Summary Perspectives: First International Workshop on Chromosomes in Solid Tumors Jeffrey M. Trent
INTRODUCTION The First Workshop on Chromosomes in Solid Tumors p r o v i d e d a m u c h n e e d e d forum for discussing recent advances in this d e v e l o p i n g area of cancer research. The combination of invited and poster presentations given at this workshop and p u b l i s h e d in this v o l u m e attest to the e x p a n d i n g n u m b e r of basic and clinical studies involving solid tumor cytogenetics. There were five major sessions during this workshop, w h i c h will be reviewed briefly in turn. For clarity, this discussion will attempt to synthesize both oral and poster presentations. The goal of this s u m m a r y is not to provide an exhaustive and detailed review of each session, but rather to bring emphasis to those studies that appear to be of greatest current interest. Scientific Session I: A d v a n c e s in Cell Culture
Chairperson: Gordon Sato, Ph.D. The use of in vitro cell culture for studies of solid tumors is particularly critical for successful cytogenetic analysis. Accordingly, recent attempts to improve the success rate of solid t u m o r karyology have been based on i m p r o v e m e n t s in techniques for growth of cells in short-term culture. Discussion in this session first centered on the use of specialized cell culture m e d i u m (Dr. Sato) to enhance growth of selected tumor types. The p h i l o s o p h i c a l suggestion was m a d e that in vitro data needs to be v i e w e d at the organismal level, rather than merely the cellular level. However, questions arose as to the clinical relevance of utilizing in vitro data, w h i c h perhaps introduces bias by selecting only those m i n o r s u b p o p u l a t i o n s that can adapt to in vitro growth. In~ answer to this question, progress in several areas of cell culture research was reported, and h e l p e d in part to address the concerns of in vitro selection and clinical relevance. First, significant e n h a n c e m e n t of in vitro growth was reported with use of specialized media, serum, growth factors, and tumor disaggregation techniques, as well as conditions for varying h y p o x i a (Drs. Sato, Leibovitz, Gazdar). The potential clinical relevance of in vitro m o d e l s was suggested by studies of lung cancer (Dr. Gazdar), where results indicated that a subset of lung cancers From the University of Arizona Collegeof Medicine, Department of Radiation Oncology,Cancer Center Division, Tucson, AZ. Address requests for reprints to Dr. Jeffrey M. Trent, University of Arizona College of Medicine, Department of Radiation Oncology, Cancer Center Division, Tucson, AZ 85724. Received August 5, 1985; accepted August 19, 1985.
191 © 1986 Elsevier Science Publishing Co., Inc. 52 VanderbiltAve., New York, NY 10017
Cancer Geuet Cytogenet 1~:191-197 (1086) 0165-4608/86/$03.50
J.M. Trent could be identified with a distinct morphologic pattern in vitro, which correlated with specific cytogenetic change, elevated expression of various cellular oncogenes of the myc family, and a poor clinical prognosis. Evidence was also presented indicating that in vitro clonogenic assays may provide useful information, particularly in the area of new drug screening (Dr. Salmon). Additionally, the usefulness of this in vitro culture system in defining aspects of cellular differentiation was addressed in studies of the MCF-7 breast cancer cell line, where markers of differentiation appeared to become increasingly expressed in transit end cell compartments (Dr. Salmon).
Scientific Session II: Solid Tumor Karyology
Chairperson: Jeffrey M. Trent, Ph.D. Recent application of advances in cell culture and chromosome harvesting techniques have unquestionably facilitated karyotypic analysis of solid tumors. However, despite significant recent advances in this field, a paucity of data (particularly when compared with the wealth of knowledge in human leukemias) currently exists regarding specific or "recurring" chromosomal abnormalities in human solid tumors. Despite technical difficulties remaining in this area, results were presented at this workshop that clearly identified a clustering of chromosome alterations to specific sites within the human genome. Professor Felix Mitelman (Department of Genetics, University of Lund, Sweden) issued a renewed call for unpublished data on cytogenetic alterations of solid tumors in order to facilitate updating of his extremely useful Catalog of Chromosome Aberrations in Cancer (Progress and Topics in Cytogenetics, Volume 5, Series editor: Avery Sandberg, Alan R. Liss, Inc. New York). This appears to be a particularly worthwhile undertaking, and it is hoped that an increasing number of investigators will contribute to this increasingly important data base. A second and very exciting area of research suggested that the presence of sporadic chromosome alterations in peripheral blood lymphocytes (PBL) might assist in detecting a propensity for development of malignant disease (Dr. Pathak}. For example, the recognition of chromosomal errors in normal PBL, which involve regions known to be frequently altered in tumors cells (e.g., chromosome 3p alterations in PBL of patients at risk for renal cell carcinoma) may be of potential clinical significance. At present, the true value of these observations remains to be confirmed and extended. Additional studies are certainly warranted. Consideration of the importance of combining molecular biologic analysis with tumor karyology was also discussed, particularly in reference to anaplastic Wilms' tumors (Dr. Slater). It was pointed out that in anaplastic Wilms' tumor, results of karyotypic analysis allowed cytologic documentation of alterations of chromosome 11p in roughly 50% of all patients studied to date. However, when DNA analysis of anaplastic Wilms' tumor patients (using relevant restriction fragment length polymorphism probes for chromosome 11p) were utilized, almost 100% of the patients could be shown to display an alteration of 11p. Clearly, submicroscopic alterations in tumor cells do exist and may be of germinal significance in the genesis and progression of human cancers. Additionally, these results suggest that the merging of the disciplines of cytogenetics and molecular biology will become of increasing importance in future years.
Scientific Session III: Molecular Cytogenetics
Chairperson: Mary E. Harper, Ph.D. Two relatively specific areas were discussed in this session: first, the identification and localization of DNA or RNA sequences using in situ hybridization and, second, the characteristics, frequency, and consequences of gene amplification. Both areas are extremely important and timely subjects for research based on the recent correlations between the sites of specific chromosome rearrangements and the loci of human cellular oncogenes. Results were presented confirming that in situ hybridization techniques are becoming sufficiently sensitive to allow unequivocal localization of single copy gene sequences on human chromosomes (Dr. Emanuel). Additionally, results were presented that suggest the cytologic recognition of low abundance mRNA using in situ cytohybridization may be feasible (Dr. Harper). Both techniques hold tremendous promise for furthering our understanding of the basic biology of the molecular/chromosomal changes of importance to human cancer. In particular, RNA in situ cytohybridization for identifying cells infected with human T-cell leukemia retrovirus III (HTLV-III) appears to have significant clinical application. The recent explosion of molecular biologic research involving gene amplification in human cancer traces its origin to a series of cytogenetic observations. Interest in the possible amplification of cellular genes was clearly fostered by the work of Biedler et al., suggesting that double minutes (DM) or homogeneously staining regions (HSR) may have significant biologic significance. At the workshop, reports were presented that clearly documented the adverse clinical consequences of amplification of either drug resistance sequences or cellular oncogenes in human tumors (Drs. Brodeur and Meltzer). Especially exciting studies were presented in human neuroblastoma (Dr. Brodeur), suggesting that amplification of the cellular oncogene N-myc might be of importance in the clinical prognostication of this disease. Specifically, cytogenetic or molecular biologic identification of N-myc amplification appears to identify a subset of patients with an extremely poor prognosis. The essentially one-to-one correlation between cytologically recognizable chromosome change (DM or HSR) with N-myc amplification (identified by Southern blots) in human neuroblastoma further supports the notion of combining both cytogenetic and molecular biologic approaches. In support of this viewpoint, a report was presented that provided a conceptual framework for combining molecular and cytogenetic analysis of human tumors (Dr. Meltzer). The strategy presented suggested that cytogenetic prescreening of tumor lines was a logical "first line screen" to identify sites of recurrent breakage (for molecular breakpoint mapping) as well as to identify targets for molecular analysis of tumors with amplified domains. The "second line screen" involved use of DNA renaturation gels to confirm the presence of an amplified DNA domain at the molecular level. At this point, screening with appropriate available DNA probes (e.g., cellular oncogenes) or cloning directly from DNA renaturation gels can be performed. This strategy appears to provide a directed approach to identify tumors harboring amplified domains likely to have biological relevance. Scientific Session V: Tumor Biology
Chairperson: Sandra Wolman, M.D. This session, more than any other, aroused significant discussion at the workshop and provided a forum to discuss areas of research with relevance to cancer cell biology. Presentations confirmed and expanded the finding of tumor heterogeneity with respect to differentiation and proliferative potential as a component of a vari-
J.M. Trent ety of human tumors (Drs. Balaban, Buick, Davis, and Wolman). The recognition of biologic heterogeneity extends not only to changes in frequency distributions of subpopulations (Drs. Balaban and Buick), but also the suggestion by Wolman et al. of a significant role for karyotypically normal cells participating in early stage lesions of some human tumors (particularly breast cancer). Discussion of this general area was lively and by no means yielded a simple or definitive answer. Whether the presence of karyotypically normal cells in human tumors is merely representative of contaminating normal cellular infiltration, or in fact represents a subpopulation of the tumor with submicroscopic genetic alterations remains to be determined. This area appears open to further examination and should receive significant further study in the future. As a possible corollary to the work on human tumors, results were presented describing a significant technical advance in analyzing the karyotype of murine cancers of the skin (Dr. Aldaz). This in vivo model system has particular value because it allows examination of the progression through multiple stages of tumorigenesis, including availablility of papillomatous tissue, primary tumor, and metastatic tumor populations. Results of this murine model, indeed, may be applicable to the study of human solid tumors (particularly squamous cell carcinoma, cervix, or lung).
Scientific Session Vh Clinical Correlations of Chromosome Change in Solid Tumors
Chairperson: Avery A. Sandberg, M.D. This session also entertained two basic issues. First, the possible association of fragile sites (Dr. Glover) and sporadic translocations (Dr. Scheres) with neoplasia and, second, the clinical correlation of chromosome change in solid tumors (Drs. Levin, Sandberg, and Whang-Peng). Results were presented that strongly implicated chromosome fragility with possible predisposition to neoplasia. This association, although of great potential interest, is a subject in need of further study to confirm and clearly establish (in a prospective fashion) the clinical relevance of these in vitro findings. However, because of the recently reported associations between the loci of fragile sites (as well as sporadic translocations) and chromosomal loci of human cellular oncogenes, this area appears especially worthy of further investigation. The clinical correlation of chromosome change in acute leukemia has clearly demonstrated the utility of cytogenetic examination in human cancer. Results from many institutions are unequivocal and suggest that when major risk factors are considered for predicting survival duration in most human leukemias, only the specific karyotype classification consistently represents an independent prognostic variable. Of significance, karyotype alone is capable of predicting survival even when age, initial platelet count, presenting peripheral blood blasts, or FAB classifications are considered. Although chromosome analysis has been shown to be clinically useful in the diagnosis and evolution of hematopoietic tumors, unfortunately there is insufficient data to determine if cytogenetic analysis of solid tumors will be equally informative. Results were presented at this first workshop suggesting that, in colon cancer (Dr. Levin), bladder cancer (Drs. Sandberg and Falor), ovarian cancer, and perhaps small round cell tumors (Dr. Whang-Peng) clinical correlation of chromosome change, indeed can provide a useful adjunct to routine histopathology. This area appears particularly fertile for further analysis and it is hoped that sufficient enthusiasm for examining the clinical significance of chromosome change in solid tumors will be fostered in the scientific community to allow appropriate funding of this type of research.
CONCLUDING REMARKS General Overview New d e v e l o p m e n t s in science and technology often lead to more n e w questions than answers. After reviewing the presentations at the First International Workshop on Chromosomes in Solid Tumors, it is clear that recent studies of solid tumor karyology have already suggested a n u m b e r of areas for future exploration. There is now definite evidence to d o c u m e n t the value of cytogenetic analysis in basic and clinical investigations of h u m a n solid tumors, in a d d i t i o n to the well established value of this m e t h o d o l o g y in the h e m a t o p o i e t i c cancers. It seems imperative, however, that an increasing focus must be placed on cytogenetic examination of solid tumors (particularly carcinomas), w h i c h represent up to 80% of the h u m a n cancers. I for one have little doubt that a significant increase in our knowledge of karyotypic and m o l e c u l a r biologic alterations in a variety of h u m a n cancers will occur before the next s c h e d u l e d w o r k s h o p in early 1987. However, I am also aware that many difficult problems r e m a i n to be overcome associated with aspects of methodology and interpretation of cytogenetic studies of solid tumors. Some of the most obvious problems remaining to be a d d r e s s e d in this d e v e l o p i n g area of cancer are listed in Table 1, and they must be a d d r e s s e d by a host of laboratory researchers. In this regard, it is h o p e d that at the next w o r k s h o p reports of significant technical advances in m a n y of these areas will be addressed. Although m a n y difficulties u n q u e s t i o n a b l y remain, evidence was p r o v i d e d at the workshop to d o c u m e n t the biologic and clinical value of cytogenetic analyses of solid tumors. Table 2 lists some of the areas w h e r e tumor karyology can provide important information of value in u n d e r s t a n d i n g the basic cellular and molecular biology of cancer. A d d i t i o n a l l y , Table 3 lists some areas of potential clinical value for solid t u m o r karyology. I hasten to point out that I have not attempted to be comprehensive in m y treatment of either the problems or potentials of solid tumor karyology. However, it seems clear that if the full potential of cytogenetic analysis is to be realized, significant a d d i t i o n a l w o r k in this important area must occur. In the last analysis, e m p i r i c a l testing of solid tumor karyology (rather than merely analogy to the h u m a n leukemias) will provide the only true measure of its value in clinical cancer research. Although very w o r t h w h i l e achievements in m a n y promising areas have already occurred, m a n y obstacles remain to be overcome. It is h o p e d that major emphasis will be placed by granting agencies in supporting this type of work, but it is clearly up to all investigators in this field to provide w i t h i n the next decade a realization of the full potential of this work.
Problems associated with cytogenetic analysis of h u m a n solid tumors
Number of cases studied is still very small Not all tumor types are readily amenable to cytogenetic analysis Proportion of completely analyzable cases is low
Normal versus abnormal mitoses Primary versus secondary chromosome change Tumor heterogeneity and genetic instability
Interlaboratory variability Complexity of tumor karyotypes
196 Table 2
J.M. Trent Potential biologic value of solid t u m o r karyology
Identification of specific chromosomal alterations
Correlation with loci of cellular oncogenes
Cytologic identification of amplified cellular DNA
Correlation with disease progression or drug resistance
Role of karyotypic alterations in cellular transformation
Modulation of gene expression
Cytokinetic analysis of solid tumors
Use of BrdU-labeling techniques
Potential clinical value of solid t u m o r karyology
Tumor detection and staging
Detection of karyotypically abnormal cells in body fluids
Karyotype as an independent prognostic factor
Analogous to the human leukemias
Identification of recurring sites(s) of karyotypic change
Useful in patients with unknown primary cancers
Monitoring disease progression
Acquisition of drug resistance
A Look to the F u t u r e I w o u l d like to conclude m y comments regarding this first w o r k s h o p by providing you with a glimpse of the evolution of the m o d e r n - d a y cytogeneticist. In the past, the highly specialized, labor-intensive, and extremely t i m e - c o n s u m i n g study of tumor karyology has been p r i m a r i l y performed in laboratories whose major emphasis has a p p r o p r i a t e l y been on technical aspects of c h r o m o s o m e preparation. However, with the recent advances in m o l e c u l a r genetics, as well as the necessity of utilizing specialized cell culture techniques prior to the studying of chromosomes in solid tumors, the cytogeneticist must increasingly collaborate with other research specialists in order to optimize the o p p o r t u n i t y for success. This figure depicts m y idea of "The Ultimate Cytogeneticist," a h y b r i d being possessing the m u l t i p l e attributes required of the m o d e r n - d a y cytogeneticist. Although there appears little reason to believe that such a being exists, this cartoon hopefully illustrates the increasing i m p o r t a n c e of collaborative,studies if cytogeneticists are to continue to p l a y a major role in tumor biology. The bringing together of specialists from m a n y diverse areas at w o r k s h o p s and conferences of the type documented in this volume, in m y opinion, is crucial to p r o v i d i n g the encouragement and fortification necessary to continue the pursuit of knowledge in this area. The future appears to be particularly bright for this area of cancer research. We should be p r e p a r e d for surprises! The First International Workshop on Chromosomes in Solid Tumors was supported in part by PHHS Grant CA-40716 awarded by the National Cancer Institute, National Institutes of Health, Bethesda, MD.
computer Radioactive ~ b e ~ material •
Cell Culture syringe Medical Oncologist
pq forms ~