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Molecular Diagnostics

Headings
molecular diagnosis, hereditary cancers

After approximately two decades of molecular studies, little doubt remains that cancer is a genetic disease. In fact, more than one hundred genetic elements, known to be altered in tumor cells, have been identified so far, and many others are likely to be discovered in the next future. Most mutations occur somatically, and it is actually through the accumulation of a certain number of these mutations that the cell achieves a fully malignant phenotype (1). In some individuals mutations in cancer related genes may be present at the constitutional level, either spontaneously arisen "in utero", or inherited from one of the parents. As a consequence, these individuals have a much higher risk than the general population for certain types of cancers. Moreover, they show a tendency to develop multiple primary tumours.

Already one hundred years ago, the observation of anomalous tumour clusterings led to speculate that in certain families cancer may be heritable (2). However, it was only with the isolation of the genetic elements associated with the predisposition to tumor development, and with the demonstration that these genes are mutated in affected family members, that this hypothesis could be fully demonstrated. The first such gene to be isolated, in 1986, was the RB1 gene responsible of the familial form of retinoblastoma, a rare childhood ocular malignancy (3). Since then, about 20 different genes have been reported to be linked to the known human familial cancer syndromes.

The vast majority of familial cancer syndromes are transmitted as autosomal dominant diseases. This is to say that the inheritance of a single mutated allele is sufficient to confer an elevated risk for cancer development. Yet, most of the genes associated to these syndromes act as tumour suppressors, in that both alleles of these genes must be inactivated at the somatic level for the cells to become malignant. In individuals carrying germline predisposing mutations the single inactivation event required at the somatic level has a very high likelihood to occur, thus explaining the dominant nature of most familial cancer syndromes. Exceptions are represented by Xeroderma Pigmentosum (XP), where only homozygously mutated individuals show a tendency higher than in the general population to develop skin malignancies (4) and by the RET gene, associated with the Multiple Endocrine Neoplasia type 2 (MEN2) syndrome, that behaves as a dominant oncogene also at the cellular level (5).

According to their genetic and phenotypic characteristics, familial cancer syndromes can be divided into two categories. The first one, from now on referred as "type I", includes those syndromes that can be considered as "genetically homogeneous" diseases, since they associate with mutations in a single locus (Table I). With the exception of Neurofibromatosis type I (NF1), which affects approximately 1 in 3.500 newborns (6), type I syndromes are relatively rare in the population, with frequencies ranging from approximately 1 in 10^-4, as in the case of Familial Adenomatous Polyposis (FAP) (7), to 1 in 10^-6, as for WAGR syndrome (8). Remarkably, many type I cancer predisposing syndromes display specific non-malignant or pre-malignant phenotypes. This is, for example, the case of the developmental abnormalities, typical of the Beckwith-Wiedemann syndrome (BWS) (9) or of the thyroid C-cell hyperplasia in MEN2 patients (10). Where present, these phenotypic characters facilitate the diagnosis of hereditary cancer, that can be done on a clinical and/or pathological basis.

Table I. Familial cancer syndromes (type I)

*gene mapped, not yet cloned

In contrast with type I syndromes, hereditary cancer belonging to the type II group are "genetically heterogeneous" diseases, in that the same phenotype may be due to mutations in more than one gene (Table II). For example, Hereditary Non-Polyposis Colorectal Cancer (HNPCC) syndrome patients have been found to carry mutations in at least four different DNA mismatch repair (MMR) genes (11).

Table II. Familial cancer syndromes (type II).

*Gene mapped, not yet cloned

Some type II hereditary cancers are thought to be relatively common. According to different epidemiological studies, hereditary colon and breast cancers may account to 7-13% and to 5%, respectively, of all cancers of the same histotypes (11, 12). However, the exact frequencies of these pathologies in the general population have not yet been established with certainty. This is mainly because type II syndromes lack of defined phenotypic hallmarks, such as specific tumour-associated features or extra-tumoral conditions and, therefore, in the absence of molecular analyses, they can be diagnosed only "tentatively" on the basis of family history.

The latter observation is of particular interest, since it illustrates a first important contribution of the molecular diagnosis to the management of familial tumours. Knowing the incidence of a pathology in a given population is of great value, because it provides useful information as to the social relevance of the pathology itself. As indicated, in the case of the familial syndromes predisposing to some of the most common types of cancer, this incidence can be inferred only from molecular investigations. In fact, in these tumours only the molecular analyses is able to distinguish family aggregations occurring just by chance from those that are actually due to the inheritance of predisposing mutations. In addition, molecular investigations are useful in identifying "de novo" mutations, that otherwise could be unrecognised, since they are not usually associated with a positive family history. Unfortunately, molecular genetic analyses are hampered not only by the already mentioned genetic heterogeneity of the most common types of hereditary cancers, by also by the lack of mutational "hot spots" in the associated genes. Because of these problems, different "indirect" screening methods are being applied to the search of constitutional mutations associated with hereditary cancers, including SSCP (single strand conformation polymorphism) analysis, IVSP (in vitro synthesized protein) assay, also referred as PTT (protein truncation test) and ASO (allele specific oligonucleotide) hybridization (13). In addition, "functional" assays have been developed, that identify inactivating mutations in cancer predisposing genes by in vitro testing of the activity of their products (14).

A second relevant application of the molecular diagnosis is the recognition of tumor patients carrying germline predisposing mutations. Although some of the most common types of hereditary cancers display a tendency to associate preferentially with specific clinical and pathological characteristics (15, 16), they are usually indistinguishable from sporadic cases. Yet, their identification is of great importance in establishing treatment regimens, since, as stated above, many individuals with germline mutations tend to develop multiple tumours. For example, women with a breast cancer due to constitutional mutations in the BRCA1 gene have a very high risk for controlateral tumours and for ovarian carcinomas (17).

A third point that is worth mentioning with reference to molecular diagnosis is the identification of at-risk family members. In fact, once a cancer predisposing mutation has been identified in an individual, his, or her, relatives may be screened for the presence of the same mutation. In such a way, subjects negative to the test will benefit from molecular analysis both in terms of psychological relief and avoidance of unnecessary clinical testing. On the other hand, mutation carriers, if still unaffected, could be addressed to the adequate programs of prevention and surveillance. At present, it is a matter of much debate which interventions are better in these individuals. However, it has to be noted that, thanks to the molecular diagnosis, it is now possible, in contrast with the past, to test the efficacy of cancer preventing strategies on groups of genetically characterized subjects.

Finally, the molecular analyses have revealed that in different familial cancer syndromes some genetic lesions are implicated in specific phenotypic variants of the disease. These genotype-phenotype correlations have important consequences for the prognosis of affected individuals. For example, FAP patients with germline mutations in the 3' half of the APC gene have a much higher probability than patients with mutations in the 5' half, to develop desmoid tumours, that are a severe complication of this syndrome (18). Analogously, only HNPCC patients with mutations in the MSH2 gene show a significantly increased relative risk of cancers of the urinary tract, stomach and ovaries, in contrast with those with mutations in the MLH1 gene (19). On the other hand, it has been observed that identical mutations may be associated with a variable expression of the disease in different families, as in the case of the common mutation in BRCA1 exon 2 (185delAG), which is present in approximately 1% of Ashkenazi Jews (20). Besides external agents, such as smoking and environmental carcinogens, which are known to be important elements in inducing cancer development, it has been proposed that this phenomenon might also be due to the presence of "modifying" genes, that could variably modulate the effect of cancer predisposing mutations in different individuals. It is not difficult to predict that the search for these modifying genes, one of which, the mouse locus Mom-1, has been already identified (21), will represent one of the more exiting challenge of the molecular genetics of cancer in the next future.

In summary, it can be concluded that molecular genetic analyses in familial cancer syndromes may represent a valuable tool to: i) increase our knowledge as to the incidence of these pathologies in the general population, ii) identify tumour cases due to predisposing germline mutations, iii) provide pre-symptomatic diagnosis in relatives of gene carriers and iv) improve the clinical management of these diseases through the identification of defined genotype-phenotype correlations. Moreover, it has to be remarked that molecular studies on hereditary cancers may have a more general relevance as to the elucidation of the mechanisms of tumour development, since most genes conferring an inheritable susceptibility to cancers have been found to be altered, at the somatic level, also in sporadic cases (22).

It is expected that the use of molecular analyses to identify individuals with inherited susceptibility to cancer development will soon have a rapid and widespread diffusion. This is bound to have important ethical, legal and phychosocial implications (23). In addition, most tests remain complex, costly and often unable to identify 100% of cases, especially where not yet identified genes or mutations are present. For these reasons, only qualified institutions, that can guarantee the adequate support in terms of genetic counselling, confidentiality, clinical options and psychological assistance, should be allowed to provide molecular diagnosis to at-risk individuals, as part of research protocols.

Acknowledgements

The author thanks Dr. Marco A. Pierotti for critical reading and Mrs. Anna Grassi for secretarial assistance.

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