Eur Respir J 1989, 2. 461-469

REVIEW

The molecular genetics of human lung cancer

R.J.C. Slebos, S. Rodenhuis

The molecular genetics of human lung cancer. RJ.C. Slebos, S. Rodenhuis. ABSTRACT: With the development of molecular biological techniques the search for genetic alterations in cancer cells has resulted In the beginning of a molecular description of cellular transformation. Most of these genetic changes occur in genes which have a role in the control of cellular growth and development, the proto oncogenes. In the last decade, it has become clear that the myc and ras oncogene families are important In the carcino­genesis of human lung cancers. The m ye oncogenes are usually found to be altered In small cell lung cancer (SCLC), and these alterations appear to correlate with rapid growth and progression. Mutations in the Kras gene are specific for adenocarcinoma, a subclass of non small cell lung cancer (NSCLC). Kras gene mutations are closely associated with tobacco smok­ing, since all were found in adenocarcinomas from patient.; with a history of smoking. The erhB oncogene, which encodes the epidermal growth factor receptor, Is often highly expressed in epidermoid carcinomas. The roles for other oncogenes, such as raj or myh, as well as those of "suppressor" genes remain to be investigated, but may be of paramount Importance. The study of alterations in proto oncogenes may aid In the (sub)classificatlon and diagnosis of lung cancer, and may yield useful prognostic Information In the near future.

Divisions of ExperimenLal Therapy and Medical Oncology, ·me Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

Keywords: Human lung cancer; proto oncogcne; suppressor genes.

Supported by grant NKI 87-15 from the Nether­lands Cancer Foundation K.W.F.

Eur Respir J., 1989, 2, 461-469.

Most human lung cancers can be classified as one of four major types: small cell carcinoma, epidermoid car­cinoma, adenocarcinoma, and large cell carcinoma. The latter three types are also referred to as non small cell lung carcinoma (NSCLC) [1]. Clinically, the distinction between small cell. lung carcinoma (SCLC) and non small cell lung carcinoma is important because SCLC is treated mostly with chemotherapy while the treatment of choice in limited NSCLC is surgery. For all types of lung cancer, however, treatment results are disappointing, and the outlook for patients whose tumours cannot be completely resected remains grim. After curative resection 5 yr survival is about 30% in NSCLC, but the majority of the patients are already inoperable at time of presentation and their 2 yr survival is only a few percent [2).

It is in this context that one might hope that a better understanding of the biology of human lung cancer will eventually lead to urgently required new treatment strate­gies. The study of cellular genes involved in human cancer has progressed rapidly since viral genes from acutely transforming animal retroviruses were shown to have cellular counterparts [3]. When introduced into mouse fibroblasts in vitro, the genes were able to transform these cells [4] and were called proto oncogenes. More than 40 different proto oncogcnes have been idcn1illcd 10 date [5-7]. Most of these genes are thought 10 be of c ritical importance in the regulation of normal cellular growth and development. In the normal situation, the expression

of proto oncogenes is strictly regulated and the gene products are able to rulfill their essential roles in cellular physiology. The name "proto oncogenes" is. thus. more infonnative about the way in which these genes have been discovered than about their normal functions in the cell. It is only when these genes are activated by specific structural alterations or by a disturbance of their genetic expression that their relationship with tumorigenesis becomes evident. After such an activation the product, which is now called an oncogene, acquires transforming properties.

Extensive investigations in this decade have revealed several mechanisms by which a proto oncogene can become activated, and some of these changes have also been found to have a role in the development of primary human malignancies. One of the best studied mechanisms by which a proto oncogcne can become activated is by amplification , a process in which the proto oncogene region in the deoxyribonucleic acid (DNA) is replicated several times within the cellular genome [8]. This proc­ess usually results in an increased amount of messenger ribonucleic acid (RNA) and hence of protO oncogene encoded protein within the cell. If the amount of ampli­fication is substantial, the over-expressed normal proto oncogene product may exert transforming activity.

Chromosomal translocation is another mechanism by which a proto oncogene may become activated. In this process, which is a common event in genetically

462 R.J.C. SLEBOS, S. RODENHUIS

unstable tumour cells, parts of chromosomes are deleted or translocated to other chromosomes. This may either result in loss of DNA sequences which control genetic expression of other genes or may induce reciprocal translocation of two different genes. The latter case may result in a combination of two protein encoding DNA segments and hence lead to an aberrant fusion protein which may exert transforming activity.

A more subtle change which can activate a proto oncogene is that of a single base pair alteration, a point mutation, in the protein encoding domain of the gene. This mechanism is seen in the ras family of proto onco­genes (see below). If a point mutation occurs at a spe­cific position in the DNA sequence, the resulting altered ras protein acquires transforming activity.

In table I a summary is given of the proto oncogenes which have been reported to be activated in one or more type of human lung cancer. Of these genes the best studied examples are the myc and ras genes, genes that were amongst the first pro to oncogenes to be discovered. Apart from these, other genes might be involved in the carcino­genesis of human lung cancer e.g. the erbB, myb and raf genes which appear to be altered in their expression in human lung cancers.

An exciting new concept in the moleculflr genetics of cancer is that of the "suppressor" genes or "anti onco­genes" which are thought to suppress other genes which, if uninhibited, may contribute to malignant transforma­tion. Chromosomal deletion may result in loss of such a gene which may disclose a null mutation in the other allele. Deletion in the short arm of chromosome 3 which has been described for human lung cancer might reflect the loss of a "suppressor" gene.

The novel insights into the pathogenesis of human (lung) cancer generated by the discovery of the cellular proto oncogenes, should ultimately find clinical applica­tions. Firstly, the detection of activated oncogenes in malignancies might lead to a beuer (sub)c1assification of certain types of tumours. Secondly, activated oncogenes might serve as targets for newly developed antineoplas­tic drugs or for novel genetic modalities of therapy. This paper reviews the detection of activated cellular onco­genes, their apparent role in human lung cancer, and their possible clinical relevance.

The myc proto oncogenes

There is little doubt that proto oncogenes of the myc family have a definite role in the pathogenesis of human cancer. This family consists of at least three closely related genes which are called cmyc, Nmyc, and Lmyc. All encode nuclear proteins with DNA-binding properties involved in the regulation of the cell cycle [6]. Activation of cmyc is invariably found in Burkitt lymphoma, in which cmyc is translocated to one of the immunoglobulin loci which results in an aberrant expression of the gene [9]. Evi­dence for the direct involvement of the translocation in the pathogenesis of Burkitt lymphoma comes from stud­ies with transgenic mice, in which the integration of an activated cmyc gene in the genome results in a high incidence of lymphomas [10, 11]. The Nmyc gene is found amplified and over-expressed in neuroblastoma cell lines [12], whilst amplification of this gene is also frequently observed in primary neuroblastomas [13]. In this tumour, amplification of Nmyc is an early event associated with poor prognosis and hence over represented in the ad­vanced stage of the disease [14]. To date, this is one of the few examples of a clinically applicable result of oncogene research. The third gene, Lmyc, was first de­tected in an SCLC cell line [15]; its genetic activity is regulated by a complex mechanism [16].

In lung cancer, the patterns of activation of the myc genes are more complicated and less clear than in Burkitt lymphoma or neuroblastoma. Most work has been car­ried out with cell lines derived from SCLCs, which can now be established with relative ease, and are thus available for cytogenetic and molecular genetic studies. Amplification of cmyc in five of eight SCLC cell lines was first reported by the group of LmLE et al., at the National Cancer Institute (NCI) [17]. Interestingly, all cmyc amplifications were found in cell lines which dif­fered from the "classical" SCLC in their biochemical properties and morphology and are referred to as "vari­ant" SCLC. In later studies at NCI and in other labora­tories, the association between an amplified cmyc gene (or sometimes high cmyc expression without amplifica­tion) and SCLC cell lines could be confirmed [18, 19]. In SCLC cell lines, N- and Lmyc do sometimes appear to be amplified also [20]. At NCI, the analysis of 44

Table 1. - Properties of cellular proto oncogenes involved in lung cancer

Oncogen Mechanism Location Function

cm ye Nmyc Amplification Nucleus DNA-binding Lmyc Hr as Inner Kras Point-mutation cell GTP-binding Nras membrane erbB Amplification Cell membrane Growth factor rcceptor myb Deletion Nucleus DNA-binding raJ Rearrangement Cytoplasm Serine/threonine kinase

DNA: deoxyribonucleic acid; GTP: guanosine triphosphate.

THE MOLECULAR GENETICS OF HUMAN LUNG CANCER 463

SCLC cell lines revealed five cmyc, four Nmyc, and four Lmyc amplifications [21], whilst a British study demon­strated four Nmyc and two Lmyc amplifications in eight SCLC cell lines [19]. Amplifications of N- and Lmyc were not restricted to the "variant" SCLC cell lines, but were also found in "classical" SCLC cell lines.

Cell lines of the "variant" type have a different mor­phology in culture, faster growth rates, higher cloning effkiency in soft agar, resistance to radiation in vitro, and a decreased expression of neuroendocrine markers such as L-dopa decarboxylase, neuroendocrine granules, and peptide hormones related to bombesin [22, 23]. There is evidence that comparable changes occur in patients during the course of the disease.

In about 20% of all SCLC patients a change to a non small cell histology is apparent during tumour pro­gression [24]. Recent observations suggest that small cell carcinomas with a distinct admixture of large cells are even more aggressive than other SCLCs [25]. Further­more, amplifications of myc genes appear to be more frequent in tumours which relapse after chemotherapy than in tumours from untreated patients [21].

An obvious question arising from these data is whether or not the enhanced expression levels of cmyc are the direct cause of the "variant" SCLC cell line phenotype. This was investigated by the introduction of an active cmyc gene into a "classical" SCLC cell line [26]. As a result of this transference, the cell line acquired some, but not all "variant" characteristics: altered morphology, faster growth rate and a resistance to radiation, but no loss of neuroendocrine markers. Thus, it is clear that cmyc over-expression in SCLC cell lines is responsible for some of the "variant" characteristics, but that its amplification is not the only step in the progression from the "classical" to the "variant" phenotype.

Although frequem in SCLC cell Lines, amplification of the cmyc proto oncogene in fresh tumour specimens has only occasionally been found . The &rroup from NCI did not demonstrate cmyc amplifications in 20 tumour samples obtained <ll autopsy [18], whilst WoNo et al. [271 de­tected two cmyc and three Nmyc gene amplifications in 96 DNA preparations of paraffin embedded tissues from 45 patients. Furt11ermorc, when SCLC cells are cultured for only a limited period (up to a few monlhs) myc gene amplif ications are rare: an amplified cmyc or Nmyc could be detected in none of ten SCLC cell llnes [28].

It can be concluded from these data that amplification of myc family genes does not appear to be a primary event in the pathogencsis of SCLC. Amplification of these genes may render a growth advantage to an already lr<~nsformed ceJI , which can subsequenlly become the dominant population in a tumour. Indications for the relevance of such a mechanism is the increase in cmyc copy number which was observed when an SCLC cell line was maintained as a xenograft in nude mice [18]. The clinically important question, whether or not myc amplified tumours have a worse prognosis, was investi­gated at NCI: 44 cell lines derived from 227 patients with SCLC were s tudied [21]. It was found that patients whose tumours could successfully be cultured in vitro had a significantly poorer prognosis than others. An

amplified myc gene was found in only 2 out of 19 cell lines derived from untreated patients (1 Nmyc and 1 Lmyc), whilst myc amplifications were present in cell lines from 11 out of 22 patients who had relapsed after chemotherapy (5 cmyc, 3 Nmyc, and 3 Lmyc). Those patients from whom amplified myc containing cell lines had been established had a poorer survival (and thus presumably more aggressive tumours) than others.

In another study, 7 out of 15 primary SCLCs had an increased expression of Nmyc as determined by in situ hybridization on paraffin embedded, formalin fixed speci­mens [29). These patients had a shorter survival and were less responsive to chemotherapy than patient<; with low Nmyc expression. Tn another study, a good response to chemotherapy in 2 out of 3 SCLCs which had cmyc gene amplifications was reported 1271. Thus, although fiequent in SCLC cell lines, only a minority of newly diagnosed SCLCs have amplified myc genes. It therefore seems likely that myc gene amplifications render a growth advantage to already transformed cells , particularly under conditions of cell culture and in patients who received chemotherapy.

Since establishing cell lines from NSCLCs is more difficult than from SCLCs, only a limited number of cell lines have been available for the study of proto oncogene activations so far. In two NSCLC cell lines derived from two independent giant cell carcinomas of the lung an amplified cmyc gene, accompanied by gene rearrange­ment was detected [30].

In one adenocarcinoma cell line a cmyc amplification was detected, while four NSCLC cell lines investigated had normal c-, N-, and Lmyc gene copy numbers [31]. One case of a large cell lung carcinoma xenograft main­tained in nude mice has been described, which contained an amplified cmyc gene together with a mutationally activated Kras gene [32].

In primary NSCLC, aberrations of the myc proto oncogencs have also been described, but do not appear to be frequent. Amplification of cmyc has been detected in 1 out of 3 adenocarcinomas [331, 1 out of 2 broncho alveolar carcinomas [34], 3 out of 26 NSCLCs [35], and none of 24 NSCLCs [36]. In our series of DNAs, we detected two cases of cmyc amplification, but no N- or Lmyc amplilications in 52 specimens of untreated NSCLC [31J. Another study could not demonstrate any c-or Lmyc amplification in 25 NSCLCs tested, but revealed a highly ampli fied Nmyc gene in one adenocarcinoma L37]. The c linical or biological significance of amplified myc genes in clinical samples is not clear. Since Lbese aberrations arc only very infrequently found, it is conceivable !.hat Lhey do not have an imponant role in t.he early carcino­genesis of NSCLC. Amplification of myc genes occur­ring at a late r stage, for instance during treatment of the tumour, cannot be excluded since comprehensive studies of this question with autopsy specimens have not yet been performed. Furthermore, the known myc genes may not be the only ones involved in lung carcinogenesis: some reportS indicate thal add itional members of the myc proto oncogenc family will be identiiied in the future [18, 37]. Although knowledge of myc gene amplification or over-expression could possibly be used as an

464 R.J.C. SLEBOS, S. RODENHUIS

additional prognostic test, the clinical implications of these findings appear to be limited at present. Since amplifica­tion or over-expression of the myc genes seems to be an event associated with progression and not with primary transformation, these genes do not appear a suitable target for therapeutic strategies. Even if the suppression of these genes or their products would be successful, this might only lead to reversion to a less undifferentiated pheno­type, which would still constitute a lethal disease. A single study has attempted to exploit the presence of myc over­expression in lung cancer for diagnostic imaging [38]. A radio iodine labelled monoclonal antibody against the cmyc gene product was used. In 12 out of 14 lung cancer patients good tumour locali7..ation was possible, includ­ing 2 out of 3 epithelial carcinomas and 2 out of 3 adenocarcinomas. The scans only detected large lesions, probably because a degree of necrosis was necessary to make some of the nuclear proteins accessible for the antibody.

The ras proto oncogenes

In the search for transforming DNA sequences, genes of the ras family have frequently been encountered. This family includes at least three closely related genes which are called H-, K-, and Nras [39]. The first two ras genes were isolated from the Harvey and Kirsten murine sarcoma viruses, whilst Nras was discovered in a neu­roblastoma. Homologues of ras can be found in many different eukaryotic organisms, from yeast to mammals, suggesting a fundamental role in cellular processes. In humans, all three genes encode closely related 21 kD proteins (called p21) which are located at the inner cell membrane, which can bind guanosine triphosphate (GTP) and guanosine diphosphate (GDP), and which possess GTPase activity [39, 40]. The proteins share these bio­chemical and also some structural properties with G proteins, which have a key role in the transduction of signals from membrane bound receptors to the adenylate cyclase pathway. Thus, it has been postulated that the ras p21 proteins may serve as mediators of external signals, such as those generated by growth factors [41].

Although the exact biological function of the ras p21 proteins is still not completely clear, their oncogenic activation does appear to depend on the loss of their GTPase activity. Activating point mutations in codons 12, 13 and 61, all in or near the GTP-binding domain [42], reduces the p-21ras GTPase activity by about lOO­fold. Without this activity, the biologically active GTP bound state persists even without an external stimulus, and a signal is transduced that is, in fact, not there. Another mechanism of activation of the ras genes con­sists of enhanced expression, which in human tumours is usually the result of gene amplification [43]. This mecha­nism of activation is still poorly understood, but it may be hypothesized that enhanced levels result in sufficient p21 in an active (GTP bound) state to induce malignant transformation without altering the ratio between active and non-active p21 molecules. Expression of ras genes has been encountered in a wide variety of normal adult tissues as well as in many tumour types [44-46]. In human

malignancies, mutationally activated ras genes are found in about 15% of a ll cases, whilst the incidence of ampli­fied ras genes appears to be about 1% [ 40]. Mutations in ras genes are particularly frequent in colorectaJ carcino­mas [47], haematological malignancies [48], and lung carcinomas [49). Recently, pancreatic carcinomas were shown to contain point mutated Kras genes in more than 90% of all tumours [SOJ.

In human lung cancer, the ras mutations were first reported to occur in cell lines derived from NSCLC [51, 52]. In a recent review a total of 13 lung cancer cell lines with ras mutations were listed [39]. Most of these cell lines were derived from lung adenocarcinomas harbour­ing a mutation in codon 12 of Kras.

In uncultured human lung carcinomas, a specific mutation in codon 12 of Kras could be detected using an Sstl restriction fragment length polymorphism (RFLP) [53]. In this case, the Sstl restriction endonuclease can only recognize and cut the mutated sequence. The en­zyme thus generates a specific DNA fragment which is not generated when only the normal DNA sequence is present. With this technique, only one of the possible point mutations can be detected, one that appears to be infrequent in human lung cancer [54].

Recently, a method employing the polymerase chain reaction (PCR) followed by oligonucleotide hybridiza­tion, was developed that allows routine screening of tumour DNA samples for the activation of ras genes by point mutation [55]. Using this method, a set of 36 human NSCLC specimens obtained at thoracotomy was analysed by our group in collaboration with RoDENHUls et al. [56]. Of these, only 5 specimens (14%) had an activated ras gene, al l having a point mutation in codon 12 of Kras. This frequency was in good agreement with other studies including the results obtained with NSCLC cell lines. Unexpectedly, however, all mutations were found in 5 of the 10 adenocarcinomas, whilst none were detected in either the epidermoid or large cell carcinomas investi­gated. This finding raised the question as to whether the Kras oncogene might have been activated by amplifica­tion in the 5 mutation negative adenocarcinomas, or whether as yet unknown factors had complemented for the Kras mutation. Subsequent Southern blot analysis revealed that the Kras proto oncogene was amplified in none of the primary lung tumours, a finding which is in agreement with the results of an earlier study on lung carcinomas [36].

One conclusion at this point was that the Kras point mutation was an essential step in the carcinogenesis of lung adenocarcinoma, and that in the mutation negative tumours another similar event had taken place. An alter­native explanation for our observations could be that the Kras mutation was a secondary event which had been induced in one or more already transformed cells, which had subsequently overgrown the rest of the tumour population.

In an effort to find clues to answer these questions, the clinical information on these 10 adenocarcinomas were examined, but no histological differences were observed between the two groups. One striking difference, how­ever, was noted between the smoking histories: all Kras

THE MOLECULAR GENETICS OF HUMAN LUNG CANCER 465

mutation pos•uve tumours were from heavy smokers, whilst in the mutation negative group three patients had never smoked or had stopped smoking long before. A second, larger series of lung adenocarcinomas essentially confinned these results: mutations being found in about one third of all adenocarcinomas, all from patients with a history of smoking [49].

These data, taken together with the association of tobacco smoking with the occurrence of lung tumours [S7J, lead to Lhe attractive hypolhesis that the Kras mutations in Lhe tumours of smokers have been induced by some carcinogenic component in tobacco smoke. Indeed, ras mutations are known to represent links be­tween carc inogen exposure and oncogene activation: in experimental animals mutations in ras genes can be induced by a wide variety of carcinogens and are asso­ciated wilh the occurrence of several tumour types [58] .

The association between carcinogen induced Kras mutations and adenocarcinoma of the lung is also known from animal studies. In 12 out of 14 mice, Kras codon 12 point mutation containing lung tumours could be in­duced by benzo(a)pyrene [59], a potent carcinogen known to be one of the components of tobacco smoke [60]. Other carcinogens may also activate Kras in lung tu­mours of experimental animals [58, 61 ]. Exposure to radioactive plutonium caused lung tumours containing mutated Kras in all of eight dogs [62]. In some inbred mice strains the occurrence of lung adenocarcinomas is closely correlated with an aberrant Kras gene r63), whilst in 6 out of 8 transgenic mice with a point mutated Nras gene, lung adenocarcinomas were found [11). The latter findings suggest Lhat the normal tissue specificity can be overcome by introducing a constitutive promotor in the transgene, and that in Lhis case the expression of the mutated Hras p21 protein alone is suflicient to induce tumour development.

Although these results may not be directly applicable to the situation in humans it is tempting to speculate that carcinogens in tobacco smoke arc the inducers of the Kras point mutations and that, by a still unknown mecha­nism, Lhese mutations specifically lead to tumours of the adenocarcinoma type. The size of our second series of lung tumours permitted us to examine the correlations between other clinical parameters and the Kras point mutations. Statistical analysis revealed that the Kras positive adenocarcinomas were significantly smaller and were less likely to have spread to the local lymph nodes at the time of diagnosis than negative ones [49]. Due to the still short follow up the relevance of this finding remains to be assessed.

Proto oncogenes other than myc or ras

Activational patterns of other proto oncogenes Lhan Lhe myc or ras genes witll potential roles in the carcinogene­sis of human lung cancer are now beginning to emerge. One major candidate is t.hc erbB prmo oncogene, which encodes the epidermal growth factor receptor (EGFR). This gene can be activated by either amplification or rearrangement , both of which have been encountered in

human glioblastomas [64]. Over-expression of erbB has been described in 6 out of 11 lung carcinoma cell lines, of which two, derived from adenocarcinomas, had an amplified erbB gene [65). Jn uncultured NSCLCs, the situation is unclear: amplification of erbB was found in 5 out of 47 NSCLCs, whilst 20 out of 47 showed loss of a genomic fragment encoding the tyrosine kinase domain of the EGFR [66]. Others have found amplification of erbB in 6 out of 27 NSCLCs [35], whilst in a further study of 10 epidermoid carcinomas tested two contained an amplified erbB gene, but none of eighl adenocarcino­mas [671. Tn our series of DNAs, which included 13 epidermoid, 21 adeno, and 9 large cell carcinomas, no elevated erbB copy numbers were detected [31 ). Others have reported absence of amplification in 28 lung carci­nomas of all four types [37], or in 9 squamous and 15 adenocarcinomas [68). The latter study, however, reports over-expression of the gene in 4 out of 14 NSCLCs, indkating that in these tumours Lhe regulation of erbB gene expression had been altered. Immunohistochemical studies of EGFR expression showed a strong staining in 11 out of 11 epidermoid lung carcinomas but in only 1 out of 8 adenocarcinomas and none of 2 SCLCs [69]. The correlation between epidennoid carcinomas and EGFR staining, however, was less obvious in a subse­quent study: 28 out of 36 epidermoid carcinomas were positive when the same monoclonal antibody was em­ployed f70). Fifteen out of 15 SCLC were negative in this study. Another large sn1dy of NSCLC specimens revealed enhanced protein levels in 39 out of 109 NSCLC tumours, of which 27 were epidermoid carcino­mas [671. Thus, it seems that activation of the erbB gene by amplification, accounts for only some of the elevated EGFRs in NSCLC, and that an altered regulation of the expression of the gene may be at least an equally important activational mechanism in human lung carci­nomas.

Furlher investigations should reveal whclher EGFR positive tumour cells differ in Lhelr clinical behaviour from EGFR negative ones. The nucJear protein encoding proto oncogene myb has been found to be altered in 2 lung adenocarcinomas as a resuJt of loss of one of the t.wo alleles normally prc$ent in tJ1e genome, or as a result of ampl ification of the gene in another adenocarcinoma [351. One swdy report.cd over expression of the myb gene in SCLC [71 ]. Another proto oncogenc, called mf, is trnnscriptionaJiy active in SCLCs [20). The significance of these Cindings remains to be investigated.

Tumour suppressor genes

Activation of cellular oncogenes is only one of the possible events which may lead to tumorigenesis, and olher genetic alterations may be equally important (72). lo several hereditary cancers, loss of genetic information can predispose for Lhc development of malignancy. The best documented example of such a situation is the case of familial retinoblastoma. In patients with this disease, a germ line mutation is present which affects one of the two alleles of the retinoblastoma (Rb) gene. The

466 R.J.C. SLEBOS, S. RODENHUIS

likelihood of a somatic mutation occurring in the other allele of one of the developing retinal cells is high, and such an event is followed by the development of a retinal tumour at an early age [73). The Rb locus is now known to reside on the long arm of chromosome 13, and cloned DNA sequences from this area are thought to represent a tumou~ "suppressor" gene (74). The Rb gene is ex­pressed m many tumour cells and also in foetal retina but not in retinoblastomas. Nevertheless, it remains to b~ shown that the gene can revert some of the malignant properties of retinoblastoma cells.

In human lung cancer, several studies have reported non-random chromosomal deletions and the molecular analysis of the involved chromosomal regions is under way. In SCLC, a consistent deletion in the short arm of chromosome 3, with a shortest region of overlap at 3p 14 distal p23, has been reported by several authors [19, 28, 75, 7~], althou.gh this abnormality was not apparent in all cell hnes studted [28, 76]. Submicroscopic evidence for 3p deletions can be obtained by using molecular cloned DNA fragments as probes for this region. The most widely used for this purpose is pH2H3 [77]. This probe detects a restriction fragment length polymorphism (RFLP) bc­~w~~ the two alleles of the locus in a heterozygous tndtvtdual. If only one of the two alleles is present in the tumour DNA, one of the normally present fragments on a DNA blot will be lost Several authors have used the pH3H2 probe for the analysis of DNA isolated from normal lymphocytes and from the tumour of the same patients .. This work revealed that the defect is invariably present m SCLC [78- 80], but somewhat diverging re­sults have been reported for NSCLC.

Some studies suggest allelic loss in some but not all t~mo~s .[78, 81], another study suggests a tight associa­tion Stmtlar to that in SCLC [79]. The putative tumour "suppressor" gene might, thus, be important not only for SCLC but also for at least some cases of NSCLC. Fur­thermore, in all tumours studied so far the deletion was found to be present in only one of the two chromosomes. It is at present not clear whether or not other, more subtle defects reside in the other alJele of this locus. Recent ~or.k on extrapulmonary small cell cancer, histologically Stmtlar to. SCLC, revealed loss of chromosome 3p sequences m only 1 out of 5 of these tumours [82]. 1l is o~ considerable interest that a deletion at 3p21 detected wtth pH3H2 has also been observed in renal cancer (83). However, another study of renal cancers indicates that involvement of 3pl2-14 in chromosomal abnormalities is pr?bab~y more common in this tumour type [84). Al­terattons m 3p are not restricted to lung and renal can­cers: in ovarian adenocarcinomas clonal abnormalities in the 3p21-25 region have been reported [85]. Molecular cloning of DNA sequences in or very near the 3p2llocus should allow isolation and subsequent characterization of the putative gene in the future. These results must be await~d before the role of 3p21 sequences in the patho­genests of human lung cancer and the specificity for this type of tumour can be adequately assessed.

Apart from chromosome 3 deletions, other chromoso­mal a.lteratio~s have been reported in lung cancer. Cyto­geneuc studtes show that loss of chromosome 13 is a

common finding in SCLC cell lines [28, 76), whilst the use of several RFLPs revealed the loss of chromosome 11 sequences in NSCLC [86). In the latter study loss of heterozygosity was associated with progression of the tumours, and this might, therefore, reflect their genetic instability.

It may be expected that both cytogenetic and molecu­lar studies will disclose additional (micro )deletions, which will aid in the molecular characterization of the patho­genesis of lung cancer.

Conclusions

The study of the mechanisms which underlie the trans­f? rmation of lung epithelial cells has led to the descrip­Lton of several genetic abnormalities in human lung cancer, involving proto oncogenes as well as specific chro~osomal deletions. Although these findings are of great Importance for the understanding of the molecular processes which direct cellular transformation, their clini­cal ~elevance is limited at present. Amplification of myc family genes appears to be associated with tumour pro­gression but is mainly found in in vitro cultured SCLC cells .. In p~m~ tu.mours of SCLC patients the frequency of th•s acllvauon 1s too low to be of use as a possible prognostic factor. The use of potentially valuable infor­mation when used as a marker for SCLC tumour pro­gression, is hampered by the fact that no treatment alternatives are presently available. The Kras point mutations specifically found in lung adenocarcinomas might be of use to distinguish these tumours from aden­ocarcinomas of different origin [87). The role of this activation fnr prognosis or classification remains to be investigated. Over-expression of the EGFR, at first as­sumed to be more or less specific for epidermoid carcinomas, does not seem to be restricted to this NSCLC subtype, and contributes little in the distinction between SCLC and NSCLC. No data arc available concerning possible differences in clinical or biological properties of EGFR positive and negative tumours.

Research investigating the potential of anti EGF anti­bodies for diagnostic imaging and even for the therapy of ~SC~C is now in progress. Only the first steps in the eluc•datton of the molecular basis of lung cancer have been made. The rapid progress in identifying the genes and processes involved in the pathogenesis of this devastating disease is encouraging and exciting for both fundamental scientists and clinicians.

Rererences

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THE MOLECULAR GENETICS OF HUMAN LUNG CANCER 467

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87. Slebos RJC, De Graeff A, Van Zandwijk N. Mooi WJ, Bos JL, Rodenhuis S. - Recurrent breast cancer and an aden­ocarcinoma of the lung occurring in one patient: c-myc ampli­fication and K-ras codon 12 point mutation as tumor markers. Eur J Cancer Clin Oncol, 1988, 24, 1529- 1530.

Genetiques moleculaires du cancer du poumon chez l'homme. RJ.C. Slebos, S. Rodenhuis. RESUME: Grace au dcveloppement des techniques de biologic molcculairc, la recherche d'alterations genetiques dans les cel­lules cancereuses a permis un debut de description des lrans­forrnations moleculaircs au niveau cellulaire. Beaucoup de ces modifications gcnctiques se produiscnt au niveau des genes, qui jouent un role dans le control c. de la croissance et du devel­oppemcnt cellulaire, les proto-oncogcnes. Au cours de la der­nicrc decennie, i1 est apparu que les families oncogenes m ye et ras sont importantes dan~ la carcinogcnese des tumeurs pulmonaires humaines. Les oncogcnes myc apparaissent hab­ituellement altercs dans le cancer a petites cellules: ces altcra­tions sont en correlation avec un developpement rapide et une progression tumorale. Les mutations du gene K-ras sont spcci­Ciquc.s de l'adcno-carcinome, une sous-classe des cancers pulmonnires autres qu'a pctites ccllulcs. Les mutations des genes K-ras ont ete decelecs chcz les adcno-carcinomes des grands fumeurs, tandis que dans le groupe sans mutation, les patients etaient des non fumeurs ou des ex-fumeurs de longue date. Les mutations du gene K-ras sont done elroitemcnt associees a la fumce de tabac. L'oncogene erbB, qui encode le recepteur du facteur de croissance epiderrnique, est souvent tres manifeste dans les carcinomes epiderrnoi'des. Les rOles d'aulres oncogenes, comme raf ou myb, de meme que ceux des genes suppresseurs, doivent encore etre investigues, mais pourraicnt etre d'impor­tance essentielle. L'etude des altetations des proto-oncogenes pourraint aider a la subclassification et au diagnostic du cancer pulmonaire, et pourrait foumir des informations pronostiques utiles au cours des prochaines annees. Eur Respir J., 1989, 2, 462-469.