Neoplasia – tumour precursors and development

Learning objectives

By the end of this CAL you will be able to:

  • Describe the precursors of malignancy
  • Document the cellular and molecular mechanisms behind carcinogenesis

Tumour precursors Part 1 of 9

Cancer does not develop suddenly, it is the end result of multiple steps.  In many organs, we can see changes in the epithelium that precede invasive malignancy, sometimes called pre-cancerous precursors.

Development of cancer in the cervix will be used as an example to illustrate this process.

Development of cancer in the uterine cervix

Precancerous precursor stages are described as intra-epithelial neoplasms (UK) or intra-epithelial lesions (USA). Older terms include dysplasia or carcinoma in-situ.

These abnormal neoplastic cells have not invaded (hence are benign) – they stay on the epithelial side of the basement membrane and are intra-epithelial (within the epithelium).

A good example is the cervix, where such precursor lesions are called either Cervical Intra-epithelial Neoplasia (CIN – in the UK) or Squamous Intra-epithelial Lesions (SIL – in the USA). They form a spectrum: low grade to high grade. CIN 1 is equivalent to Low-Grade SIL, whereas CIN 2 and CIN 3 are equivalent to High-Grade SIL.

Morphological features

Intra-epithelial neoplasms have a proliferating epithelium, which has the morphological features of neoplasia:

  • abnormal nuclei (showing pleomorphism and hyperchromasia)
  • abnormal mitosis
  • loss of nuclear polarity
  • loss of differentiation

Pleomorphism refers to variation in size and shape of the nuclei. Hyperchromasia (or hyperchromatism) refers to increased staining colour (the nuclei look darker).

Clinical importance and progression

These precursor conditions show a sequential progression in neoplastic development to invasive cancer. Identification of precursor lesions can allow early treatment that prevents the development of malignancy. This is one of the rationales behind the screening program.

With reference to the development of cervical cancer, precursor lesions can be detected by cytological sampling (cervical smears/scrapes or liquid-based cytology – stained with the Papanicolaou stain or “Pap Smear”) and treated effectively, preventing progression to invasive cancer – as part of the cervical screening programme.

©Mark Arends, University of Edinburgh 2017 CC BY-SA
“Pap Smear” from cervix showing cluster of neoplastic cells (middle of image) with enlarged and pleomorphic nuclei (hyperchromatic – darkly staining), compared with the surrounding normal cells with small oval regular nuclei in cells with green (intermediate keratinocytes) or pink (superficial keratinocytes) cytoplasm.
©Robin Crawford, Addenbrookes Hospital, Cambridge
Cancer of the cervix with surface bleeding.

Precursor lesions in the colon

In the large intestine, a majority of adenocarcinomas are preceded by a precursor or dysplastic stage called an adenoma.

All adenomas have dysplastic glandular epithelium and this is graded into low-grade or high-grade dysplasia, depending on the severity of the morphological features of neoplasia (as described above). Regions of high-grade dysplasia within an adenoma (benign) may sometimes evolve into an invasive adenocarcinoma (malignant) showing early signs of invasion through the muscularis mucosae.

Screening for bowel cancers and adenomas tests for occult/hidden blood in the stool and if this is positive the individual has bowel endoscopy/colonoscopy to look for adenomas (polyps) or cancers.

Most invasive cancers develop from an adenoma, as a result of progression from an adenoma to adenocarcinoma – “adenoma-carcinoma” sequence.

©Margaret Stanley, University of Cambridge
Cancer of rectum (towards the right of image) with some surface ulceration.

Tumours and mutations Part 2 of 9

Neoplasms are the result of the escape of tumour cells from the normal homeostatic mechanisms that control the maintenance of organ and tissue architecture and function. These mechanisms are complex; corruption or breakdown of the results from loss or errors in key controls, and the downstream effects from this.

  • The “errors” are direct damage to DNA – mutations
  • The targets for mutations are genes controlling proliferation, death, and genomic stability
  • The transition from a normal, growth-controlled cell through to a malignant cancer cell requires several mutations

Mechanisms of mutation


Carcinogens are agents that induce cancer in man or animals.

Carcinogenesis is the process of cancer induction.

Classes of carcinogen

  • Chemical – synthetic or naturally occurring molecules
  • Physical – UV or ionising radiation
  • Biological- viruses, bacteria, parasites

Cancer as a multi-step process Part 3 of 9

The separate steps that occur in carcinogenesis are studied using:

  • Animal models of carcinogenesis
  • In vitro carcinogenesis – cell transformation
  • Replicative Senescence, Immortalisation & Telomeres
  • Inherited cancers in humans
  • Molecular genetic analysis of cancers and their precursor lesions

Animal models and chemical carcinogenesis Part 4 of 9

In the early 1900s, experimental tumours were induced in rabbits by painting tar on the ears.

The chemical identity of the active molecules in the tar was determined in the 1930s to be polycyclic hydrocarbons such as 3-benzpyrene and 3’methylcholanthrene – potent carcinogens.

The availability of pure carcinogens stimulated animal experimentation, and some important concepts of carcinogenic activity were defined:

  • Dose-response – there is a linear relationship between the amount of carcinogen delivered in a single dose and the number of tumours that develop
  • Latent period – there is a time lag between the administration of a carcinogen and the appearance of macroscopic tumours. The length of this time lag is dose-dependent – high doses reduce it, low doses extend it
  • Threshold dose – there is a threshold dose of carcinogen, below which no tumours form. However, if some secondary non-carcinogenic growth-promoting stimulus (e.g. wounding, chemicals such as Phorbol Esters) is applied to the site after this subthreshold dose of carcinogen, then tumours develop
  • Initiation and promotion – the observations above gave rise to the concept of 2 stages in carcinogenesis: initiation and promotion
  • Progression – sometimes added as a third stage

Initiation of carcinogenesis

Initiation is the alteration of a normal cell to a potentially cancerous cell. Carcinogens cause this, and it irreversible. Carcinogens are mutagens.


A process which permits the clonal amplification of the initiated cell. Promoters are not carcinogens, they induce proliferation (“fix the mutation”*). A benign neoplasm (e.g. skin papilloma) forms.

(* – “fix the mutation” in this context means to make the mutation permanent – not repair it).


Progression is the acquisition of further mutations within the neoplastic clone drive progression to a malignant neoplasm.

In vitro carcinogenesis Part 5 of 9

Much of our understanding of the molecular and genetic mechanisms of carcinogenesis has come from studies on cells in tissue culture (in vitro) exploiting the phenomenon of cell transformation.

Transformation is where the cultured cells change from growing in a flat monolayer of cells attached to the culture flask surface to transformed cells. These have altered morphological appearances, pile up in foci (many piled on top of one another), grow in reduced serum (serum provides grow factors to stimulate normal cells to grow, but lower concentrations of growth factors are needed for transformed cells), can often grow in suspension (unattached to the flask surface), or can form invasive tumours when injected into test mice (immunosuppressed so that the cells are not rejected).

Replicative senescence, immortalisation and telomeres Part 6 of 9

Replicative senescence

Primary diploid cells in tissue culture exhibit the phenomenon of replicative senescence.

  • Cells can only undergo a defined number of cell divisions in tissue culture – the Hayflick number (~50 in humans)
  • They then undergo “senescence”. This involves cell cycle arrest (held in G0), eventually death by apoptosis
  • At low frequency, apparently senescent rodent cells in culture may escape from senescence and become immortal (see below – TERT)
  • Cells from long-lived animals such as humans, in contrast to cells from mice or rats, very rarely undergo spontaneous immortalisation in tissue culture
  • The viral oncogenes of DNA tumour viruses eg SV40 T, adenovirus E1A and E1B, HPV 16 E6 and E7, can immortalise primary human cells (chemical carcinogens rarely do so), usually by inactivating RB1 and TP53


©Mark Arends, University of Edinburgh 2017 CC BY-SA
The relationship between telomere length, senescence and immortalisation

Immortalisation is usually due to expression of the telomerase enzyme complex (TERT with RNA component) that is capable of synthesising telomere DNA sequences to maintain the ends of the chromosomes with repetitive telomere sequences (these are lost every time the cell divides as the normal DNA synthesis process cannot replicate the DNA end).

High levels of telomerase activity have been detected in >90% of cancers studied so far and is also found in a fraction of precancers and in germ cells, stem cells and some other somatic tissues (at low level).

Telomeres: chromosome ends have repeated sequences

Telomeres are repetitive sequences (TTAGGG) at ends of chromosomes. These form loops to protect chromosome ends so that they do not appear as DNA double-strand breaks and lead to end-to-end fusions.

©Samulili CC-BY-SA-3 via Wikimedia Commons
Telomere caps ‘protect’ the chromosomes from shortening during chromosome replication.

There is an associated “DNA end replication problem” as there is no opposite strand of sequence for the primer to bind to after the end of the strand. Therefore a specialised enzyme complex (telomerase) is needed that provides its own RNA primer as part of the complex.

Replicative senescence:  telomere hypothesis

In normal germ and stem cells, telomerase is expressed and active, maintaining telomere length.

In most somatic cells, there is no active telomerase, such that telomere length is reduced with every cell division.

Eventually, the cell ‘runs out’ of the repetitive telomere sequence so that it is no longer possible to form the telomere loop. The cell reacts to an apparent DNA double-strand break, leading to senescence and eventually apoptotic death.

Cancer cells re-express telomerase so that telomere length is maintained, meaning that the cell becomes immortal.

Inherited cancers in humans Part 7 of 9

In rare instances, tumour mutations can be inherited in the germline giving rise to an inherited predisposition to a particular cancer.

Studies on familial cancer have:

  • Reinforced the concept of cancer as a multi-step process
  • Identified genes central to the pathogenesis of cancer


Retinoblastoma is a very rare childhood cancer of the retinoblasts in the retina of the eye, with a peak incidence at 3-4 years of age in children. It can be both sporadic (no family history) and inherited (with a family history).

The American geneticist Knudson hypothesised that, in both situations, the tumours developed because of a 2 step mutational process (2-hit model), implying that both copies of the retinoblastoma susceptibility gene (RB1) must be mutated an inactivated.

©Aerts et al CC BY 2.0 (, via Wikimedia Commons
Ocular fundus view of retinoblastoma.

Genetic basis

In retinoblastoma, the mutations affect both alleles of the gene RB1. The mutations result in loss or inactivation of the wildtype RB1 gene product. The tumour cells show loss of heterozygosity as evidence of loss of one RB1 allele.

(Loss of heterozygosity, LOH, refers to a test to look for the presence of two different-sized (or heterozygous) bands on a gel, one band for each RB1 allele. If one of the bands is lost and only the other band remains, it is interpreted as showing LOH indicating loss of a piece of DNA).

Knudson: 2 step mutational process

In the familial form, the child inherits one mutant RB1 (may be written as Rb- to indicate inactivation) allele and one normal RB1 allele. Thus, all retinoblasts only need to mutate a single gene to have loss or inactivation of both alleles. This occurs relatively frequently as there are around one million retinoblasts in which this may happen (some children get 3 or 4 retinoblastoma tumours often affecting both eyes).

In the sporadic form, the child inherits two normal RB1 alleles. Only rarely will there be mutations that hit both of the two RB1 alleles in the same cell, leading to tumour formation. Hence, the sporadic form is very rare and patients only get a single tumour in one eye.

Inherited cases have: (1) pre-zygotic mutation (inherited mutation), and (2) post-zygotic mutation (acquired during life – called a somatic / acquired mutation)

Sporadic cases have: (1) and (2) both steps post-zygotic (acquired / somatic)

This theoretical framework turned out to be correct.

Familial                  Sporadic

Parent                  Rb / RB1               RB1 / RB1

Child                     Rb / RB1               RB1 / RB1

Retinoblastoma  Rb / Rb                Rb/ Rb

Other cancer inheritance disorders

There are several rare syndromes that have the same inheritance pattern as retinoblastoma (autosomal dominant with a 50% probability of inheriting the mutated gene).

The affected individuals in these families inherit the mutant gene, or, in a very small percentage of families, a family member may be the first to spontaneously acquire a mutant gene (then pass it on).

The individuals are heterozygous for this gene in all their somatic cells (wildtype allele / mutant allele).

The cancers that arise in these individuals have mutated the second allele in the target cell type that is susceptible to tumour formation following the loss of both alleles’ functions.

The tumour cells are homozygous for the mutant gene (mutant/mutant).

Syndromes in which the heterozygotes express the tumour phenotype

Tumour syndrome Gene Gene function
Retinoblastoma RB1 Cell cycle checkpoint control
Familial adenomatous polyposis coli APC Signal transduction
Li Fraumeni TP53 Cell cycle control/DNA damage
Hereditary non-polyposis colorectal cancer / lynch MLH1, MSH2 DNA Mismatch repair
Familial breast and ovarian cancer BRCA-1/BRCA-2 DNA repair – d/s break
Basal cell naevus Ptch Signal transduction

The affected individual (proband) has the genotype wildtype/mutant. Autosomal dominant inheritance (so inherit 1st hit). The tumour cells acquire 2nd hit, and so have the genotype mutant/mutant.

Syndromes in which the homozygotes express the tumour phenotype

In a second group of inherited syndromes, the pattern of inheritance of the mutant gene is as a classical Mendelian recessive inheritance. The affected individual inherits 2 mutant alleles, one from each parent. These individuals have an increased risk of developing cancer.

The proband has the genotype mutant/mutant. They are homozygous for the mutant gene, inheriting two mutant alleles, one from each parent. Parents are often unaffected carriers. Autosomal recessive inheritance (inherit both 1st & 2nd hits).

Summary Part 8 of 9

What do these studies of inherited cancer tell us?

  • Cancer involves genetic changes – the initiating event is a somatic mutation in a single cell
  • More than one mutation is necessary for progression
  • Maintaining error-free DNA is crucial
  • Controls restricting cellular lifespan must be overcome for tumour progression
  • Cancer is a multi-stage process

Questions Part 9 of 9