The Treatment and Prevention of Asbestos Diseases Volume 15 of the Sourcebook on Asbestos Diseases Edited by GEORGE A. PETERS, J.D., C.S.P., P.E. Registered Professional Engineer (California, Safety and Quality) Licensed Psychologist (Medical Board of California) Counselor at Law (California and US. Supreme Courts) Fellow, The Royal Society of Health Fellow, American Association for the Advancement of Science Santa Monica, California, U.S.A. BARBARA J. PETERS, J.D. Peters & Peters Santa Monica, California, U.S.A. LEXISe Law Publishing6 Customizing Cancer Treatment Options PAUL HEROUX, PH.D. Department of Occupational Health Faculty of Medicine McGill University Montreal, PQ. Canada Synopsis BIOGRAPHY EDITORS’ COMMENTARY INTRODUCTION CLINICAL OUTLOOK FOR MESOTHELIOMA NEW METHODS OF CANCER TREATMENT STRATEGY IN VITRO MODELING IMPLEMENTATION INSPECTIONS OF ANIMATIONS BY THERAPIST AUTOMATED IMAGE ANALYSIS OTHER APPLICATIONS OF THE SYSTEM CONCLUSION REFERENCES ME MOM DOW132 Sourcebook on Asbestos Diseases BIOGRAPHY Professor Paul Héroux teaches Toxicology and Health Effects of Electromagnetism & Acoustics at the department of Occupa- tional Health, Faculty of Medicine, McGill University, in Mont- real, Canada. He is also a medical scientist at the department of Surgery, Royal Victoria Hospital, in Montreal. He has long held strong interests in the health effects of Physical Agents and has been a pioneer in the development of many automated, computer- based systems supporting health research. He developed an elec- tromagnetic dosimeter used in epidemiological studies of the interactions between electromagnetic fields and human health. In a different area, he contributed a diagnostic tool for assessing the edematous state of biological tissues by sensing cell mem- brane capacitance and tissue compartment spaces based on in vivo electrical impedance measurements (“Electrical Impedance Spectroscopy”). His current interests concentrate on developing methods for the monitoring of human cells growing under labo- ratory conditions. At his laboratory at the Royal Victoria Hospi- tal, a computer-vision system is currently being developed as a real-time toxicology system designed to assess the proliferation of human cells under the influence of various agents. Dr. Héroux is a member of the Society of Toxicology, of the International Commission on Occupational Health (ICOH), and of many com- mittees of the Institute of Electrical and Electronics Engineers dealing with health impacts. His intimate knowledge of physics and engineering brings a different approach to health problems and to the measurement of toxic reactions. Professor Héroux may be contacted at McGill University Faculty of Medicine, 1130 Pine Ave West, Montreal, Quebec, Canada, H3A 1A3. Telephone: (514) 398-6988, Fax: (514) 398-7435, E-mail: cxhx@musica.megill.ca. http://libra.rvh.mcegill.ca.Customizing Cancer Treatment Options 133 EDITORS’ COMMENTARY As important as clinical research has proven to be, it some- times seems rather slow, costly, and limited by the human subjects available at a given time. Laboratory testing also might be labori- ous, time-consuming, and even error prone. The ideal would be some type of sophisticated automated system, capable of many simultaneous experiments, following accepted scientific proce- dures, and yielding individualized information useful for immedi- ate therapeutic purposes. It would be objective, with controls to assure minimal uncontrolled variability of independent and dependent variables, yet have the flexibility necessary for flexible, economic, and timely experimental designs. Absent the ideal, something better than rank speculation from uncontrolled testing would be helpful. Methodology is important and a useful topic in this book series The following chapter presents a relatively new methodology of considerable potential value for both research and immediate therapeutic assistance. The focus is on imaging living human cells undergoing changes induced by chemotherapeutic, immunologic, radiologic, angiogenic, and other agents, singly or in combination, that may be helpful in the eradication or remission of major disease processes. The use of the techniques described may be quite help- ful to many of those engaged in medical research, particularly in the troublesome area of asbestos-induced disease. A. INTRODUCTION A system for the chronic assessment of human cells in vitro has been under development at the Royal Victoria Hospital for134 Sourcebook on Asbestos Diseases some years. The system has potentially wide-ranging applications in clinical and research work. It can form a permanent record of cell behaviour under numerous culture conditions simultaneously. The system is being developed primarily to support the treatment of intractable cancers. We describe below how such a system could contribute to improving the clinical outlook for victims of mesothe- lioma, and to advancing research on the disease. B. CLINICAL OUTLOOK FOR MESOTHELIOMA Prognosis for the cancer primarily attributed to asbestos exposure, malignant (diffuse) mesothelioma, is very poor. Typical survivals are only 1 year post-diagnosis. Traditional methods of treatment such as surgery, radiotherapy and chemotherapy have proven largely ineffective. Surgery can be used with some success in patients with limited disease (early stage) at the time of diag- nosis, but is associated with significant morbidity and mortality. For the disease generally, curative surgery is of controversial value. Both surgery and radiation therapies are limited to treating cancers that are spatially confined. As well, radiotherapy and com- bination chemotherapy are generally ineffective against mesothe- lioma. Chemotherapy has been a mainstay in the treatment of malignancies, a major advantage being its ability to treat the wide- spread or metastatic cancers typical of mesothelioma. The ability of chemotherapeutic agents to preferentially kill cancer cells throughout the body while doing little harm to most other body cells, the specificity of treatment, has been pivotal to their success. In mesothelioma chemotherapy, many drugs are often used in com- bination. The preference for multiple agents is based upon the belief that tissue adaptation to toxic challenges is more difficult if the tumour is solicited by aggressors simultaneously addressing many physiological mechanisms. Confidence in combination che- motherapy is also inspired by the success rates achieved by the method in dealing with other tumours, such as Hodgkin’s disease. The large majority of chemotherapeutic drugs kill cancer cells by affecting DNA synthesis or function: alkylating agents (such as cyclophosphamide and cisplatin), nitrosoureas (such as car- mustine), antimetabolites (such as 6-mercaptopurine), antitumor antibiotics (such as doxorubicin and mitomycin) and plant alka- loids (such as vincristine) all act in this way. Steroid hormones are also useful in treating some types of tumours by modifying the growth of hormone-dependent cancers.Customizing Cancer Treatment Options 135 Combination chemotherapies are generally highly toxic and are administered to the patient’s limit of tolerance. Drug regimens are tailored by coordinating the state of the patient with the known toxicities of specific drugs. The aim is to avoid compromis- ing further vulnerable organ systems. Another important factor is the experience of various centers in dealing with the drug’s tox- icities. The number of drugs used alone or in combination in the treatment of mesothelioma is quite extensive (see Table 6-1). The number of choices available,* as well as the limited data available for comparing the success rates among so many different treat- ments, places clinicians in difficult situations. Table 6-1 Some of the drugs used in the treatment of mesothelioma eee eee DRUG COMBINATIONS | DRUGS COMBINED DRUGS USED ALONE WITH DOXORUBICIN 5-Fluorouraeil Cisplatin S-Azacytidine 5-Fluorouracil Semustine Cisplatin ‘incristine Cyclophosphamide Detorubicin |_Vinblastine Bleomycin | _Razoxane (| _—__—‘Hypericin——_—| Methotrexate Folinic-acid Vincristine Ifosfamide isplatin Methotrexate Folinic-acid Mechlorethamine Vincristine Vinblastine Bleomycin Cyclophosphamide Melphalan senate asi lophosphamide lophosphamide Methotrexate wstotinats Copinin | O*Gpuatmiame | Meticizomte Cyclophosphamide Dacarbazine Mitomycin-C Vinbiastine 5. Fluorouracil Cyclophosphamide | __ Mitomyein-c | Cisplatin Mitomycin-C Cyclophosphamide Dacarbazine Vincristine Paclitaxel Vincristine 5-Fluorouracil Cyclophosphamide Dacarbazine Vincristine Procarbazine Vincristine omycin-D Cyclophosphamide 5-Fluorouracil Vinblastine | Vincristine Methotrexate m-tetrahydroxyphenylchlori: Carmustine Methotrexate Folinic-acid ‘lophosphamide lophosphamide Thio-tepa vinggeiget 5-Flucrouracil metoiophose Etoposide p Methotrexate Vincristine Cyclophosphamide Dacarbazine Vincristine Vinorelbine Vincristine Dacarbazine Cyclophosphamide Actinomycin-D ‘Actinomycin-D136 Sourcebook on Asbestos Diseases The overall strategy of chemotherapy is to incite tumour cells to go into a cycling phase, where their nuclear membrane is dis- solved, the DNA unwound and the mitotic apparatus active. In that state, cells are relatively vulnerable to toxins. Drug combi- nations might be administered periodically (three drugs each three weeks would be typical of a cycle) for a number of cycles which depends on patient and tumour responses. Because mesothelioma itself has many histological subtypes (epithelial, mixed and sarcomatoid‘), and because potential chemo- therapies are so numerous, optimizing the choice of drugs repre- sents a difficult problem. Assuming some drugs are potent against some types of mesothelioma, the task of gathering sufficient clini- cal evidence to clarify the best combinations would be a difficult and laborious process requiring decades of clinical work. This per- spective has incited many investigators to revise strategies. C. NEW METHODS OF CANCER TREATMENT Molecular biologists hope to improve on classical chemother- apy by homing in on oncogenes which are at the root of many tumours. These approaches would improve the specificity of treat- ments by addressing genetic characteristics of cancer cells that set them apart. In cancers of the colon and of the pancreas, where the ras oncogene is often key to tumour formation, the blockage of the enzyme farnesyltransferase may prevent ras protein from exerting its proliferative effect on the cells.® A similar strategy involves the p53 gene. When this gene fails because of mutation, the functional p53 protein product is depleted. Cells then proliferate uncontrollably, because p53 protein is an endogenous brake on cell division. The treatment involves the administration of a lethal virus which activates only in cells with mutated p53.° While these examples provide exciting possibilities, enthusi- asm is tempered by the fact that tumours are not genetically homogeneous. The genetic specificities addressed may not be ef- fective for all cells in a tumour, or for all tumours. In mesothe- lioma, for example, loss of allele on the short arm of chromosome 8p has displayed diversity.’ Resistance through cell adaptation or selection may develop under these therapies in the same way that it does in conventional chemotherapy. : Although neither ras nor p53° seem to be of significance in mesothelioma, and in spite of the absence of a well-defined genetic target, an innovative attempt to link gene and chemical therapies together is being tried.° The approach is based on insertion intoCustomizing Cancer Treatment Options 137 tumour cells of a specially designed gene which enhances sensitiv- ity to the drug ganglicovir. In the treatment, the herpes simplex thymidine kinase gene is inserted into a common cold virus (ade- novirus), which is then administered intrapleurally to the tumour. Preliminary experiments indicate efficient tumour cell infection. These cells are sensitised to ganglicovir, hopefully sparing normal cells while killing cancer cells. Other innovative approaches to fighting tumours involve immune methods,” featuring specificity, as well as angiogenic con- trol," featuring low toxicity. Even as cancer specialists work at developing these innovative treatments, one frequent sobering comment from the developers themselves is that combinations of methods are likely to be needed to improve clinical outcomes. So resistant are some tumours that almost every narrative on the development of a particular tech- nique is a roller coaster of a tale where encouraging successes mix with discouraging failures. Specificity of treatment (the ability to address cancer cells to the exclusion of normal cells) has long been the focus of cancer research. But specificity will not by itself pro- vide a complete solution, because tumours exhibit diversity. With their high mutation rates, cancer cells are a heterogeneous group that can adapt rapidly to environmental changes. Therefore, the most threatening characteristic of resistant tumours is their plas- ticity. In spite of these difficulties, it is apparently possible to survive mesothelioma, although such cases are rare. Clinical studies men- tion occasional remissions in patients treated early for the dis- ease.” However, the impact of the treatment itself is partly in doubt, because untreated long-term survivors also occur occasion- ally." It is immaterial whether natural immunity or chemotherapy rid the body of the disease. It remains that it is possible to do so in a limited number of cases. Can this breach be expanded? Per- haps the simplest explanation is that the survivors represent a spe- cial subset of cases, destined to fare well because of the particular nature of their disease. The opposite view would be that from a group of nondescript initial tumours, any can be eradicated if prop- erly challenged by the right medication. Remission without thera- peutic intervention would be seen as a particular instance of endogenous immune treatment. Although clinical intervention does not generally appear to improve survival in mesothelioma, there have been many instances of favourable clinical responses and transient tumour regressions using combinations of chemo- therapeutic agents. Possibly, more substantial inroads would be made against these tumours if treatments and treatment admini- strations were more successfully optimised. The ability of tumour138 Sourcebook on Asbestos Diseases tissue to adapt to treatments and neutralise them may be a pri- mary explanation for the small number of favourable outcomes. This view is supported by the observation that after an initial bout of chemotherapeutic treatment, cancer cells surviving the chemi- cal blast often become resistant to another round of chemotherapy, and the cancer is almost always rapidly lethal. Dividing tumour cell populations continually diversify, almost insuring that some sub-fraction will be positioned to sustain tumour growth. Designers of therapies seem to withdraw from the concept of a silver bullet that kills all cells in a tumour. Many mention co- existence of the host with the tumour over time. This suggests that it may be possible to build upon a long series of modest advantages gained against the tumor through medication. Detailed informa- tion on tumour behaviour may be necessary to sustain these advantages. Eradication of the tumour may then present as a slow attrition that would mirror in reverse the slow growth of the tumour in healthy tissue. D. STRATEGY Our own research effort against resistant tumours does not focus on a particular regime of chemotherapy or on any specific method. Rather, we deal with the complexity and plasticity of tumours by accumulating a large amount of information on them. This consists of a series of observations through time for numerous therapeutic alternatives applied separately to groups of cells in vitro. We believe that these historical records of the interaction- between the patient’s own tumour cells and potential therapies should help in guiding treatment selection. Such an exercise yields: 1. permanent record of tumour cell behaviour in vitro under various therapies, 2. possible technique to help the clinician in the choice of thera- peutic alternatives, as well as 3. research tool to resolve scientific issues surrounding resistant tumours. Running in vitro simulations of chemotherapeutic options broadens the range of therapies that can be explored for any spe- cific tumour. Our experimental set-up also attempts to deal with particular characteristics of tumours. First is the recognition of the diversity of tumours and of can- cer cell types within a tumour, attended by using biopsy material.Customizing Cancer Treatment Options 139 Second is adaptability over time, a basic property of tumour biology, which is attended by experimenting over a suitably long period. This could mean test durations ranging from one week to one month. Third is recognition that optimised therapy may be complex and primarily dependant on experimental evidence, attended by running as many therapeutic simulations as possible. To document in detail the reaction of cancer cells to therapeu- tic agents, the most basic information relates to cell division and survival. At any time, cells are either cycling, non-cycling or arrested (the case of most somatic cells). To determine whether agents produce shifts in the repartition of cells between these three compartments, it is necessary to recognise not only changes in overall cell count, but also how a given cell-count change is gen- erated from various cell sub-groups. There is a need for accurate fixes on the behaviour of cell sub-fractions, requiring a substantial number of observations. This requirement already points to the necessity of automated counting techniques for appropriate sys- tem performance. E. IN VITRO MODELING There have been previous attempts to measure in vitro the sen- sitivity of tumour cells to chemotherapy,"* but the clinical use- fulness of the technique has been limited until now to guiding decisions on management of patients who relapsed after receiving multiple forms of chemotherapy (i.e., determining to which drugs tumours have become resistant). One difficulty related to the fact that the test was implemented as a clonogenic assay, measuring the ability of cells to form colonies while under the influence of various drugs. In lung cancer, only a very small number (signifi- cantly less than 1%) of cells in a tumour could form such colonies, restricting the significance of the test to this small, hardy fraction. If vigorously dividing clones are used for testing, there is danger of drift in the DNA content of the cells, resulting in an altered specimen. Many clinical specimens also contain fibroblasts and stromal cells, sub-fractions that should ideally be distinguished from the truly malignant fraction. Also, many of the cells one would need to monitor in order to gain insight into treatment effectiveness have long doubling times ranging from days to weeks. Prolonged observation and accurate counting are therefore necessary. Previous investigators have also observed that although pre- dictive ability for resistance is excellent, correspondence with in140 Sourcebook on Asbestos Diseases vivo sensitivity is only about 60%." This may be because sensitivity determinations are more dependant on cell sub-fraction composi- tion.* An alternative explanation would involve the change in environment from in vivo to in vitro systems. Because the drugs used in chemotherapy are mostly directly cytotoxic, however, it is not expected that biotransformation would play a significant role in determining sensitivity. Influence of the immune system on out- come is a possibility. ) If interesting data can be obtained from challenging cultured cells with chemo-therapeutic agents, more clinically relevant in- formation is generated if the cells being challenged are fresh from the patient’s own tissues. The representativity of the biopsy is of importance. The genetic expression of cells taken out of the body is known to alter over time in culture. Therefore, using recently biopsied cells is of value, perhaps to the point of repeatedly refresh- ing test-cell stocks (and re-initiating tests) as clinical treatment proceeds. Our method of assessing cells is not dependant on colony formation, but only assumes that a viable and representative sus- pension of tumour cells can be obtained from tumour tissue. Ex- periments could be carried out pre-treatment, in order to suggest the best initial drug therapy, as well as concurrently with treat- ment with new cell stocks in order to update strategies continu- ously over the chronic course of tumour evolution, Although reasonable success has been recorded in other laboratories using conventional culture techniques (RPMI 1640 with 10-20% fetal calf serum) for about half of the biopsies, use of the patient’s own plasma should be considered. Optimisation of drug selection and concentration inevitably involves a large number of tests performed simultaneously. An advanced system including real-time assessment of results would allow the performance of flexible tests which would automatically adapt the drug strategy according to recent observations. Envis- aging this level of plurality and complexity in biological testing inescapably leads to inclusion of computer intervention. EF IMPLEMENTATION The detailed recording of the behaviour of tumour cells under numerous therapeutic conditions implies an intimate marriage between cell-culture techniques and computer control. Data acqui- sition is essentially based on computer-vision. There is a literature dealing with cell tracking under the microscope,’"” but we believe ourselves to be pioneers in using large-scale computer imaging of living cells in therapeutic experimentation. Computer-based imageCustomizing Cancer Treatment Options 141 analysis is superior to the human brain in quantifying image dot (pixel) intensity, but is rather poor (slow) in shape recognition.”** There are delicate adjustments needed to accomplish the micro- processor-intensive tasks needed for the system’s operation within reasonable execution times. : To make exploitation of the system practical, a high level of automation is needed so that tests do not monopolise a team of specialists. As is shown in Figure 6-1, the Front-End hardware includes a CO, incubator which contains microscope optics, CCD camera and a highly accurate XYZ motorised stage under com- puter control. The motorised stage holds a multi-well dish on which 77 wells have been seeded with tumour cell suspension. Inverted Microscope within . water-jacketed COs Incubator Scan path for 96-well dish 4 > dX} re ~, ey Ss, o> rSrr5 Soo , Naa Figure 6-1 Front-End hardware supporting the documentation of 77 therapeutic alternatives. The 96-well dish is a conventional dish used routinely in many laboratories for diagnostic studies. Some wells are used to stock drugs, rather than cells. Each well bottom (lower right) is filled with contiguous images, called “patches.” While the tumour cells mature in the incubator, the motorised stage-microscope-camera combination repeatedly scans the dish and relays images of the tumour cells to a computer. Each of 77 wells is subdivided into 64 patches (or images) when using a 100x magnification (the system is functional up to 400x), corresponding to a maximum of 4,928 image sites for the whole dish. Depending on the requirements of each experiment, the individual wells can be injected with various combinations and concentrations of thera- peutic agents. While this can be done manually, equipment and programs are being developed to administer drugs automatically142 Sourcebook on Asbestos Diseases ) over time from the free wells in Figure 6-1 to the individual wells that contain tumour cells, in a simulation of bolus drug admini- stration. We also plan to include facilities that circulate culture me- dium in the wells, in order to include realistic toxicokinetics in the experiment. The images gathered are relayed to a vision card. Some images are saved to hard disk to produce animations to be viewed by hu- man observers. Others are optimised for automated analysis by computer-vision algorithms. Because of the processor-intensive nature of the procedure, the tasks are actually shared between various computers linked together through a network, as shown in Figure 6-2. HUB Figure 6-2 Computer configuration supporting image acquisition, stor- age and interpretation over time. The Front-End processor manages multi-well dish movements, image grabbing and saving (in the future, fluids management and microscopy adjustments), using custom Visual C software. The Imaging station runs specialised software interpreting the images being stored by the system, for example, count, position and mor- phology of the individual cells found in the images. A more advanced determination is the state of vitality of individual cells. The Monitoring computer integrates and displays in real-time critical data from the experiment. Such data include test execution variables and evolution over time of the number of cells in the vari- ous wells. A sample of such a determination obtained with trans- formed mouse macrophages activated with growth factor is shown in Figure 6-3.Customizing Cancer Treatment Options 143 Transformed Mouse Macrophages with Growth Factor Number of Cells in Image Figure 6-3 Evolution over time of cell counts in a single image of a multi-well dish. Cells within a frame may number as many as 600. The counts from many images or “patches” can be integrated to form a common data pool. In the future, the Monitoring processor will also host artificial intelligence routines capable of relaying instructions to the Front- End to modify test progress according to observed results. From the images generated by the system, some evaluations are possible which are highly processor-intensive, complex, or in need of direct human intervention, and must be performed “off-line” (develop- ment of new system capabilities). For this purpose, the system is fitted with a CD recordable disk drive which allows the data from a given experiment to be written to a group of compact disks. These disks can then be analysed by remote computers. G. INSPECTION OF ANIMATIONS BY THERAPIST The most primitive function that the system can accomplish is the simple storage of stacks of images, each representing the evolution of tumour cells under the effect of a combination of therapeutic agents. The scope of this activity is limited only by con- siderations of image compression time and disk storage space. Typical stacks are formed from many hundreds of images recorded at approximately one-hour intervals. Once the experiment is ter-144 Sourcebook on Asbestos Diseases minated, the stacks of images can be transformed into computer animations viewable by a human therapist. Although the multi- well dishes presently in use contain only 77 separate experiments, technology exists to produce multi-well dishes that would accom- modate more than a thousand separate tests (but with fewer individual images). An early goal, at this stage of system development, is for oncologists to gain insight into preferred treatment options by inspection of these numerous animations. However, the system characteristics point to a need for semi-automated data interpre- tation, so that human specialists can be guided as to where to focus, within the mass of the data available. H. AUTOMATED IMAGE ANALYSIS In order to provide such support, some of the images grabbed by the system are enhanced, binarised, interpreted and displayed as integrated results ongoingly as a function of time (see Figure 6-4). The software is also used to monitor evolution of the 77 experiments in real time (the curve in Figure 6-3 is updated as the test proceeds). 1. Digitized images 2. Binarized images Lose TL Figure 6-4 Automated image analysis procedure used for test monitor- ing and to aid in cell animations inspection. This procedure needs deli- cate coordination of sample illumination with image enhancement algorithms. Bitmap Processing Software can recognise individual cells with high reliability under certain illuminations, and if it has been “trained” for rec- ognition of a particular cell type with features that are reasonably constant. We have assessed the performance of the system in rec- ognising such haematological workhorses as HL-60 cells, which have relatively simple shapes. Recognition accuracy results were better than 95 % for the image shown in Figure 6-5. More workCustomizing Cancer Treatment Options 145 ition of HL-60 cells. From an enhanced image, algorithms are used to circle and characterize with 10 parame- ters (not shown) objects that represent living cells (dark halo). Dead cells and decaying material could be tracked independently in this micrograph. Accuracy in recognizing live cells is better than 95%. is needed before the recognition rate for mixed cell mixtures is as high, but it must be realised that even fairly inaccurate cell rec- ognition can be used effectively for the purpose of guiding manual inspection. Beyond aiding the human brain in sifting through large arrays of experimental results, computer-based image analysis may prove even more valuable when further developed. If tumour cells can be reliably delimited, all of the classic microscopy-based determi- nations can in principle be used to study large cell populations under numerous conditions. First to come to mind are the 10 mor- phological cell parameters which we already compiled in our HL- 60 experiments, as well as point-pattern analyses using known positions of cells. But one should remember that any fluorescence- based determination is directly amenable to massive automated investigation under this system. One remarkable characteristic of the system is its potential ability to follow large number of cells over time, each cell being146 Sourcebook on Asbestos Diseases individually known to the computer. These exhaustive chronic in- vestigations of small cell sub-populations cannot realistically be performed using human workers, but are precisely the tasks at which computers excel: undivided sustained attention, tracking millions of objects over long periods. I, OTHER APPLICATIONS OF THE SYSTEM The system can find other uses, beyond sifting through large numbers of alternate therapies for resistant cancers such as mesothelioma. 1. Research into the process of carcinogenicity of fibers: the interaction between asbestos fibers and macrophages. Both cells and fibers could be tracked by the system over time (see Figure 6-6). Figure 6-6 The micrograph shows HL-60 cells dispersed among asbestos crocidolite fibers. Simple enhancements algorithms produced this false image that can be used to track the respective positions of fibers (straight segments) and cells (circles).Customizing Cancer Treatment Options 147 2. Research on the proliferative activity of exposed cells having important prognostic significance in several types of cancers such as non-Hodgkin’s lymphoma, neuroblastoma, breast can- cer, colon cancer, lung cancer, cancer of the ovaries and bladder cancer, as summarised in Table 6-2. Table 6-2 Human cancers associated with agents whose major action is induction of increased cell proliferation. Hepatitis B virus Schistosoma hematobium Bladder Schistosoma japonicum Clonorchis siniensis, Opisthorchis viverrini Epstein-Barr virus Bile and pancreatic juice Small intestine Betel nut, lime Oral cavit Physical or mechanical trauma Asbestos Mesothelioma Chronic impulse noise [Tropical ulcers Sin 3. Toxicological investigations could be conducted for chemicals that produce hyperplasia in humans (see Table 6-3). This could lead to tests for cancer promotion, in the same way that the Ames test addresses mutagenicity. Ames has recently pointed to the importance of cell proliferation as an indication of promoter action.”148 Sourcebook on Asbestos Diseases (Tabl le 6-3 Chemicals that produce hyperplasia in humans. Agent Bock (1968) Klein-Szanto (1989) Schmahl (1977) Hydantoin Gingiva-Lymphoid Tissues 4. Farther areas of application are drug development and ageing research. J. CONCLUSION The long-term payoff of sophisticated experiments conducted automatically by computers to analyse tumour cell behaviour and guide therapists to optimal treatment of a disease so resistant as mesothelioma may be fairly distant. But there is an opportunity for the approach described above to make more modest contribu- tions to the improvement of cancer therapy relatively soon. The recording of numerous animations of tumour cells illlus- trating the effect of a wide variety of chemo-therapeutic possibili- ties is a realistic short-term goal. More experience will have to be gained before the relevance of these results to clinical outcome is confirmed. Preparation techniques for cell suspensions appropri- ate for the conduct of the experiments is critical. Our research in developing the automated cell-monitoring tool described above has attempted to address the most uncertain ele- ments of the technique early, when feasible. But we have yet to uncover any insurmountable obstacle. The method is likely to greatly expand our understanding of tumour cell behaviour, and may eventually benefit the victims of intractable cancers such as mesothelioma.Customizing Cancer Treatment Options 149 REFERENCES 10. 11. Scannell JG. Mesothelioma. In: Thoracic oncology. Raven Press, 1983. — ws : Roggli V, Sanfilippo F, Shelburne JD. Mesothelioma. 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