Biomedical subjects
Publications and source records attributed to M M Lieber.
Genitourinary filariasis is a well-known entity in most tropical areas. However, filariasis cannot be eliminated from the differential diagnosis of testicular, epididymal, or spermatic cord masses in nontropical climes. We report a case of filariasis of the spermatic cord that manifested as an unexplained scrotal mass in a patient in Rochester, Minnesota.
Use of the Hamburger-Salmon soft agar assay method for in vitro chemotherapy sensitivity testing of samples of renal cell carcinoma has been somewhat limited by a relatively low proliferation/evaluability rate for this tumor type (approximately 50%). The tritiated thymidine ([ 3H]-TdR) incorporation assay method of Tanigawa et al. (Cancer Res., 42: 2159, 1982) was compared to a standard optical colony counting assay technique. Fifty-seven different primary and five metastatic fresh samples of human renal cell carcinoma were studied. Evaluability rate by the [3H]-TdR assay was 90% (greater than or equal to 300 cpm control). In comparison, evaluability rate by optical colony counting was 43% for this group of tumors. [3H]-TdR incorporation increased with increasing tumor grade and increasing stage. Spindle cell tumors showed significantly higher cpm than other cell types. Twenty-three primary tumors were evaluable by both [3H]-TdR and colony counting methods. The correlation coefficient ("r") for regression lines for drug sensitivity data points (optical counting vs. [3H]-TdR) of these individual experiments ranged from 0.50 to 0.99 with a mean r +/- S.D. of 0.76 +/- 0.15. For all 260 paired drug response observations of 23 tumors exposed to different drugs, the correlation was very good with r = 0.71. Since the [3H]-TdR assay has an evaluability rate of approximately 90% for renal cell carcinoma, gives drug sensitivity information which correlates well with the colony counting endpoint and yields chemotherapy sensitivity information four days after sample accession, the [3H]-TdR assay may be a more useful method for study of human renal cell carcinoma in vitro chemotherapy sensitivity testing than standard colony counting techniques.
Technical methods for assessing the growth and chemotherapy sensitivity of human tumor cells growing in soft-agar culture have been less than ideal. Within the past year, there have been reports of studying the extent of growth of human tumor cells in these cultures by quantitating the change in cumulative volume for the growth units observed. The present report describes the results of computer-assisted volume analysis applied to soft-agar cultures of cells from 74 primary renal cell carcinomas, 14 primary transitional cell carcinomas of the renal pelvis, and four different human renal cell carcinoma xenografts. The extent of growth in vitro observed for cells from freshly excised human renal tumors showed the expected and statistically significant relationship to tumor grade and stage. The renal cell carcinoma xenografts proliferated to a much greater extent in vitro than the cells from freshly excised human renal carcinomas. The fundamental growth limit of 10(9) micron. cumulative growth unit volume per plate was confirmed by this series of experiments. Computer-assisted volume analysis appears to be a useful method to study the growth of freshly excised human renal carcinoma cells in vitro.
Flow cytometry was performed on 51 typical specimens of renal oncocytoma. Nuclei were extracted from paraffin-embedded archival material and isolated nuclei were stained with propidium iodide. Of the 51 available tissue blocks 86 per cent were evaluable and 50 per cent of these samples showed a deoxyribonucleic acid (DNA) histogram that was approximately the same as normal renal parenchyma. Of the oncocytoma samples 39 per cent showed a marked increase (more than 10 per cent of the nuclei) in the tetraploid DNA peak, while 11 per cent showed a distinct DNA aneuploid peak. Among 21 evaluable grade 2 oncocytic renal tumors 33 per cent showed a normal DNA histogram, 43 per cent showed a marked increase in the DNA tetraploid peak and 24 per cent showed a DNA aneuploid peak. The common presence of polyploid nuclei containing double quantities of chromosomal DNA may correlate with the long-standing pathological observation that oncocytic tumors often contain a distinct population of large nuclei. Indeed, 86 per cent concurrence was seen between the detection of an abnormal DNA content by flow cytometry and the histopathological presence of large abnormal nuclei in these specimens. Since renal oncocytomas (grade 1 oncocytic tumors) rarely, if ever, metastasize and are relatively noninvasive locally, their markedly abnormal flow cytometry patterns are of considerable interest. Moreover, DNA polyploidy has not been identified previously in renal tumors. The biological significance and mechanism of DNA polyploidy, and the relationship of DNA polyploidy and DNA aneuploidy to the pathogenesis of oncocytic renal tumors require further laboratory investigation. The clinical use of flow cytometry to classify and to predict the behavior of renal tumors will be complicated, since renal oncocytomas commonly have polyploid and aneuploid DNA histograms.
Bisantrene, a clinically active anticancer drug with limited solubility at physiologic pH, was delivered by selective injection into the internal iliac artery of male calves. The percutaneous transfemoral angiographic techniques used in the calves were identical to those used in adult human patients. Directed intravascular precipitation of bisantrene at the maximal tolerable clinical dose for intravenous administration (260 mg/m2) caused severe tissue damage in 5 of 10 animals that received these intra-arterial injections. (One calf in this study group died of unknown causes 10 days after the drug infusion). A reduced intra-arterial dose (50 mg/m2) was used in seven calves, and no local tissue damage was evident on gross or microscopic examination. Nevertheless, resultant concentrations of bisantrene deposited in the ipsilateral bladder wall were 10- to 100-fold those concentrations found after intravenous administration of a dose 5 times higher. These animal toxicology and pharmacology data support initiation of a phase I clinical trial of directed intravascular precipitation of bisantrene in humans. This clinical trial will be developed for patients with advanced refractory cancers of the anatomic true pelvis, such as those originating in the urinary bladder, prostate, rectum, and uterine cervix.
Nuclear DNA ploidy studies were performed by flow cytometry on extracted nuclei from 12 oncocytic tumours of the salivary gland, 65 oncocytic tumours of the kidney, and 37 oncocytic tumours of the thyroid gland from the pathology archives of the Mayo Clinic. In order to provide an interesting clinical spectrum, three different classes of well-differentiated oncocytic tumours were selected for examination. Salivary gland oncocytic tumours were chosen for their generally benign behaviour. Oncocytic thyroid cancers exhibiting malignant potential because of local invasion, were thought to represent the opposite extreme of aggressiveness. Renal oncocytic tumours were known to demonstrate an intermediate degree of malignancy. All of the oncocytic salivary gland tumours showed a 'normal' DNA histogram and had a benign clinical course. For the oncocytic tumours of the kidney, 45% of DNA histograms were normal, 40% exhibited a significant increase in the DNA tetraploid/polyploid (4C) peak, and 15% showed a DNA aneuploid peak. Three patients with a DNA tetraploid pattern developed tumour metastasis and two have died from metastatic renal cancer. Among the oncocytic thyroid cancers, 27% were normal, 22% exhibited an increased DNA tetraploid peak, and 51% had a distinct DNA aneuploid peak. None of the thyroid tumour patients with a normal DNA pattern or with an increased DNA tetraploid peak died as a result of thyroid malignancy. In contrast, 58% of patients whose thyroid tumours showed a DNA aneuploid peak subsequently died from thyroid cancer.
The proteins of solid lung tumours (15 adenocarcinomas and 10 squamous cell carcinomas) were examined by high resolution two-dimensional electrophoresis (2-DE) and compared with the proteins of adjacent lung tissue which demonstrated no histological evidence of malignant transformation, and with the proteins of other malignant tumours and normal tissues. To investigate tumour cell-specific protein synthesis, we isolated malignant and normal cells enzymatically with collagenase, elastase, and DNase. Tissue and tumour cells were enriched in an additional step on a Percoll gradient. The 2-DE gel patterns derived from entire tissue and enriched tissue cell preparations were compared. No specific differences were found between the 2-DE protein patterns from adenocarcinomas and squamous cell carcinomas of the lung, but three proteins identified on the 2-DE gels appeared to be tumour-associated. Spot A is present in non-neoplastic and neoplastic epithelial tissues. Spot B is pronounced in 2-DE gels of sarcomas, but is also present in preparations of other malignant tissues. Spot C is present in all malignant cell preparations. These three spots were also demonstrated in 2-DE protein patterns from tissue cultures of malignant cell lines. Spot B and spot C were also present in some normal tissues.
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Several problems have been pointed out in colony formation assay (CFA) developed by Hamburger and Salmon. We studied those using 87 fresh renal carcinoma, comprising of 72 renal cell carcinoma and 15 transitional cell carcinoma of renal pelvis. Results are as follows: Pure single cell suspension was difficult to be yielded from these carcinoma. Our result suggests that cell suspension contains various sized growth unit as well as greater than or equal to 60 microns growth unit. Presence of substantial number of greater than or equal to 60 microns growth unit in plating cell suspension was revealed to influence on the cell growth, ie, the increase of newly formed greater than 60 microns growth unit. This indicates that initial growth unit count is mandatory for "quality control" in CFA. The current study in extent of growth for renal carcinoma revealed three different patterns, one of which did not seem suitable for in vitro chemosensitivity test with CFA. This finding brings for us the fact to prepare "proliferation control" which can check periodically the growth extent of renal carcinoma.
Nuclear deoxyribonucleic acid (DNA) ploidy studies with paraffin-embedded archival tumor specimen blocks were performed by flow cytometry on extracted nuclei from 75 pheochromocytomas. Clinical details, specifically including histologic findings, biochemical studies, and ultimate fate, were investigated. Preparation of paraffin-embedded tissue specimens was carried out by the technique of Hedley et al. and stained with propidium iodide according to the method of Vindeløv et al. Twenty-three tumors showed a normal DNA histogram, 31 showed significant increase in the 4C (DNA tetraploid) peak, and 21 exhibited a DNA aneuploid peak. To define a subset of patients who had either died as a result of pheochromocytoma or had been followed for a minimum of 10 years, 13 patients were excluded. Of the remaining 62 patients, all of the 18 patients with a normal DNA histogram followed a benign clinical course, including normal fractionated urinary catecholamines. However, eight (31%) of the 26 patients classified DNA tetraploid/polyploid and seven (39%) of the 18 patients exhibiting a DNA aneuploid peak had evidence of malignancy; these two groups had significantly more malignant tumors (p less than 0.05 and p less than 0.02, respectively) than the normal DNA group. Flow cytometric DNA ploidy measurements of isolated nuclei seem to provide useful prognostic information for patients with pheochromocytoma.
Targeting of anticancer drugs to specific organs by directed intravascular precipitation was studied in calves using 9-10 anthracenedicarboxyaldehyde bis [(4,5-dihydro-lH-imidazol-2-yl) hydrazone] dihydrochloride (Bisantrene), a clinically active anticancer drug with limited solubility at physiological pH. Rapid injection of Bisantrene in solution at pH 4.5 into the internal iliac artery resulted in concentrations of drug in the urinary bladder wall supplied by the artery that were more than 1000 times those in the same tissue following injection of the same dose of drug IV, the route of administration used clinically. Localization of the orange fluorescent drug to the ipsilateral bladder wall was easily seen. Fluorescence microscopy revealed deposits of drug along the walls of the arteriolar and capillary bed supplied by the artery into which it had been injected. Concentrations of drug in the systemic circulation and in tissues not supplied by the internal iliac artery used for drug injection were lower after intraarterial (IA) drug administration than after IV administration. Pathological studies of the tissues of calves sacrificed at intervals up to four weeks following rapid injection into the internal iliac artery of the same doses of Bisantrene used IV in cancer patients did not reveal evidence of extensive cytotoxicity to the infused organs.
Sarcomas of the bladder occur rarely in adults. As a result, the natural history of these unusual tumors and the best methods of treatment are uncertain. We reviewed our experience with 13 patients presenting between 1970 and 1980 (5 had leiomyosarcoma, 5 had carcinosarcoma and 3 had rhabdomyosarcoma). Gross hematuria nearly always was the presenting symptom. Patients treated by partial cystectomy fared poorly, while 7 treated by radical cystectomy and supravesical urinary diversion had an excellent prognosis: 6 (86 per cent) have been free of disease for more than 1 year, including 4 who have survived more than 45 months without evidence of recurrence or metastasis.
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Growth in soft-agar bilayer cultures of human tumour cells derived from 4 in vitro continuous cell lines, from 21 xenografts carried in athymic mice, and from 197 samples of fresh human solid tumours of various histologic types was analyzed by computer-assisted image analysis. Replicate cultures for each specimen were assessed on successive days of incubation for the number and volume of growth units within multiple size categories. Our results confirm the recent finding of others that there is an upper limit of approximately 10(9) microns 3 to the cumulative growth unit volume obtainable in a 2 ml bilayer soft agar culture system. Since this upper limit to the carrying capacity of the closed culture system exists, the extent of growth within the cultures is determined in a fundamental way by the cumulative volume of growth units initially inoculated into cultures. A growth index of greater than or equal to 16-fold was only seen when initial cumulative growth unit volume was less than 10(7) microns 3 per culture dish. Computer-assisted volume analysis (CAVA) appears to be a useful quantitative method to study the growth of human tumour cells in soft agar cultures.
In vitro drug sensitivity testing, both by optical colony counting and by a [3H]-TdR incorporation assay, was performed on human tumour cells proliferating in soft agar cultures. Cells from two different human tumour cell lines, 5 different human tumour xenografts, and 94 different primary human tumour specimens of various histologic types were studied. Regression analysis comparing the results of the colony counting assay and the [3H]-TdR assay revealed good to excellent correlations between the two assay endpoints for quantitating the effect of in vitro anticancer drug exposure for a large number of different agents. The presence of pre-existing tumour cell aggregates complicates the performance of the optical colony counting assay. The [3H]-TdR incorporation assay is more sensitive and reproducible than the colony counting assay when performed on samples containing a large number of initially seeded tumour cell aggregates.
The applicability of a human tumor colony-forming assay to drug screening was investigated in terms of feasibility, validity, and potential for discovering new antitumor drugs. Feasibility was addressed in a pilot study during which basic methods, appropriate assay quality controls, and a standardized protocol for screening were developed. Considerable variability was noted in the availability and colony growth of different tumor types. The majority of the evaluable experiments utilized breast, colorectal, kidney, lung, melanoma, or ovarian tumors. For many tumor types, little evidence of growth was observed, or only rare specimens formed colonies. Colony-forming efficiencies ranged from 0.05 to 0.11% for the six most useful tumors listed above. A set of quality control measures was developed to address technical problems inherent in the assay. Testing of standard agents in the pilot study established that most of these agents could be detected as active. However, it also identified three assay limitations: compounds requiring systemic metabolic activation are inactive; medium constituents may block the activity of certain antimetabolites; and compounds without therapeutic efficacy may be positive in the assay. The assay categorized nontoxic clinically ineffective agents as true negatives with 97% accuracy. Of 79 compounds which were negative in the current National Cancer Institute prescreen (leukemia P388), 14 were active in the assay. Several demonstrated outstanding in vitro activity and are structurally unrelated to compounds already in development or in clinical trials. A subset of these active compounds were found to lack activity in a P388 in vitro colony-forming assay. This indication of differential cytotoxicity to human tumor cells makes this subset of compounds particularly interesting as antitumor drug leads. The demonstrated sensitivity to most standard agents, discrimination of nontoxic compounds, reproducibility of survival values within assays and between laboratories, and evidence of ability to identify active compounds which were negative in the in vivo prescreen suggest that the human tumor colony-forming assay may be a valuable tool for antitumor drug screening. However, because of technical limitations inherent in the current assay methodology, this must be confined to selected tumor types and limited to screening on a moderate scale.
The photosensitizing/cytotoxic effects of four porphyrin compounds derived from hematoporphyrin were compared using two human tumor cell lines with a soft-agar colony-forming assay. Reproducible dose- and time-dependent increases in reproductive cell death were observed for each porphyrin tested. Two fractions derived from hematoporphyrin derivative (HPD) were found to be better photosensitizers than HPD itself, indicating the potential value of this in vitro assay for detecting the most active component from a heterogeneous mixture.
If soft agar colony formation assays for primary human tumor cells are going to be performed and the results assessed by optical analysis of colony formation, then we believe it is mandatory in such assay techniques to objectively count control plates on the day of culture initiation using stained plates, use universal cytotoxic control compounds which should uniformly eliminate all viable cell proliferation, and use a vital stain such as the tetrazolium dye INT to document the presence or absence of viable cell colonies when cultures are assessed. There is no question, however, after careful observation of thousands of soft agar cultures of primary human tumor cells in my laboratory, that significant proliferation of small tumor cell aggregates often takes place in vitro and can in fact be used to assess cytotoxic drug effects in vitro. We do not believe that this is demonstrably stem cell or clonal growth. Nevertheless, it almost certainly is in vitro tumor cell proliferation. With very careful controls, we believe that optical methods can be used reliably to evaluate drug effects on this soft agar proliferative capacity of primary human tumor cells. However, it may eventually prove more useful to study human tumor cell proliferation in vitro (even soft agar colony growth) by other methods, such as incorporation of radiolabeled bases into newly synthesized DNA or other macromolecules, or by the simple use of vital dyes, as discussed elsewhere in this volume.(ABSTRACT TRUNCATED AT 250 WORDS)