Stroma

The neoplastic cells are embedded in and supported by a connective tissue framework called the stroma (from the Greek word meaning a mattress), which provides mechanical support and nutrition to the neoplastic cells. The process of stroma formation, called a desmoplastic reaction when it is particularly fibrous, is due to induction of connective tissue proliferation by growth factors in the immediate tumour environment.

The stroma always contains blood vessels which perfuse the tumour (Fig. 11.4). The growth of a tumour is dependent upon its ability to induce blood vessels to perfuse it, for unless it becomes permeated by a vascular supply its growth will be limited by the ability of nutrients to diffuse into it, and the tumour cells will cease growing when the nodule has attained a diameter of no more than 1–2mm (Fig. 11.5). Angiogenesis in tumours is induced by factors such as vascular endothelial growth factor (VEGF). This action is opposed by factors such as angiostatin and endostatin which have potential in cancer therapy.

Fig. 11.4 Vascular stroma. Increased vascularity of a malignant tumour in a kidney, revealed by radiology after intra-arterial injection of X-ray contrast fluid.

Fig. 11.5 Tumour angiogenesis. Neoplastic transformation of a single cell results in the growth of a tumour nodule, limited by the ability of nutrients to diffuse into it, to a diameter of 1–2 mm. Production of angiogenic factors (AF) stimulates the proliferation and ingrowth of blood vessels, enabling tumour growth to be supported by perfusion. Eventually, the tumour outgrows its blood supply, and areas of necrosis appear, resulting in slower growth.

Fibroblasts and the matrix they secrete give some mechanical support to the tumour cells and may in addition have nutritive properties. Stromal myofibroblasts are often abundant, particularly in carcinomas of the breast; their contractility is responsible for the puckering and retraction of adjacent structures.

The stroma often contains a lymphocytic infiltrate of variable density. This may reflect a host immune reaction to the tumour (Ch. 9), a hypothesis supported by the observation that patients whose tumours are densely infiltrated by lymphocytes tend to have a better prognosis.

Tumour shape and correlation with behaviour

The gross appearance of a tumour on a surface (e.g. gastrointestinal mucosa) may be described as sessile, polypoid, papillary, fungating, ulcerated or annular (Fig. 11.6). The behaviour of a tumour (i.e. whether it is benign or malignant) can often be deduced from its gross appearance: polypoid tumours are generally benign, i.e. unlikely to spread beyond the tissue of origin (Fig. 11.7); ulceration is more commonly associated with aggressive behaviour because invasion is the defining feature of malignancy (Fig. 11.8).

Fig. 11.6 Tumour shapes. Sessile, polypoid and papillary tumours are usually benign. Fungating, ulcerated or annular tumours are more likely to be malignant. Annular tumours encircling a tubular structure (e.g. intestine) are common in the large bowel, where they often cause intestinal obstruction.

Fig. 11.7 Adenomatous polyp of the colon. This common lesion has a clearly visible stalk. Although benign, these lesions are precursors of adenocarcinoma of the large bowel. When seen endoscopically, adenomatous polyps can be removed by snaring the stalk.

Fig. 11.8 Squamous cell carcinoma of the cervix. This uterus is invaded by a carcinoma arising in and destroying the cervix. The small round tumours in the myometrium are benign leiomyomas (‘fibroids’).

Ulcerated tumours can often be distinguished from non-neoplastic ulcers, such as peptic ulcers in the stomach, because the former tend to have heaped-up or rolled edges.

The shape of connective tissue neoplasms can be misleading. Although circumscription by a clearly defined border is one of the characteristics of benign epithelial tumours, some malignant connective tissue tumours are also well circumscribed.

Tumours are usually firmer than the surrounding tissue, causing a palpable lump in accessible sites such as the breasts. Extremely hard tumours are often referred to as ‘scirrhous’. Softer lesions are sometimes called ‘medullary’; they occur in the thyroid and breast.

The cut surfaces of malignant tumours are often variegated due to areas of necrosis and degeneration, but some, such as lymphomas and seminomas, appear uniformly bland.

Benign tumours

Non-invasive and remain localised

Slow growth rate

Close histological resemblance to parent tissue

Benign tumours remain localised. They are slowly growing lesions that do not invade the surrounding tissues or spread to other sites in the body. They are often enveloped by a thin layer of compressed connective tissue (i.e. encapsulated).

When a benign tumour arises in an epithelial or mucosal surface, the tumour grows away from the surface, because it cannot invade, often forming a polyp which may be either pedunculated (stalked) or sessile; this non-invasive outward direction of growth creates an exophytic lesion (Fig. 11.10). Histologically, benign tumours closely resemble the parent cell or tissue.

Fig. 11.10 Benign and malignant tumours growing on surfaces (e.g. skin, bowel wall), showing the principal differences in their gross appearances.

Although benign tumours are, by definition, confined to their site of origin, they may cause clinical problems due to:

Malignant tumours

Invasive and thus capable of spreading directly or by metastasis

Relatively rapid growth rate

Variable histological resemblance to the parent tissue

Malignant tumours are, by definition, invasive. They are typically rapidly growing and poorly circumscribed. Histologically, they resemble the parent cell or tissue to a lesser extent than do benign tumours. Malignant tumours encroach on and destroy the adjacent tissues (Fig. 11.10), enabling the neoplastic cells to penetrate the walls of blood vessels and lymphatic channels and thereby disseminate to other sites. This important process is called metastasis and the resulting secondary tumours are called metastases. Patients with widespread metastases are often said to have carcinomatosis.

Not all tumours categorised as malignant exhibit metastatic behaviour. For example, basal cell carcinoma of the skin (rodent ulcer) rarely forms metastases, yet is regarded as malignant because it is highly invasive and destructive.

Malignant tumours on epithelial or mucosal surfaces may form a protrusion in the early stages, but eventually invade the underlying tissue; this invasive inward direction of growth gives rise to an endophytic tumour. Ulceration is common.

Malignant tumours in solid organs tend to be poorly circumscribed, often with strands of neoplastic tissue penetrating adjacent normal structures. The resemblance of the cut surface of these lesions to a crab (Latin: cancer) gives the disease its popular name. Malignant tumours often show central necrosis because of inadequate vascular perfusion.

The considerable morbidity and mortality associated with malignant tumours may be due to:

Histological grade (degree of differentiation)

The extent to which the tumour resembles histologically its cell or tissue of origin determines the tumour grade (Fig. 11.11) or degree of differentiation. Benign tumours are not usually further classified in this way because they nearly always closely resemble their parent tissue and grading the degree of differentiation offers no further clinical benefit in terms of choosing the most appropriate treatment. However, the degree of differentiation of malignant tumours is clinically useful both because it correlates strongly with patient survival (prognosis), and because it often indicates the most appropriate treatment. Thus, malignant tumours are usually graded either as well, moderately or poorly differentiated, or numerically, often by strict criteria, as grade 1, grade 2 or grade 3.

Fig. 11.11 Histological grading of differentiation. Well-differentiated adenocarcinoma of the colon characterised by glandular structures similar to those in normal mucosa. Poorly differentiated adenocarcinoma of the colon characterised by a more solid growth pattern with little evidence of gland formation.

A well-differentiated tumour more closely resembles the parent tissue than does a poorly differentiated tumour, while moderately differentiated tumours are intermediate between these two extremes. Poorly differentiated tumours are more aggressive than well-differentiated tumours.

A few tumours are so poorly differentiated that they lack easily recognisable histogenetic features. There may even be great difficulty in deciding whether they are carcinomas or lymphomas, for example, although immunocytochemistry and genetic analysis often enable a distinction to be made. Tumours defying precise histogenetic classification are often referred to as ‘anaplastic’, or by some purely descriptive term such as ‘spindle cell’ or ‘small round cell’ tumour. Fortunately, advances in diagnostic histopathology have resulted in considerably fewer unclassifiable tumours and these descriptive terms are rapidly becoming obsolete.

Benign epithelial tumours

Benign epithelial tumours are either:

A papilloma is a benign tumour of non-glandular or non-secretory epithelium, such as transitional or stratified squamous epithelium (Fig. 11.12). An adenoma is a benign tumour of glandular or secretory epithelium (Fig. 11.13). The name of a papilloma or adenoma is incomplete unless prefixed by the name of the specific epithelial cell type or glandular origin; examples include squamous cell papilloma, transitional cell papilloma, colonic adenoma and thyroid adenoma.

Fig. 11.12 Histology of a benign tumour of non-secretory epithelium: squamous cell papilloma. The tumour is non-invasive and grows outwards from the skin surface (i.e. it is exophytic). The tumour cells closely resemble those of the normal epidermis. This benign tumour is commonly caused by a human papillomavirus.

Fig. 11.13 Histology of a benign tumour of secretory epithelium: adenoma of the colon. The tumour cells closely resemble those of the normal colonic epithelium and contain mucin within their cytoplasm.

Malignant epithelial tumours

Malignant tumours of epithelium are always called carcinomas. Carcinomas of non-glandular epithelium are always prefixed by the name of the epithelial cell type; examples include squamous cell carcinoma and transitional cell carcinoma. Malignant tumours of glandular epithelium are always designated adenocarcinomas, coupled with the name of the tissue of origin; examples include adenocarcinoma of the breast, adenocarcinoma of the prostate and adenocarcinoma of the stomach.

Carcinomas should be further categorised according to their degree of differentiation: their resemblance to the tissue of origin.

Carcinoma in situ

The term carcinoma in situ refers to an epithelial neoplasm exhibiting all the cellular features associated with malignancy, but which has not yet invaded through the epithelial basement membrane separating it from potential routes of metastasis—blood vessels and lymphatics (Fig. 11.14). Complete excision at this very early stage will guarantee a cure. Detection of carcinomas at the in situ stage, or of their precursor lesions, is the aim of population screening programmes for cervical, breast and some other carcinomas. The phase of in situ growth may last for several years before invasion commences.

Fig. 11.14 Evolution of an invasive squamous cell carcinomafrom the precursor lesions of dysplasia and carcinoma in situ (usually grouped together as intra-epithelial neoplasia). Note that the tumour cells cannot reach routes of metastasis such as blood vessels and lymphatics until the basement membrane has been breached.

Carcinoma in situ may be preceded by a phase of dysplasia, in which the epithelium shows disordered maturation short of frank neoplasia. Some dysplastic lesions are almost certainly reversible. As there are other applications of the word ‘dysplasia’, as well as some difficulty in reliably distinguishing between carcinoma in situ and dysplasia in biopsies, the term is now less favoured. The term ‘intra-epithelial neoplasia’, as in cervical intra-epithelial neoplasia (CIN), is used increasingly to encompass both carcinoma in situ and the precursor lesions formerly known as dysplasia.

Benign connective tissue and mesenchymal tumours

Benign mesenchymal tumours are named after the cell or tissue of origin suffixed by ‘-oma’, as follows:

Malignant connective tissue and mesenchymal tumours

Malignant tumours of mesenchyme are always designated sarcomas, prefixed by the name that describes the cell or tissue of origin. Examples include:

As with carcinomas, sarcomas can be further categorised according to their grade or degree of differentiation (Fig. 11.15).

Fig. 11.15 Osteosarcoma. Histology showing pleomorphic tumour cells sufficiently differentiated to produce the amorphous pink-stained osteoid (arrow) lying between them.

Teratomas

A teratoma is a neoplasm formed of cells representing all three germ cell layers: ectoderm, mesoderm and endoderm. In their benign form, these cellular types are often easily recognised; the tumour may contain teeth and hair, and, on histology, respiratory epithelium, cartilage, muscle, neural tissue, etc. In their malignant form, these representatives of ectoderm, mesoderm and endoderm will be less easily identifiable.

Teratomas are of germ cell origin. They occur most often in the gonads, where germ cells are abundant. Although all cells in the body contain the same genetic information, arguably in germ cells this information is in the least repressed state and is therefore capable of programming such divergent lines of differentiation. Supporting evidence for a germ cell origin for teratomas comes from karyotypic analysis of their sex chromosome content. Teratomas in the female are always XX, whereas only 50% of those in the male are XX and the remainder XY; this correlates with the sex chromosome distribution in the germ cells of the two sexes.

Ovarian teratomas are almost always benign and cystic; in the testis, they are almost always malignant and relatively solid. As germ cells in the embryo originate at a site remote from the developing gonads, teratomas arise occasionally elsewhere in the body, usually in the midline, possibly from germ cells that have been arrested in their migration. These extragonadal sites for teratomas include the mediastinum and sacro-coccygeal region.

Embryonal tumours: the ‘blastomas’

Some types of tumour occur almost exclusively in the very young, usually in those below 5 years of age, and bear a histological resemblance to the embryonic form of the organ in which they arise. Examples include:

Mixed tumours

Mixed tumours show a characteristic combination of cell types. The best example is the mixed parotid tumour (pleomorphic salivary adenoma); this consists of glands embedded in a cartilaginous or mucinous matrix derived from the myoepithelial cells of the gland. Another common mixed tumour is the fibroadenoma of the breast, a lobular tumour consisting of epithelium-lined glands or clefts in a loose fibrous tissue matrix.

The occurrence of mixed tumours in an individual organ can sometimes be predicted from its embryology. This is illustrated by the Müllerian tract tumours that occur in the female genital tract; these often contain a mixture of carcinomatous and sarcomatous elements reflecting the intrinsic capacity of the tissue for divergent differentiation.

A tumour may also have a mixed appearance because of metaplasia within it. For example, transitional cell carcinomas of the bladder sometimes have foci of glandular or squamous differentiation.

Carcinosarcomas combine the appearances of carcinoma and sarcoma in one tumour, as a result of either collision of adjacent carcinoma and sarcoma or divergent carcinomatous and sarcomatous differentiation from one original transformed cell.

Neuroendocrine tumours

Neuroendocrine tumours are derived from peptide hormone-secreting cells scattered diffusely in various epithelial tissues. These cells are sometimes referred to as APUD cells; this acronym signifies their biochemical properties (amine content and/or precursor uptake and decarboxylation). (For this reason, the tumours have been called ‘apudomas’.)

The name of those neuroendocrine tumours producing a specific peptide hormone is usually derived from the name of the hormone, together with the suffix ‘-oma’. For example, the insulin-producing tumour originating from the beta-cells of the islets of Langerhans is called an insulinoma. There are exceptions: for example, the calcitonin-producing tumour of the thyroid gland is called a ‘medullary carcinoma of the thyroid gland’ because it was described as a specific entity before calcitonin had been discovered.

Neuroendocrine tumours of the gut and respiratory tract that do not produce any known peptide hormone are called carcinoid tumours. The appendix is the commonest site, but, here, these tumours are usually an incidental finding of little clinical significance. Carcinoids arising elsewhere (the small bowel is the next commonest site) often metastasise to mesenteric lymph nodes and the liver. Extensive metastases lead to the carcinoid syndrome (tachycardia, sweating, skin flushing, anxiety and diarrhoea) due to excessive production of 5-hydroxytryptamine and prostaglandins.

Many neuroendocrine tumours are functionally active, and clinical syndromes often result from excessive secretion of their products (Table 11.4).

Table 11.4 Some neuroendocrine tumours and their associated clinical syndromes

These neoplasms often pursue an indolent course, growing relatively slowly and metastasising late. Their behaviour cannot always be predicted from their histological features.

Some individuals inherit a familial predisposition to develop neuroendocrine tumours; they have a multiple endocrine neoplasia (MEN) syndrome (Ch. 17).

Hamartomas

A hamartoma is a tumour-like lesion, the growth of which is co-ordinated with the individual; it lacks the autonomy of a true neoplasm. Hamartomas are always benign and usually consist of two or more mature cell types normally found in the organ in which the lesion arises. A common example occurs in the lung, where a hamartoma typically consists of a mixture of cartilage and bronchial-type epithelium (the so-called ‘adenochondroma’; Ch. 14). Pigmented naevi or ‘moles’ (Ch. 24) may also be considered as hamartomatous lesions. Their clinical importance is:

Cysts

A cyst is a fluid-filled space lined by epithelium. Cysts are not necessarily tumours or neoplasms but, because they may have local effects similar to those produced by true tumours and some tumours are typically cystic, it is pertinent to consider them here. Common types of cyst are:

The only type of cyst whose aetiology merits its inclusion within this chapter is the neoplastic cyst. This is seen most commonly in the ovary, where it may be either a benign cystic teratoma, filled with sebaceous material, or a cystadenoma or cystadenocarcinoma, each of which may be filled with either serous fluid or mucus depending on the secretory properties of the lining epithelium.

Tumour products

The major types of tumour product are:

Some tumour products are useful as markers for diagnosis or follow-up (Table 11.5). They can be detected in histological sections or their concentrations measured in the blood. Rising blood levels suggest the presence of tumour; falling levels indicate a sustained response to therapy (Fig. 11.16).

Table 11.5 Tumours secreting markers used in diagnosis or follow-up

Epidemiological evidence

Some types of cancer are more common in certain countries, regions or communities within them (Fig. 11.17). Epidemiology has proved to be a fruitful source of information about the causes of tumours. Tumour incidence is more important than mortality data in this regard, because only a proportion of tumours prove fatal and the precise causes of death may not be well documented. It is thus essential to survey populations thoroughly for tumour incidence; in countries with well-developed health services, investigators can usually rely on diagnostic records and cancer registries, but elsewhere it may be necessary to visit and examine the population under study. Variations in tumour incidence may genuinely be due to environmental factors, but the data must first be standardised to eliminate the effect of, for example, any differences in the age distribution. The long latency between exposure to a carcinogen and the appearance of the tumour makes it necessary to consider also the effect of population movement. This effect can be used to distinguish between racial (hereditary) and environmental factors in determining cancer incidence in migrants.

Fig. 11.17 World map showing countries in which there is a relatively high incidence of specific types of cancer. Colorectal cancer. Oesophageal cancer. Low-incidence countries may conceal high-incidence regions or communities; for example, oesophageal carcinoma is more common among blacks in the USA (lighter shaded area). Note that colorectal cancer is much commoner in countries whose inhabitants eat a more refined diet. Dietary associations with oesophageal cancer are less well defined.

Having found a high tumour incidence in a population, comparisons of lifestyle, diet and occupational risks with those of a low tumour-incidence control population often leads to specific causative associations being identified.

The following examples illustrate how carcinogens can be identified in this way.

Occupational and behavioural risks

Certain types of cancer are, or have been, more common in people engaged in specific activities. The discovery in a given community of a link between a higher risk of developing a cancer and the person’s occupation helps to separate general environmental causes of carcinogenesis from those that are probably specific to an individual person or group in the community.

Lung carcinoma

Lung carcinoma is a major public health problem in many countries. In the UK, more then 30000 deaths are attributed to this cause annually; the actual incidence is only marginally higher because this form of cancer has an extremely poor prognosis. The unarguable association with cigarette smoking was established by meticulous epidemiological research. The problem, a common one for epidemiologists, was that people who smoke are commonly exposed to many other possible risks: they tend to live in cities, inhale atmospheric pollutants from motor vehicles, domestic fires and industry, be fond of alcoholic drinks, etc. However, careful analysis of environmental factors showed that cigarette smoking correlated most strongly with the incidence of lung carcinoma. There is an almost linear dose–response relationship between the number of cigarettes smoked daily and the risk of developing lung cancer (Fig. 11.18). Furthermore, the incidence of lung carcinoma declined in those groups of people, such as British male doctors, whose tobacco consumption fell substantially.

Fig. 11.18 Approximate dose–response relationship between cigarette consumption and the relative risk of developing lung cancer. Smoking at the rate of 10 cigarettes per day increases the risk of developing lung cancer 10-fold. (1 = non-smoker.)

Direct evidence

It is fortunately a rare event for someone to be knowingly exposed to a single agent that causes cancer. Sadly, such happenings are often the adverse result of diagnostic or therapeutic medical intervention, but we must not overlook the opportunity to learn much from them.

Thyroid carcinoma and radiation in children

The thyroid gland is vulnerable to the carcinogenic effects of external irradiation and of the radioactive isotopes of iodine (the latter are concentrated by the thyroid gland in the synthesis of thyroid hormone). For example, in April 1986 a nuclear reactor exploded at Chernobyl in Ukraine, releasing a large quantity of radioactive material into the atmosphere. The material released included radioactive iodine. After a 4-year latent interval, there has been a dramatic increase in the local incidence of thyroid carcinoma in children (Fig. 11.19). To minimise this risk, non-radioactive iodine is usually given to people immediately after any accidental exposure to radioactive iodine to compete with the latter for uptake by the thyroid gland.

Fig. 11.19 Rising incidence of thyroid carcinoma in children (0–14 years) in Belarus. Thyroid carcinoma in children is relatively uncommon, but since the nuclear reactor explosion in 1986 at Chernobyl there has been a significantly increased local incidence.

(Based on data in Mahoney MC, Lawvere S, Falkner KL et al 2004 Thyroid cancer incidence trends in Belarus. International Journal of Epidemiology 33: 1025–1033.)

Experimental testing

Carcinogens are not united by any common physical or chemical properties; it is therefore considered necessary to screen all new drugs, food additives and potential environmental pollutants in non-human systems before they are introduced for human use. Three types of test system for carcinogenic or mutagenic activity are employed:

None of these is perfect; animals and isolated cell cultures often metabolise the agent being tested in a way that differs from normal human metabolic pathways, and mutagenicity in bacterial DNA may not correspond to carcinogenicity. In addition, the dynamics of these test systems are very different from that of clinical cancer; cancer in humans is a chronic process often lasting decades, whereas tests for carcinogenic activity in experimental systems usually seek more immediate effects. Nevertheless, despite these limitations, it is still appropriate to investigate possible carcinogens in this way.

Chemical carcinogens

No common structural features

Most require metabolic conversion into active carcinogens

Major classes include polycyclic aromatic hydrocarbons, aromatic amines, nitrosamines, azo dyes, alkylating agents

Many chemical carcinogens have now been identified. The main categories are shown in Table 11.6.

Table 11.6 Examples of proven or suspected chemical carcinogens and the tumours with which they are associated

The carcinogenic risk cannot be predicted from the structural formula alone; even apparently closely related compounds can have different effects.

Some agents act directly, requiring no metabolic conversion. Others (procarcinogens) require metabolic conversion into active carcinogens (ultimate carcinogens) (Fig. 11.21). If the enzyme required for conversion is ubiquitous within tissues, tumours will occur at the site of contact or entry; for example, polycyclic aromatic hydrocarbons induce skin tumours if painted on to the skin, or lung cancer if inhaled in tobacco smoke. Other agents require metabolic conversion by enzymes confined to certain organs, and thus often induce tumours remote from the site of entry; for example, aromatic amines require hydroxylation in the liver before expressing their carcinogenic effects. In a few instances the carcinogen is synthesised in the body from ingredients in the diet; thus, carcinogenic nitrosamines are synthesised by gut bacteria utilising dietary nitrates and nitrites.

Fig. 11.21 Summary of some metabolic pathways for conversion of chemical procarcinogens into the active ultimate carcinogens. Polycyclic aromatic hydrocarbons. Aromatic amines. Nitrates and nitrites. (See text for details.)

Oncogenic viruses

Clusters of cancer cases in space and time suggest a viral aetiology

Tumours associated with viruses tend to be more common in youth

Immunosuppression favours viral oncogenesis

Viruses implicated in human carcinogenesis include Epstein–Barr virus (Burkitt’s lymphoma) and human papillomavirus (cancer of the cervix)

Oncogenic DNA viral genome is directly incorporated into host cell DNA

Oncogenic RNA viral genome is transcribed into DNA by reverse transcriptase prior to incorporation (oncogenic retrovirus)

Viruses were first implicated as carcinogenic agents through the experiments of Rous (in 1911) and Shope (in 1932), who studied fowl sarcomas and rabbit skin tumours, respectively. They showed that it was possible to transmit the tumours from one animal to another, in the manner of an infectious disease; tumours could be induced by injecting a cell-free filtrate of each tumour. The only possible transmissible agent was considered to be a virus, because the pores of the filter were too fine to permit the passage of bacteria or whole tumour cells. The study of oncogenic retroviruses in laboratory animals has had a seminal effect on our understanding of the molecular basis of tumour development and has led to the discovery of oncogenes (Fig. 11.22 and Table 11.12, p. 251).

Fig. 11.22 Simplified mechanisms of integration of oncogenic viral genes, or DNA transcripts, into the host cell DNA. Oncogenic DNA virus: the viral genome is integrated into host cell DNA; neoplastic transformation is a postulated consequence. ‘Acute’ transforming oncogenic RNA virus: transduction into the host cell of an RNA transcript of a cellular oncogene picked up from another cell. ‘Slow’ transforming oncogenic RNA virus: insertion of a viral promoter gene next to a cellular oncogene. In both and , DNA transcripts are made from the RNA conveyed by the virus using the enzyme reverse transcriptase and, in contrast to cellular oncogenes, they lack introns.

Many human tumours are now known to be associated with viruses (Table 11.7).

Table 11.7 Viruses implicated in human tumours

Inherited predisposition

Some individuals inherit an increased risk of developing certain tumours (Table 11.8). There is, for example, an inherited predisposition to breast cancer; a woman whose mother and one sister have developed breast cancer has a c. 50% probability of developing it herself. Genes responsible for this inherited risk (BRCA1 on chromosome 17 and BRCA2 on chromosome 13) have been identified. Sometimes the inherited risk is well defined, as in the condition xeroderma pigmentosum, a deficiency of DNA repair enzymes. Polyposis coli is an autosomal dominant inherited predisposition to develop multiple adenomatous polyps of the large bowel; consequently there is an increased risk of carcinoma of the colon and rectum arising in these polyps. Retinoblastoma, a malignant tumour of the eye in children, is familial and often bilateral in approximately one-third of cases; in these patients there is usually an abnormality of the RB1 gene on chromosome 13.

Table 11.8 Examples of inherited cancer risks

Attributed to mutated BRCA1 gene on chromosome 17

Age

The incidence of cancer increases with age. There are several possible explanations: the cumulative risk of exposure to carcinogens with increasing age; the long latent interval between exposure to the initiating carcinogenic agent and the clinical appearance of the resulting tumour means that there is inevitably a tendency for most tumours to begin to appear only after a few decades of life have elapsed; accumulating genetic lesions (mutations) may render the ageing cell more sensitive to carcinogenic effects. Finally, it may be that incipient tumours developing in young individuals are recognised and eliminated by some innate defence system, such as natural killer cells, and that this protective effect is lost with age.

Gender

Breast cancer is at least 200 times commoner in women than in men. This is probably due to the greater mammary epithelial volume and to the promoting effects of oestrogens in females. It is more common in women who are nulliparous or who have not breast-fed their children, and those who have experienced an early menarche and/or late menopause. Endocrine factors are undoubtedly important.

Associations with gender occur in other cancers, but these are more often due to, for example, smoking habits than to hormonal factors.

Latency

Part of the reason for the long latent interval between exposure to a carcinogen and clinical recognition of the tumour is the fact that tumours result from the clonal proliferation of single cells; it takes an appreciable time for this transformed single cell to grow into a nodule of cells large enough to cause signs and symptoms. However, another important factor is that, with the possible exceptions of ionising radiation and of some fast-transforming oncogenic retroviruses, the change from a normal cell into a growing and potentially fatal neoplasm is thought to entail more than one genetic event.

Initiation, promotion and progression

Experimental carcinogenesis has revealed two major steps— initiation and promotion—in the transformation of cells from normal to neoplastic and a further step—progression— resulting in the malignant phenotype.

A frequently cited example of this sequence is the effect of successive applications of methylcholanthrene and croton oil on mouse skin. A single application of methylcholanthrene results in a visible tumour only if it is followed by repeated painting of the site with non-carcinogenic croton oil. Methylcholanthrene is the initiator inducing lesions in the DNA of the target cell, and croton oil promotes the growth of the initiated cell; further mutational events then cause the lesion to progress to malignancy (Fig. 11.25).

Fig. 11.25 Initiation, promotion and progression, as illustrated by the multiple steps involved in experimental chemical carcinogenesis in the epidermis. Latency is represented by the time interval between exposure to the initiating agent and the growth of a detectable neoplasm. (See text for details.)

Such experiments cannot, of course, be performed in humans, but there are many malignant tumours that develop from observable precursor lesions such as epithelial dysplasia or benign adenomas; for example, the adenoma–carcinoma sequence is well characterised in the large bowel.

This sequential development of neoplasms is associated with genetic abnormalities which drive the uncontrolled proliferation of tumour cells and, in malignant tumours, their progression to invasive behaviour.

Chromosomal abnormalities

The simplest technique for examining the genome of cells is chromosomal (karyotypic) analysis. This involves culturing the cells in the presence of colchicine, which blocks formation of the mitotic spindle and arrests mitosis in metaphase. On exposure to a hypotonic medium, the osmotic shock causes the cells to explode and spill their chromosomes onto the surface of a glass slide where they can be stained, counted and examined in detail. Unfortunately, at this relatively crude level of analysis in molecular terms, very few recurring patterns of chromosomal abnormality have been found in tumours. Abnormalities such as additional chromosomes and translocation of part of one chromosome to another are very common, but few are constant even among a single tumour type (Table 11.10). A notable exception is the Philadelphia chromosome; this 9;11 translocation resulting in the bcr–abl fusion gene is one of the most consistent chromosomal abnormalities yet discovered, and is commonly found in chronic myeloid (granulocytic) leukaemia. More recently, however, chromosomes have been studied by in situ hybridisation, a technique that enables determination of the number and location of specific DNA sequences (Fig. 11.26).

Table 11.10 Examples of non-random chromosomal abnormalities in neoplastic diseases

Fig. 11.26 Fluorescent in situ hybridisation (FISH) of nuclei from an adult germ cell tumour of testis. This reveals, as green dots, extra copies of the short arm of chromosome 12 (12p). The fewer red dots mark the normally represented long arm of chromosome 12 (12q). The extra copies of 12p are in the form of isochromosomes (i(12p)), a useful diagnostic marker of adult germ cell tumours.

(Courtesy of Jill Elliott, Sheffield Regional Cytogenetics Service, Sheffield Children’s Hospital.)

Genetic mechanisms in carcinogenesis

Genetic alterations are the root cause of neoplastic cellular behaviour. Research has revealed that a minimum of three genetic alterations are needed to transform a normal cell into a neoplastic cell (Fig. 11.27):

Fig. 11.27 Essential steps in neoplastic transformation. Three key genetic events are the minimum needed to convert a normal human cell into a neoplastic cell. Telomerase expression prevents telomeric shortening with each cell division and thus thwarts cellular senescence. Inactivation of tumour suppressor gene function in the immortalised cells removes inhibition of growth control. Oncogene activation sets up autocrine growth stimulation; the cell now produces a growth factor for which it already has a receptor or expresses a receptor for a growth factor it normally produces. The cell is now fully transformed.

(Based on observations by Hahn WC, Counter CM, Lundberg AS et al 1999 Creation of human tumor cells with defined genetic elements. Nature 400: 464–468)

Telomerase expression confers immortalisation on the cells. Cells lacking telomerase (most cells in the body) have only a limited replicative ability. Our chromosomal telomeres shorten as we age. Eventually the telomeres become so short with each cell division that replication cannot start, and cellular senescence and death ensues.

Tumour suppressor gene inactivation and abnormal oncogene expression work in concert (Fig. 11.28) to drive cells from their normal state of regulated growth to the dysregulated and uncontrolled growth that characterises neoplastic cells.

Fig. 11.28 Oncogenes and tumour suppressor genes. Abnormal expression of oncogenes drives normal cells towards the neoplastic state. Loss of tumour suppressor gene function enables neoplastic transformation by permitting mutations.

Tumour suppressor genes

Clues to the existence of inhibitory genes came from observations on the behaviour of transformed cells that were fused with untransformed cells; the resulting hybrid cells behaved like untransformed cells until specific chromosomes bearing the inhibitory genes were lost, causing the cells to revert to their transformed state.

The existence of these inhibitory genes was also postulated by Alfred Knudson in 1971. Using a statistical approach to familial cancer incidence he formulated a two-hit hypothesis. The first ‘hit’ is the inheritance of a defective (mutant) allele of a tumour suppressor gene, the other allele being normal (wild) and expressing sufficient suppressive effect. The second ‘hit’ is the mutational loss of function of the normal allele, thus now fully depriving the cell of the suppressive effect of that tumour suppressor gene.

‘Caretakers’ and ‘gatekeepers’

Tumour suppressor genes are categorised further according to their mechanism of action:

• caretaker genes maintain the integrity of the genome by repairing DNA damage

• gatekeeper genes inhibit the proliferation or promote the death of cells with damaged DNA.

Examples and the tumour susceptibilities with which inherited abnormalities of these genes are associated are given in Table 11.11.

Table 11.11Tumour suppressor genes: functional categories and tumour associations

The RB gene was the first inhibitory gene to have been well characterised, and is associated with retinoblastomas. Retinoblastomas are malignant tumours derived from the retina; they occur almost exclusively in children. In some cases they are hereditary, occurring bilaterally and also in some of the patient’s siblings. In other cases they are sporadic, occurring unilaterally and without any familial associations. Individuals with hereditary retinoblastomas show a germline deletion on chromosome 13, corresponding to the known site of the RB1 gene. Therefore, only one further mutational loss of the paired gene in the target retinal cell is required for the tumour to develop. Sporadic retinoblastoma cases have a normal chromosome 13 and therefore require two mutational losses before the tumour can develop (Fig. 11.29).

Fig. 11.29 Loss of tumour suppressor gene function and inherited retinoblastoma. Loss of functional tumour suppressor genes permits tumour development. Individuals with an inherited risk of retinoblastoma are born with a predisposing germline mutation in one of the paired alleles of the RB1 suppressor gene; mutational loss of the remaining RB1 allele is required for retinoblastomas to develop. Normal individuals without an inherited germline mutation of the RB1 gene have a low incidence of retinoblastoma because acquired mutations in both alleles have to occur in the same cell or its daughters; sporadic retinoblastoma is, therefore, a rare event.

The tumour suppressor gene p53, situated on the short arm of chromosome 17, is the gene most frequently mutated and extensively studied in human cancer. The normal functions of p53 are to enable:

• repair of damaged DNA before S-phase in the cell cycle by arresting the cell cycle in G₁ until the damage is repaired

• apoptotic cell death if there is extensive DNA damage.

The p53 levels rise in cells that have sustained DNA damage, until either the damage is repaired or the cell undergoes apoptosis. This prevents propagation of possibly mutated genes. This important function of p53 results in it being called ‘the guardian of the genome’.p53 can lose its normal function by a variety of mechanisms:

• mutations that either render the gene unreadable (nonsense mutations) or encode for a defective protein (missense mutations)

• complexes of normal p53 and mutant p53 (in heterozygous individuals or cells) inactivating the function of the normal allele

• binding of normal p53 protein to proteins encoded by oncogenic DNA viruses (e.g. human papillomavirus, polyomaviruses).

These events have major implications. Cells with damaged DNA, possibly with mutated oncogenes, undergo mitotic replication rather than apoptotic death (Fig. 11.30). Also, cytotoxic chemotherapy against the tumour may be less effective if the cells fail to respond by apoptosis.

Fig. 11.30 Role of p53 in cells with damaged DNA. In the presence of normal p53, cells with a mutation resulting from a potentially carcinogenic stimulus are arrested in G₁ of the cell cycle until either the mutation is repaired or, if the damage is severe, apoptosis occurs. If the p53 is defective, as a result of mutation or binding, the cells proceed to S phase and the mutation is propagated to daughter cells, possibly eventually leading to a tumour.

Inherited germline (present in all cells) mutations of p53 occur in the rare Li–Fraumeni syndrome. Affected individuals have an inherited predisposition to a wide range of tumours. At birth they are heterozygous for the defective gene (only very rarely are the maternal and paternal alleles both defective). Eventually, the normal allele is itself lost or mutated (loss of heterozygosity) in a variety of cells, thus enabling their neoplastic transformation.

Oncogenes

Oncogenes are genes driving the neoplastic behaviour of cells. Originally proposed as a hypothesis, oncogenes were discovered as a result of studies of oncogenic RNA retroviruses. These are RNA viruses that have the ability to transfer their genome, or parts of it, to the genome of the cells they infect. Normally the transfer of genomic information is in the opposite direction: DNA sequences are transcribed into RNA, which then determines the amino acid sequence of a peptide or protein. However, retroviruses contain an enzyme, reverse transcriptase, that enables the viral RNA to be transcribed into complementary DNA which is then incorporated into the infected cell’s genome. In the case of oncogenic retroviruses, these genes were called oncogenes.

The next major discovery was of the presence of DNA sequences almost identical to viral oncogenes (v-oncogenes) in the genome of normal cells (cellular or proto-oncogenes). Numerous oncogenes have now been identified. However, in normal cells these oncogenes are present at the frequency of only one copy per haploid genome, and their transcription is tightly controlled as required for cell growth and differentiation.

They are present in the genome of even the most primitive protozoa and metazoa; this high degree of evolutionary conservation implies a function indispensable to normal life. The result of much research now leads us to conclude that these cellular oncogenes are essential for normal cell and tissue growth and differentiation, particularly during embryogenesis and healing. But when they are aberrant or inappropriately expressed they result in the growth of a tumour.

Normal or partially transformed cell cultures can be fully transformed by the addition of DNA bearing oncogenes, a process known as transfection. Alternatively, oncogenic (or carcinogenic) retroviruses can transform cells by transferring oncogenes from another cell, a process known as transduction.

Oncogenes can be classified into five groups according to the function of the gene product (oncoprotein):

Abnormalities of oncogene expression are crucial for the growth and behaviour of tumour cells.

Abnormalities of oncogene expression in tumours

Oncogenes can have tumour-promoting effects by either:

• mutation resulting in an oncoprotein molecule altered in such a way that it is excessively active

• excessive production of a normal oncoprotein because of gene amplification or enhanced transcription.

Increased expression of oncogenes has been found in most tumours. The mechanisms are summarised in Figure 11.31. Increased expression may be detected by:

• the presence of more of the oncogene product (oncoprotein) within or on the cells

• increased production of mRNA transcripts of the oncogene

• increased numbers of copies of the oncogene in the genome.

Fig. 11.31 Mechanisms of oncogene activation. Translocation of an oncogene from an untranscribed site to a position adjacent to an actively transcribed gene; e.g. simplified chromosomal translocation in Burkitt’s lymphoma, in which the c-myc oncogene is often translocated from chromosome 8, its normal location, to chromosome 14, where it is placed adjacent to one of the immunoglobulin genes and is thus inappropriately transcribed. Point mutation (in this case in codon 12 of the ras oncogene), in which the substitution of a single base in the oncogene is translated into an amino acid substitution in the oncoprotein causing it to be hyperactive. Amplification by the insertion of multiple copies of the oncogene (in this case, c-myc in neuroblastoma), resulting in cellular proliferation stimulated by excessive quantities of the oncoprotein. Increased oncogene expression by gene insertion (insertional mutagenesis) resulting in proximity of an oncogene to a promoter or enhancing gene; this is one mechanism of retroviral carcinogenesis.

Increased numbers of oncogene copies may result from infection by a retrovirus which causes reverse transcription of its RNA and insertion of multiple copies of the resulting DNA into the DNA of the host cell genome. A more common occurrence in human tumours is gene amplification resulting in multiple copies, such as in the myc family of oncogenes in neuroblastoma; this can be recognised in chromosome preparations from tumour cells by the presence of homogeneously staining regions and double minute chromosomes.

Increased transcription can occur if a normally silent (i.e. not transcribed) oncogene is moved to another part of the genome where active transcription is occurring. This is often evident from the karyotype; part of one chromosome which is known to bear an oncogene may be translocated to another chromosome where a gene known to be actively transcribed is situated. Specific examples include:

• translocation of the c-abl gene from chromosome 9 to chromosome 22, an event that results in the formation of the Philadelphia chromosome and expression of a bcr–ablfusion gene product in chronic myeloid leukaemia

• translocation of the c-myc oncogene from chromosome 8 to chromosome 14, where its expression is assured by juxtaposition with one of the immunoglobulin genes which will be actively transcribed in the B-cell that is the origin of Burkitt’s lymphoma.

Alternatively, the cellular oncogene may undergo a point mutation resulting in a gene product, such as a protein kinase, with increased or inappropriate activity.

Autocrine stimulation of neoplastic cell growth

Oncogene products play an important role in cellular growth and behaviour (Table 11.12). By their expression in inappropriate circumstances a cell can become autonomous, proliferating without the usual requirement for external signals (Fig. 11.32). For example, an oncogene product may be a receptor for a growth factor (Ch. 5) already normally produced by that cell; the cell then responds to stimulation by its own growth factor. Alternatively, the oncogene product may be a growth factor for which the cell already normally bears a specific receptor. In both cases the result is autocrine stimulation of growth.

Table 11.12 Examples of oncogenes and the function of their products

Oncogene	Function of oncoprotein	Abbreviated from
abl	Protein-tyrosine kinase activity	Abelson mouse leukaemia
myc	Binds to DNA, directly stimulating synthesis	Myelocytomatosis
sis	Growth factor (platelet-derived growth factor)	Simian sarcoma
erbB	Receptor for epidermal growth factor	Avian erythroblastosis (also erbA)
ras	Acts on intracellular signalling (cyclic nucleotides)	Rat sarcoma

Fig. 11.32 Self-stimulation of neoplastic cell proliferation mediated by oncoproteins. (A) Direct stimulation of DNA synthesis by an oncoprotein that binds to DNA, (B) Synthesis of receptors () for a growth factor (red triangles) already present in the extracellular environment. (C) Synthesis of a growth factor (green circles), receptors for which () are normally present on the cell. (D) Interference with intracellular signalling between the cell membrane and the nucleus.

Other oncogene products act directly within the nucleus to stimulate mitosis or on intracytoplasmic second messengers, such as cyclic nucleotides, thus modulating intracellular signalling.

Invasion

The invasiveness of malignant neoplasms is determined by the properties of the neoplastic cells within them. Factors influencing tumour invasion are:

Cellular motility is abnormal in that the cells are not only more motile than their normal counterparts (which may not move at all), but also show loss of the normal mechanism that arrests or reverses normal cellular migration: contact inhibition of migration.

Metastasis

Metastasis is the process whereby malignant tumours spread from their site of origin (the primary tumour) to form other tumours (secondary tumours) at distant sites. The total tumour burden resulting from this process can be very great indeed, and the total mass of the secondary tumours invariably exceeds that of the primary lesion; it is not uncommon at autopsy to find a liver weighing several kilograms more than normal, laden with metastases. The word carcinomatosis is used to denote extensive metastatic disease.

Sometimes, metastases can be the presenting clinical feature. Bone pain or fractures due to skeletal metastases can be the first manifestation of a clinically occult internal malignancy. Palpable lymph nodes, due to metastatic involvement, may appear before the signs and symptoms of the primary tumour.

The metastatic cascade

Neoplastic cells must successfully complete a cascade of events before forming a metastatic tumour (Fig. 11.33). Only a proportion of the neoplastic cells in a malignant tumour may have the full repertoire of properties necessary for completion of the cascade. Many tumours studied experimentally in animals consist of metastatic and non-metastatic clones, and metastatic tumours in humans often appear histologically less well differentiated than the primary lesion, suggesting that there is clonal evolution of the metastatic phenotype. There is experimental evidence for the inactivation of ‘anti-metastatic’ genes (‘metastogenes’), such as nm23, in neoplastic cells capable of metastasis, but their precise role in the metastatic cascade in human tumours is uncertain.

Fig. 11.33 Metastatic cascade. The spread of tumour cells from the site of origin, the primary tumour, to form secondary tumours in other locations requires completion of a logical sequence of events mediated by tumour–host interactions.

The sequential steps involved in the metastatic cascade are:

1. detachment of tumour cells from their neighbours

2. invasion of the surrounding connective tissue to reach conduits for metastasis (blood and lymphatic vessels)

3. intravasation into the lumen of vessels

4. evasion of host defence mechanisms, such as natural killer cells in the blood

5. adherence to endothelium at a remote location

6. extravasation of the cells from the vessel lumen into the surrounding tissue.

On reaching the site of metastasis there is a recapitulation of the events that were required for primary tumour growth. The tumour cells must proliferate and, if they are to grow to form a nodule larger than a few millimetres in diameter, the ingrowth of blood vessels must be elicited by angiogenic factors.

Alterations in cell adhesion molecules are important at several points in the metastatic cascade; these affect cell–cell and cell–substrate adhesion. Studies on experimental and human tumours show that reduced expression of cadherins, which are involved in adhesion between epithelial cells, correlates positively with invasive and metastatic behaviour. Increased expression of integrins appears to be important for the invasive migration of neoplastic cells into connective tissues.

Routes of metastasis

The routes of metastasis are (Fig. 11.34):

Fig. 11.34 Routes of metastasis exemplified by a carcinoma of the bowel.

Carcinomas tend to prefer lymphatic spread, at least initially, while sarcomas prefer haematogenous spread. However, exceptions to these tendencies are common, and carcinomas often generate blood-borne metastases.

Haematogenous metastasis

Bone is a site favoured by haematogenous metastases from five carcinomas—lung, breast, kidney, thyroid and prostate. Other organs commonly involved by haematogenous metastases are lung, liver and brain (Fig. 11.35). The metastases are frequently multiple, whereas primary tumours arising in the affected organs are usually solitary. Curiously, tumours rarely metastasise to skeletal muscle or to the spleen, despite their lavish blood supply.

Fig. 11.35 Liver metastases. Liver from an autopsy on a patient who died from carcinomatosis due to carcinoma of the breast.

Lymphatic metastasis

Tumour cells reach the lymph node through the afferent lymphatic channel. The tumour cells settle and grow in the periphery of the node, gradually extending to replace it (Fig. 11.36).Lymph nodes involved by metastatic tumours are usually firmer and larger than normal. Groups of involved lymph nodes may be matted together by both tumour tissue and the connective tissue reaction to it. Lymph node metastases often interrupt lymphatic flow, thus causing oedema in the territory that they drain.

Fig. 11.36 Lymph node metastasis. The lymph node is partly replaced by a deposit of metastatic adenocarcinoma (arrowed) from a primary in the stomach.

Clinically, it is necessary to be cautious in interpreting the significance of enlarged lymph nodes draining tumours because the enlargement could simply be due to reactive changes.

Local effects

Tumours exert local effects through compression and displacement of adjacent tissues and, if malignant, through their destruction by actual invasion. These effects can be clinically inconsequential if the organ is large relative to the size of the tumour or if no vital structure is threatened. However, even benign tumours can have life-threatening effects on neighbouring structures; for example, a functionally inactive adenoma of the pituitary gland may obliterate the adjacent functioning pituitary tissue, such is the confined space in which the gland sits, resulting in hypopituitarism.

Malignant neoplasms obviously have more serious local effects because they invade and destroy local structures. This may be rapidly fatal if a vital structure is eroded, for example a pulmonary artery by a carcinoma of the lung. In the case of basal cell carcinoma of the skin (‘rodent ulcer’), its local effects are sufficient to justify the label ‘carcinoma’ because, although the tumour rarely metastasises, its invasiveness can be very disfiguring.

Malignant tumours on mucosal surfaces are often ulcerated. Blood can ooze from these lesions; this blood loss can beoccult in the case of gastrointestinal tumours and this is a very important cause of anaemia. Ulcerated surfaces also expose the patient to the risk of infection.

Metabolic effects

The metabolic effects of tumours can be subdivided into those specific to individual tumours and those common to many tumours.

Tumour type

The tumour type is usually determined from the growth pattern of the tumour and its relationship to the surrounding structures from which a direct origin may be evident. Thus, a gland-forming neoplasm in the breast is most likely to be a primary adenocarcinoma of the breast, particularly if carcinoma cells are also present within the breast ducts near the tumour (ductal carcinoma in situ). A squamous cell carcinoma is often recognisable from the production of keratin, and it may be in continuity with adjacent squamous epithelium that may show carcinoma in situ.

Some types of tumour need to be subclassified because variants with differing behaviour exist. Malignant lymphomas, for example, are subclassified into Hodgkin’s and non-Hodgkin’s lymphoma, each of which is then further subclassified by detailed appraisal of the histology (Ch. 22).

Genetic analysis or immunohistology may be necessary to type tumours that do not have obvious differentiated features detectable on routine light microscopy.

Tumour grade

The grade of a tumour is an assessment of its degree of malignancy or aggressiveness. This can be inferred from its histology. The most important features contributing to the assessment of tumour grade are:

Grading systems have been devised for many types of tumour,and most involve an assessment of the above features. Tumours are often heterogeneous, and the grading should be performed on what appears to be the least differentiated area as this is likely to contain the most aggressive clone or clones of tumour cells.

Tumour stage

The stage of a tumour is the extent of spread. This is determined by histopathological examination of the resected tumour and by clinical assessment of the patient, often involving imaging techniques. Perhaps the best-known staging system is that devised in the 1930s by Cuthbert Dukes for colorectal carcinomas (Ch. 15):

The most generally applicable staging system is the TNM system (Fig. 11.37):

Fig. 11.37 Summary of TNM system for staging of tumours. This concept (tumour, nodes and distant metastases) is the basis of most tumour staging systems.

For example, a T1 breast carcinoma is equal to, or less than, 20mm in diameter; large numbers denote large tumours. N0 denotes no nodal metastases, N1 one or few nodal metastases, and N2 many nodal metastases. M0 denotes an absence of metastases, and M1 and greater denotes increasing extent of distant metastases.

For many tumours, the TNM status is used to derive a stage score. Typically, a stage 1 tumour is confined to the organ of origin and a stage 4 tumour has disseminated widely.