- Email: [email protected]
Quantitative Changes in DNA and RNA Metabolism in Retinoblastoma
VOL. 67, NO. 5
737
SKIN-TEST REACTIVITY IN UVEITIS
lationship may be expressed by a quadratic equation. 4. There is a striking parallel between the curve expressing the frequency of uveitis at different ages and that for the relationship between skin-test reactivity and age. 1100 West Michigan Street (46202) ACKNOWLEDGMENTS
We thank James A. Norton, Jr., Ph.D., for sta tistical advice, and Hal Lowe, for programming as sistance. REFERENCES
1. Woods, A. C. and Wahlen, H. E. : The prob able role of benign histoplasmosis in the etiology of granulomatous uveitis. Am. J. Ophth. 49 :205, 1960.
2. Schlaegel, T. F., Jr. : The Indiana method of surveying uveitis patients. In Aronson, S. B., Gam ble, C. N., Goodner, E. K , O'conner, G. R. (ed.). Clinical Methods in Uveitis. St. Louis, Mosby, 1968, p. 66. 3. Woods, A. C. : Endogenous Uveitis. Baltimore, Williams & Wilkins, 1956, p. 195. 4. Freeman, M. F. and Tukey, J. W. : Transfor mations related to the angular and the square root. Ann. Math. Stat. 21:607, 1950. 5. Giannini, D. and Sloan, R. S. : A tuberculin survey of 1,285 adults with special reference to the elderly. Lancet 1:52S, 1957. 6. Kligman, A. M. : Poison ivy (rhus) dermati tis. Arch. Derm. 77:149, 1958. 7. Waldorf, D. S., Willkens, R. F. and Decker, J. L. : Impaired delayed hypersensitivity in an aging population. JAMA 203 :831, 1968. 8. Editorial : Transfer factor and leprosy. New England J. Med. 278 :333, 1968.
QUANTITATIVE CHANGES IN DNA AND RNA IN RETINOBLASTOMA GEORGE A. H Y M A N , M.D.,
METABOLISM
AND BRUNO F I N G E R H U T ,
M.D.
New York Autoradiographic studies, valuable in the evaluation of many neoplastic processes, have been under-utilized in the study of reti noblastoma. 1 O u r group at the ColumbiaPresbyterian Medical Center has a unique opportunity for such studies because of the relatively large number of new patients seen with this rare tumor of infancy and child hood. W e are indebted to Drs. Algernon B. Reese and Robert M. Ellsworth of the Insti tute of Ophthalmology for making 29 pa tients available for this study. The tumor tissue was obtained only from patients with imperative clinical indications for enucleation and consisted of two groups : ( A ) the patients initially seen with massive tumors endangering their lives (without
prior therapy—23 cases) ; and ( B ) those in whom tumors had become resistant to ther apy and were growing out of control (six cases). T h e technique involved the use of tritiated thymidine for DNA-labeling and tritiated cytidine for RNA-labeling in a method mod ified from Rubini, Keller, and Cronkite. 2 I n the retinoblastoma group, labeling studies were performed in 12 cases with D N A alone, 11 cases with D N A and R N A , and six cases with R N A alone. Control tissue in cluded human retina, normal rat retinal tis sue and normal and carcinomatous bladder mucosa, with which considerable experience with D N A - and RNA-labeling had been ob tained.
From the Departments of Medicine and Urology, College of Physicians and Surgeons, Columbia Uni versity, and the Medical Service of the ColumbiaPresbyterian Medical Center. This work was sup ported in part by USPHS Research Grant CA 02332 from the National Cancer Institute, The Hyman Goldburg Memorial Fund and the Alfred H. Drewes Memorial Fund for Medical Research. Presented in part at the annual meeting of the American Society of Clinical Oncology, April 10, 1968, Atlantic City, New Jersey.
Immediately upon enucleation, in the operating room, retinoblastoma tissue was dissected free, cut in two equal parts, and placed in a mixture containing 20 \>.c of 3 H thymidine in 1.0 cc of Puck's medium and a mixture containing 10 \>.c of 3 H-cytidine in 1.0 cc of Puck's medium and incubated at
METHODS
AMERICAN JOURNAL OF OPHTHALMOLOGY
738
± 2 0 grains per cell. One hundred percent of those cells were labeled with 3H-cytidine (RNA) with approximately ± 1 2 grains per cell. Because of long experience with the la beling of bladder mucosa and bladder tumor we used these tissues for additional con trols. Comparable changes were found in the difference of labeling of normal and carcinomatous bladder mucosa (table 1). Thus, in normal bladder mucosa there was 2% label ing with thymidine and 40 grains per cell (DNA) in each case, and 100% labeling with cytidine and 12 grains per cell (RNA), whereas in neoplastic mucosa there was 10%-30% of cells labeled with 100 grains per cell with thymidine (DNA) and 100% of cells labeled with 100 grains per cell with cytidine (RNA). 3 · 4 An idealized sketch of the DNA- and RNA-labeling is seen in Figure 1 and the actual labeling of bladder mucosa and bladder cancer is seen in Fig ures 2 and 3.
37°C for 30 minutes. The specific activity of the 3 H-thymidine used was 1.9 c/mmole and that of the 3H-cytidine used was 3.0 c/mmole. At the end of the 30 minutes, the tissue was washed in a phosphate buffer of 7.0 pH for five minutes, then fixed in Bouin's solution for eight hours. After the fixation, the tissue was pro cessed histologically. The material was dehy drated, using graded ethyl alcohol from 50% to absolute, embedded in paraffin, sectioned and stained with hematoxylin and erythrosin (which does not wash out) and then left to air-dry. The stained slides were dipped in Kodak NTB2 emulsion, stored in dark boxes at 4°C, and all exposed for seven days, after which they were developed with Dextol for one minute, rinsed and fixed in Kodafix for two minutes, then washed in running water for 20 minutes, dehydrated again through graded ethyl alcohol from 50% to absolute and two changes of xylene, then coverslipped, read and photographed. A minimum of 200 cells were counted microscopically and, where background radioactivity interferred with the analysis, the section was eliminated. Treatment of the normal retinal tissue was identical to that of the retinoblas toma tissue.
B. RETINOBLASTOMA
DNA-labeling. An idealized sketch of the results of labeling of normal retinal cells and retinoblastoma cells is seen in Figure 1. DNA-labeling occurred in 36% ( ± 1 1 ) * of cells in retinoblastoma tissue, averaging 79 ( ± 2 9 ) * grains per cell in untreated eyes (table 2), a 10-20 fold increase over normal in percentage labeled and 4-fold increase in grain count. These findings compare to 36% of cells labeled with 73 grains per cell+ in the radiation-treated eyes, a difference in
RESULTS A. NORMAL
RETINA,
NORMAL
MAY, 1969
AND NEO-
P L A S T I C BLADDER MUCOSA
DNA- and RNA-Labeling. Normal rat and human retina show 1% to 2% cells la beled with 3H-thymidine (DNA) averaging
* Standard deviation. t Too few studies for standard deviation.
TABLE 1 C E L L LABELING AND GRAIN COUNTS EMPLOYING 3 H-THYMIDINE AND 3 H-CYTIDINE
Substance
Normal rat retina Human retina Normal bladder Cancer—bladder
>H-thymidi ine (DNA) % Cells Labeled 1-2 1-2 2 10-30
]So.
Grains per Cell 20 20 40 ±100
3
% Cells Labeled 100 100 100 100
H-cytidine (RNA) No. Grains per Cell 12 12 12 ±100
VOL. 67, NO. 5
739
METABOLISM IN RETINOBLASTOMA
A. TRITIATED THYMIDINE INCORPORATION (H3-DNA) RETINOBLASTOMA CELL
NORMAL RETINAL CELL Fig. 1 (Hyman and Fingerhut). A. (DNA). Idealized sketch to demonstrate the increased percent age of cells labeled with thymidine showing a higher grain count in the nucleus on right (retinoblastoma cell) as compared to normal retinal cell on left. B. (RNA). Labeled with cytidine. In the retinoblastoma cell, the grains are concentrated in the nucleus with very few in the cy toplasm, as compared to the large number of cytoplasmic granules in the normal retinal cell on the left, and a smaller number in the nucleus. More of the retinoblastoma cells than normal retinal cells are labeled and the grain count is increased.
1-2% ±20
AVERAGE CELLS LABELED — 3 6 « AVERAGE GRAINS PER CELL- 79± 29
_ TRITIATED CYTIDINE INCORPORATION (H3-RNA) NORMAL RETINAL CELL
RETINOBLASTOMA CELL
AVERAGE CELLS LABELEO 100% AVERAGE GRAINS PER CELL 86 ± 2 4
DNA-Iabeling which is not significant. T h e actual labeling process in retinal tissue, be nign and malignant, is seen in Figures 4 and 5. C.
RETINOBLASTOMA
RNA-labeling. T h e data obtained by RNA-labeling with 3 H-cytidine from 14 pre viously untreated enucleated eyes ( G r o u p A ) and three previously treated eyes ( G r o u p B ) are indicated in Chart 1. In Group A (enucleation without prior radiotherapy), it can be seen that 100% ( ± 0 ) * of the cells were labeled in each case with approximately 86 ( ± 2 4 ) * grains per cell. In Group B ( e n u cleation after failure of radiation t h e r a p y ) , * Standard deviation.
the percentage of cells labeled decreased to an average of 5 8 % t with an average grain count of 30+ (table 2 ) . In the retinoblastoma tumor cells there was an intense concentra tion of RNA-labeled granules over the nu cleus and only a scattering of granules over the cytoplasm, as previously reported in blad der cancer cells and leukemic cells 4 (figs. 1, 4 and S ) . These were distinct qualitative differences from the normal retinal cells in which RNA-labeled granules were present primarily in the cytoplasm. T h e findings in the retinoblastoma tumor cells were comple mented by the quantitative increases of the percentage of cells labeled ( R N A ) and n u m ber of grains per cell in retinoblastoma. t Too few studies for standard deviation.
740
AMERICAN JOURNAL OF OPHTHALMOLOGY
MAY, 1969
Fig. 2 (Hyman and Fingerhut). DNA-labeling of normal and cancer bladder mucosal cells (Normal— 2% labeled, 40 grains per cell; cancer—10% to 30% labeled, 100 grains per cell.) (A) Normal DNA, χ125, hematoxylin-erythrosin. (B) Cancer DNA, X125, hematoxylin-erythrosin. (C) Normal bladder DNA, χ900, hematoxylin-erythrosin. Note the small number of cells labeled. (D) Cancer bladder DNA, χ900, hematoxylin-erythrosin. Note the increased number and intensity of the labeling procedure. DISCUSSION
A n important and consistent finding was that D N A - and RNA-labeling of retinoblastoma tumor tissue by radioautography was intense and generally far greater than that seen in normal tissue and in most other tu mors. This suggests a high rate of cell dupli cation and activity, a fact possibly explain ing the radiocurability of many of these tu mors when diagnosed at an early stage. These findings complemented those of P o lack, 1 employing tritiated thymidine alone, when he studied the eyes of nine patients
with retinoblastoma, labeling five with tri tiated thymidine. H e found that the D N A labeling rate was far greater than in mela noma and suggested that, since the labeling rate is far higher than the mitotic rate, the labeling technique is superior for estimating tumor reproductive capacity. O n e hundred percent of normal retinal cells were labeled with 12 grains per cell la beled, and were located uniformly in the nu cleus and cytoplasm. In retinoblastoma cells, the number of labeled cells were 100%, with 86 ± 24 grains per cell, and most were lo-
VOL. 67, NO. 5
METABOLISM IN RETINOBLASTOMA
741
Fig. 3 (Hyman and Fingerhut). RNA-labeling of normal and cancer bladder mucosal cells. (Normal— 100% labeled, 12 grains per cell; cancer—100% labeled, 100 grains per cell.) (A) Normal RNA, χ400, hematoxylin-erythrosin. (B) Cancer RNA, X400, hematoxylin-erythrosin. (C) Normal RNA, X900, hematoxylin-erythrosin. Note the smaller number of labeled cells and the presence of grains in the cyto plasm and not over the nucleus. (D) Cancer RNA, X900, hematoxylin-erythrosin. Note that few grains are distributed in the background but that there is an extremely dense concentration of grains in the cytoplasm of the labeled cells. cated intranuclearly. Possible explanations are : ( 1 ) a partial block exists in the transfer of R N A from nucleus to cytoplasm in these neoplastic cells ; ( 2 ) there is exceedingly rapid intranuclear R N A t u r n o v e r ; ( 3 ) in creased production or impaired degradation of intranuclear R N A ; or ( 4 ) there is a very
active intranuclear pool which does not leave the nucleus in these tumor cells. A predictive value may be present in these studies when the findings are correlated with response to therapy. O t h e r findings indicate that, in spite of the regrowth of the tumor tissue, radiation
742
AMERICAN JOURNAL OF OPHTHALMOLOGY
MAY, 1969
Fig. S (Hyman and Fingerhut). ( A ) RNA-labeling of normal human retinal cells, 100% labeled, 12 grains per cell (X900, hematoxylin-erythrosin). Note the smaller number of cells labeled and the presence of grains in the cytoplasm and not over the nucleus. ( B ) Untreated retinoblastoma tumor cells, 100% labeled, 90 grains per cell ( x 9 0 0 , hematoxylin-erythrosin). Note that few grains are distributed in the background but that there is an extremely dense concentration of grains over the nucleus, with fewer grains in the cytoplasm of the labeled cells.
TABLE 2 C E L L LABELING AND GRAIN COUNTS IN RETINOBLASTOMA EMPLOYING A H-THYMIDINE AND ' H - C Y T I D I N E 3
No. Cases
A. 23
Retinoblastoma
a. Enucleation without prior therapy
H-cytidine (RNA)
% Cells Labeled
No. Grains per Cell
% Cells Labeled
36±11*
79±29*
100±0*
Number of studies b. Enucleation after radiation therapy
3
H- thy midi ne (DNA)
19 36f
No. Grains per Cell 86 ± 2 4 * 14
731
58f
30t
Number of studies * Standard deviation. t Too few studies for standard deviation.
Fig. 4 (Hyman and Fingerhut). ( A ) DNA-labeling of normal human retinal cells, 1% to 2 % labeling, 20 grains per cell ( χ 9 0 0 , hematoxylin-erythrosin). Note the small number of cells labeled. ( B ) Untreated retinoblastoma cells, 30% labeled, 67 grains per cell (X900, hematoxylin-erythrosin). Note the increased number and intensity of the labeling procedure.
and/or chemotherapy still have an effect on the RNA metabolism when untreated eyes and radiation-treated eyes are compared. Thus there was no significant change noted
in DNA-labeling of active tumors with or without prior radiotherapy, but there was a decrease in RNA-labeling postradiotherapy. The difference in the results of RNA- and
7777/r
Chart 1 (Hyman and Fingerhut). RNA-labeling data obtained from 14 previously untreated enucleated eyes and three previously treated eyes.
RETINOBLASTOMA
00
WITH
00
RNA
LABELING - 17 PTS. 00
00
o°o°o
X
000u
90 βο 70 M -1 -1 kl
ϋ
60 50
Û Id 40 -1 U
a < 30 -1 « 20 0 = NO PRIOR TREATMENT X = PRIOR TREATMENT
10
10
20
30 40 SO NO. OF GRAINS PER
βθ CELL
70
80
90
100
744
AMERICAN JOURNAL OF OPHTHALMOLOGY
DNA-labeling revealed in Table 2 indicates that the greater sensitivity of the RNA-labeling techniques yield more meaningful data. This is the technique being concen trated upon at present in tritiated labeling studies of retinoblastoma and other tumors. Evaluation of the prognostic indications of the labeling data is in progress in retino blastoma and other neoplasms.4·5 In the reti noblastoma group, the results of the labeling in the enucleated eyes have been combined with the postenucleated clinical findings in an attempt to yield prognostic information. The time period of these studies is too short for evaluation of the so-called "five-year cure" at present. The data on DNA-labeling of the enucleated eyes of 11 patients, fol lowed for more than two years, are not ade quate to yield prognostic information except for the two extremes, where the patient with the lowest labeling had had an excellent fol low-up and the patient with the highest label ing had a very short course. Considerably further follow-up time, a larger number of studies and the use of expanded RNA data may be helpful in this regard, in combination with newly reported genetic data6 which con cludes that "sporadic bilateral retinoblastoma must now be considered to be due to germi nal mutations in 100% of cases, in contrast to unilateral retinoblastoma when the penetrance is less than 50%." SUMMARY
Autoradiographic studies were performed on the enucleated eyes of 29 patients with retinoblastoma, employing tritiated thymi-
MAY, 1969
dine and cytidine. The enucleations were re quired because of uncontrolled tumor activ ity, which was endangering the life of the patient, in eyes previously untreated or aftef radiotherapy. There was an increase in DNA-labeling of tumor cells to 36%, with 79 grains per cell labeled, when compared to normal cells. RNA studies with tritiated cy tidine revealed an increased percentage of tumor cells labeled and an intranuclear con centration of grains compared to normal cells. 99 Fort Washington Avenue (10032) ACKNOWLEDGMENTS
Figures 2 and 3 are reprinted with the permission of the editors of Cancer, 21:3S7, 1968. We thank Mrs. Marion Butler for her technical assistance and Mrs. Florence Lefcourt for her assistance in the preparation of this manuscript. REFERENCES
1. Polack, F. M. : Incorporation of tritiated thymidine by ocular tumors in vitro. Am. J. Ophth. 60 :46, 1965. 2. Rubini, J. R., Keller, S. and Cronkite, E. P. : In vitro DNA labeling of bone marrow and leukemic blood leukocytes with tritiated thymidine. J. Lab. Clin. Med. 66 :483, 1965. 3. Veenema, R. J., Fingerhut, B. and Girgis, A. S. : Histochemistry : A possible guide to therapy of bladder tumors. J. Urol. 90:736, 1963. 4. Hyman, G. A., Fingerhut, B. and Tiburcio. A. C. : Possible defect in RNA metabolism in leukemic cells. Cancer 21:357, 1968. 5. Fingerhut, B., Veenema, R. J. and Graff, S. : Observations on RNA synthesis in bladder cancer. Presented at the Internat. Urological Meeting, Munich, Germany, July, 1967. 6. Kitchin, F. D., Hyman, G. A., Ellsworth, R. M. and Reese, A. B. : Newer genetic findings in retinoblastoma (abstract). 4th annual meeting, American Society of Clinical Oncology, Atlantic City, April 10, 1968.
737
SKIN-TEST REACTIVITY IN UVEITIS
lationship may be expressed by a quadratic equation. 4. There is a striking parallel between the curve expressing the frequency of uveitis at different ages and that for the relationship between skin-test reactivity and age. 1100 West Michigan Street (46202) ACKNOWLEDGMENTS
We thank James A. Norton, Jr., Ph.D., for sta tistical advice, and Hal Lowe, for programming as sistance. REFERENCES
1. Woods, A. C. and Wahlen, H. E. : The prob able role of benign histoplasmosis in the etiology of granulomatous uveitis. Am. J. Ophth. 49 :205, 1960.
2. Schlaegel, T. F., Jr. : The Indiana method of surveying uveitis patients. In Aronson, S. B., Gam ble, C. N., Goodner, E. K , O'conner, G. R. (ed.). Clinical Methods in Uveitis. St. Louis, Mosby, 1968, p. 66. 3. Woods, A. C. : Endogenous Uveitis. Baltimore, Williams & Wilkins, 1956, p. 195. 4. Freeman, M. F. and Tukey, J. W. : Transfor mations related to the angular and the square root. Ann. Math. Stat. 21:607, 1950. 5. Giannini, D. and Sloan, R. S. : A tuberculin survey of 1,285 adults with special reference to the elderly. Lancet 1:52S, 1957. 6. Kligman, A. M. : Poison ivy (rhus) dermati tis. Arch. Derm. 77:149, 1958. 7. Waldorf, D. S., Willkens, R. F. and Decker, J. L. : Impaired delayed hypersensitivity in an aging population. JAMA 203 :831, 1968. 8. Editorial : Transfer factor and leprosy. New England J. Med. 278 :333, 1968.
QUANTITATIVE CHANGES IN DNA AND RNA IN RETINOBLASTOMA GEORGE A. H Y M A N , M.D.,
METABOLISM
AND BRUNO F I N G E R H U T ,
M.D.
New York Autoradiographic studies, valuable in the evaluation of many neoplastic processes, have been under-utilized in the study of reti noblastoma. 1 O u r group at the ColumbiaPresbyterian Medical Center has a unique opportunity for such studies because of the relatively large number of new patients seen with this rare tumor of infancy and child hood. W e are indebted to Drs. Algernon B. Reese and Robert M. Ellsworth of the Insti tute of Ophthalmology for making 29 pa tients available for this study. The tumor tissue was obtained only from patients with imperative clinical indications for enucleation and consisted of two groups : ( A ) the patients initially seen with massive tumors endangering their lives (without
prior therapy—23 cases) ; and ( B ) those in whom tumors had become resistant to ther apy and were growing out of control (six cases). T h e technique involved the use of tritiated thymidine for DNA-labeling and tritiated cytidine for RNA-labeling in a method mod ified from Rubini, Keller, and Cronkite. 2 I n the retinoblastoma group, labeling studies were performed in 12 cases with D N A alone, 11 cases with D N A and R N A , and six cases with R N A alone. Control tissue in cluded human retina, normal rat retinal tis sue and normal and carcinomatous bladder mucosa, with which considerable experience with D N A - and RNA-labeling had been ob tained.
From the Departments of Medicine and Urology, College of Physicians and Surgeons, Columbia Uni versity, and the Medical Service of the ColumbiaPresbyterian Medical Center. This work was sup ported in part by USPHS Research Grant CA 02332 from the National Cancer Institute, The Hyman Goldburg Memorial Fund and the Alfred H. Drewes Memorial Fund for Medical Research. Presented in part at the annual meeting of the American Society of Clinical Oncology, April 10, 1968, Atlantic City, New Jersey.
Immediately upon enucleation, in the operating room, retinoblastoma tissue was dissected free, cut in two equal parts, and placed in a mixture containing 20 \>.c of 3 H thymidine in 1.0 cc of Puck's medium and a mixture containing 10 \>.c of 3 H-cytidine in 1.0 cc of Puck's medium and incubated at
METHODS
AMERICAN JOURNAL OF OPHTHALMOLOGY
738
± 2 0 grains per cell. One hundred percent of those cells were labeled with 3H-cytidine (RNA) with approximately ± 1 2 grains per cell. Because of long experience with the la beling of bladder mucosa and bladder tumor we used these tissues for additional con trols. Comparable changes were found in the difference of labeling of normal and carcinomatous bladder mucosa (table 1). Thus, in normal bladder mucosa there was 2% label ing with thymidine and 40 grains per cell (DNA) in each case, and 100% labeling with cytidine and 12 grains per cell (RNA), whereas in neoplastic mucosa there was 10%-30% of cells labeled with 100 grains per cell with thymidine (DNA) and 100% of cells labeled with 100 grains per cell with cytidine (RNA). 3 · 4 An idealized sketch of the DNA- and RNA-labeling is seen in Figure 1 and the actual labeling of bladder mucosa and bladder cancer is seen in Fig ures 2 and 3.
37°C for 30 minutes. The specific activity of the 3 H-thymidine used was 1.9 c/mmole and that of the 3H-cytidine used was 3.0 c/mmole. At the end of the 30 minutes, the tissue was washed in a phosphate buffer of 7.0 pH for five minutes, then fixed in Bouin's solution for eight hours. After the fixation, the tissue was pro cessed histologically. The material was dehy drated, using graded ethyl alcohol from 50% to absolute, embedded in paraffin, sectioned and stained with hematoxylin and erythrosin (which does not wash out) and then left to air-dry. The stained slides were dipped in Kodak NTB2 emulsion, stored in dark boxes at 4°C, and all exposed for seven days, after which they were developed with Dextol for one minute, rinsed and fixed in Kodafix for two minutes, then washed in running water for 20 minutes, dehydrated again through graded ethyl alcohol from 50% to absolute and two changes of xylene, then coverslipped, read and photographed. A minimum of 200 cells were counted microscopically and, where background radioactivity interferred with the analysis, the section was eliminated. Treatment of the normal retinal tissue was identical to that of the retinoblas toma tissue.
B. RETINOBLASTOMA
DNA-labeling. An idealized sketch of the results of labeling of normal retinal cells and retinoblastoma cells is seen in Figure 1. DNA-labeling occurred in 36% ( ± 1 1 ) * of cells in retinoblastoma tissue, averaging 79 ( ± 2 9 ) * grains per cell in untreated eyes (table 2), a 10-20 fold increase over normal in percentage labeled and 4-fold increase in grain count. These findings compare to 36% of cells labeled with 73 grains per cell+ in the radiation-treated eyes, a difference in
RESULTS A. NORMAL
RETINA,
NORMAL
MAY, 1969
AND NEO-
P L A S T I C BLADDER MUCOSA
DNA- and RNA-Labeling. Normal rat and human retina show 1% to 2% cells la beled with 3H-thymidine (DNA) averaging
* Standard deviation. t Too few studies for standard deviation.
TABLE 1 C E L L LABELING AND GRAIN COUNTS EMPLOYING 3 H-THYMIDINE AND 3 H-CYTIDINE
Substance
Normal rat retina Human retina Normal bladder Cancer—bladder
>H-thymidi ine (DNA) % Cells Labeled 1-2 1-2 2 10-30
]So.
Grains per Cell 20 20 40 ±100
3
% Cells Labeled 100 100 100 100
H-cytidine (RNA) No. Grains per Cell 12 12 12 ±100
VOL. 67, NO. 5
739
METABOLISM IN RETINOBLASTOMA
A. TRITIATED THYMIDINE INCORPORATION (H3-DNA) RETINOBLASTOMA CELL
NORMAL RETINAL CELL Fig. 1 (Hyman and Fingerhut). A. (DNA). Idealized sketch to demonstrate the increased percent age of cells labeled with thymidine showing a higher grain count in the nucleus on right (retinoblastoma cell) as compared to normal retinal cell on left. B. (RNA). Labeled with cytidine. In the retinoblastoma cell, the grains are concentrated in the nucleus with very few in the cy toplasm, as compared to the large number of cytoplasmic granules in the normal retinal cell on the left, and a smaller number in the nucleus. More of the retinoblastoma cells than normal retinal cells are labeled and the grain count is increased.
1-2% ±20
AVERAGE CELLS LABELED — 3 6 « AVERAGE GRAINS PER CELL- 79± 29
_ TRITIATED CYTIDINE INCORPORATION (H3-RNA) NORMAL RETINAL CELL
RETINOBLASTOMA CELL
AVERAGE CELLS LABELEO 100% AVERAGE GRAINS PER CELL 86 ± 2 4
DNA-Iabeling which is not significant. T h e actual labeling process in retinal tissue, be nign and malignant, is seen in Figures 4 and 5. C.
RETINOBLASTOMA
RNA-labeling. T h e data obtained by RNA-labeling with 3 H-cytidine from 14 pre viously untreated enucleated eyes ( G r o u p A ) and three previously treated eyes ( G r o u p B ) are indicated in Chart 1. In Group A (enucleation without prior radiotherapy), it can be seen that 100% ( ± 0 ) * of the cells were labeled in each case with approximately 86 ( ± 2 4 ) * grains per cell. In Group B ( e n u cleation after failure of radiation t h e r a p y ) , * Standard deviation.
the percentage of cells labeled decreased to an average of 5 8 % t with an average grain count of 30+ (table 2 ) . In the retinoblastoma tumor cells there was an intense concentra tion of RNA-labeled granules over the nu cleus and only a scattering of granules over the cytoplasm, as previously reported in blad der cancer cells and leukemic cells 4 (figs. 1, 4 and S ) . These were distinct qualitative differences from the normal retinal cells in which RNA-labeled granules were present primarily in the cytoplasm. T h e findings in the retinoblastoma tumor cells were comple mented by the quantitative increases of the percentage of cells labeled ( R N A ) and n u m ber of grains per cell in retinoblastoma. t Too few studies for standard deviation.
740
AMERICAN JOURNAL OF OPHTHALMOLOGY
MAY, 1969
Fig. 2 (Hyman and Fingerhut). DNA-labeling of normal and cancer bladder mucosal cells (Normal— 2% labeled, 40 grains per cell; cancer—10% to 30% labeled, 100 grains per cell.) (A) Normal DNA, χ125, hematoxylin-erythrosin. (B) Cancer DNA, X125, hematoxylin-erythrosin. (C) Normal bladder DNA, χ900, hematoxylin-erythrosin. Note the small number of cells labeled. (D) Cancer bladder DNA, χ900, hematoxylin-erythrosin. Note the increased number and intensity of the labeling procedure. DISCUSSION
A n important and consistent finding was that D N A - and RNA-labeling of retinoblastoma tumor tissue by radioautography was intense and generally far greater than that seen in normal tissue and in most other tu mors. This suggests a high rate of cell dupli cation and activity, a fact possibly explain ing the radiocurability of many of these tu mors when diagnosed at an early stage. These findings complemented those of P o lack, 1 employing tritiated thymidine alone, when he studied the eyes of nine patients
with retinoblastoma, labeling five with tri tiated thymidine. H e found that the D N A labeling rate was far greater than in mela noma and suggested that, since the labeling rate is far higher than the mitotic rate, the labeling technique is superior for estimating tumor reproductive capacity. O n e hundred percent of normal retinal cells were labeled with 12 grains per cell la beled, and were located uniformly in the nu cleus and cytoplasm. In retinoblastoma cells, the number of labeled cells were 100%, with 86 ± 24 grains per cell, and most were lo-
VOL. 67, NO. 5
METABOLISM IN RETINOBLASTOMA
741
Fig. 3 (Hyman and Fingerhut). RNA-labeling of normal and cancer bladder mucosal cells. (Normal— 100% labeled, 12 grains per cell; cancer—100% labeled, 100 grains per cell.) (A) Normal RNA, χ400, hematoxylin-erythrosin. (B) Cancer RNA, X400, hematoxylin-erythrosin. (C) Normal RNA, X900, hematoxylin-erythrosin. Note the smaller number of labeled cells and the presence of grains in the cyto plasm and not over the nucleus. (D) Cancer RNA, X900, hematoxylin-erythrosin. Note that few grains are distributed in the background but that there is an extremely dense concentration of grains in the cytoplasm of the labeled cells. cated intranuclearly. Possible explanations are : ( 1 ) a partial block exists in the transfer of R N A from nucleus to cytoplasm in these neoplastic cells ; ( 2 ) there is exceedingly rapid intranuclear R N A t u r n o v e r ; ( 3 ) in creased production or impaired degradation of intranuclear R N A ; or ( 4 ) there is a very
active intranuclear pool which does not leave the nucleus in these tumor cells. A predictive value may be present in these studies when the findings are correlated with response to therapy. O t h e r findings indicate that, in spite of the regrowth of the tumor tissue, radiation
742
AMERICAN JOURNAL OF OPHTHALMOLOGY
MAY, 1969
Fig. S (Hyman and Fingerhut). ( A ) RNA-labeling of normal human retinal cells, 100% labeled, 12 grains per cell (X900, hematoxylin-erythrosin). Note the smaller number of cells labeled and the presence of grains in the cytoplasm and not over the nucleus. ( B ) Untreated retinoblastoma tumor cells, 100% labeled, 90 grains per cell ( x 9 0 0 , hematoxylin-erythrosin). Note that few grains are distributed in the background but that there is an extremely dense concentration of grains over the nucleus, with fewer grains in the cytoplasm of the labeled cells.
TABLE 2 C E L L LABELING AND GRAIN COUNTS IN RETINOBLASTOMA EMPLOYING A H-THYMIDINE AND ' H - C Y T I D I N E 3
No. Cases
A. 23
Retinoblastoma
a. Enucleation without prior therapy
H-cytidine (RNA)
% Cells Labeled
No. Grains per Cell
% Cells Labeled
36±11*
79±29*
100±0*
Number of studies b. Enucleation after radiation therapy
3
H- thy midi ne (DNA)
19 36f
No. Grains per Cell 86 ± 2 4 * 14
731
58f
30t
Number of studies * Standard deviation. t Too few studies for standard deviation.
Fig. 4 (Hyman and Fingerhut). ( A ) DNA-labeling of normal human retinal cells, 1% to 2 % labeling, 20 grains per cell ( χ 9 0 0 , hematoxylin-erythrosin). Note the small number of cells labeled. ( B ) Untreated retinoblastoma cells, 30% labeled, 67 grains per cell (X900, hematoxylin-erythrosin). Note the increased number and intensity of the labeling procedure.
and/or chemotherapy still have an effect on the RNA metabolism when untreated eyes and radiation-treated eyes are compared. Thus there was no significant change noted
in DNA-labeling of active tumors with or without prior radiotherapy, but there was a decrease in RNA-labeling postradiotherapy. The difference in the results of RNA- and
7777/r
Chart 1 (Hyman and Fingerhut). RNA-labeling data obtained from 14 previously untreated enucleated eyes and three previously treated eyes.
RETINOBLASTOMA
00
WITH
00
RNA
LABELING - 17 PTS. 00
00
o°o°o
X
000u
90 βο 70 M -1 -1 kl
ϋ
60 50
Û Id 40 -1 U
a < 30 -1 « 20 0 = NO PRIOR TREATMENT X = PRIOR TREATMENT
10
10
20
30 40 SO NO. OF GRAINS PER
βθ CELL
70
80
90
100
744
AMERICAN JOURNAL OF OPHTHALMOLOGY
DNA-labeling revealed in Table 2 indicates that the greater sensitivity of the RNA-labeling techniques yield more meaningful data. This is the technique being concen trated upon at present in tritiated labeling studies of retinoblastoma and other tumors. Evaluation of the prognostic indications of the labeling data is in progress in retino blastoma and other neoplasms.4·5 In the reti noblastoma group, the results of the labeling in the enucleated eyes have been combined with the postenucleated clinical findings in an attempt to yield prognostic information. The time period of these studies is too short for evaluation of the so-called "five-year cure" at present. The data on DNA-labeling of the enucleated eyes of 11 patients, fol lowed for more than two years, are not ade quate to yield prognostic information except for the two extremes, where the patient with the lowest labeling had had an excellent fol low-up and the patient with the highest label ing had a very short course. Considerably further follow-up time, a larger number of studies and the use of expanded RNA data may be helpful in this regard, in combination with newly reported genetic data6 which con cludes that "sporadic bilateral retinoblastoma must now be considered to be due to germi nal mutations in 100% of cases, in contrast to unilateral retinoblastoma when the penetrance is less than 50%." SUMMARY
Autoradiographic studies were performed on the enucleated eyes of 29 patients with retinoblastoma, employing tritiated thymi-
MAY, 1969
dine and cytidine. The enucleations were re quired because of uncontrolled tumor activ ity, which was endangering the life of the patient, in eyes previously untreated or aftef radiotherapy. There was an increase in DNA-labeling of tumor cells to 36%, with 79 grains per cell labeled, when compared to normal cells. RNA studies with tritiated cy tidine revealed an increased percentage of tumor cells labeled and an intranuclear con centration of grains compared to normal cells. 99 Fort Washington Avenue (10032) ACKNOWLEDGMENTS
Figures 2 and 3 are reprinted with the permission of the editors of Cancer, 21:3S7, 1968. We thank Mrs. Marion Butler for her technical assistance and Mrs. Florence Lefcourt for her assistance in the preparation of this manuscript. REFERENCES
1. Polack, F. M. : Incorporation of tritiated thymidine by ocular tumors in vitro. Am. J. Ophth. 60 :46, 1965. 2. Rubini, J. R., Keller, S. and Cronkite, E. P. : In vitro DNA labeling of bone marrow and leukemic blood leukocytes with tritiated thymidine. J. Lab. Clin. Med. 66 :483, 1965. 3. Veenema, R. J., Fingerhut, B. and Girgis, A. S. : Histochemistry : A possible guide to therapy of bladder tumors. J. Urol. 90:736, 1963. 4. Hyman, G. A., Fingerhut, B. and Tiburcio. A. C. : Possible defect in RNA metabolism in leukemic cells. Cancer 21:357, 1968. 5. Fingerhut, B., Veenema, R. J. and Graff, S. : Observations on RNA synthesis in bladder cancer. Presented at the Internat. Urological Meeting, Munich, Germany, July, 1967. 6. Kitchin, F. D., Hyman, G. A., Ellsworth, R. M. and Reese, A. B. : Newer genetic findings in retinoblastoma (abstract). 4th annual meeting, American Society of Clinical Oncology, Atlantic City, April 10, 1968.