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Measurement of Proliferation Indices in Non Hodgkin's Lymphoma—Is it Useful? a
b
a
K. J. Tusenius , P. J. M. Bakker & M. H. J. Van Oers a
Department of Haematology, Academic Medical Center, Amsterdam, The Netherlands
b
Department of Oncology, Academic Medical Center, Amsterdam, The Netherlands Published online: 01 Jun 2015.
To cite this article: K. J. Tusenius, P. J. M. Bakker & M. H. J. Van Oers (1992) Measurement of Proliferation Indices in Non Hodgkin's Lymphoma—Is it Useful?, Leukemia & Lymphoma, 7:3, 181-187, DOI: 10.3109/10428199209053621 To link to this article: http://dx.doi.org/10.3109/10428199209053621
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Leukemia and Lymphoma, Vol. 7, pp. 181-187 Reprints available directly from the publisher Photocopying permitted by license only
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Measurement of Proliferation Indices in Non Hodgkin’s Lymphoma-Is it Useful? K. J. TUSENIUS’, P. J. M. BAKKER,, and M. H. J. VAN OERS’ Departments of Haematology and Oncology’, Academic Medical Center, Amsterdam, The Netherlands
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(Received 21 November 1991)
In this review we discuss the possible value of additional information concerning growth characteristics of NHL‘s for the prognosis of the individual patient. Techniques for measuring cell cycle kinetic parameters (e.g. S-phase assessment) and proliferation related antigens (e.g. anti-transferrin receptor and Ki67) are shortly reviewed. This is followed by an overview of proliferation studies in NHL. Until now in most of these studies overall measurements of tumour tissue have been performed. Moreover the parameters that have been assessed are related to proliferation state rather than proliferation rate. None of the methods used has a convincing superiority with regard to clinical outcome. In the future, tumour specific assessment (by means of double-labeling techniques) of proliferation rate parameters might provide information, more relevant to the prognosis of the individual patient with NHL. KEY WORDS:
I INTRODUCTION
Non Hodgkin’s Lymphoma proliferation related antigens
cell cycle kinetic studies prognosis
tion and selection of therapy. Knowledge of the growth characteristics of tumours might be a useful addition to the already existing classifications5. In this report we will give a brief overview of several new techniques which measure proliferative activity of NHL. Their possible value for the assessment of prognosis, when used alone or in combination with other parameters will also be discussed.
Early classifications of non Hodgkin’s Lymphoma’s (NHL) were based on morphology and enzyme histochemistry while later classifications incorporated both immunophenotype and differentiation state of the NHL2*3*.In an effort to correlate histopathological and survival characteristics of NHL, the so called Working Formulation (W.F.)4was developed during the early nineteen eighties. It is a compromise between the many and confusing classifications and its main emphasis is on clinical 2 CELL PROLIFERATION utility. However a serious shortcoming of the W.F. is that it fails to identify NHL‘s by cell lineage, and it Based on studies in the 1950s, using 32P incorporadoes not distinguish the immunological variants nor tion and autoradiography, the concept of the cell cycle the individual lymphomas by biological behaviour. was postulated6. Pulse labeling of growing cells by This is a major drawback for accuracy in prognostica- radiolabeled DNA precursors, including 3H-thymidine’ enabled subdivision of the cell cycle into several phases (mitotic or M-phase, synthetic or S-phase, and Address for correspondence: K. J. Tusenius, Academic Medical Center, F4-224, Meibergdreef 9, 1105 AZ Amsterdam, The two gap-phases G, and G,) and calculation of the Netherlands. duration of the cell cycle time. Further studies showed
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that the increase in tumour mass observed in vivo was invariably less than expected from the measured cell cycle duration and this finding gave rise to the idea that only a proportion of the tumour cell population was actively proliferating. Non-proliferating cells may temporarily leave the cell cycle (Gophase cells) or may differentiate and ultimately die. Moreover, it became evident that there is considerable cell loss within a tumour, due to insufficient vascularization, immunological responses of the host, errors of DNA replication etc. In rapidly growing tumours cell death frequently comprises up to 90% of the cell production rate5. In histological sections this can be seen as areas of necrosis. Tumours are thus comprised of cycling and non-cycling cells, the latter consisting of both Go cells and doomed cells. The ratio of cycling to cycling plus non-cycling cells has been defined as the proliferative or growth fraction'. Based on this concept of the cell cycle several approaches have been used to determine the proliferative activity of tumours. They include the measurement of the number of cells in certain phases of the cell cycle, measurement of cell cycle duration, and assessment of proliferation associated antigens, which are expressed during one or more cell cycle phases. We will briefly describe the principles of the most relevant methods for assessment of proliferative activity. This will be followed by a critical review of studies performed in NHL.
3.2 Labeling of cells in DNA synthesis
3.2.I Thymidine labeling The thymidine labeling technique was introduced by Taylor13 and has since that time gained an important place in the study of cell cycle kinetics5. The basic method is simple. TritiumC3H] or carbon-14[ 14C] labeled thymidine is incorporated in cells actively synthesizing DNA. The incorporated radioactive labels can be detected by means of autoradiography. The type of information obtained with this method depends on whether the labels are administered as a pulse, as a (semi-) continuous infusion, or as a combination of both and whether multiple biopsies can be taken from a tumour. The two most commonly used cell kinetic assays based on autoradiography are the determination of the labeling index (L.I., the fraction of labeled cells) and the fraction of pulse labeled mitoses (PLM)14. For the assessment of the L.1, cells are pulse labeled with [3H]thymidine. L.I. is dependent on both the fraction of proliferating cells and the duration of the S-phase. The L.I. is often used as a measure of proliferative activity. In the PLM procedure in analogy to the L.I., cells are exposed to a pulse label of C3H] thymidine. Thereafter at various intervals series of samples of the tumour are taken. From the rise and fall of the number of labeled mitoses at various time intervals several important cell proliferative parameters can be derived e.g. duration of the cell cycle time and of the different cell cycle phases. For the analysis of cell cycle kinetic parameters in human tumours the use of radioactive 3 DETERMINATION OF PROLIFERATION thymidine combined with the autoradiography INDICES BY MEASURING CELL CYCLE technique has however several limitations inherent to KINETIC PARAMETERS the use of radioactive compounds, the cumbersome and time consuming radiographic procedure and the 3.1 Assessment of mitotic index (M.I.) requirement of multiple biopsies. Therefore this This was the first kinetic technique used. It is a simple method has been largely abandoned. quantitative method using microscopically observable characteristics of cells. The number of mitoses is a 3.2.2 Non-radioactive thymidine analogues function of both the number of proliferating cells and This method is rapid and easy to perform and thus of the duration of the observable part of the cell facilitates the study of cell cycle kinetics in human division. As a general rule when the mitotic index is tumours. The method uses the thymidin analogs high, proliferation is rapid and vice versa. Usually the bromodeoxyuridine (BrdUrd) or iododeoxyuridine fraction of metaphase cells is small (0.5-2%). (IdUrd) which are incorporated in cells actively Therefore many cells have to be examined in order to synthesizing DNA. The incorporated labels can be derive an accurate value of M.I. M.I. assessment can detected by quenching the dye Hoechst 33258 or by be improved by treating a proliferating cell population the use of a monoconal antibody15*16.The labewith an agent which arrests all cells in metaphase (e.g. lings index is simply measured as the proportion of In addition cells that have taken up BrdUrd or IdUrd. This colcemid, vincristine or ~inblastine)~*". the application of flow cytometry allows a more method has the advantage that by means of the simultaneous measurement of DNA content and accurate measurement of M.1.' '*12.
PROLIFERATION INDICES IN LYMPHOMA-USEFUL?
BrdUrd fluorescence from a single sample taken a few hours after pulse labeling DNA synthesis time (Ts) and the potential doubling time (Tpot) of a tumour can be calculated”. Two recently developed labelingand staining procedures using either BrdUrd and IdUrd or IdUrd and chlorodeoxyuridine (CldUrd) as a label even further expand the possibilities of detailed cell cycle kinetic studies in human t ~ m o u r s ’ ~ * ’ ~ .
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3.3 Distribution of cellular DNA content The introduction of flow cytometry20.21*22 has provided a rapid method for measuring the distribution of cells over the different cell cycle phases. After preparing a single cell suspension, cells are stained with a DNA specific fluorescent dye e.g. ethidiumbromide, Hoechst 33258, chromomycin or propidiumiodide, The relative DNA content of each cell is thus measured in several thousands of cells and the derived DNA histogram can be analyzed through one of many computer programs. It allows the assessment of the fractions of cells in G , , S and G, + M phases respectively. The presence of aneuploidy complicates the assessment of the S-phase, but with special mathematical analysis this can be estimated23. The disadvantage of this method is that no information is obtained about actual phase durations or growth fractions. Therefore the actual cell cycle kinetic information which can be obtained is limited.
4 MEASUREMENT OF PROLIFERATION RELATED ANTIGENS The development of immunocytochemical techniques produced a new interesting area of proliferation studies. Several antigens have been described, which are expressed in or on the nucleus, in the cytoplasm or on the membrane of cells in association with proliferation. Some of them, especially those applied to NHL will be discussed below. 4.1 Transferrin Receptor (TfR) Iron is essential for every human cell. It plays a central role in all oxidative energy metabolism and in cellular growth and p r ~ l i f e r a t i o nThis ~ ~ . probably reflects the iron dependency of ribonucleotide reductase, the enzyme responsible for synthesis of deoxyribonucleotides from ribon~cleotides~~. Studies with immature red cells showed that iron uptake occurred via the
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binding of the serum iron transport protein transferrin to a specific cell surface receptor26. Transferrin-mediated iron uptake has been shown to be an absolute requirement for growth of many different cell types”. The number of TfR’s on the cell surface is related to the iron uptake and reflects the cellular iron requirement. Resting lymphocytes hardly bind any transferrinZ8.However after stimulation the number of TfR’s is increased to about 80.000 per cell29.This is caused by externalisation of intracellular receptor^,^. With the upregulation of TfR’s resting cells progress from the G, phase into S-phase3’. Therefore modulation of TfR activity is important for regulation of the proliferative state3 Recently the monoclonal antibodies (moab’s) against the TfR were clustered as CD71. The TfR is now well characterized biochemically and at the DNA level,’. A drawback for the use of the TfR measurement as a proliferation parameter in malignancies, is its expression on monocytes/macrophages, and on nonmalignant reactive lymphocyte^^^*'^.
’.
4.2 Ki-67 In 1983 the production of the mouse monoclonal antibody termed Ki-67, recognizing a nuclear antigen present in proliferating cells was described3’. Recently the antigen was characterized immunobiochemically and by molecular biological technique^^^. Its expression was found to be restricted to G , , s, G, and M-phase of the cellcycle, and absent in Go phase.Thus, the Ki-67 moab enables determination of the growth fraction of a cell population. 4.3 Other antigens related to cell proliferation Several other antigens have been studied in relation to cell proliferation and the cellcycle. Proliferating cell nuclear antigen (PCNA) is formed at rates related to both cell proliferation and DNA synthesis34. It was found to be identical to cyclin (an auxiliary protein of DNA polymerase) a polypeptide of 36 K D with an is0 electic point of 4.8. Recently the sequence of the DNA coding for PCNA has been determined74. PCNA begins to accumulate during the G, phase, peaks during S-phase and declines in G, and M-phase. Except for the activation-associated 4F2 antigen which is expressed early in the GO-G1 phases of the ~ e l l c y c l e ~other ~ , putative proliferation associated markers like C5F1036,the p-40 protein3’ and the c-myc oncogene38 have not yet been sufficiently studied in relation to NHL to be discussed.
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NUCLEOLAR ORGANIZER REGIONS (NORs)
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the transferrin receptor in NHL have yielded conflicting results. Some authors have reported a positive correlation between the number of TfR Nucleolar organizer regions (NORs) represent loops positive cells and the histological malignancy grade of of DNA, which contain the genes for ribosomal RNA NHLS3. However, others found TfR expression in and proteins. These were first described in the only a minority of NHL (34%); and with a low 1 9 7 0 ' ~ ~ ~The *~O function . of the nucleic acid-protein densitys4. More recently in a large retrospective study complexes is not fully understood, but seems to be a on cryostat sections of 267 NHL'sSS. TfR expression regulatory one in maintaining DNA in an extended was found in a wide variety of B and T lineage NHL's configuration4' or in controlling the transcription of and to be only loosely correlated with the the genes for ribosomal RNA42. As might be expected International Working Formulation classification. In NORs, playing an important role in protein synthesis, general, high grade lymphomas expressed the TfR indicate cell activation or t r a n s f ~ r m a t i o n ~ ~ -A~ ' . more often and more intensely. However exceptions linear correlation was found between AgNOR score were found in certain histological subtypes. This per nucleus and S-phase ~ e r c e n t a g e ~ ~ . discrepancy was also found in another studys6. Similary, conflicting data have been obtained regarding the correlation between TfR expression and 6 PROLIFERATION STUDIES IN NHL p r o g n ~ s i s These ~ ~ ~ confusing ~~. data might partly be due to TfR expression on monocytes/macrophages, Because counting of mitoses is a time consuming and and on non-malignant reactive lymphocytes28929. This insufficiently standardized procedure, it has been makes TfR less specific as a marker for tumour performed in NHL in a limited scale only47. This is p r o l i f e r a t i ~ n ~ *O*n~ ~the . other hand differences in in contrast to S-phase assessment. As might be technique might explain these disagreements: memexpected pulse labeling studies using 'H thymidine4' brane-expression of the TfR as detected by flow or BrdUrd49 as well as S-phase determinations with cytometry might not be comparable to the combinaDNA histogram^'^.^ have yielded comparable re- tion of membrane- and cytoplasmatic expression as sults. In all these studies a positive correlation was detected by cryostat section immunoperoxidasestainfound between the percentage of cells in the S-phase ing". In summary, the value of TfR measurements for and the malignancy grade of the NHL, according to subclassification and assessment of prognosis of the Working Formulation. However, the correlation NHL's seems to be limited. Ki-67 expression has been studied extensively in certainly was not an absolute one and many discrepancies have been observed, thus limiting its NHL and varing correlations between the immunocyusefulness for the individual patient. Although tochemical pattern and histological grade of malmeasurement of S-phase duration (Ts) and calcula- ignancy have been found. In general a low and high tions of the potential doubling time (T pot) have been Ki67 score were obtained in low and high grade performed in vitro for many years, only recently malignant NHL's re~pectively'~-~'.Often however, techniques have become available for in vivo considerable overlap between both groups exists, procedure^'^. Therefore extensive studies on the value which precludes accurate diagnosis in individual of the measurement of these dynamic cell cycle cases. Within the group of low grade NHL a relative parameters in NHLs are lacking at the moment. In a high Ki-67 score correlated with bad o u t ~ o m e ~ ~ . ~ ~ . recent study preliminary results of this procedure in For high grade lymphoma inconsistant data about a limited number of NHL patients have been Ki-67 expression have been observed: one study reported". Although a large variation in L.I. was reporting very high Ki-67 scores correlating with observed between tumours of the same histological better survival6 while another showed the oppograde, an inverse correlation was found between site6'. The surprising fact that high grade lymphomas grade of malignancy and T pot. The number of with high Ki-67 indices did better clinically than those patients and their follow-up were still inadequate to with lower indices was postulated to be due to the allow conclusions as to its value for assessment of larger fraction of proliferative cells which are susceptible to chemotherapy. Another explanation for individual prognosis. With the development of flow cytometry, measure- discrepancies between Ki-67 reactivity and histology ment of proliferation related antigens has gained or prognosis might be the fact that many of the early considerable popularity. Studies on the expression of data on the expression of Ki-67 in relation to the cell
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cycle were obtained with phytohaemagglutinin the existing subclassifications of NHL (e.g. the (PHA)-stimulated lymphocytes. These might not be a Working Formulation). There might be several perfect model of the proliferating compartment in explanations for this lack of prognostic value. First, tumours in vivo. Several mechanisms have been in assessing proliferation of malignant cells sample suggested to be responsible for the often lower than choice and size are of the utmost importance while expected Ki-67 score in human malignant tissues: heterogeneity is one of the characteristics of nutrient deprivation, quiescent S-phase cells and neoplasm^^^.^^. This means that in a tumour, possible inclusion of dead cells or aggregates in the malignant and non malignant areas can be found. analysis. Moreover, some cycling cells may not Within the malignant area cells are in different phases of the cell cycle. Also several other factors like express the Ki-67 antigen63. Other proliferation related antigens have not yet inclusion of dead cells, quiescent S-phase cells, loss of been studied extensively in NHL. Proliferating cells antigenic expression etc. can contribute to sampling in NHL express PCNA64. The 4F2 antigen is errors. In the classical histological assessment part of expressed more strongly and more often in high grade these complicating factors can be eliminated, whereas NHL than in other s u b c l a ~ s e s ~Finally ~. the with flow cytometric analysis they have to be taken assessment of silver stained NORs (AgNORs) in into account seriously. Thus, results obtained on paraffin slides for interphase chromosomes has also been found to allow discrimination between high and low grade example, cannot simply be compared with data obtained with flow cytometry of cell suspensions. On NHL6ss66. Since virtually all proliferation markers gage the other hand the advantage of the latter method is different cell cycle phases, in many studies two or more that by means of double labeling with tumour specific proliferation parameters have been compared. Corre- markers, in heterogeneous populations, the proliferalation was found between Ki-67 expression and the tion characteristics of the tumour population can be 3H-thymidine labeling index6’ and BrdUrd in- measured specifically. However, until now most of the corporation r e ~ p e c t i v e l y ~TfR ~ . ~measurements ~. did studies with flow cytometry have been performed on not show a similar correlation with Ki-67 expres- entire cell suspensions, resulting in an undersi0n6 7 69. This was explained by the finding of a high estimation of the proliferative activity of the tumour number of TfR positive cells in low grade lymphomas. cells. Secondly, in general, all the methods used for In a study comparing Ki-67 immunostaining, NOR counting and morphology in 36 follicular NHL’s all assessment of tumour proliferation only give a partial three methods proved to be equally useful for picture of tumour kinetics, because they provide subclassification. They also predicted poor clinical percentages of proliferating cells in a certain cell cycle outcome c o n ~ i s t e n t l yIn ~ ~80 . NHL‘s Ki-67 expression phase and as such measure the proliferation and the AgNOR method displayed a good correla- state. Information about the proliferation rate is tion”. Likewise a good linear correlation between the more difficult to obtain, but might be more mean numbers of AgNOR sites per nucleus and the informative than the proliferative state. It requires percentage of S-phase cells was found, both values measurement of the rate at which cells enter a certain being high in high grade NHL and low in low grade cell cycle phase and of the time required to go through this phase. Therefore the development of double lesions46. staining DNA-BrdUrd techniques was of invaluable importance. With this procedure, information about 7 DISCUSSION AND FUTURE DIRECTIONS cell cycle kinetics including DNA synthesis time (Ts) and potential doubling time (Tpot) can be gathered Until now no convincing evidence has been presented using the data obtained in a single sample of cells. as to superiority of the assessment of either Ki-67 With special denaturation and fixation methods the expression, AgNOR, BrdUrd incorporation or other expression of membrane antigens is not suppressed. S-phase measurements as proliferation parameter. All This enables specific measurement of tumour kinetics methods allow for discrimination between low and of the lymphoma fraction. Moreover simultaneous high grade malignant NHL. Yet considerable overlap assessment of membrane antigens associated with between the categories occurs. This reduces their value proliferation can be done. Two recent studies describe an indirect fluorescence for the individual patient. As an independent prognostic factor they are hardly more powerful than double staining procedure for the simultaneous I
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detection of two different thymidine analogues in the same cell n u c l e ~ s ' ~ *By ' ~ adding . these two analogues at different points of time the rate of entry or exit of cell populations from distinct cycle compartments can be assessed, again providing data on the proliferation rate of these cells. For the future it seems justified to expect that tumour speciJic assessment (by means of double labeling techniques) of the prolgeration rate rather than state might add new information with more prognostic impact for the individual patient.
17. Begg, A. C. McNally, N. J. Shrieve, D. C. and Karcher, H. (1985) A method to measure the duration of DNA synthesis and the potential doubling time from a single sample. Cytometry, 6, 620-626. 18. Shibui, S. Hoshino, T. Vanderlaan, M. and Gray, J. W. (1989) Double labeling with iodo- and bromodeoxymidine for cell kinetic studies. J. Histochem. Cytochem., 37, 1007-101 I . 19. Bakker, P. J. M. Stap, J. Tukker, C. J. Oven van, C. H. Veenhof C. H. N. and Aten, J. (1991) An indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo- and chlorodeoxyuridine incorporated into DNA. Cytometry, 12, 366-372. 20. Kamentsky, L. A. (1973) Cytology automation. Adv. Eiol. Med. Phys., 14, 93. 21. Melamed, M. R. Mullaney, P. F. and Mendelsohn, M. L. (1979) Flow cytometry and sorting. John Wiley, and sons, New York. 22. Shapiro, H, M, (1985) Practical flow cytometry. Allan, R. Liss Inc New York. REFERENCES 23. Camplejohn, R. S. (1989). S-phase fraction in lymfoid tissue: fresh versus paraffin embedded tissue and DAPl versus PI 1. Rappaport, H, et a/.(1956) Follicular lymphoma: a reevaluation staining. Cytometry, 10, 41&6. of its position in the scheme of malignant lymphoma, based o n a 24. Editorial: (1985) Transferrin and its cellular receptor undersurvey of 253 cases. Cancer, 9, 792-821. score the importance of iron in cell growth. Nutr. Rev., 43, (2), 2. Lukes, R. J. and Collins, R. D. (1974 Immunologic 56. characterization of human malignant lymphomas. Cancer, 34, 25. Chitambar, C. e.a. (1988) Inhibition of leukemic HL60 cell growth by transferringallium: effects on ribonucleotide 1488. 3. Lennert, K. (1978) Malignant lymphomas other than Hodgkin's reductase and demonstration of drug synergy with hydroxyurea. Blood, 72, 1930. Disease. N.Y. Springer-Verlag. 4. The Non-Hodgkin's Lymphoma Pathologic Classification 26. Jandl, J. and Katz, J. (1963) The plasma to cell cycle of Group (1982) NCI sponsored study of classifications of transferrin. J. Clin. Invest., 42, 31426. non-Hodgkin's lymphomas: summary and description of a 27. Trowbridge, 1. and Shackelford, P. (1985) Structure and function of transferrin receptors and their relationship to cell Working Formulation for clinical usage. Cancer, 49, 21 12. growth. Eiochem. Soc. Symp., 51, 117. 5. Steel, G. C. (1977) Growth kinetic of tumours: Cell population kinetics in relation to the growth and treatment of cancer. 28. Galbraith, R. e.a. (1980) Transferrin binding to periferal blood lymphocytes activated by phytohemagglutinin involves a Clarondon Press, Oxford. specific receptor ligand interaction. J. Clin. Invest., 66, 1135. 6. Howard, A. and Pelc, S. R. (1985) Nuclear incorporation of 32 P as demonstrated by autoradiographs. Exp. Cell. Res., 2, 29. Weiel, J. and Hamilton, T. (1984) Quescent lymphocytes express intracellular transferrin receptors. Biochem. Eiophys. Res. 178-87. Common. 119, 598. 7. Cleaver. J. E. (1967) Thvmidine Metabolism and cell kinetics. J. Wiley, New York. 30. Larrick, J. and Logue G. (1980) Transferrin receptors on leukaemic cells. Lancet, ii, 862-863. 8. Mendelsohn, M. L. (1963) The growth fraction: A new concept 31. Huebers, H. and Finch, C. (1987) The physiology of transferrin applied to tumours. Science, 132, 1496. and transferrin receptors. Physiological Reviews, 67, 52041. 9. Tannock, I. F. (1967) A comparison of the relative efficiencies of various metaphase arrest agents, Exp. Cell Res., 47, 345-356. 32. Gerdes, J. Schwab, U. Lemke, H. and Stein, H. (1983) Production of a mouse monoclonal antibody reactive with a 10. Wright, N. A. and Appleton, D. R. (1980) The metaphase arrest technique; a critical review. Cell Tissue Kinet., 13, 643-663. human nuclear antigen associated with cell proliferation Int. J. 11. Dosik, G. W. Barlogie, B. Allen White, R. Gohdew, and Cancer., 31, 13-20, Drewinko, B. (1981) A rapid automated statmokinetic method 33. Gerdes, J. e.a. (1991) lmmunobiochemical and molecular biologic characterization of the cell proliferation associated for determination of in vitro cell cycle transit times. Cell Tissue nuclear antigen that is defined by monoclonal antibody Kinef.,14 121-134. Ki-67. Am. J. Pathol., 138, 867-873. 12. Darzynkiewicz, Z. Tragonos, F. and Kimmel. W. (1986) Assay of cell cycle kinetics by multivariate flow cytometry using the 34. Mathews, M. Bernstein, R. Franza, R. and Carrels, J. (1984) Identity of the proliferating nuclear antigen and cyprinciple of stathmokinesis. In: Gray, J. W. Darzynkiewicz, Z. Techniques in cell cycle analysis, Biological Methods, Humana clin. Nature, 309,374. Press Inc, Clifton New Jersey. USA, 291-336. 35. Salter, D. M. Krajewski, A. S. and Cunningham, S. (1988) Activation and differentiation antigen expression in B-cell 13. Taylor, J. H. Woods, P. S. and Hughes, W. L. (1957) The non-Hodgkins lymphoma. J. Pathol, 154(3), 209-22. organisation and duplication of chromosomes as revealed by autoradiograph studies using tritium-labeled thymidine. Proc. 36. Lloyd, R. V. Wilson, B. S. Varani, J. Gaur, P. K. Moline, S. and Makari, J. G. (1985) lmmunocytochemical characterisation of Natl. Acad. Sci., 43, 122. a monoclonal antibody that recognizes mitosing cells. Am. J. 14. Quastler, H. and Sherman, F. G. (1959) Cell population kinetics Pathol., 121, 275. in the intestinal epithelium of the mouse. Exp. Cell Rex, 17, 420438. 37. Chatterjee, A. Freeman, J. W. and Busch, H. (1987) Identification and partial characterisation of a Mr. 40.000 15. Bohmer, R. M. and Ellwart, J. (1981) Cell cycle analysis by nucleolar antigen associated with cell proliferation. Cancer combining the 5-bromodeoxyuridine/33258 Hoechst technique Res., 47, 1123. with DNA specific Ethidium Bromide Staining. Cytometry, 2, 38. Persson, H. Henninghausen, L. Taub, R. Degrado, W. and 31-34. Leder, P. (1984) Antibodies to human c-myc oncogens product: 16. Gratzner, H. G. (1982) Monoclonal antibody to 5-bromo and evidence of an evolutionary conserved protein induced during 5-iododeoxyuridine: a new reagent for detection of DNA cell proliferation. Science, 225, 687. replication. Science, 218, 474475. ~I
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PROLIFERATION INDICES IN LYMPHOMA-USEFUL? 39. Hsu, T. Spirito, S. and Pardue, M. (1975) Distribution of 18 + 285 ribosomal genes in mammalion genomes. Chromosoma, 53, 25. 40. Goodpasture, C. and Bloom, S. (1975) Visualisation of nucleolar organizer regions in mammalion chromosomes using silver staining organizer regions. Chromosome, 53, 37. 41. Hernandez-Verdun, D. Derenzini, M. and Bouteille, M. (1984) Relationship between AgNOR proteins and ribosomal chromatin in situ during drug induced RNA synthesis inhibition. J . Ultrastrucr. Res.. 88, 55-65. 42. DeCapoa, A. Baldini, A. Marieka, P. el al. (1985) Hormone regulated rRNA gene activity is visualised by selective staining of NOR’s. Cell Biol. Int. Rep., 9, 791-6. 43. Crocker, J. (1989)Proliferation indices in malignant lymphomas. Clin. Exp. Immunol. 77, 299-308. 44. Crocker, J. and Nar, P. (1987) NOR’s in lymphomas. J . Pathol., 151, 1 11-8. 45. Jan-Mohamed R. M. Armstrong, S. J. Crocker, J. Leyland M. J. and Hulten, M. A. (1989) The relationship between numbers of interphase NOR’s and NOR bearing chromosomes in NHL. J. Pathol., 158, 3-7. 46. Crocker, J. McCarthey, J. C. and Smith, P. J. (1988) Correlation between DNA flow cytometric and nucleolar organizer region data in non-Hodgkin’s Lymphoma’s J. Pathol., 154, 151-156. 47. Akerman, M. Brandt, L. Johnson, A. and Olson, H. (1987) Mitotic activity in NHL. Relation to the Kiel classification and to prognosis. Br. J Cancer, 55, 219-223. 48. Kvaloy, S. Marton, P. F. Kaalhaus 0, Hoie, J. FossAbrahamsen, A. and Godal. T. (1985) 3H thymidine uptake in B cell lymphomas-relationship to treatment, response and survival. Scand. J . Haematol, 34, 429. 49. Witzig Th, ea. (1989) Rapid S-phase determination of N H L with the use of an immunofluorescence bromodeoxyuridine labeling index procedure. Am. J. Clin. Pathol., 91, 298-301. 50. Bray’lan, R. C. (1980) Percentage of cells in the S-phase of the cell cycle in human lymphoma determined by flow cytometry. Correlation with labelings index and patient survival. CJjtome1r.v. I, 171. 51. Christensson B. (1989) Flow cytometric DNA analysis: a prognostic tool in NHL. Leuk. Res., 14 (4),307-14. 52. Brons, P. T. ea. (1990) Cell kinetics in acute myeloid leukemia and other hematological malignancies studied with in vivo lododeoxy uridine administration and flow cytometry. 3th N.U.C.C. symposium on recent developments in oncology. University of Nijmegen, Netherlands feb, 49-58. 53. Habeshaw, J. Lister, T. Stansfeld, A. and Graeves M. (1983) Correlation of transferrin receptor expression with histological class and outcome in NHL. Lancet. I , 498. 54. Barnett, D. e.a. (1987) Transferrin receptor expression in the leukaemias and lymphoproliferative disorders. Clin. Lab. Haemat., 9, 361-70. 55. Medeiros, L. Picker, L. Homing, S. and Warnke, R.(1988) Transferrin receptor expression by NHL. Cancer, 61, 184C51. 56. Greil. R. (1986) Growth fraction of tumor cells and infiltration density with natural killer-like (HNK I + ) cells in NHL. Br. J . Hemarol.. 62, 293-300. 57. Kvaloy S. (1984) Transferrin receptor and B-lymphoblast antigen: their relationship to DNA synthesis, histology and survival in B cell lymphomas. fnr. J. Cancer, 33, 173-177.
187
58. Gerdes J, Dallenberg, F. Lennert, K. Lemke, H. and Stein,
59.
60.
61. 62.
63.
64.
65. 66. 67. 68. 69. 70.
71.
72. 73. 74.
H. (1984)Growth fractions in malignant NHL as determined in situ with the moabKi67. Hemurol. Oncol., 2, 365. Weiss, L. Stricker, J. Medeiros, L. Gerdes, J. Stein, H. and Warnke, R. (1987) Proliferative rates of non-Hodgkin’s Lymphomas a s assessed by Ki67 antibody. Hum. Pathol., I, 8(ii), 1155-9. Veneroni, S. Costa, A. Motta, R. Giurdini, R. Rilke, F. and Silvertrini, R. (1988) Comparative analysis of [3H]-thymidine labeling index and monoclonal antibody Ki67 in non-Hodgkins lymphomas. Hemurol. Oncol., 6(1), 21-8. Hall, P. Richards, M. Gregory, W. d’Ardenne, A. Lister, T. and Stansfield, A.( 1988) The prognostic value of Ki67 immunostaining in NHL. J. Puthol., 154, 223. Gregan, Th, Lippman, S. Spier, C. Slymen, B. Rijbski, J. Rangel, C . Richter, L. and Miller Th. (1988) Independent prognostic significance of a nuclear proliferation antigen in diffuse large cell lymphomas as determined by the monoclonal antibody Ki67. Blood, 74(4), 1157-1 160. Blockstaele, P. van, Lan, J. Peetermans, M. (1990) A multiparameter flow cytometric study of Ki67 reactivity versus cell cycle analysis on normal and pathological bone marrow samples. 3th N.U.C.C. Symposium on recent developments in oncology, University of Nijmegen, Netherlands feb: 3 1 4 1 . Smetana, K. Gyorkey, F. Chan, P. K. Tan, E. and, Busch, H. (1983) Proliferating cell nuclear antigen (PCNA) and human tumor nucleolar antigens (HMTNA) in nucleoli of human hematological malignancies. Blut., 46,133 Crocker, J. and Egan, M. J. (1988) Correlation between NOR sizes and numbers in non Hodgkin’s lymphomas. J. Path., 156, 233. Crocker, J. (1989) Nucleolar organizers regions. In current topics in pathology. Springer Verlag Heidelberg. Schrape, S. Jones, D. B. and Wright, D. H. (1987) A comparison of three methods for the determination of the growth fraction in non-Hodgkin’s lymphoma. Br. J . Cuncer, 55, 283-286. Kawauchi, K. e.a. (1989) Determination of proliferative fractions detected by immunohistochemistry with the moabs to BrdU and Ki67 in NHL. Hemarol. Oncol., 7(4), 257-65. Ciball, M. e.a. (1989) The utility of Ki67 immunostaining nuclear organizer region counting and morphology in the assessment offollicular lymphomas. J. Pathol., 158, (3) 189-93. Pileri, S. Gerdes, J. Rivano, M. Tazzari, P. L. Magnani, M. Gobbi, M. and Stein, H. (1987) immunohistochemical determination of growth fractions in human permanent cell lines and lymphoid tumours: a critical comparison of the monoclonal antibodies OKT9 and Ki-67. Br. J. Haemutol., 65, 27 1-276. Hall, P. A. Crocker, J. Watts, A. and Stansfeld, A. G . (1988) A comparison of nucleolar organizer region staining and Ki-67 immunostaining in non-Hodgkin’s lymphoma. Histopathol, 12, 373-381. McFarlane, J. H. (1986) Flow cytometric analysis of DNA heterogeneity in NHL. J. Path., 149, 236a. Norton, L. (1985) Implications of kinetic heterogeneity in clinical oncology. Semin. Oncol., 12, 23149. Almendral, J. M. Huebsch D. e.a. (1987). Cloning and sequence of the human nuclear protein cyclin: homology with DNA-binding proteins. Proc. Natn. Acud. Sci. USA, 84, 1575-1579.
Leukemia & Lymphoma Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ilal20
Measurement of Proliferation Indices in Non Hodgkin's Lymphoma—Is it Useful? a
b
a
K. J. Tusenius , P. J. M. Bakker & M. H. J. Van Oers a
Department of Haematology, Academic Medical Center, Amsterdam, The Netherlands
b
Department of Oncology, Academic Medical Center, Amsterdam, The Netherlands Published online: 01 Jun 2015.
To cite this article: K. J. Tusenius, P. J. M. Bakker & M. H. J. Van Oers (1992) Measurement of Proliferation Indices in Non Hodgkin's Lymphoma—Is it Useful?, Leukemia & Lymphoma, 7:3, 181-187, DOI: 10.3109/10428199209053621 To link to this article: http://dx.doi.org/10.3109/10428199209053621
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Measurement of Proliferation Indices in Non Hodgkin’s Lymphoma-Is it Useful? K. J. TUSENIUS’, P. J. M. BAKKER,, and M. H. J. VAN OERS’ Departments of Haematology and Oncology’, Academic Medical Center, Amsterdam, The Netherlands
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(Received 21 November 1991)
In this review we discuss the possible value of additional information concerning growth characteristics of NHL‘s for the prognosis of the individual patient. Techniques for measuring cell cycle kinetic parameters (e.g. S-phase assessment) and proliferation related antigens (e.g. anti-transferrin receptor and Ki67) are shortly reviewed. This is followed by an overview of proliferation studies in NHL. Until now in most of these studies overall measurements of tumour tissue have been performed. Moreover the parameters that have been assessed are related to proliferation state rather than proliferation rate. None of the methods used has a convincing superiority with regard to clinical outcome. In the future, tumour specific assessment (by means of double-labeling techniques) of proliferation rate parameters might provide information, more relevant to the prognosis of the individual patient with NHL. KEY WORDS:
I INTRODUCTION
Non Hodgkin’s Lymphoma proliferation related antigens
cell cycle kinetic studies prognosis
tion and selection of therapy. Knowledge of the growth characteristics of tumours might be a useful addition to the already existing classifications5. In this report we will give a brief overview of several new techniques which measure proliferative activity of NHL. Their possible value for the assessment of prognosis, when used alone or in combination with other parameters will also be discussed.
Early classifications of non Hodgkin’s Lymphoma’s (NHL) were based on morphology and enzyme histochemistry while later classifications incorporated both immunophenotype and differentiation state of the NHL2*3*.In an effort to correlate histopathological and survival characteristics of NHL, the so called Working Formulation (W.F.)4was developed during the early nineteen eighties. It is a compromise between the many and confusing classifications and its main emphasis is on clinical 2 CELL PROLIFERATION utility. However a serious shortcoming of the W.F. is that it fails to identify NHL‘s by cell lineage, and it Based on studies in the 1950s, using 32P incorporadoes not distinguish the immunological variants nor tion and autoradiography, the concept of the cell cycle the individual lymphomas by biological behaviour. was postulated6. Pulse labeling of growing cells by This is a major drawback for accuracy in prognostica- radiolabeled DNA precursors, including 3H-thymidine’ enabled subdivision of the cell cycle into several phases (mitotic or M-phase, synthetic or S-phase, and Address for correspondence: K. J. Tusenius, Academic Medical Center, F4-224, Meibergdreef 9, 1105 AZ Amsterdam, The two gap-phases G, and G,) and calculation of the Netherlands. duration of the cell cycle time. Further studies showed
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that the increase in tumour mass observed in vivo was invariably less than expected from the measured cell cycle duration and this finding gave rise to the idea that only a proportion of the tumour cell population was actively proliferating. Non-proliferating cells may temporarily leave the cell cycle (Gophase cells) or may differentiate and ultimately die. Moreover, it became evident that there is considerable cell loss within a tumour, due to insufficient vascularization, immunological responses of the host, errors of DNA replication etc. In rapidly growing tumours cell death frequently comprises up to 90% of the cell production rate5. In histological sections this can be seen as areas of necrosis. Tumours are thus comprised of cycling and non-cycling cells, the latter consisting of both Go cells and doomed cells. The ratio of cycling to cycling plus non-cycling cells has been defined as the proliferative or growth fraction'. Based on this concept of the cell cycle several approaches have been used to determine the proliferative activity of tumours. They include the measurement of the number of cells in certain phases of the cell cycle, measurement of cell cycle duration, and assessment of proliferation associated antigens, which are expressed during one or more cell cycle phases. We will briefly describe the principles of the most relevant methods for assessment of proliferative activity. This will be followed by a critical review of studies performed in NHL.
3.2 Labeling of cells in DNA synthesis
3.2.I Thymidine labeling The thymidine labeling technique was introduced by Taylor13 and has since that time gained an important place in the study of cell cycle kinetics5. The basic method is simple. TritiumC3H] or carbon-14[ 14C] labeled thymidine is incorporated in cells actively synthesizing DNA. The incorporated radioactive labels can be detected by means of autoradiography. The type of information obtained with this method depends on whether the labels are administered as a pulse, as a (semi-) continuous infusion, or as a combination of both and whether multiple biopsies can be taken from a tumour. The two most commonly used cell kinetic assays based on autoradiography are the determination of the labeling index (L.I., the fraction of labeled cells) and the fraction of pulse labeled mitoses (PLM)14. For the assessment of the L.1, cells are pulse labeled with [3H]thymidine. L.I. is dependent on both the fraction of proliferating cells and the duration of the S-phase. The L.I. is often used as a measure of proliferative activity. In the PLM procedure in analogy to the L.I., cells are exposed to a pulse label of C3H] thymidine. Thereafter at various intervals series of samples of the tumour are taken. From the rise and fall of the number of labeled mitoses at various time intervals several important cell proliferative parameters can be derived e.g. duration of the cell cycle time and of the different cell cycle phases. For the analysis of cell cycle kinetic parameters in human tumours the use of radioactive 3 DETERMINATION OF PROLIFERATION thymidine combined with the autoradiography INDICES BY MEASURING CELL CYCLE technique has however several limitations inherent to KINETIC PARAMETERS the use of radioactive compounds, the cumbersome and time consuming radiographic procedure and the 3.1 Assessment of mitotic index (M.I.) requirement of multiple biopsies. Therefore this This was the first kinetic technique used. It is a simple method has been largely abandoned. quantitative method using microscopically observable characteristics of cells. The number of mitoses is a 3.2.2 Non-radioactive thymidine analogues function of both the number of proliferating cells and This method is rapid and easy to perform and thus of the duration of the observable part of the cell facilitates the study of cell cycle kinetics in human division. As a general rule when the mitotic index is tumours. The method uses the thymidin analogs high, proliferation is rapid and vice versa. Usually the bromodeoxyuridine (BrdUrd) or iododeoxyuridine fraction of metaphase cells is small (0.5-2%). (IdUrd) which are incorporated in cells actively Therefore many cells have to be examined in order to synthesizing DNA. The incorporated labels can be derive an accurate value of M.I. M.I. assessment can detected by quenching the dye Hoechst 33258 or by be improved by treating a proliferating cell population the use of a monoconal antibody15*16.The labewith an agent which arrests all cells in metaphase (e.g. lings index is simply measured as the proportion of In addition cells that have taken up BrdUrd or IdUrd. This colcemid, vincristine or ~inblastine)~*". the application of flow cytometry allows a more method has the advantage that by means of the simultaneous measurement of DNA content and accurate measurement of M.1.' '*12.
PROLIFERATION INDICES IN LYMPHOMA-USEFUL?
BrdUrd fluorescence from a single sample taken a few hours after pulse labeling DNA synthesis time (Ts) and the potential doubling time (Tpot) of a tumour can be calculated”. Two recently developed labelingand staining procedures using either BrdUrd and IdUrd or IdUrd and chlorodeoxyuridine (CldUrd) as a label even further expand the possibilities of detailed cell cycle kinetic studies in human t ~ m o u r s ’ ~ * ’ ~ .
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3.3 Distribution of cellular DNA content The introduction of flow cytometry20.21*22 has provided a rapid method for measuring the distribution of cells over the different cell cycle phases. After preparing a single cell suspension, cells are stained with a DNA specific fluorescent dye e.g. ethidiumbromide, Hoechst 33258, chromomycin or propidiumiodide, The relative DNA content of each cell is thus measured in several thousands of cells and the derived DNA histogram can be analyzed through one of many computer programs. It allows the assessment of the fractions of cells in G , , S and G, + M phases respectively. The presence of aneuploidy complicates the assessment of the S-phase, but with special mathematical analysis this can be estimated23. The disadvantage of this method is that no information is obtained about actual phase durations or growth fractions. Therefore the actual cell cycle kinetic information which can be obtained is limited.
4 MEASUREMENT OF PROLIFERATION RELATED ANTIGENS The development of immunocytochemical techniques produced a new interesting area of proliferation studies. Several antigens have been described, which are expressed in or on the nucleus, in the cytoplasm or on the membrane of cells in association with proliferation. Some of them, especially those applied to NHL will be discussed below. 4.1 Transferrin Receptor (TfR) Iron is essential for every human cell. It plays a central role in all oxidative energy metabolism and in cellular growth and p r ~ l i f e r a t i o nThis ~ ~ . probably reflects the iron dependency of ribonucleotide reductase, the enzyme responsible for synthesis of deoxyribonucleotides from ribon~cleotides~~. Studies with immature red cells showed that iron uptake occurred via the
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binding of the serum iron transport protein transferrin to a specific cell surface receptor26. Transferrin-mediated iron uptake has been shown to be an absolute requirement for growth of many different cell types”. The number of TfR’s on the cell surface is related to the iron uptake and reflects the cellular iron requirement. Resting lymphocytes hardly bind any transferrinZ8.However after stimulation the number of TfR’s is increased to about 80.000 per cell29.This is caused by externalisation of intracellular receptor^,^. With the upregulation of TfR’s resting cells progress from the G, phase into S-phase3’. Therefore modulation of TfR activity is important for regulation of the proliferative state3 Recently the monoclonal antibodies (moab’s) against the TfR were clustered as CD71. The TfR is now well characterized biochemically and at the DNA level,’. A drawback for the use of the TfR measurement as a proliferation parameter in malignancies, is its expression on monocytes/macrophages, and on nonmalignant reactive lymphocyte^^^*'^.
’.
4.2 Ki-67 In 1983 the production of the mouse monoclonal antibody termed Ki-67, recognizing a nuclear antigen present in proliferating cells was described3’. Recently the antigen was characterized immunobiochemically and by molecular biological technique^^^. Its expression was found to be restricted to G , , s, G, and M-phase of the cellcycle, and absent in Go phase.Thus, the Ki-67 moab enables determination of the growth fraction of a cell population. 4.3 Other antigens related to cell proliferation Several other antigens have been studied in relation to cell proliferation and the cellcycle. Proliferating cell nuclear antigen (PCNA) is formed at rates related to both cell proliferation and DNA synthesis34. It was found to be identical to cyclin (an auxiliary protein of DNA polymerase) a polypeptide of 36 K D with an is0 electic point of 4.8. Recently the sequence of the DNA coding for PCNA has been determined74. PCNA begins to accumulate during the G, phase, peaks during S-phase and declines in G, and M-phase. Except for the activation-associated 4F2 antigen which is expressed early in the GO-G1 phases of the ~ e l l c y c l e ~other ~ , putative proliferation associated markers like C5F1036,the p-40 protein3’ and the c-myc oncogene38 have not yet been sufficiently studied in relation to NHL to be discussed.
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NUCLEOLAR ORGANIZER REGIONS (NORs)
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the transferrin receptor in NHL have yielded conflicting results. Some authors have reported a positive correlation between the number of TfR Nucleolar organizer regions (NORs) represent loops positive cells and the histological malignancy grade of of DNA, which contain the genes for ribosomal RNA NHLS3. However, others found TfR expression in and proteins. These were first described in the only a minority of NHL (34%); and with a low 1 9 7 0 ' ~ ~ ~The *~O function . of the nucleic acid-protein densitys4. More recently in a large retrospective study complexes is not fully understood, but seems to be a on cryostat sections of 267 NHL'sSS. TfR expression regulatory one in maintaining DNA in an extended was found in a wide variety of B and T lineage NHL's configuration4' or in controlling the transcription of and to be only loosely correlated with the the genes for ribosomal RNA42. As might be expected International Working Formulation classification. In NORs, playing an important role in protein synthesis, general, high grade lymphomas expressed the TfR indicate cell activation or t r a n s f ~ r m a t i o n ~ ~ -A~ ' . more often and more intensely. However exceptions linear correlation was found between AgNOR score were found in certain histological subtypes. This per nucleus and S-phase ~ e r c e n t a g e ~ ~ . discrepancy was also found in another studys6. Similary, conflicting data have been obtained regarding the correlation between TfR expression and 6 PROLIFERATION STUDIES IN NHL p r o g n ~ s i s These ~ ~ ~ confusing ~~. data might partly be due to TfR expression on monocytes/macrophages, Because counting of mitoses is a time consuming and and on non-malignant reactive lymphocytes28929. This insufficiently standardized procedure, it has been makes TfR less specific as a marker for tumour performed in NHL in a limited scale only47. This is p r o l i f e r a t i ~ n ~ *O*n~ ~the . other hand differences in in contrast to S-phase assessment. As might be technique might explain these disagreements: memexpected pulse labeling studies using 'H thymidine4' brane-expression of the TfR as detected by flow or BrdUrd49 as well as S-phase determinations with cytometry might not be comparable to the combinaDNA histogram^'^.^ have yielded comparable re- tion of membrane- and cytoplasmatic expression as sults. In all these studies a positive correlation was detected by cryostat section immunoperoxidasestainfound between the percentage of cells in the S-phase ing". In summary, the value of TfR measurements for and the malignancy grade of the NHL, according to subclassification and assessment of prognosis of the Working Formulation. However, the correlation NHL's seems to be limited. Ki-67 expression has been studied extensively in certainly was not an absolute one and many discrepancies have been observed, thus limiting its NHL and varing correlations between the immunocyusefulness for the individual patient. Although tochemical pattern and histological grade of malmeasurement of S-phase duration (Ts) and calcula- ignancy have been found. In general a low and high tions of the potential doubling time (T pot) have been Ki67 score were obtained in low and high grade performed in vitro for many years, only recently malignant NHL's re~pectively'~-~'.Often however, techniques have become available for in vivo considerable overlap between both groups exists, procedure^'^. Therefore extensive studies on the value which precludes accurate diagnosis in individual of the measurement of these dynamic cell cycle cases. Within the group of low grade NHL a relative parameters in NHLs are lacking at the moment. In a high Ki-67 score correlated with bad o u t ~ o m e ~ ~ . ~ ~ . recent study preliminary results of this procedure in For high grade lymphoma inconsistant data about a limited number of NHL patients have been Ki-67 expression have been observed: one study reported". Although a large variation in L.I. was reporting very high Ki-67 scores correlating with observed between tumours of the same histological better survival6 while another showed the oppograde, an inverse correlation was found between site6'. The surprising fact that high grade lymphomas grade of malignancy and T pot. The number of with high Ki-67 indices did better clinically than those patients and their follow-up were still inadequate to with lower indices was postulated to be due to the allow conclusions as to its value for assessment of larger fraction of proliferative cells which are susceptible to chemotherapy. Another explanation for individual prognosis. With the development of flow cytometry, measure- discrepancies between Ki-67 reactivity and histology ment of proliferation related antigens has gained or prognosis might be the fact that many of the early considerable popularity. Studies on the expression of data on the expression of Ki-67 in relation to the cell
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cycle were obtained with phytohaemagglutinin the existing subclassifications of NHL (e.g. the (PHA)-stimulated lymphocytes. These might not be a Working Formulation). There might be several perfect model of the proliferating compartment in explanations for this lack of prognostic value. First, tumours in vivo. Several mechanisms have been in assessing proliferation of malignant cells sample suggested to be responsible for the often lower than choice and size are of the utmost importance while expected Ki-67 score in human malignant tissues: heterogeneity is one of the characteristics of nutrient deprivation, quiescent S-phase cells and neoplasm^^^.^^. This means that in a tumour, possible inclusion of dead cells or aggregates in the malignant and non malignant areas can be found. analysis. Moreover, some cycling cells may not Within the malignant area cells are in different phases of the cell cycle. Also several other factors like express the Ki-67 antigen63. Other proliferation related antigens have not yet inclusion of dead cells, quiescent S-phase cells, loss of been studied extensively in NHL. Proliferating cells antigenic expression etc. can contribute to sampling in NHL express PCNA64. The 4F2 antigen is errors. In the classical histological assessment part of expressed more strongly and more often in high grade these complicating factors can be eliminated, whereas NHL than in other s u b c l a ~ s e s ~Finally ~. the with flow cytometric analysis they have to be taken assessment of silver stained NORs (AgNORs) in into account seriously. Thus, results obtained on paraffin slides for interphase chromosomes has also been found to allow discrimination between high and low grade example, cannot simply be compared with data obtained with flow cytometry of cell suspensions. On NHL6ss66. Since virtually all proliferation markers gage the other hand the advantage of the latter method is different cell cycle phases, in many studies two or more that by means of double labeling with tumour specific proliferation parameters have been compared. Corre- markers, in heterogeneous populations, the proliferalation was found between Ki-67 expression and the tion characteristics of the tumour population can be 3H-thymidine labeling index6’ and BrdUrd in- measured specifically. However, until now most of the corporation r e ~ p e c t i v e l y ~TfR ~ . ~measurements ~. did studies with flow cytometry have been performed on not show a similar correlation with Ki-67 expres- entire cell suspensions, resulting in an undersi0n6 7 69. This was explained by the finding of a high estimation of the proliferative activity of the tumour number of TfR positive cells in low grade lymphomas. cells. Secondly, in general, all the methods used for In a study comparing Ki-67 immunostaining, NOR counting and morphology in 36 follicular NHL’s all assessment of tumour proliferation only give a partial three methods proved to be equally useful for picture of tumour kinetics, because they provide subclassification. They also predicted poor clinical percentages of proliferating cells in a certain cell cycle outcome c o n ~ i s t e n t l yIn ~ ~80 . NHL‘s Ki-67 expression phase and as such measure the proliferation and the AgNOR method displayed a good correla- state. Information about the proliferation rate is tion”. Likewise a good linear correlation between the more difficult to obtain, but might be more mean numbers of AgNOR sites per nucleus and the informative than the proliferative state. It requires percentage of S-phase cells was found, both values measurement of the rate at which cells enter a certain being high in high grade NHL and low in low grade cell cycle phase and of the time required to go through this phase. Therefore the development of double lesions46. staining DNA-BrdUrd techniques was of invaluable importance. With this procedure, information about 7 DISCUSSION AND FUTURE DIRECTIONS cell cycle kinetics including DNA synthesis time (Ts) and potential doubling time (Tpot) can be gathered Until now no convincing evidence has been presented using the data obtained in a single sample of cells. as to superiority of the assessment of either Ki-67 With special denaturation and fixation methods the expression, AgNOR, BrdUrd incorporation or other expression of membrane antigens is not suppressed. S-phase measurements as proliferation parameter. All This enables specific measurement of tumour kinetics methods allow for discrimination between low and of the lymphoma fraction. Moreover simultaneous high grade malignant NHL. Yet considerable overlap assessment of membrane antigens associated with between the categories occurs. This reduces their value proliferation can be done. Two recent studies describe an indirect fluorescence for the individual patient. As an independent prognostic factor they are hardly more powerful than double staining procedure for the simultaneous I
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detection of two different thymidine analogues in the same cell n u c l e ~ s ' ~ *By ' ~ adding . these two analogues at different points of time the rate of entry or exit of cell populations from distinct cycle compartments can be assessed, again providing data on the proliferation rate of these cells. For the future it seems justified to expect that tumour speciJic assessment (by means of double labeling techniques) of the prolgeration rate rather than state might add new information with more prognostic impact for the individual patient.
17. Begg, A. C. McNally, N. J. Shrieve, D. C. and Karcher, H. (1985) A method to measure the duration of DNA synthesis and the potential doubling time from a single sample. Cytometry, 6, 620-626. 18. Shibui, S. Hoshino, T. Vanderlaan, M. and Gray, J. W. (1989) Double labeling with iodo- and bromodeoxymidine for cell kinetic studies. J. Histochem. Cytochem., 37, 1007-101 I . 19. Bakker, P. J. M. Stap, J. Tukker, C. J. Oven van, C. H. Veenhof C. H. N. and Aten, J. (1991) An indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo- and chlorodeoxyuridine incorporated into DNA. Cytometry, 12, 366-372. 20. Kamentsky, L. A. (1973) Cytology automation. Adv. Eiol. Med. Phys., 14, 93. 21. Melamed, M. R. Mullaney, P. F. and Mendelsohn, M. L. (1979) Flow cytometry and sorting. John Wiley, and sons, New York. 22. Shapiro, H, M, (1985) Practical flow cytometry. Allan, R. Liss Inc New York. REFERENCES 23. Camplejohn, R. S. (1989). S-phase fraction in lymfoid tissue: fresh versus paraffin embedded tissue and DAPl versus PI 1. Rappaport, H, et a/.(1956) Follicular lymphoma: a reevaluation staining. Cytometry, 10, 41&6. of its position in the scheme of malignant lymphoma, based o n a 24. Editorial: (1985) Transferrin and its cellular receptor undersurvey of 253 cases. Cancer, 9, 792-821. score the importance of iron in cell growth. Nutr. Rev., 43, (2), 2. Lukes, R. J. and Collins, R. D. (1974 Immunologic 56. characterization of human malignant lymphomas. Cancer, 34, 25. Chitambar, C. e.a. (1988) Inhibition of leukemic HL60 cell growth by transferringallium: effects on ribonucleotide 1488. 3. Lennert, K. (1978) Malignant lymphomas other than Hodgkin's reductase and demonstration of drug synergy with hydroxyurea. Blood, 72, 1930. Disease. N.Y. Springer-Verlag. 4. The Non-Hodgkin's Lymphoma Pathologic Classification 26. Jandl, J. and Katz, J. (1963) The plasma to cell cycle of Group (1982) NCI sponsored study of classifications of transferrin. J. Clin. Invest., 42, 31426. non-Hodgkin's lymphomas: summary and description of a 27. Trowbridge, 1. and Shackelford, P. (1985) Structure and function of transferrin receptors and their relationship to cell Working Formulation for clinical usage. Cancer, 49, 21 12. growth. Eiochem. Soc. Symp., 51, 117. 5. Steel, G. C. (1977) Growth kinetic of tumours: Cell population kinetics in relation to the growth and treatment of cancer. 28. Galbraith, R. e.a. (1980) Transferrin binding to periferal blood lymphocytes activated by phytohemagglutinin involves a Clarondon Press, Oxford. specific receptor ligand interaction. J. Clin. Invest., 66, 1135. 6. Howard, A. and Pelc, S. R. (1985) Nuclear incorporation of 32 P as demonstrated by autoradiographs. Exp. Cell. Res., 2, 29. Weiel, J. and Hamilton, T. (1984) Quescent lymphocytes express intracellular transferrin receptors. Biochem. Eiophys. Res. 178-87. Common. 119, 598. 7. Cleaver. J. E. (1967) Thvmidine Metabolism and cell kinetics. J. Wiley, New York. 30. Larrick, J. and Logue G. (1980) Transferrin receptors on leukaemic cells. Lancet, ii, 862-863. 8. Mendelsohn, M. L. (1963) The growth fraction: A new concept 31. Huebers, H. and Finch, C. (1987) The physiology of transferrin applied to tumours. Science, 132, 1496. and transferrin receptors. Physiological Reviews, 67, 52041. 9. Tannock, I. F. (1967) A comparison of the relative efficiencies of various metaphase arrest agents, Exp. Cell Res., 47, 345-356. 32. Gerdes, J. Schwab, U. Lemke, H. and Stein, H. (1983) Production of a mouse monoclonal antibody reactive with a 10. Wright, N. A. and Appleton, D. R. (1980) The metaphase arrest technique; a critical review. Cell Tissue Kinet., 13, 643-663. human nuclear antigen associated with cell proliferation Int. J. 11. Dosik, G. W. Barlogie, B. Allen White, R. Gohdew, and Cancer., 31, 13-20, Drewinko, B. (1981) A rapid automated statmokinetic method 33. Gerdes, J. e.a. (1991) lmmunobiochemical and molecular biologic characterization of the cell proliferation associated for determination of in vitro cell cycle transit times. Cell Tissue nuclear antigen that is defined by monoclonal antibody Kinef.,14 121-134. Ki-67. Am. J. Pathol., 138, 867-873. 12. Darzynkiewicz, Z. Tragonos, F. and Kimmel. W. (1986) Assay of cell cycle kinetics by multivariate flow cytometry using the 34. Mathews, M. Bernstein, R. Franza, R. and Carrels, J. (1984) Identity of the proliferating nuclear antigen and cyprinciple of stathmokinesis. In: Gray, J. W. Darzynkiewicz, Z. Techniques in cell cycle analysis, Biological Methods, Humana clin. Nature, 309,374. Press Inc, Clifton New Jersey. USA, 291-336. 35. Salter, D. M. Krajewski, A. S. and Cunningham, S. (1988) Activation and differentiation antigen expression in B-cell 13. Taylor, J. H. Woods, P. S. and Hughes, W. L. (1957) The non-Hodgkins lymphoma. J. Pathol, 154(3), 209-22. organisation and duplication of chromosomes as revealed by autoradiograph studies using tritium-labeled thymidine. Proc. 36. Lloyd, R. V. Wilson, B. S. Varani, J. Gaur, P. K. Moline, S. and Makari, J. G. (1985) lmmunocytochemical characterisation of Natl. Acad. Sci., 43, 122. a monoclonal antibody that recognizes mitosing cells. Am. J. 14. Quastler, H. and Sherman, F. G. (1959) Cell population kinetics Pathol., 121, 275. in the intestinal epithelium of the mouse. Exp. Cell Rex, 17, 420438. 37. Chatterjee, A. Freeman, J. W. and Busch, H. (1987) Identification and partial characterisation of a Mr. 40.000 15. Bohmer, R. M. and Ellwart, J. (1981) Cell cycle analysis by nucleolar antigen associated with cell proliferation. Cancer combining the 5-bromodeoxyuridine/33258 Hoechst technique Res., 47, 1123. with DNA specific Ethidium Bromide Staining. Cytometry, 2, 38. Persson, H. Henninghausen, L. Taub, R. Degrado, W. and 31-34. Leder, P. (1984) Antibodies to human c-myc oncogens product: 16. Gratzner, H. G. (1982) Monoclonal antibody to 5-bromo and evidence of an evolutionary conserved protein induced during 5-iododeoxyuridine: a new reagent for detection of DNA cell proliferation. Science, 225, 687. replication. Science, 218, 474475. ~I
Downloaded by [Deakin University Library] at 01:47 10 August 2015
PROLIFERATION INDICES IN LYMPHOMA-USEFUL? 39. Hsu, T. Spirito, S. and Pardue, M. (1975) Distribution of 18 + 285 ribosomal genes in mammalion genomes. Chromosoma, 53, 25. 40. Goodpasture, C. and Bloom, S. (1975) Visualisation of nucleolar organizer regions in mammalion chromosomes using silver staining organizer regions. Chromosome, 53, 37. 41. Hernandez-Verdun, D. Derenzini, M. and Bouteille, M. (1984) Relationship between AgNOR proteins and ribosomal chromatin in situ during drug induced RNA synthesis inhibition. J . Ultrastrucr. Res.. 88, 55-65. 42. DeCapoa, A. Baldini, A. Marieka, P. el al. (1985) Hormone regulated rRNA gene activity is visualised by selective staining of NOR’s. Cell Biol. Int. Rep., 9, 791-6. 43. Crocker, J. (1989)Proliferation indices in malignant lymphomas. Clin. Exp. Immunol. 77, 299-308. 44. Crocker, J. and Nar, P. (1987) NOR’s in lymphomas. J . Pathol., 151, 1 11-8. 45. Jan-Mohamed R. M. Armstrong, S. J. Crocker, J. Leyland M. J. and Hulten, M. A. (1989) The relationship between numbers of interphase NOR’s and NOR bearing chromosomes in NHL. J. Pathol., 158, 3-7. 46. Crocker, J. McCarthey, J. C. and Smith, P. J. (1988) Correlation between DNA flow cytometric and nucleolar organizer region data in non-Hodgkin’s Lymphoma’s J. Pathol., 154, 151-156. 47. Akerman, M. Brandt, L. Johnson, A. and Olson, H. (1987) Mitotic activity in NHL. Relation to the Kiel classification and to prognosis. Br. J Cancer, 55, 219-223. 48. Kvaloy, S. Marton, P. F. Kaalhaus 0, Hoie, J. FossAbrahamsen, A. and Godal. T. (1985) 3H thymidine uptake in B cell lymphomas-relationship to treatment, response and survival. Scand. J . Haematol, 34, 429. 49. Witzig Th, ea. (1989) Rapid S-phase determination of N H L with the use of an immunofluorescence bromodeoxyuridine labeling index procedure. Am. J. Clin. Pathol., 91, 298-301. 50. Bray’lan, R. C. (1980) Percentage of cells in the S-phase of the cell cycle in human lymphoma determined by flow cytometry. Correlation with labelings index and patient survival. CJjtome1r.v. I, 171. 51. Christensson B. (1989) Flow cytometric DNA analysis: a prognostic tool in NHL. Leuk. Res., 14 (4),307-14. 52. Brons, P. T. ea. (1990) Cell kinetics in acute myeloid leukemia and other hematological malignancies studied with in vivo lododeoxy uridine administration and flow cytometry. 3th N.U.C.C. symposium on recent developments in oncology. University of Nijmegen, Netherlands feb, 49-58. 53. Habeshaw, J. Lister, T. Stansfeld, A. and Graeves M. (1983) Correlation of transferrin receptor expression with histological class and outcome in NHL. Lancet. I , 498. 54. Barnett, D. e.a. (1987) Transferrin receptor expression in the leukaemias and lymphoproliferative disorders. Clin. Lab. Haemat., 9, 361-70. 55. Medeiros, L. Picker, L. Homing, S. and Warnke, R.(1988) Transferrin receptor expression by NHL. Cancer, 61, 184C51. 56. Greil. R. (1986) Growth fraction of tumor cells and infiltration density with natural killer-like (HNK I + ) cells in NHL. Br. J . Hemarol.. 62, 293-300. 57. Kvaloy S. (1984) Transferrin receptor and B-lymphoblast antigen: their relationship to DNA synthesis, histology and survival in B cell lymphomas. fnr. J. Cancer, 33, 173-177.
187
58. Gerdes J, Dallenberg, F. Lennert, K. Lemke, H. and Stein,
59.
60.
61. 62.
63.
64.
65. 66. 67. 68. 69. 70.
71.
72. 73. 74.
H. (1984)Growth fractions in malignant NHL as determined in situ with the moabKi67. Hemurol. Oncol., 2, 365. Weiss, L. Stricker, J. Medeiros, L. Gerdes, J. Stein, H. and Warnke, R. (1987) Proliferative rates of non-Hodgkin’s Lymphomas a s assessed by Ki67 antibody. Hum. Pathol., I, 8(ii), 1155-9. Veneroni, S. Costa, A. Motta, R. Giurdini, R. Rilke, F. and Silvertrini, R. (1988) Comparative analysis of [3H]-thymidine labeling index and monoclonal antibody Ki67 in non-Hodgkins lymphomas. Hemurol. Oncol., 6(1), 21-8. Hall, P. Richards, M. Gregory, W. d’Ardenne, A. Lister, T. and Stansfield, A.( 1988) The prognostic value of Ki67 immunostaining in NHL. J. Puthol., 154, 223. Gregan, Th, Lippman, S. Spier, C. Slymen, B. Rijbski, J. Rangel, C . Richter, L. and Miller Th. (1988) Independent prognostic significance of a nuclear proliferation antigen in diffuse large cell lymphomas as determined by the monoclonal antibody Ki67. Blood, 74(4), 1157-1 160. Blockstaele, P. van, Lan, J. Peetermans, M. (1990) A multiparameter flow cytometric study of Ki67 reactivity versus cell cycle analysis on normal and pathological bone marrow samples. 3th N.U.C.C. Symposium on recent developments in oncology, University of Nijmegen, Netherlands feb: 3 1 4 1 . Smetana, K. Gyorkey, F. Chan, P. K. Tan, E. and, Busch, H. (1983) Proliferating cell nuclear antigen (PCNA) and human tumor nucleolar antigens (HMTNA) in nucleoli of human hematological malignancies. Blut., 46,133 Crocker, J. and Egan, M. J. (1988) Correlation between NOR sizes and numbers in non Hodgkin’s lymphomas. J. Path., 156, 233. Crocker, J. (1989) Nucleolar organizers regions. In current topics in pathology. Springer Verlag Heidelberg. Schrape, S. Jones, D. B. and Wright, D. H. (1987) A comparison of three methods for the determination of the growth fraction in non-Hodgkin’s lymphoma. Br. J . Cuncer, 55, 283-286. Kawauchi, K. e.a. (1989) Determination of proliferative fractions detected by immunohistochemistry with the moabs to BrdU and Ki67 in NHL. Hemarol. Oncol., 7(4), 257-65. Ciball, M. e.a. (1989) The utility of Ki67 immunostaining nuclear organizer region counting and morphology in the assessment offollicular lymphomas. J. Pathol., 158, (3) 189-93. Pileri, S. Gerdes, J. Rivano, M. Tazzari, P. L. Magnani, M. Gobbi, M. and Stein, H. (1987) immunohistochemical determination of growth fractions in human permanent cell lines and lymphoid tumours: a critical comparison of the monoclonal antibodies OKT9 and Ki-67. Br. J. Haemutol., 65, 27 1-276. Hall, P. A. Crocker, J. Watts, A. and Stansfeld, A. G . (1988) A comparison of nucleolar organizer region staining and Ki-67 immunostaining in non-Hodgkin’s lymphoma. Histopathol, 12, 373-381. McFarlane, J. H. (1986) Flow cytometric analysis of DNA heterogeneity in NHL. J. Path., 149, 236a. Norton, L. (1985) Implications of kinetic heterogeneity in clinical oncology. Semin. Oncol., 12, 23149. Almendral, J. M. Huebsch D. e.a. (1987). Cloning and sequence of the human nuclear protein cyclin: homology with DNA-binding proteins. Proc. Natn. Acud. Sci. USA, 84, 1575-1579.
