlements near the Semipalatinsk Test Site (SNTS) in northeastern Kazakhstan were exposed to radioactive fallout during 1949-1962. Thyroid disease prevalence among 2994 residents of eight villages was ascertained by ultrasound screening. Malignancy was determined by cytopathology. Individual thyroid doses from external and internal radiation sources were reconstructed from fallout deposition patterns, residential histories and diet, including childhood milk consumption. Point estimates of individual external and internal dose averaged 0.04 Gy (range 0-0.65) and 0.31 Gy (0-9.6), respectively, with a Pearson correlation coefficient of 0.46. Ultrasound-detected thyroid nodule prevalence was 18% and 39% among males and females, respectively. It was significantly and independently associated with both external and internal dose, the main study finding. The estimated relative biological effectiveness of internal compared to external radiation dose was 0.33, with 95% confidence bounds of 0.09-3.11. Prevalence of papillary cancer was 0.9% and was not significantly associated with radiation dose. In terms of excess relative risk per unit dose, our dose-response findings for nodule prevalence are comparable to those from populations exposed to medical X rays and to acute radiation from the Hiroshima and Nagasaki atomic bombings.

Estimates of fallout in the continental U.S. from Nevada weapons testing based on gummed-film monitoring data.

During periods of weapons testing at the Nevada Test Site (NTS) between 1951 and 1958, the Environmental Measurements Laboratory (EML) monitored daily fallout at about 100 sites in the U.S. using gummed-film collectors. These gummed-film data represent the only comprehensive set of actual measurements of fallout during this period for areas outside the immediate vicinity of the NTS. The measured beta activities originally reported by EML have been reviewed and reevaluated. This reevaluation corrected a number of errors in the original data set and allowed fairly accurate estimates to be made of specific radionuclide depositions from individual NTS shots. Estimates of the geographical and temporal variations in cumulative 137Cs and 131I depositions from all NTS shots through 1957 are presented, as well as estimates of the relative impact of particular shots and test series. The revised gummed-film estimates of total NTS fallout depositions are compared with estimates based on contemporary and historical soil sample analyses. These reevaluated gummed-film fallout deposition estimates are being extensively utilized in a number of ongoing programs to reconstruct the radiation exposure of the U.S. population from Nevada weapons testing.

Models of radioiodine transport to populations within the continental U.S.

A methodology is being developed to estimate the exposure of Americans to 131I originating from atmospheric nuclear weapons tests carried out at the Nevada Test Site (NTS) during the 1950s and early 1960s. Since very few direct environmental measurements of 131I were made at that time, the assessment must rely on estimates of 131I deposition based on meterological modeling and on measurements of total beta activity from the radioactive fallout deposited on gummed-film collectors that were located across the country. The most important source of human exposure from fallout 131I was due to the ingestion of cows' milk. The overall methodology used to assess the 131I concentration in milk and the 131I intake by people on a county basis for the most significant atmospheric tests is presented and discussed. Certain aspects of the methodology are discussed in a more detailed manner in companion papers also presented in this issue. This work is carried out within the framework of a task group established by the National Cancer Institute.

National registry of cervical cytologic diagnoses in The Netherlands.

The national cervical cytology registry being developed in the Netherlands is described. A large-scale screening program for cervical cancer has been in effect since 1975 in the region of the cities of Nijmegen, Utrecht and Rotterdam. At the start of the pilot projects, laboratories agreed upon a uniform protocol for reporting cytologic findings and recommendations for follow-up examinations in cases of abnormalities. Based on the results of the three pilot projects, in 1985 the Dutch government decided to organize a nationwide screening program for cervical cancer. All pathology laboratories involved in this national screening program are using the same screening protocol and the same coding system for cytologic and histologic diagnoses. By the end of 1989, all pathology laboratories will be linked to a central pathology diagnosis data base (PALGA). Linkage of screening results to previous and follow-up cytologic and histologic findings will enable epidemiologic studies on a regional or national level. Each physician who has submitted specimens will, next to the cytology reports, periodically receive reviews of the number of smears submitted, the cellular composition (quality) of those smears and the follow-up findings. The execution of requests for follow-up examination will be supervised by the participating pathology laboratories. The national cervical cytology registry will enable registration of all relevant cytologic and histologic diagnoses in a uniform way, but will also establish a unique high-quality national data bank, which will be of great value in the analysis of the effectiveness of the national screening program for cervical cancer. It will enable measurement of the impact of various screening protocols and give insight into the behavior of cervical cancer and the progressive or regressive character of its early stages. It will also offer the opportunity to initiate and evaluate quality control protocols.

DNA cytometry of pure dysgerminomas of the ovary.

The value of DNA cytometry for predicting the malignant potential of pure dysgerminomas of the ovary was investigated. Feulgen-thionin DNA image cytometry and propidium iodide DNA flow cytometry were performed in isolated nuclei from paraffin-embedded tissue of 25 dysgerminomas. Nineteen were in clinical stage Ia1, and six were in stage III (two in IIIa and four in IIIb). Eight patients showed recurrences during a 5-year follow-up period and one patient died of the disease. The DNA index (DI; the modal DNA value compared with that of normal cells) of the tumor cells was computed from the image and flow DNA histograms. Comparable DI values, ranging from 1.29 and 3.23, were found with both types of cytometry. No correlation was found with the clinical stage or the recurrence state. Furthermore, it was striking that, although values were found strongly deviating from the normal diploid content (DI = 1.0) suggesting an unfavorable prognosis, the survival rate was relatively high. It can be concluded that prediction of the clinical course of pure dysgerminomas by DNA cytometry does not seem feasible.

Comparison of tissue disaggregation techniques of transitional cell bladder carcinomas for flow cytometry and chromosomal analysis.

DNA index (DI) measurements and chromosomal analysis of 42 transitional cell carcinomas were done after mechanical and enzymatical disaggregation of the tumor specimens. The results obtained with these different disaggregation techniques were compared in the 33 cases (79%) that showed recognizable chromosomes. The enzymatically obtained cell suspensions could not be used for chromosomal analysis after short-term culture of 24 hours. In four cases, the DI after enzymatical treatment could not be estimated. In most cases, the DI obtained from the tumor cells was similar for both aggregation techniques, with the exception of four cases of enzymatically treated cell suspensions in which the DI could not be estimated. The average DI of the aneuploid tumors was 13% higher than the corresponding chromosome count. In 19% of the aneuploid tumors the proportion of aneuploid cells could not be measured after enzymatical treatment. In the remaining suspensions the proportion of diploid cells was higher after enzymatical disaggregation than after mechanical treatment. It is concluded that for flow cytometric and direct chromosomal analysis of bladder tumors, the mechanical disaggregation technique is most suitable.

Application of antibodies to intermediate filament proteins as tissue-specific probes in the flow cytometric analysis of complex tumors.

The flow cytometric (FCM) analysis of carcinomas is often hampered by the presence of stromal and inflammatory cells in the cell suspensions obtained from such neoplasms. Therefore, an FCM method was developed to distinguish epithelial from nonepithelial cells by using polyclonal and monoclonal antibodies to (cyto)keratins, the epithelial type of intermediate filament proteins. Using a model system of cultured bladder carcinoma (T24) and leukemia (MOLT-4) cells, we tested our hypothesis and procedures by labeling cell mixtures with these antibodies. After incubation with an appropriate intermediate filament antibody and propidium iodide staining, the DNA content and distribution of T24 cells could be analyzed separately from MOLT-4 cells. When applied to cell suspensions of endometrial carcinomas, bladder carcinomas and Grawitz tumors, only the epithelial (primarily carcinoma) cells were stained for cytokeratin; these cells could thus be analyzed separately from stromal, inflammatory and other nonepithelial cells. In this way, a more accurate FCM analysis of the malignant fraction within a tumor can be achieved.

Extraction of nuclei from selected regions in paraffin-embedded tissue.

A method for the extraction of nuclei from selected regions in paraffin-embedded tissue is described. Fifty-micrometer sections are cut, dewaxed, and rehydrated. For the final handling, the sections are manually transferred from one tray to another. The sections are put on a slide under a dissection microscope and the region of interest is isolated by scraping off the irrelevant region with a scalpel. An optimal number of single nuclei is obtained by incubation in a protease solution with intermediate syringing. The nuclei are washed and can be used for flow cytometry. Resuspension of the nuclei in foetal calf serum and cytocentrifugation results in preparations suited for image analysis. DNA cytometric measurements of nuclei in a carcinoma in situ and an invasive carcinoma region in breast tissue present in the same tissue block and in a severe dysplasia/carcinoma in situ (CIN III) region of the cervix are presented.

DNA and nuclear protein measurement in columnar epithelial cells of human endometrium.

Propidium iodide DNA flow cytometry, Feulgen-DNA, and nuclear light green protein scanning cytometry were performed in columnar epithelial cells of normal, nonmalignant human endometrium and endometrial adenocarcinomas. Columnar cells were identified by immunohistochemical staining for cytokeratin 18, an intermediate filament protein specifically present in columnar cell epithelium. DNA measurements derived from flow and scanning cytometry showed comparable results. The DNA content of the G0/G1 fraction of the adenocarcinomas had a considerable overlap with that of normal endometrium, with that of the carcinomas shifted toward higher values. For the carcinomas, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas. Most of the clinical stage I tumors showed a DNA content in the normal diploid region. Three of the four carcinomas of clinical stage II and higher had an increased DNA content. For the carcinomas, the percentage of cells in the proliferative fraction, as determined from scanning cytometric derived DNA histograms, was comparable to that of normal endometrium, or higher. No correlation was found with the histological grade. Tumors of clinical stage II and higher had intermediate values compared to carcinomas of lower stages. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. Within the tumor population, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas, and clinical stage. Based on the aforementioned parameters, no discrimination could be obtained between normal and malignant endometrium. However, when the DNA content of the G0/G1 fraction was combined with the coefficient of variation of the nuclear protein/DNA ratio, a clear discrimination could be obtained with only two false-positive cases.

Tissue-specific markers in flow cytometry of urological cancers. II. Cytokeratin and vimentin in renal-cell tumors.

Nine primary human renal-cell tumors (RCT), one lymph-node metastasis, 4 human xenografts of a RCT in nude mice and a rat RCT line were analyzed by flow cytometry (FCM) using propidium iodide for DNA analysis and antibodies to cytokeratin and vimentin in the indirect immunofluorescence technique for labelling of specific tumor-cell populations. By means of 2-dimensional FCM analysis, vimentin- and cytokeratin-positive (tumor) cells were compared and their DNA content and proliferative fraction analyzed separately from those of cytokeratin-negative stromal and inflammatory cells. In primary human RCT, 2 subpopulations of cells were detected and analyzed separately. Small numbers of tumor cells with an abnormal DNA stemline were also detected. In addition, co-expression of intermediate filament proteins of both the cytokeratin and the vimentin types was detected in the aneuploid cell population. Comparison of 2 model systems of RCT with primary human RCT revealed a similar pattern of tumor-cell subfractions within these tumors. The 2-parameter FCM analysis permits the detection of subpopulations in complex cell suspensions and the quantification of these fractions, as well as analysis of their cellular DNA content.

Tissue-specific markers in flow cytometry of urological cancers: cytokeratins in bladder carcinoma.

Thirty-eight transitional-cell carcinomas (TCC) were analyzed by flow cytometry (FCM) using propidium iodide for DNA analysis and antibodies to cytokeratin by indirect immunofluorescence. By means of two-dimensional FCM analysis, cytokeratin-positive tumor cells could be analysed separately from cytokeratin-negative stromal and inflammatory cells. This resulted in an 18% increase in sensitivity of FCM detection of aneuploidy (10/38 samples with one-parameter DNA analysis versus 15/38 samples with two-parameter DNA and cytokeratin analysis). In addition, S-phase could be determined in the 15 aneuploid samples by means of two-parameter analysis where this was not possible using only DNA content because of the overlap of diploid and aneuploid populations. FCM analysis allowed quantification of the percentage of tumor cells expressing cytokeratin 18 which has previously been shown to correlate quantitatively with higher grade, higher stage TCC. The quantitative measurement of tumor-cell expression of cytokeratin 18 by FCM analysis appears to provide additional information of potential prognostic value, independent of tumor-cell ploidy and proliferative fractions.

Flow cytometric analysis and sorting of human endometrial cells after immunocytochemical labeling for cytokeratin using a monoclonal antibody.

Endometrial cells in suspension were stained with propidium iodide and a monoclonal antibody against a cytokeratin intermediate filament protein specific for glandular and columnar cells (RGE 53). In this way columnar epithelial cells of the normal endometrium and of adenocarcinomas can be distinguished and separated by flow cytometry from non-epithelial cells (fibroblasts and inflammatory cells) and squamous epithelial cells, all of which are negative for RGE 53. This makes it possible to analyse and also sort pure fractions of this particular tissue type for further studies. The use of propidium iodide allows simultaneous DNA flow cytometry of these columnar epithelial cells. Therefore, the use of antibodies to cytokeratin in combination with propidium iodide can be of help in analyzing and sorting pure fractions of both normal and malignant cells. This allows a more refined examination of complex cell mixtures using flow cytometry.

Radiation exposures in Utah from Nevada Nuclear Tests.

The exposure of the population of Utah to external gamma-radiation from the fallout from nuclear weapons tests carried out between 1951 and 1958 at the Nevada Test Site has been reconstructed from recent measurements of residual cesium-137 and plutonium in soil. Although the highest exposures were found in the extreme southwest part of Utah, as expected, the residents of the populous northern valleys around Provo, Salt Lake City, and Ogden received a higher mean dose and a significantly greater population dose (person-rads) than did the residents of most counties closer to the test site. However, population doses from external exposure throughout Utah were far too low to result in any statistically observable health effects.