Critical Reviews in Clinical Laboratory Sciences
ISSN: 1040-8363 (Print) 1549-781X (Online) Journal homepage: http://www.tandfonline.com/loi/ilab20
Chemosensitivity Testing: A Critical Review Andrew B. Cramer, Eugene A. Woltering, Basil T. Doumas & Tai-Wing Wu To cite this article: Andrew B. Cramer, Eugene A. Woltering, Basil T. Doumas & Tai-Wing Wu (1991) Chemosensitivity Testing: A Critical Review, Critical Reviews in Clinical Laboratory Sciences, 28:5-6, 405-413 To link to this article: http://dx.doi.org/10.3109/10408369109106871
Published online: 27 Sep 2008.
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Date: 06 November 2015, At: 08:41
Volume 28, Issue 5,6 (1991)
405
Chemosensitivity Testing: A Critical Review Andrew B. Cramer, M.D. and Eugene A. Woltering, M.D.
Key Words: chemotherapy, chemosensitivity, assays, cloning, clonogenic assays
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1. INTRODUCTION Chemotherapy is the cornerstone of treatment in patients who suffer from metastatic cancer. For the more common types of cancer, current treatment is most often based on the results of prospective randomized trials performed in patients who have had similar types and stages of disease. These treatment protocols have shown that despite more aggressive therapy and the development of new agents and combinations, only a limited number of patients respond. It is quite difficult to predict which specific patients will benefit from chemotherapeutic interventions as assessed by degree or duration of response. In 1957, Wright et al. and colleagues described their attempts to culture tumor cells in v i m and to test their response to various chemotherapeutic agents.' Since their initial work, investigators have developed several types of human tumor assays for the purpose of testing tumors against chemotherapeutic agents. These assays, also known as chemosensitivity assays, in theory are much like bacterial culture and sensitivity testing. The techniques for performing such assays have undergone marked changes over the last 30 years such that in the last 2 to 3 years, significant advances have been achieved in this field. Surprisingly enough, medical oncologists and surgeons have been very reluctant to accept the idea of chemosensitivity testing. This has perhaps been related to the difficulties previously encountered with successfully cloning the tumor. Significant advances have been made in improving cloning assay techniques such that cloning success rates are now over 90%. In addition, recent work indicates that the problems of correlating in vivo pharmacokinetics to in vitro testing is now largely solved. Finally, a growing body of literature indicates that chemosensitivity testing has significant predictive value in cancer patients. Because of these recent advances with current assay techniques, it is now becoming apparent that the sensitivity and specificity of chemosensitivity assays approaches or exceeds that of both bacterial 'culture and sensitivity testing as well as estrogen receptor and progesterone receptor testing. Chemosensitivity testing also has potential for future development. Recent advances by Rotman et al. indicate that our technology in this area remains in its infancy. For example, only recently have in v i m tumor colony cultures been routinely performed that have the capability of monitoring tumor survival without sacrificing the viable cell culture.3 This allows much greater flexibility for drug testing in terms of timing and variabilities in drug regimens. These newer techniques need further evaluation by prospective clinical trials in order to assess their role in patient care. In particular, since chemotherapy has usually been shown to be most effective when multiple agents are used, prospective studies correlating assays to clinical efficacy using multimodality testing will need to be designed and performed.
E. A. Woltering, B.A., M.D.,Dept. of SurgerylOncology, L224A - Baird Hall 3056, Oregon Health Sciences University, 3181 S.W. S a m Jackson Park Road, Portland, OR 97201-3980; A. B. Cramer, Dept. of Surgery, MacKenzie Hall, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201.
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Two studies have been completed and one is currently being performed at this time to evaluate this problem. However, participation in these studies by health-care providers has been so poor that accrual of adequate numbers of patients has limited the significance of these studies. A critical review of chemosensitivity testing follows, beginning with a description of the major techniques and a review of the studies that correlate these techniques with clinical trials. We have chosen to highlight these three assays based on the differences in the techniques utilized to accomplish the goal of accurately assessing chemosensitivity. These data are then reviewed in light of physician nonacceptance of tumor assays and the current attempts to respond to these criticisms.
II. TUMOR ASSAY METHODS A. The Clonogenic Assay In 1976 Hamburger and Salmon reported in vitro tests of the human tumor cloning ~ y s t e m .Their ~ . ~ technique has become the one most often used in laboratories that perform in vitro clonogenic assays. In this in vitro system, solid tumors, bone marrow containing tumor cells, malignant effusions, bronchoscopy washings, bladder washings, and even cerebral spinal fluid can be used as a source of tumor cells. Solid tumors, obtained during surgical excision, are minced rapidly and placed in cold tissue culture media with heat-inactivated fetal calf serum. These tumors are then transported to the laboratory where the assays are to be performed or to an interim laboratory where they are further prepared for transport. Tumors are prepared for transport by sterilely crossblade mincing until fragments are 1 to 2 mm3 in size.6 Asscites, pleural or pericardial effusions, are obtained by aspirating the fluid that is then placed in sterile containers with 10 units of preservative-free heparin per milliliter of malignant fluid. Bone marrows containing tumor cells are collected in a similar manner. These specimens are placed in transport tubes and the tubes are securely closed. The tumors are transported on wet ice (not frozen) to the appropriate reference laboratory. Upon arrival at the reference laboratory, solid tumors are mechanically and enzymatically dissociated to form single-cell suspensions. Cells are washed and then incubated in either a control tissue culture media or in a tissue culture media containing the desired chemotherapeutic drug. The concentration of drug utilized in most studies has been one tenth of the peak plasma level or, alternatively, one tenth of the concentration times time value, i.e., the area under the drug concentration curve.5 Cells are incubated with the drug of interest for 1 h and then washed twice in tissue culture media. They are then cultured in a top layer of agar over a bottom layer of agar and an enriched tissue culture media.6 Cell culture plates are incubated at 37°C and in 7.5% CO, humidified atmosphere. After 10 to 14 d, the colonies that have formed can be counted using an inverted microscope. The number of colonies growing on drug-treated plates is compared with the number of colonies on the control plate, and the percentage decrease in tumor colony forming units (TCFUs) caused by a drug can be B. Subrenal Capsule Assay An in vivo method, the subrenal capsule assay (SCA), was developed in response to the need for a rapid in vivo test system for screening drugs against transplantation-established human tumor xenografts (transplants across species) in the athymic nude mouse. This method was later developed to utilize fresh human tumors as first-generation transplant xenografts in the immunocompetent mouse for drug testing, raising the possibility of its use as a predictive assay for chemosensitivity.lo
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Preparation of the solid tumor cells is accomplished in a similar fashion to that of clonogenic assays. Specifically, surgically obtained solid tumors are minced rapidly and placed in cold tissue media with heat-inactivated fetal calf serum. These are then transported to the laboratory where the assay is performed or to an interim laboratory where they are further prepared for transport. The specimens are then minced into fragments of 1 mm3 in size. These 1 mm3 fragments are then transported to the appropriate laboratory on wet ice. These 1 mm3 tumor fragments are then surgically placed into the subrenal capsule of normal immunocompetent mice. Initial and final body weights are obtained, and initial and final in situ tumor sizes are determined. On days 1 to 5 , the mice are exposed to the various chemotherapeutic agents in differing dose regimens. On day 6 , the animals are sacrificed and determination of tumor sizes obtained. This is done as a direct measurement and is made possible by the fact that the renal capsule of the mouse is nearly transparent. Tissues are therefore not frxed for histologic staining. The tumor is measured with respect to its length and width using ocular micrometer units (OMU), with magnification calibrated so that 10 OMU are equal to 1 mm’. True size and weight are determined using these measurements and drug activity is reported as either a change in tumor size or as a percent change based on tumor weight. Drug doses are chosen that are appropriate for the mouse because of the accelerated metabolism of mice compared with humans. While the clonogenic assay utilizes a single cell suspension of tumor stem cells to determine drug sensitivity of single-cell lines in vifro, the subrenal capsule assay utilizes tumor fragments to accomplish the same result. These fragments allow the subrenal capsular assay to maintain cell membrane integrity, cell to cell contact, and a heterogeneous tumor cell population. This is accomplished by the use of tumor fragments rather than single cell suspensions, and hence offers a theoretical advantage over the clonogenic assay.” Conversely, the use of tumor fragments may generate new problems. Tissue fragments may, by nature, include antibodies, growth factors, macrophages, neutrophils, and other preformed immune effector cells. These factors may affect the growth of tumor fragments.
C. Fluorescent Cytoprint Assay Recently, a new predictive chemosensitivity assay has been developed by Rotman and c o - w o r k e r ~ . ~The * ~ ~Rotman * ’ ~ in v i m chemosensitivity assay (RIVCA), also known as the in vitro fluorescent cytoprint assay (FCA), is a new tissue culture system whose unique features include culture of tumor cell clusters rather than cloning single cell lines. In addition, it depends on the activation of a fluorogenic substrate, fluorescein mono- or diacetate. The ability of viable human tumor cells to transport, hydrolyze, and retain fluorescein (fluorochromasia) is used to select and measure the viability of tumor clusters. Tumor specimens are obtained and transported in a similar fashion to the clonogenic assay specimens. Once the 1 mm3fragments are obtained, the tumor is subjected to shearing forces in a tissue homogenizer resulting in a suspension. Some tumors with poor cellularity such as breast tumors are minced and then placed in a medium with collagenase and incubated. In these instances, treatment with the tissue homogenizer may be omitted. The tumor is then resuspended, washed, and incubated in the presence of fluorescein to visualize viable microorgans for subsequent culturing. Viable cells rapidly accumulate intracellular fluorescein as a result of enzymatic hydrolysis of lipophilic fluorogenic substrates such as fluorescein esters of fatty acids. Dead cells do not retain fluorescein due to their lack of cell membrane integrity. After incubation, the cells are spun down, resuspended in regular culture media, and examined under blue light. Viable fluorescein-laden cells are visible by the naked eye. Viable tumor fragments are then cultured on metal grids covered with porous sterile “tea bag” paper and collagen solution in a “sandwich” fashion. The plate is kept in a 95% air 5% CO, atmosphere to keep a neutral pH, and, after 3 d of culture, drug exposure is performed.
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The FCA incubates tumor fragments in media and drug for 48 h. Drug concentrations are chosen to reflect peak plasma levels, and photographic records of fluorescent cytoprints are obtained. Cytotoxicity is assessed by comparing photographic records before and after drug treatment, i.e., day 2 and day 7. The FCA system allows more flexibility in testing drug combinations and variations of drug exposure time than either the SCA or clonogenic assay techniques. Time from specimen collection until result reporting averages 7 to 8 d, which compares well with the SCA technique. More importantly, the percent of specimens that can be evaluated is much higher with the FCA technique than in either of the other two techniques. Specifically, reports indicate that 95 to 98% of tumors that do not have microbial contamination are able to be e v a l ~ a t e dThis . ~ compares with an evaluation rate of 40 to 60% for the clonogenic assay and of 80 to 90% for the subrenal capsule a s ~ a y . ~ . ~Thus, . ' ' one of the major criticisms of chemosensitivity testing, that of the inability to culture a large proportion of tumors, appears to no longer be an issue. However, critics of this assay contend that there is a potential hazard of stromal cell contamination altering the overall assay results.
111. ASSAY TO CLINICAL CORRELATIONS The usefulness of predictive assays is ultimately determined by how well assay results correlate with clinical response. Most of the work in this area has utilized the clonogenic assay results. Results from the subrenal capsule assay (although introduced more recently) are encouraging. More work needs to be done with this assay. Finally, the fluorescent cytoprint assay (FCA), although it has some theoretic advantages, has not been available long enough for large prospective studies correlating its predictive capabilities to have been performed. Retrospective results, however, indicate that the patterns of sensitivity and resistance to drugs in vitro correlates well with clinical response. In a review of the literature regarding the clonogenic assay, Von Hoff et al. summarized 54 different in vitro to in vivo clinical correlation trials occurring in 35 institutions over the There were 2300 attempts at in vitro to in vivo correlations. In this review, last 12 years. the clonogenic assay successfully predicted that a chemotherapeutic agent would work 5 12 times and erroneously predicted an agent would work 226 times. This correlates to a 69% true positive rate. The assay also successfully predicted resistance to the drug 1427 times and erroneously predicted that the chemotherapy would work when it clinically did not 135 times. This correlates to a true negative rate of 91%. These data mean that the sensitivity rate (true positivedtme positives plus false negatives) is 79% and the specificity of the test (true negatives/tme negatives plus false positives) is 86%. Although these results are a compendium of multiple studies, they are very significant. For example, it is well known that the estrogen and progesterone receptor assay has only a 60 to 70% in vitro to in vivo correlation yet it is currently standard practice to perform this assay to predict responsiveness to hormonal manipulation in patients with breast cancer. Similarly, another in vitro to in vivo test that is routinely used in the clinical setting is in vitro antibiotic sensitivity testing. It is actually very difficult to obtain good data on the quality of the in vitro to in vivo correlation for bacterial culture and sensitivity testing. Abboud and Waisbren reported a favorable clinical response in 60% of patients whose infections were treated with antibiotics shown to be appropriate by assay testing." Like chemosensitivity testing, antibiotic assays are much more effective at predicting resistance rather than sensitivity. Yet, in spite of little data and only modest results, the clinician rarely questions the validity of either the antibiotic assay or estrogen receptor testing results. There are less data available that compare the predictive results of the subrenal capsule assay with clinical efficacy. This is due in part from its more recent introduction as a viable
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technique for chemosensitivity testing. In a review of the literature in 1986, Bogden and Cobb quoted that in approximately 1400 solid tumor specimens, the SRC assay was evaluable 90% of the time.18 In these evaluable specimens, the assay accurately predicted clinical sensitivity 91% of the time, and it predicted clinical resistance 73% of the time. This correlated to a false negative rate of 3% and a false positive rate of 19%. In another study by Bogden and Von Hoff, the SRC assay and the clonogenic assay were compared with respect to their ability to predict clinical efficacy of chemotherapeuticagents." Of 84 tumors, 75 (89%) were evaluable by the SRC assay and only 33 (42%) by the clonogenic assay. Because not all tumors were also clinically evaluable, only 23 SRC assay to clinical correlations and only 10 clonogenic assay to clinical correlations could be made. Nevertheless, the SRC assay predicted clinical sensitivity 100% of the time (3 out of 3), and clinical resistance in 80% (16 out of 20). The clonogenic assay predicted clinical resistance 100% of the time (lO/lO), and no drug-sensitive tumors were observed. Of the other 23 evaluable clonogenic assay specimens, excellent correlation was found between it and the SRC assay. The number of comparisons presented in these studies is small and few definitive statements can be made. However, it does appear that the SCA assay may have value as an assay for predicting the clinical effectiveness of chemotherapy. Even less data are available to assess the predictive value of the FCA technique of chemosensitivity testing. Leone et al. reported a retrospective study of 73 tumor patients in whom the FCA was utilized.13They noted a specificity of 98% and a sensitivity of 81% for the assay. This correlates well with other chemosensitivity assays as well as with either antibiotic or hormone receptor assays. Follow up reports continue to be encouraging.2o Prospective randomized studies are needed to c o n f m these results.
IV. NONACCEPTANCE OF PREDICTIVE ASSAYS Given the value of chemosensitivity testing for predicting clinical sensitivity and clinical resistance, it is surprising that there is marked resistance to the clinical use of these techniques. This resistance has been grounded in five main issues: (1) failure to consistently grow tumor, (2) quality control, (3) matching of in vivo pharmacokinetics to lab technique, (4) theoretic objections, and ( 5 ) lack of well-designed clinical trials. Perhaps the most important reason for physician nonacceptance of predictive assays has been the general finding that not all tumors can be grown, and therefore be tested. The worst growth performance has been the clonogenic assay. Until recently, the clonogenic assay has grown evaluable tumor 30 to 40% of the time.I4 More recent results indicate that with improved techniques, the tumor can be grown up to 77%of the time.2' The subrenal capsule assay, as described earlier, is much more successful in terms of growing tumor. Authors report an 80 to 90% success rate'8.19.22 in growing solid tumor, and Stratton and others have reported techniques for growing tumor from malignant effusions and hematopoietic tumors .23 Although this evaluability rate is impressive for the subrenal capsule assay, the fluorescent cytoprint assay appears to provide superior results. Rotman reported that of 469 surgical specimens without microbial contamination, 95%of cervical and ovarian tumors were grown; 98%of colon tumors were grown with an average growth rate for all tumors of 96%. Three more recent reports confim these results." These are clearly exceptional results and approximate those for other routinely utilized diagnostic techniques such as bacterial culture. A second objection physicians have had regarding tumor chemosensitivity assays has been quality control. Specifically, many have been critical of difficulties with cell clumping artifacts in the clonogenic assay that could lead to erroneous drug sensitivity test results.24 This objection has not been supported by the fact that serial testing at multiple laboratories indicate the assay is consistently reproducible.25 In addition, studies evaluating the possibility of cell clumping have been performed at the National Cancer Institute, which indicated that
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cell clumping was not a significant problem.25Other work has been done in this area. Of course, this issue is not a problem with either the subrenal capsule assay or with the FCA technique that, by the nature of their methodology, rely on whole tissue or microorgan specimens. A third problem with chemosensitivity testing has been the difficulty in accurately modeling the in vivo pharmacokinetics of an anti-cancer agent in the assay technique. With respect to the clonogenic assay and hence the FCA techniques, excellent progress has been made in recent years to mimic in vivo pharmacokinetics.2The subrenal capsule assay has a unique problem with pharmacokineticsdue to its interface with mouse physiology. In general, higher doses of chemotherapeutic agents are required in mice to mimic the relevant human dose. However, there is a large body of literature that is helpful in this area obtained from chemotherapeutic tumor trials in the mouse. Mouse trials are currently used as the gold standard for screening new chemotherapeutic agents for the NCI, and a large body of information exists comparing the results of mouse tumor drug trials to clinical drug dose and usefulness. Finally, in spite of these objections, the results of well-designed clinical prospective trials will aid in determining the drug levels in virro that correlate best with in vivo response. A fourth problem identified with chemosensitivity testing has been the theoretic limitations of predicting a relatively rare event, i.e., chemosensitivity of a tumor. Rozencweig and Staquet have pointed out that the predictive value of a test is mathematically limited by the probability of a response.26Because the sensitivity of a tumor to single-agent chemotherapy has a low probability (i.e., between 0 to 30%), then the likelihood that a positive result would be accurate will be low irrespective of the test itself. Therefore, to enhance the predictive value of chemosensitivity testing, either more efficacious single agents must be identfied or comparisons with multimodality therapy (which have a higher response rate) must be performed. The fluorescent cytoprint assay offers distinct flexibility and advantages in this respect since the culture is not sacrificed during assessment of drug response and should more easily allow multidrug testing. The most significant impediment to physician acceptance of chemosensitivity assays, however, has been in obtaining data from carefully controlled prospective trials. All but two of the clinical correlation studies to date have been retrospective studies or single arm prospective studies. There is only one prospective randomized study currently being performed. This study, and the two completed studies, look only at the clonogenic assay. No prospective randomized studies of either the subrenal capsule assay or the assay with perhaps the most potential efficacy, the fluorescent cytoprint assay, are currently in progress. The completed prospective randomized trial of the clonogenic assay was reported by Von Hoff et al. and performed at the University of Texas Health Sciences Center in San Ant~nio.~’ They randomized 133 patients with advanced metastatic cancer between either their clinician’s choice of a single agent vs. a single agent therapy selected by the clonogenic assay. Response rate for the clonogenic assay was 21% vs. a 3% response rate in patients receiving the clinician’s choice. This had a P value of 0.04. These benefits were related to partial response and there was no difference in survival seen between the two arms of the study. Only patients with advanced metastatic cancer were entered. In another prospective randomized trial of the clonogenic assay, Welander and colleagues at Bowman Gray conducted a trial randomizing patients with ovarian cancer between standard chemotherapy, (cyclophosphamide and cisplatinum and doxorubicin) or the most active agents selected by the clonogenic assay.28 The response rates were 67.7% for standard chemotherapy (20.6% complete response rate) and 80.7% for the clonogenic assay (35.5% complete response rate). However, because of the small number of patients entered into the study (65), there is no statistically significant difference in response rate between the groups. However, statistical significance ( p
ISSN: 1040-8363 (Print) 1549-781X (Online) Journal homepage: http://www.tandfonline.com/loi/ilab20
Chemosensitivity Testing: A Critical Review Andrew B. Cramer, Eugene A. Woltering, Basil T. Doumas & Tai-Wing Wu To cite this article: Andrew B. Cramer, Eugene A. Woltering, Basil T. Doumas & Tai-Wing Wu (1991) Chemosensitivity Testing: A Critical Review, Critical Reviews in Clinical Laboratory Sciences, 28:5-6, 405-413 To link to this article: http://dx.doi.org/10.3109/10408369109106871
Published online: 27 Sep 2008.
Submit your article to this journal
Article views: 8
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ilab20 Download by: [University of California Santa Barbara]
Date: 06 November 2015, At: 08:41
Volume 28, Issue 5,6 (1991)
405
Chemosensitivity Testing: A Critical Review Andrew B. Cramer, M.D. and Eugene A. Woltering, M.D.
Key Words: chemotherapy, chemosensitivity, assays, cloning, clonogenic assays
Downloaded by [University of California Santa Barbara] at 08:41 06 November 2015
1. INTRODUCTION Chemotherapy is the cornerstone of treatment in patients who suffer from metastatic cancer. For the more common types of cancer, current treatment is most often based on the results of prospective randomized trials performed in patients who have had similar types and stages of disease. These treatment protocols have shown that despite more aggressive therapy and the development of new agents and combinations, only a limited number of patients respond. It is quite difficult to predict which specific patients will benefit from chemotherapeutic interventions as assessed by degree or duration of response. In 1957, Wright et al. and colleagues described their attempts to culture tumor cells in v i m and to test their response to various chemotherapeutic agents.' Since their initial work, investigators have developed several types of human tumor assays for the purpose of testing tumors against chemotherapeutic agents. These assays, also known as chemosensitivity assays, in theory are much like bacterial culture and sensitivity testing. The techniques for performing such assays have undergone marked changes over the last 30 years such that in the last 2 to 3 years, significant advances have been achieved in this field. Surprisingly enough, medical oncologists and surgeons have been very reluctant to accept the idea of chemosensitivity testing. This has perhaps been related to the difficulties previously encountered with successfully cloning the tumor. Significant advances have been made in improving cloning assay techniques such that cloning success rates are now over 90%. In addition, recent work indicates that the problems of correlating in vivo pharmacokinetics to in vitro testing is now largely solved. Finally, a growing body of literature indicates that chemosensitivity testing has significant predictive value in cancer patients. Because of these recent advances with current assay techniques, it is now becoming apparent that the sensitivity and specificity of chemosensitivity assays approaches or exceeds that of both bacterial 'culture and sensitivity testing as well as estrogen receptor and progesterone receptor testing. Chemosensitivity testing also has potential for future development. Recent advances by Rotman et al. indicate that our technology in this area remains in its infancy. For example, only recently have in v i m tumor colony cultures been routinely performed that have the capability of monitoring tumor survival without sacrificing the viable cell culture.3 This allows much greater flexibility for drug testing in terms of timing and variabilities in drug regimens. These newer techniques need further evaluation by prospective clinical trials in order to assess their role in patient care. In particular, since chemotherapy has usually been shown to be most effective when multiple agents are used, prospective studies correlating assays to clinical efficacy using multimodality testing will need to be designed and performed.
E. A. Woltering, B.A., M.D.,Dept. of SurgerylOncology, L224A - Baird Hall 3056, Oregon Health Sciences University, 3181 S.W. S a m Jackson Park Road, Portland, OR 97201-3980; A. B. Cramer, Dept. of Surgery, MacKenzie Hall, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201.
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Two studies have been completed and one is currently being performed at this time to evaluate this problem. However, participation in these studies by health-care providers has been so poor that accrual of adequate numbers of patients has limited the significance of these studies. A critical review of chemosensitivity testing follows, beginning with a description of the major techniques and a review of the studies that correlate these techniques with clinical trials. We have chosen to highlight these three assays based on the differences in the techniques utilized to accomplish the goal of accurately assessing chemosensitivity. These data are then reviewed in light of physician nonacceptance of tumor assays and the current attempts to respond to these criticisms.
II. TUMOR ASSAY METHODS A. The Clonogenic Assay In 1976 Hamburger and Salmon reported in vitro tests of the human tumor cloning ~ y s t e m .Their ~ . ~ technique has become the one most often used in laboratories that perform in vitro clonogenic assays. In this in vitro system, solid tumors, bone marrow containing tumor cells, malignant effusions, bronchoscopy washings, bladder washings, and even cerebral spinal fluid can be used as a source of tumor cells. Solid tumors, obtained during surgical excision, are minced rapidly and placed in cold tissue culture media with heat-inactivated fetal calf serum. These tumors are then transported to the laboratory where the assays are to be performed or to an interim laboratory where they are further prepared for transport. Tumors are prepared for transport by sterilely crossblade mincing until fragments are 1 to 2 mm3 in size.6 Asscites, pleural or pericardial effusions, are obtained by aspirating the fluid that is then placed in sterile containers with 10 units of preservative-free heparin per milliliter of malignant fluid. Bone marrows containing tumor cells are collected in a similar manner. These specimens are placed in transport tubes and the tubes are securely closed. The tumors are transported on wet ice (not frozen) to the appropriate reference laboratory. Upon arrival at the reference laboratory, solid tumors are mechanically and enzymatically dissociated to form single-cell suspensions. Cells are washed and then incubated in either a control tissue culture media or in a tissue culture media containing the desired chemotherapeutic drug. The concentration of drug utilized in most studies has been one tenth of the peak plasma level or, alternatively, one tenth of the concentration times time value, i.e., the area under the drug concentration curve.5 Cells are incubated with the drug of interest for 1 h and then washed twice in tissue culture media. They are then cultured in a top layer of agar over a bottom layer of agar and an enriched tissue culture media.6 Cell culture plates are incubated at 37°C and in 7.5% CO, humidified atmosphere. After 10 to 14 d, the colonies that have formed can be counted using an inverted microscope. The number of colonies growing on drug-treated plates is compared with the number of colonies on the control plate, and the percentage decrease in tumor colony forming units (TCFUs) caused by a drug can be B. Subrenal Capsule Assay An in vivo method, the subrenal capsule assay (SCA), was developed in response to the need for a rapid in vivo test system for screening drugs against transplantation-established human tumor xenografts (transplants across species) in the athymic nude mouse. This method was later developed to utilize fresh human tumors as first-generation transplant xenografts in the immunocompetent mouse for drug testing, raising the possibility of its use as a predictive assay for chemosensitivity.lo
Downloaded by [University of California Santa Barbara] at 08:41 06 November 2015
Volume 28, Issue 5,6 (1991)
407
Preparation of the solid tumor cells is accomplished in a similar fashion to that of clonogenic assays. Specifically, surgically obtained solid tumors are minced rapidly and placed in cold tissue media with heat-inactivated fetal calf serum. These are then transported to the laboratory where the assay is performed or to an interim laboratory where they are further prepared for transport. The specimens are then minced into fragments of 1 mm3 in size. These 1 mm3 fragments are then transported to the appropriate laboratory on wet ice. These 1 mm3 tumor fragments are then surgically placed into the subrenal capsule of normal immunocompetent mice. Initial and final body weights are obtained, and initial and final in situ tumor sizes are determined. On days 1 to 5 , the mice are exposed to the various chemotherapeutic agents in differing dose regimens. On day 6 , the animals are sacrificed and determination of tumor sizes obtained. This is done as a direct measurement and is made possible by the fact that the renal capsule of the mouse is nearly transparent. Tissues are therefore not frxed for histologic staining. The tumor is measured with respect to its length and width using ocular micrometer units (OMU), with magnification calibrated so that 10 OMU are equal to 1 mm’. True size and weight are determined using these measurements and drug activity is reported as either a change in tumor size or as a percent change based on tumor weight. Drug doses are chosen that are appropriate for the mouse because of the accelerated metabolism of mice compared with humans. While the clonogenic assay utilizes a single cell suspension of tumor stem cells to determine drug sensitivity of single-cell lines in vifro, the subrenal capsule assay utilizes tumor fragments to accomplish the same result. These fragments allow the subrenal capsular assay to maintain cell membrane integrity, cell to cell contact, and a heterogeneous tumor cell population. This is accomplished by the use of tumor fragments rather than single cell suspensions, and hence offers a theoretical advantage over the clonogenic assay.” Conversely, the use of tumor fragments may generate new problems. Tissue fragments may, by nature, include antibodies, growth factors, macrophages, neutrophils, and other preformed immune effector cells. These factors may affect the growth of tumor fragments.
C. Fluorescent Cytoprint Assay Recently, a new predictive chemosensitivity assay has been developed by Rotman and c o - w o r k e r ~ . ~The * ~ ~Rotman * ’ ~ in v i m chemosensitivity assay (RIVCA), also known as the in vitro fluorescent cytoprint assay (FCA), is a new tissue culture system whose unique features include culture of tumor cell clusters rather than cloning single cell lines. In addition, it depends on the activation of a fluorogenic substrate, fluorescein mono- or diacetate. The ability of viable human tumor cells to transport, hydrolyze, and retain fluorescein (fluorochromasia) is used to select and measure the viability of tumor clusters. Tumor specimens are obtained and transported in a similar fashion to the clonogenic assay specimens. Once the 1 mm3fragments are obtained, the tumor is subjected to shearing forces in a tissue homogenizer resulting in a suspension. Some tumors with poor cellularity such as breast tumors are minced and then placed in a medium with collagenase and incubated. In these instances, treatment with the tissue homogenizer may be omitted. The tumor is then resuspended, washed, and incubated in the presence of fluorescein to visualize viable microorgans for subsequent culturing. Viable cells rapidly accumulate intracellular fluorescein as a result of enzymatic hydrolysis of lipophilic fluorogenic substrates such as fluorescein esters of fatty acids. Dead cells do not retain fluorescein due to their lack of cell membrane integrity. After incubation, the cells are spun down, resuspended in regular culture media, and examined under blue light. Viable fluorescein-laden cells are visible by the naked eye. Viable tumor fragments are then cultured on metal grids covered with porous sterile “tea bag” paper and collagen solution in a “sandwich” fashion. The plate is kept in a 95% air 5% CO, atmosphere to keep a neutral pH, and, after 3 d of culture, drug exposure is performed.
Downloaded by [University of California Santa Barbara] at 08:41 06 November 2015
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Critical Reviews in Clinical Laboratory Sciences
The FCA incubates tumor fragments in media and drug for 48 h. Drug concentrations are chosen to reflect peak plasma levels, and photographic records of fluorescent cytoprints are obtained. Cytotoxicity is assessed by comparing photographic records before and after drug treatment, i.e., day 2 and day 7. The FCA system allows more flexibility in testing drug combinations and variations of drug exposure time than either the SCA or clonogenic assay techniques. Time from specimen collection until result reporting averages 7 to 8 d, which compares well with the SCA technique. More importantly, the percent of specimens that can be evaluated is much higher with the FCA technique than in either of the other two techniques. Specifically, reports indicate that 95 to 98% of tumors that do not have microbial contamination are able to be e v a l ~ a t e dThis . ~ compares with an evaluation rate of 40 to 60% for the clonogenic assay and of 80 to 90% for the subrenal capsule a s ~ a y . ~ . ~Thus, . ' ' one of the major criticisms of chemosensitivity testing, that of the inability to culture a large proportion of tumors, appears to no longer be an issue. However, critics of this assay contend that there is a potential hazard of stromal cell contamination altering the overall assay results.
111. ASSAY TO CLINICAL CORRELATIONS The usefulness of predictive assays is ultimately determined by how well assay results correlate with clinical response. Most of the work in this area has utilized the clonogenic assay results. Results from the subrenal capsule assay (although introduced more recently) are encouraging. More work needs to be done with this assay. Finally, the fluorescent cytoprint assay (FCA), although it has some theoretic advantages, has not been available long enough for large prospective studies correlating its predictive capabilities to have been performed. Retrospective results, however, indicate that the patterns of sensitivity and resistance to drugs in vitro correlates well with clinical response. In a review of the literature regarding the clonogenic assay, Von Hoff et al. summarized 54 different in vitro to in vivo clinical correlation trials occurring in 35 institutions over the There were 2300 attempts at in vitro to in vivo correlations. In this review, last 12 years. the clonogenic assay successfully predicted that a chemotherapeutic agent would work 5 12 times and erroneously predicted an agent would work 226 times. This correlates to a 69% true positive rate. The assay also successfully predicted resistance to the drug 1427 times and erroneously predicted that the chemotherapy would work when it clinically did not 135 times. This correlates to a true negative rate of 91%. These data mean that the sensitivity rate (true positivedtme positives plus false negatives) is 79% and the specificity of the test (true negatives/tme negatives plus false positives) is 86%. Although these results are a compendium of multiple studies, they are very significant. For example, it is well known that the estrogen and progesterone receptor assay has only a 60 to 70% in vitro to in vivo correlation yet it is currently standard practice to perform this assay to predict responsiveness to hormonal manipulation in patients with breast cancer. Similarly, another in vitro to in vivo test that is routinely used in the clinical setting is in vitro antibiotic sensitivity testing. It is actually very difficult to obtain good data on the quality of the in vitro to in vivo correlation for bacterial culture and sensitivity testing. Abboud and Waisbren reported a favorable clinical response in 60% of patients whose infections were treated with antibiotics shown to be appropriate by assay testing." Like chemosensitivity testing, antibiotic assays are much more effective at predicting resistance rather than sensitivity. Yet, in spite of little data and only modest results, the clinician rarely questions the validity of either the antibiotic assay or estrogen receptor testing results. There are less data available that compare the predictive results of the subrenal capsule assay with clinical efficacy. This is due in part from its more recent introduction as a viable
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technique for chemosensitivity testing. In a review of the literature in 1986, Bogden and Cobb quoted that in approximately 1400 solid tumor specimens, the SRC assay was evaluable 90% of the time.18 In these evaluable specimens, the assay accurately predicted clinical sensitivity 91% of the time, and it predicted clinical resistance 73% of the time. This correlated to a false negative rate of 3% and a false positive rate of 19%. In another study by Bogden and Von Hoff, the SRC assay and the clonogenic assay were compared with respect to their ability to predict clinical efficacy of chemotherapeuticagents." Of 84 tumors, 75 (89%) were evaluable by the SRC assay and only 33 (42%) by the clonogenic assay. Because not all tumors were also clinically evaluable, only 23 SRC assay to clinical correlations and only 10 clonogenic assay to clinical correlations could be made. Nevertheless, the SRC assay predicted clinical sensitivity 100% of the time (3 out of 3), and clinical resistance in 80% (16 out of 20). The clonogenic assay predicted clinical resistance 100% of the time (lO/lO), and no drug-sensitive tumors were observed. Of the other 23 evaluable clonogenic assay specimens, excellent correlation was found between it and the SRC assay. The number of comparisons presented in these studies is small and few definitive statements can be made. However, it does appear that the SCA assay may have value as an assay for predicting the clinical effectiveness of chemotherapy. Even less data are available to assess the predictive value of the FCA technique of chemosensitivity testing. Leone et al. reported a retrospective study of 73 tumor patients in whom the FCA was utilized.13They noted a specificity of 98% and a sensitivity of 81% for the assay. This correlates well with other chemosensitivity assays as well as with either antibiotic or hormone receptor assays. Follow up reports continue to be encouraging.2o Prospective randomized studies are needed to c o n f m these results.
IV. NONACCEPTANCE OF PREDICTIVE ASSAYS Given the value of chemosensitivity testing for predicting clinical sensitivity and clinical resistance, it is surprising that there is marked resistance to the clinical use of these techniques. This resistance has been grounded in five main issues: (1) failure to consistently grow tumor, (2) quality control, (3) matching of in vivo pharmacokinetics to lab technique, (4) theoretic objections, and ( 5 ) lack of well-designed clinical trials. Perhaps the most important reason for physician nonacceptance of predictive assays has been the general finding that not all tumors can be grown, and therefore be tested. The worst growth performance has been the clonogenic assay. Until recently, the clonogenic assay has grown evaluable tumor 30 to 40% of the time.I4 More recent results indicate that with improved techniques, the tumor can be grown up to 77%of the time.2' The subrenal capsule assay, as described earlier, is much more successful in terms of growing tumor. Authors report an 80 to 90% success rate'8.19.22 in growing solid tumor, and Stratton and others have reported techniques for growing tumor from malignant effusions and hematopoietic tumors .23 Although this evaluability rate is impressive for the subrenal capsule assay, the fluorescent cytoprint assay appears to provide superior results. Rotman reported that of 469 surgical specimens without microbial contamination, 95%of cervical and ovarian tumors were grown; 98%of colon tumors were grown with an average growth rate for all tumors of 96%. Three more recent reports confim these results." These are clearly exceptional results and approximate those for other routinely utilized diagnostic techniques such as bacterial culture. A second objection physicians have had regarding tumor chemosensitivity assays has been quality control. Specifically, many have been critical of difficulties with cell clumping artifacts in the clonogenic assay that could lead to erroneous drug sensitivity test results.24 This objection has not been supported by the fact that serial testing at multiple laboratories indicate the assay is consistently reproducible.25 In addition, studies evaluating the possibility of cell clumping have been performed at the National Cancer Institute, which indicated that
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Critical Reviews in Clinical Laboratory Sciences
cell clumping was not a significant problem.25Other work has been done in this area. Of course, this issue is not a problem with either the subrenal capsule assay or with the FCA technique that, by the nature of their methodology, rely on whole tissue or microorgan specimens. A third problem with chemosensitivity testing has been the difficulty in accurately modeling the in vivo pharmacokinetics of an anti-cancer agent in the assay technique. With respect to the clonogenic assay and hence the FCA techniques, excellent progress has been made in recent years to mimic in vivo pharmacokinetics.2The subrenal capsule assay has a unique problem with pharmacokineticsdue to its interface with mouse physiology. In general, higher doses of chemotherapeutic agents are required in mice to mimic the relevant human dose. However, there is a large body of literature that is helpful in this area obtained from chemotherapeutic tumor trials in the mouse. Mouse trials are currently used as the gold standard for screening new chemotherapeutic agents for the NCI, and a large body of information exists comparing the results of mouse tumor drug trials to clinical drug dose and usefulness. Finally, in spite of these objections, the results of well-designed clinical prospective trials will aid in determining the drug levels in virro that correlate best with in vivo response. A fourth problem identified with chemosensitivity testing has been the theoretic limitations of predicting a relatively rare event, i.e., chemosensitivity of a tumor. Rozencweig and Staquet have pointed out that the predictive value of a test is mathematically limited by the probability of a response.26Because the sensitivity of a tumor to single-agent chemotherapy has a low probability (i.e., between 0 to 30%), then the likelihood that a positive result would be accurate will be low irrespective of the test itself. Therefore, to enhance the predictive value of chemosensitivity testing, either more efficacious single agents must be identfied or comparisons with multimodality therapy (which have a higher response rate) must be performed. The fluorescent cytoprint assay offers distinct flexibility and advantages in this respect since the culture is not sacrificed during assessment of drug response and should more easily allow multidrug testing. The most significant impediment to physician acceptance of chemosensitivity assays, however, has been in obtaining data from carefully controlled prospective trials. All but two of the clinical correlation studies to date have been retrospective studies or single arm prospective studies. There is only one prospective randomized study currently being performed. This study, and the two completed studies, look only at the clonogenic assay. No prospective randomized studies of either the subrenal capsule assay or the assay with perhaps the most potential efficacy, the fluorescent cytoprint assay, are currently in progress. The completed prospective randomized trial of the clonogenic assay was reported by Von Hoff et al. and performed at the University of Texas Health Sciences Center in San Ant~nio.~’ They randomized 133 patients with advanced metastatic cancer between either their clinician’s choice of a single agent vs. a single agent therapy selected by the clonogenic assay. Response rate for the clonogenic assay was 21% vs. a 3% response rate in patients receiving the clinician’s choice. This had a P value of 0.04. These benefits were related to partial response and there was no difference in survival seen between the two arms of the study. Only patients with advanced metastatic cancer were entered. In another prospective randomized trial of the clonogenic assay, Welander and colleagues at Bowman Gray conducted a trial randomizing patients with ovarian cancer between standard chemotherapy, (cyclophosphamide and cisplatinum and doxorubicin) or the most active agents selected by the clonogenic assay.28 The response rates were 67.7% for standard chemotherapy (20.6% complete response rate) and 80.7% for the clonogenic assay (35.5% complete response rate). However, because of the small number of patients entered into the study (65), there is no statistically significant difference in response rate between the groups. However, statistical significance ( p
