Growth fraction of human tumors: Assays and complications

Growth Fraction of Human Tumors: Assays and Complications PeggyL. Olive,Jean C. LeRiche, and Stewart M.Jackson

he fraction of cells actively proliferating in human tumors is expected to be useful in predicting tumor response to therapy. 1,2 There are several excellent reviews 3-5 and a comprehensive monograph 6 describing both static and flow cytometry assays developed to measure growth fraction. This article introduces a new method, and in describing our results using clinical samples, provides a basis for understanding why this information is useful or necessary and identifies factors that may limit our ability to define growth fraction accurately for different tumor types. The doubling time of a tumor has long been known to be a valuable prognostic indicator. As an obvious generalization, patient survival is shorter when the tumor grows more rapidly, although tumor size and doubling time can vary independently] The recent application of accelerated radiotherapy to overcome rapid tumor cell repopulation a is in large part responsible for the current interest in categorizing tumors into slowly and rapidly proliferating. 9 There are three major kinetic factors contributing to tumor doubling time: growth fraction (GF), cell cycle time, and cell loss rate. l~ Although no method is able to accurately determine cell loss rate in human tumors, the two remaining factors are used to describe potential tumor doubling time (Tpot), which is proportional to the S phase duration (T~) divided by the labeling index (LI), or alternatively, the cell cycle duration divided by the GF. II It has been argued that Tpot is more informative than GF alone in predicting tumor response to accelerated radiotherapy because Tpo~ includes a measure of rate of cell proliferation (Ts) as well as state of proliferation (LI). However, when analyzing the components that determine Tpo, it is common for tumors of the same tissue of origin

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From the British Columbia CancerResearch Centre, Vancouver. Supported by the National Cancer Institute of Canada with funds provided by the Canadian CancerSociety. Address reprint requests to Peggy L. Olive, PhD, Medical Biophysics Department, British Columbia Cancer Research Centre, 601 W lOth Ave, Vancouver,B.C Canada, V5Z 1L3. Copyright 9 1993 by WB. Saunders Company 1053-4296/93/0302-0004505.00/0

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to have similar "Is values, 12 which argues that the main contributor to differences in Tpot is GF. GF can vary considerably between different tumor types, within tumor groups, and even within different parts of the same tumor. IH4 Analysis of GF in tumor cells can be accomplished using a variety of antibodies that detect proteins associated with proliferating cells (ie, Ki-67, DNA polymerase delta, PCNA/cyclin, nucleolar antigens p120 and p145, and topoisomerase II), 3 or which detect proteins associated with the nonproliferating state (eg, statin)? 6 Histone H3 mRNA measured by in situ hybridization may also be an effective method to detect proliferating cells in histological material. 17 Alternatively, 3H-thymidine and BrdUrd incorporation into S phase cells have been used as functional assays of cell proliferation. 3H-thymidine labeling index measured in tumor samples removed at the time of surgery has been of prognostic value in cancer of the breast, ovaries, and head and neck) a-2~ Examination of binding in tissue sections provides information on location of proliferating cells within the tumor cords and often allows a distinction between tumor and normal cells based on appearance (ie, nuclear shape, nuclear/cytoplasmic ratio). Intracellular location and patterns of binding of immunohistochemical stains can be determined that could allow detection of a subpopulation of evolving, rapidly growing, tumor cells within a more slowly growing tumor. For example, it has been noted that tumor GF is generally higher at the periphery than at the center of breast cancers) 3 These advantages are lost when a tumor is reduced to a single cell suspension for GF measurements because identification of normal cells can be problematic. In addition, flow cytometry signals are integrated over the whole cell, precluding measurement of intracellular localization or patterns of binding. Because information on relative cell location is lost for any monodispersed cell analysis method, average GF may underestimate growth potential. However, the ability to make use of fine needle aspirates is an important practical advantage.

Seminars in Radiation Oncology, Vo13, No 2 (April), 1993:pp 90-98

GrowthFractionofHuman Tumors

The C o m e t / E t o p o s i d e Assay to Measure GF The comet assay was introduced by Ostling and Johanson in 198421 as a method to measure DNA damage in individual cells. We have modified this method to detect and quantify DNA single and/or double-strand breaks. 22-24Sensitivity is excellent, and resolution is sufficient to allow detection of subpopulations differing in DNA damage by a factor of 3 or more. 23 Image analysis software has been developed by Dr Ralph Durand to objectively quantify damage in individual cells. The advantage of this method when applied to heterogenous populations of cells from tumors should be apparent; rather than relying on information from population averages, it is possiBle to detect subpopulations of cells that may be resistant to a specific DNA-damaging drug. One drug of particular interest in this regard is etoposide, an antitumor agent effective for testicular and lung cancer, lymphoma and leukemia. 25A major cause of toxicity is believed to be binding ofetoposide to the nuclear enzyme, topoisomerase II, and trapping of this enzyme in a "cleavable complex" with DNA2628; the resulting DNA strand breaks can be readily detected using the comet assay. 29 Topoisomerase II is an essential enzyme for cell proliferation, but is rapidly lost from nonproliferating cells. 3~ Topoisomerase II and the Ki-67 antigen share several interesting properties as discussed by Verheijin et a l y including close association of both proteins with the nuclear matrix and mitotic scaffold. Because etoposide produces DNA breaks only in proliferating cells that contain topoisomerase II, the combination of etoposide with the comet assay provides an obvious method for measuring tumor proliferative capacity; GF is simply the fraction of tumor cells showing DNA damage following exposure to etoposide. Application of the etoposide/comet assay is relatively straightforward and can be completed within 3 to 4 hours. A single cell suspension, obtained by fine needle aspiration, is incubated for 30 minutes with 5 #,g/mL etoposide. Cells are then embedded in 0.75% low gelling temperature agarose that is pipetted onto a microscope slide. Slides are placed in 1 mol/L NaCI, 0.03 mol/L NaOH, 0.1% sarkosyl for 1 hour to lyse cell membranes and release proteins that mask the sites of etoposide interaction with topoisomerase II and DNA. Slides are then rinsed in alkali to remove salt, which retards migration, and exposed to low voltage (0.6 V/cm) for 25 minutes.

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Electrophoresis draws broken DNA away from the comet "head" (ie, the cell nucleus) and into the "tail." Slides are then placed in a solution containing 2.5 Ixg/mL propidium iodide, which binds stoichiometrically to DNA and produces a strong fluorescence signal. Image cytometry is used to quantify both DNA content and DNA damage. A linear relation is found between the number of DNA single-strand breaks present in a cell and the "tail moment," which we define to be the product of tail length and amount of DNA in the tail. 22,29As shown in Fig 1, DNA damage by etoposide is readily detected in cultured human tumor cell lines and in tumor cells obtained from fine needle aspirates (FNAs). As expected, cells from FNAs show less

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Etoposide (gg/ml) F i g u r e 1. DNA damage by etoposide to human tumor cell lines (. . . . ) or human tumor cells from fine needle aspirates (--). DNA damage was measured in individual cells using the comet assay; the tail moment is proportional to the number of single strand breaks present in the cell. For these experiments, cells were exposed to etoposide for 1 hour at 37~ and dose response curves were obtained by averaging the tail moment for 40 to 200 individual comets per dose point. Lines are drawn through the data points. Human tumor cell lines included SiHa cervical, WiDr colon carcinoma, DU-145 prostate, and HT-144 melanoma. Fine needle aspirates were obtained from breast, lung, and head and neck tumors.

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etoposide-induced DNA damage than do exponentially growing cultured cell lines in which the GF is essentially 100%. Figure 2 is an example of the application of the etoposide/comet assay in a well-defined human tumor model. WiDr human colon carcinoma cells grown as spheroids are more resistant than exponentially growing WiDr monolayers to DNA damage by etoposide because the GF of the spheroids is reduced to about 30%. Whereas the decrease in the slope of the dose-response curve (Fig 2A) is an indication of a reduction in GF, the histograms shown in Fig 2B show more convincingly that a fraction of the cells (primarily those located in the inner layers of the spheroid) are resistant to etoposide, while the outer cycling cells remain sensitive. Validation of the comet/ etoposide method was achieved in WiDr spheroids by correlations with GF determined by Ki-67 binding and BrdUrd incorporation. 33 In addition, sensitivity to etoposide was seen in FrFC-conjugated antiBrdUrd antibody-labelled comets only, providing direct proof that the comet/etoposide method detects only those cells capable of incorporating BrdUrd in the 24 hours before the assay.33 A small fraction of untreated cells will also show

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DNA damage following fine needle aspiration, but it is a simple matter to calculate the proportion of treated cells that show DNA damage in excess of the untreated sample (or more accurately, 95% of the values obtained for the untreated sample). This analysis is shown for 10 human tumor samples in Fig 3. The filled bars indicate that part of the histogram that overlies the histogram for untreated cells. Resuits shown in Fig 3 indicate, as expected, considerable variation in GF from tumor to tumor. For all of the tumors shown in Fig 3, the untreated tumor shows tail moments less than 6, while cells of the etoposide treated tumor show tail moments up to 25. There is a wide variation in response of cycling cells to etoposide that may relate to the variation in topoisomerase II content observed in individual cells. 34 Another interesting possibility is that the lengthening of the cell cycle time, as some cells prepare to exit the cycle, may result in an increased resistance to etoposidefl~ This heterogeneity means that there is not always a clear distinction between proliferating and nonproliferating cells. Of the 47 tumors evaluated to date, we were unable to obtain sufficient cells for the assay from only 4 fine needle aspirates. Occasionally (about 15%

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Figure 3. Growth fraction measured in cells from fine needle aspirates. Cells were exposed for one hour to 5 Ixg/mL etoposide before measuring DNA damage using the comet assay. Black bars indicate overlap with histograms from untreated comets; 95% of comets from untreated samples appear within the black bars. The percentage of cells actively cycling (open bars) is shown for each histogram. of the samples) there was sufficient background DNA damage to prevent an accurate measurement of GF in etoposide-treated cells. This is most likely a result of the past treatment history of these tumors, DNA damage incurred during aspiration, or the effect of time between aspiration and analysis (which we attempt to minimize but which can extend to 2 hours or more). All three possibilities seem likely to play a role, as we have found using rodent tumor models. Tumor cells appear to be damaged by needle aspiration because 6 of 6 FNA samples were unable to repair DNA strand breaks in vitro. Values reported in the literature for GF or labeling index for the same tumor type are often variable and results are technique-dependent. However, regardless of the method used, a large range in values for any specific tumor type is generally observed? ~ Figure 3 indicates that GF was lower for breast tumors (representing large primary, untreated lesions) and background damage was reduced com-

pared with results for head and neck tumors. For head and neck tumors, mean LI has been reported to be 12% to 18%.9,18,35 For breast cancers, median values of 2.5% to 5% have been measured. 18,2~ Our values for this small sample compare favorably, considering that LI is typically 2 to 3 times lower than GF.

Factors Influencing Cell Sensitivity to Etoposide Several factors can influence cell sensitivity to etoposide and will potentially interfere with measurement of GF using the etoposide/comet assay. Results in Fig 1 show similar intrinsic sensitivities to this drug for a variety of tumors and cell lines, but etoposideresistance can develop, at least for cells grown in vitro. 37,38Interestingly, preliminary data indicate that DNA damage is relatively independent of temperature of incubation with etoposide; incubating FNA

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samples on ice with etoposide should help to reduce background DNA damage. Our previous results indicated that etoposide sensitivity is similar whether WiDr spheroids are incubated intact or dissociated immediately before treatment. ~3 This suggests that single cells obtained by aspiration should respond in the same way to etoposide as cells within the intact tumor. The etoposide concentration used to measure GF can influence results because even noncycling tumor cells will show some DNA damage by high concentrations of etoposide (ie, inner cells of spheroids shown in Fig 2). However, concentrations of etoposide between 2 and 10 p,g/mL appear to give the same GF estimates. 33 A similar concern exists when using BrdUrd to assess LI for tumor biopsies analyzed in vitro because it has been shown that higher BrdUrd concentrations give a higher LI in head and neck tumors. 39 Holding cells in ice-cold buffer for up to 3 hours does not significantly reduce GF measured using the etoposide/comet assay (Fig 4A). However, proliferating cells held at 37~ in the absence of serum show a progressive decrease in etoposide sensitivity, and the GF in exponentially growing cells is reduced from 100% to 60% within 4 hours (Fig 4B). Ki-67 binding is also affected by sample handling; the half-life of the antigen is less than 1 hour and cells maintained in the absence of serum show a rapid loss of Ki-67 binding, a~ Fresh cells are required for analysis of GF using ~,h,

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the etoposide/comet assay, a limitation that is shared with Ki-67 and 3H-thymidine/BrdUrd incorporation. Some proliferation-dependent antibodies, like PCNA/ cyclin and histone H3 mRNA 3,~7 can be detected in formalin-fixed tissues, which is an important advantage.

Signal Versus Noise: Separating the Response of Tumor Cells and Normal Cells Predictive assays for tumor response are continually confounded by the problem of separating the response of the tumor cells from the response of accompanying normal cells; biopsies containing 50% or more normal cells are not uncommon. ~2Normal cells, such as tumor-infiltrating lymphocytes and macrophages are also capable of proliferating and will, therefore, contribute to any measurement of GF. Conversely, large numbers of nonproliferating normal cells will lead to an underestimate of GF. This problem can be exacerbated by using fine needle aspirates that often include more circulating white blood cells than do excision biopsies. Potential ways to overcome the normal cell problem are detection of epithelial-derived tumor cells with antibodies to cytokeratin, 41 elimination of lymphocytes and macrophages by tagging with appropriate cell surface antibodies, and use of DNA content to separate diploid normal cells from aneuploid tumor cells. The latter method is easily accomplished using the comet assay by simultaneous measurement of DNA content (total fluorescence intensity) and tail moment for each comet. The bivariate distribution shown in Fig 5 clearly defines the two populations of cells in this lymphoma. The GF estimate shown in Fig 5B is based only on those cells with a neartetraploid DNA content. Notice that diploid cells constitute a significant proportion of the etoposidesensitive cells. DNA content is one of the simplest and most satisfactory ways to discriminate between tumor and normal cells, but unfortunately many tumors show a diploid (or near diploid) DNA content. Flow cytometry evaluation of cells obtained from the same fine needle aspirate can provide a more sensitive indication of DNA content than the comet assay, and this information could be useful in analysis of comets with near-diploid DNA content. For example, if 80% of the cells exhibit a DNA index of 1.5, then the 20% of comets with the lowest DNA

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content (ie, lowest total fluorescence) can be eliminated from analysis. However, this correction would only be valid if cytology confirmed the presence of a similar fraction of normal cells.

Growth Fraction: The Hidden Factors The Status of Noncycling Cells In measuring GF with any of the methods currently available, it should be recognized that the nonproliferating cells within the tumor at the time of assay may not be irrelevant to tumor response. Noncycling cells within the tumor may include not only terminally differentiated, dead, or dying cells but also cells in the "resting" compartment that can be recruited back into cycle when provided with adequate stimulus (ie, treatment induced increases in oxygen, nutrients, or growth factors). Therefore, pretreatment GF may be less useful as a predictor for certain tumors: those tumors that have a low GF but a high recruitment potential. Estimates of recruitment potential require perturbing the system. However, post-treatment measurement of GF can be complicated when uther factors (ie, cell death) come into play, 4~ and recruitment (like reoxygenation) will no

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Influence of Cell Cycle Position Results shown in Fig 5(3 indicate there is no relation between response to etoposide and DNA content, that is, that position of the cell in the cell cycle does not affect the ability to determine its proliferative status. This is an important advantage over other proliferation markers, such as p145 and p120, that show changes in levels as cells progress through the cell cycle?3 Early G1 cells show a particularly large reduction in binding of these antibodies, and in fact, p120 is almost undetectable in all of G1 phase. 43 Ki-67 binding is less variable through the cell cycle, although cells at the G1/S border show little more than background fluorescence. 44 The intracellular pattern of binding of Ki-67 is also quite heterogeneous and can serve to distinguish cells in different phases of the cell cycle.45,46PCNA/cyclin is present in all phases of the cell cycle; however, fixation leads to preferential retention of the antigen in S phase cellsY Histone H3 mRNA rapidly disappears toward the end of G2 and is absent in mitosis. 4~ Differential expression of antigens through the cell cycle is obviously a problem in obtaining an accurate mea-

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surement of GF and supports the use of (concurrent) functional assays to measure GF.

Sample Size Considerations: Tumor Heterogeneity Results obtained using fine needle aspirates or small biopsies are always subject to the concern that the sample may not be representative of the entire tumor. However, although the 105 cells obtained from a fine needle aspirate is undoubtedly a small number, it should be recognized that the cells are aspirated from a 1 to 2 cm track through the tumor (while moving the needle) so that a large area potentially contributes to the sample. One way to improve the sampling area is to combine several aspirates taken from different positions within the tumor. Generally this is most easily accomplished by using the same entry site but directing the needle at different angles within the tumor mass. The necessity of obtaining a representative tumor sample is a direct result of tumor heterogeneity. The fact that S phase duration is not greatly different for different tumors of the same type masks another important problem related to heterogeneity. In defining cell cycle rate, what is probably most important is not the average duration of S phase or the average cell cycle time, but rather the S phase duration of the most rapidly proliferating cells. It is more likely that a rapidly proliferating cell will outpace treatment and continue to grow in spite of continued therapy. Unfortunately there is no convenient way to obtain information on cycle time variability in the clinic.

Recruitment and Loss from the Proliferating Population GF measured using any of the available methods assumes that cells leaving or entering the proliferating population will be detected quickly. Loss of etoposide (or Ki-67 reactive protein) with time after removal from the tumor or incubation in the absence of serum should be expected. For PCNA, the long half-life of the antigen can result in a gross overestimation of G F . 49 Using the comet assay, increases in GF can be detected within a few hours upon disaggregation of WiDr spheroids and their return to monolayer growth. 33 Cells leaving the growth cycle, eg, when WiDr cells are allowed to form and grow as spheroids, are also detected readily. Unfortunately, this means that it is important to perform measurements of GF rapidly after biopsy or aspiration, before the

tumor cells realize they are in an inhospitable environment. Functional assays have the advantage that the cell being analyzed, while not necessarily clonogenic, is probably still viable. Other functional assays of GF include 3H-thymidine incorporation or BrdUrd incorporation. These methods are more often used to determine LI, not GF. The distinction here is that a single injection of 3H-thymidine or BrdUrd will be incorporated only by cycling cells in S phase, not by proliferating cells in other phases of the cell cycle.

The Comet/Etoposide Assay: Pros and Cons Like most other methods to measure GF, there are both advantages and disadvantages to the comet/ etoposide method. A positive feature is that GF can be measured using cells obtained by fine needle aspiration, a decided practical advantage in the clinic. DNA content is measured simultaneously, which may allow differentiation between the response of tumor and normal cells in aneuploid tumors. GF is largely independent of cell cycle position or cell type. The comet/etoposide method provides a functional assay of cell proliferation without the need to administer DNA precursors (eg, 3H-thymidine, BrdUrd). Mthough we routinely use an image analysis system, it is also possible to examine comets "by eye" to rapidly assess sensitivity or resistance to etoposide. 29 Disadvantages of the method are that analysis of single cells results in a loss of information related to the histology/geometry of the tumor, ie, the location of dividing cells within the tumor cord. It should be recognized that this is a problem for any method that analyzes single cells, and must be weighed against the advantage of obtaining information on GF using fine needle aspirates. Another limitation for the comet assay, Ki-67 binding, or 3H-thymidine incorporation is that fresh samples are required for analysis. A third consideration relates to potential cell resistance to etoposide. Although DNA damage by etoposide is relatively similar for a variety of human cell lines and for cells recovered via fine needle aspiration (Fig 3A), it is also clear that estimates of GF can be influenced by the concentration of etoposide used for producing DNA strand breaks (Fig 3B). Other than DNA content, there is little to distinguish tumor cells from normal cells in the comet assay. Knowledge of tumor growth potential/doubling time has been shown to be of benefit in predicting

Growth FractionofHuman Tumors

t r e a t m e n t o u t c o m e in p o p u l a t i o n s o f c a n c e r pat i e n t s . H o w e v e r , it is n o w i m p o r t a n t to i m p r o v e t h e ability t o c h a r a c t e r i z e g r o w t h k i n e t i c s o f i n d i v i d u a l t u m o r s to c o m b i n e t h i s i n f o r m a t i o n w i t h o t h e r p r o g n o s t i c factors. A s d i s c u s s e d by W i t h e r s et al, 5~ a shrinking tumor can mask a rapidly growing population o f s u r v i v i n g cells. W h e t h e r a n a c c u r a t e m e a s u r e m e n t o f p r e t r e a t m e n t G F will b e a v a l u a b l e p r o g n o s tic m e a s u r e m e n t , especially for t h o s e t u m o r s w i t h low p r o l i f e r a t i v e f r a c t i o n s a n d b r o a d r a n g e s o f i n t e r m i t o t i c t i m e s (eg, b r e a s t c a n c e r s ) , 42 is c u r r e n t l y u n k n o w n a n d c a n o n l y b e a d d r e s s e d by a p p r o p r i a t e clinical trials.

Acknowledgment The authors are indebted to Ralph Durand who developed the comet analysis program andJudit Banath who was responsible for performing the comet assay on the clinical samples.

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