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185: 119122 (1998)

EDITORIAL

TALES FROM THE HUMAN CRYPTINTESTINAL

STEM CELL REPERTOIRE AND THE ORIGINS OF
HUMAN CANCER
. *
University Division of Gastroenterology, Leicester General Hospital NHS Trust, Leicester LE5 4PW, U.K.

SUMMARY
A generally accepted model for the origin of human cancer is that tumours arising from the gut-lining epitheliumthe adenomas and
carcinomasare of clonal origin, i.e., they are derived from a single abnormal cell. Recent work has shed considerable light on these
matters and suggests that this model may be erroneous and that at least some adenomas are of multiclonal origin. These findings have
basic implications for more generalized models of tumourigenesis and for researchers examining the potential clinical value of gene
therapy.  1998 John Wiley & Sons, Ltd.
J. Pathol. 185: 119122, 1998.
KEY WORDScolon;

adenoma; carcinoma

Two questions have taxed gut biologists over the last

10 years: whether the repertoire of gastrointestinal stem
cells includes all the cell lineages seen in the gastric
glands or intestinal crypts; and whether the tumours that
arise from the gut-lining epithelium, the adenomas and
carcinomas, are of clonal origin. While considerable
inroads have been made into these problems by the
exploitation of mouse models using either chimaeric
animals1,2 or mice heterozygous for an X-linked marker
gene, such as glucose-6-phosphate dehydrogenase
(G6PD),3 the human gut has been strangely silent on
these issuesuntil, that is, a recent report by Novelli
et al.,4 describing a probably unique individual who not
only shows the genotype XO/XY, but who also has the
syndrome of familial polyposis coli (FAP).
In the mouse models, the intestinal crypts and gastric
glands are derived totally from one of the mouse strains
used to make the chimaera, or stain totally positive or
negative for the gene product marker; in the adult
animal, there are no mixed crypts. The important conclusion from these studies is that each crypt forms a
clonal population: each crypt is derived, ultimately at
any rate, from a single cell. This concept of a monoclonal crypt or gastric gland has led to the belief in a
single multipotential stem cell, which naturally gives
rise, ultimately at any rate, to all contained crypt cell
lineages. This view is reinforced by further studies57
where mice are given a single dose of mutagen and in the
weeks that follow, crypts appear which are entirely
composed of cells with a different, mutated phenotype,
*Correspondence to: Professor R. J. Playford, University
Division of Gastroenterology, Leicester General Hospital NHS Trust,
Gwendolen Road, Leicester LE5 4PW, U.K. E-mail: rjp13@le.ac.uk

CCC 01233417/98/06021207 $17.50

 1998 John Wiley & Sons, Ltd.

best explained by a mutation in a single stem cell from

which all lineages are then derived, and which colonize
the crypt. These observations provide strong experimental support for the unitarian hypothesis of intestinal cytogenesis, propounded by Cheng and Leblond in
1974,8 whereby all intestinal cell lineages originate in a
multipotential, undifferentiated, basally sited stem cell.
Data from human tissue are, for obvious reasons,
much more difficult to acquire. The human individual
studied by Novelli et al. was an XO/XY phenotypic male
of short stature but with no other stigmata of Turners
syndrome. By coincidence, he also had FAP for which
he had undergone a prophylactic total colectomy at 32
years of age and from which archival paraffin-embedded
material was available. This was an extremely serendipitous discovery, since the chance of finding such an
individual is predicted to be less than one in a hundred
million. The clinical diagnosis of FAP was confirmed by
mutation analysis of his adenomatous polyposis coli
(APC) gene: a protein truncation test followed by
sequencing showed a frameshift mutation (codon 1309)
in the germline. A combination of karyotyping and
fluorescent in situ hybridization (FISH) on the patients
peripheral blood lymphocytes confirmed that he was a
mosaic and also showed the Y chromosome to be
dicentric. The karyotype was 45,X/46,Xdic(Y)(Ypter<
cen<Yq11.23::Yp11.3) and approximately 20 per cent
of peripheral blood lymphocytes were XO.
By using non-isotopic in situ hybridization on histological sections of small and large intestine with Y
chromosome-specific probes, intestinal crypts were seen
to be composed exclusively of either XY or XO cells (see
Fig. 1). The patches of XO crypts were irregular in shape
and the patch size varied widely (mean patch width 185

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R. J. PLAYFORD

Fig. 1Two adjacent crypts taken from a patient with familial adenomatous polyposis and who is an
XO/XY mosaic. Non-isotopic in situ hybridization using Y chromosome-specific probes causes cells
containing the Y chromosome to have an intranuclear purple dot. The cells in the left-hand crypt all show
the presence of Y chromosome, while the cells in the right hand crypt are negativean XO crypt. Figure
reproduced from ref. 4

crypts, range 114 crypts). Crypts examined at patch

borders showed no mixed XO/XY cellularity and all
indigenous epithelial cell lineages could be directly
visualized as XO or XY. Thus, human intestinal crypts
are clonal populations, each derived ultimately from a
single stem cell. From the many thousands of crypts
examined, four mixed crypts were seen in otherwise pure
XY patches, similar to the partial loss of O-acetylated
sialomucins described in human colonic crypts9 and in
mice given mutagens.57 Hence, the occurrence of occasional mixed crypts is probably due to non-disjunction,
with loss of the Y chromosome, in a crypt stem cell.
Normal human intestinal crypts are clonal populations,
derived from a single, multipotential stem cell.
Mutational theories of tumour development suggest
that tumours arise from a series of mutations occurring
in one cell and its progeny;1012 others have argued that
tumours are not clonal in origin, but require the interaction of multiple cells;13,14 outgrowth of a dominant
clone during subsequent development accounts for their
apparent monoclonality. The patients colon contained
thousands of tubular adenomas, ranging in size from
 1998 John Wiley & Sons, Ltd.

lesions composed of a single dysplastic crypt (a monocryptal adenoma) to microadenomas of 25 mm in

diameter. The monocryptal adenomas were entirely XO
or XY in type with no mixed pattern, not surprisingly,
given the clonal nature of normal crypts. However, 13 (5
per cent) microadenomas contained a mixture of XO
and XY dysplastic crypts (see Fig. 2) and of the 17
adenomas containing XO dysplastic crypts, 76 per cent
(13) were of mixed XO/XY type and 24 per cent (4) were
purely of XO genotype. From these results it was
estimated that a minimum of 76 per cent of all the
adenomas (above monocryptal size) in this patient were
polyclonal in origin. Analysis showed that the random
collision of tumours on its own could not account for
levels of polyclonality as high as those observed.
The results of Novelli et al. therefore provide strong
evidence that colonic tumours can be polyclonal, contrary to popular models of tumourigenesis. Further
evidence for this conclusion has recently come from a
study examining the clonality of intestinal tumours in a
mouse model of FAP, the so-called min-mouse. In
these animals, the mouse homologue of the APC gene
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CLONALITY AND HUMAN CANCER

Fig. 2Microadenoma taken from the same patient. This contains

dysplastic crypts of both XY and XO genotypes. Figure reproduced
from ref. 4

(the multiple intestinal neoplasia, Min gene) is abnormal and results in the development of multiple intestinal
adenomas. The clonality of the adenomas in chimaeric
Min mice was studied and found to be polyclonal in
about 80 per cent,15 a percentage similar to that found in
the patient reported by Novelli et al.4 Studies in human
colorectal tumours using X-linked markers in females16
and the principle of X-chromosome inactivation17 have
produced conflicting results: isoenzyme studies of G6PD
from microdissected tissue in black heterozygous
females suggested that a single sporadic colonic carcinoma18 and seven colonic adenomas from three patients
with Gardners syndrome19 were polyclonal, while
X-linked restriction fragment length polymorphisms
suggested that 50 human colorectal tumours of both
familial and sporadic types were monoclonal.20 However, such X-linked markers, with biochemical or
molecular analysis of extracted tissues, are probably
inappropriate for the determination of clonality in dissected tissues from primary epithelial tumours: even
microadenomas can contain stromal elements and
entrapped normal crypts,4 which complicates the
interpretation of results. More importantly, patch size
can vary widely and unless one can directly observe
those tumours arising at the patch boundary, i.e., the
junction between crypts displaying different genotypes,
it becomes difficult, if not impossible, to make meaningful observations on tumour clonality.21,22 This means, in
effect, that only analyses where tissues can be examined
in situ can give conclusive results where tumour clonality
is concerned. This point may explain why the results in
this individual, with a small patch size for the marker
used, contrast so starkly with previous results: if the
patch size for X-inactivation markers is large, then most
tumours will appear monoclonal, since they will arise
from within the large patches. The chance of finding an
adenoma arising at the junction of a patch will be
small.22 If this result is general, then it could have
profound implications for our understanding of tumour
clonality and the origin of human tumours.
 1998 John Wiley & Sons, Ltd.

121

The work by Novelli et al. does, however, have its

limitations, as it focuses on a single patient with FAP. In
this condition, clinical tumours are numerous and
microadenomas legion. Early co-incident proliferation
of two clones, though rare, might therefore be expected
to occur. Outside such a genetically changed environment, such as occurs in FAP (and in the Min mouse
model), this would be a much rarer event. Caution must
therefore be shown in extrapolating from the results in a
single patient with FAP to assuming that sporadic
adenomas (which are far more common) are also polyclonal. It is important to note that, even if sporadic
tumours are derived from a single cell (clone), this does
not equate to remaining a homogeneous population as
they progress to the more advanced malignant state.
This is because tumours are likely to progress through a
number of subclones as further genetic mutations are
accrued. At any one time, several of these clones may
co-exist, but those with the greatest survival advantage
would prosper at the expense of the others. Clonal
selection is likely to be relevant in explaining the
resistance of recurrent tumours to chemotherapy,
despite the original tumour having previously been
found to be sensitive to the agent.
If adenomas are indeed polyclonal, it is worthwhile
speculating on possible mechanisms: field effects may
cause adenomas to cluster (non-random collision); multiple adenomatous clones may be required for (or
strongly favour) early adenomatous growth; or perhaps
more likely, early adenomas may induce adenomatous
growth in surrounding crypts. This may apply especially
in FAP, since all cells already have a single APC
mutation and perhaps some derangement of APC function. The molecular mechanisms behind these effects are,
of course, unclear. Adenomas and carcinomas are
known to produce increased amounts of growth modulatory agents such as epidermal growth factor receptor
ligands23,24 and glycine-extended gastrin.25 Such factors
might result in increased growth in the adjacent tissue,
acting via paracrine mechanisms. In addition, the presence of inflammatory cells in adenomatous and malignant tissue might result in increased local production of
several cytokines, possibly influencing growth in the
stem cells in adjacent crypts.
APC is a dominantly inherited monogenetic disease.
The exact site of mutation in patients with APC varies
between families but results in a truncated APC gene
product due to frameshifts, nonsense mutations, or
splice site changes. These changes can be detected in
asymptomatic family members using standard genetic
screening techniques.26 The defect can be considered as a
loss of a tumour suppressor gene and is therefore
potentially suitable for gene therapy to replace the
defective gene. There are, however, multiple problems to
be overcome, including how to insert the correct gene
into the intestinal stem cells. Early attempts at gene
transfer in the colonic epithelium by luminal installation
of liposomes carrying a reporter gene27 have shown that
multiple applications are necessary to maintain expression. The existence of multipotential stem cells in the
human intestinal mucosa indicates that these cells
should be targeted in future attempts at gene transfer.
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R. J. PLAYFORD

Stable transfection of retroviral constructs carrying a

reporter gene to all lineages in the colonic mucosa has
been claimed in an experimental system,28 presumably
by transfecting such stem cells. A major problem with
this approach for the treatment of APC, however, is that
whatever gene transfer technique is used, it is unlikely
that every stem cell will successfully incorporate the
construct. These non-transfected cells will remain in situ
as potential sites of tumour development and might have
a growth advantage over successfully transfected cells.
In summary, recent studies support the idea that some
adenomas (particularly those associated with FAP) are
likely to be of polyclonal origin. Research into this topic
is likely to remain a growth area for some time.
REFERENCES
1. Ponder BAJ, Schmidt GH, Wilkinson MM, Wood MJ, Mark M, Reid A.
Derivation of mouse intestinal crypts from single progenitor cells. Nature
1985; 313: 689691.
2. Thompson M, Fleming FA, Evans DJ, Fundle R, Surani MA, Wright NA.
Gastric endocrine cells share a clonal origin with other gut cell lineages.
Development 1990; 110: 477481.
3. Griffiths DFR, Davis SJ, Williams D, Williams GT, Williams ED. Demonstration of somatic mutation and colonic crypt clonality by X-linked
enzyme histochemistry. Nature 1988; 333: 461463.
4. Novelli MR, Williamson JA, Tomlinson IP, et al. Polyclonal origin of
colonic adenomas in an XO/XY patient with FAP. Science 1996; 272:
11871190.
5. Winton DJ, Ponder BA. Stem cell organization in mouse small intestine.
Proc R Soc London Biol 1990; 241: 1318.
6. Williams ED, Lowes AP, Williams D, Williams GT. A stem cell niche
theory of intestinal crypt maintenance based on a study of somatic mutation
in colonic mucosa. Am J Pathol 1992; 141: 773776.
7. Park HS, Goodlad RA, Wright NA. Crypt fission in the small intestine and
colon. A mechanism for the emergence of G6PD locus-mutated crypts after
treatment with mutagens. Am J Pathol 1995; 147: 14161427.
8. Cheng H, Leblond C. Origin, differentiation and renewal of the four main
epithelial cell types in the mouse small intestine V unitarian theory of the
origin of the four epithelial cell types. Am J Anat 1974; 141: 537562.
9. Fuller CE, Davis RP, Williams GT, Williams ED. Crypt restricted heterogenicity of goblet cell mucus glycoprotein in histologically normal human
colonic mucosa. Br J Cancer 1990; 61: 382384.

 1998 John Wiley & Sons, Ltd.

10. Armitage P, Doll R. Age distribution of cancer and multi-stage theory of

carcinogenesis. Br J Cancer 1954; 8: 112.
11. Knudson AG, Anderson MO. Mutation and human cancer. Adv Cancer Res
1973; 17: 317348.
12. Fialkow PJ. Clonal origin of human tumors. Annu Rev Med 1979; 30:
134143.
13. Rubin H. Cancer as a dynamic developmental disorder. Cancer Res 1985;
45: 29352942.
14. Alexander P. Does cancer arise from a single transformed cell or is
monoclonality of tumours a late event in carcinogenesis? Br J Cancer 1985;
51: 453457.
15. Merritt AJ, Gould KA, Dove WF. Polyclonal structure of intestinal
adenomas in apc-(min)/+ mice with concomitant loss of apc(+) from all
tumour lineages. Proc Natl Acad Sci USA 1997; 94: 1392713931.
16. Gartler SM, Linder D. Selection in mammalian mosaic cell population.
Cold Spring Harbor Symp Quant Biol 1964; XXIX: 252260.
17. Lyon MF. Sex chromatin and gene action in the mammalian
X-chromosome. Am J Hum Genet 1962; 14: 135148.
18. Beutler E, Collins Z, Iriwin L. Value of genetic variants of glucose-6phosphate dehydrogenase in tracing the origin of malignant tumors. N Engl
J Med 1967; 276: 389391.
19. Hsu SH, Luk GD, Krush AJ, Hamilton SR, Hoover HH Jr. Multiclonal
origin of polyps in Gardners syndrome. Science 1983; 221: 951953.
20. Fearon ER, Hamilton SR, Vogelstein B. Clonal analysis of human colorectal tumours. Science 1987; 238: 193197.
21. Ponder BAJ, Wilkinson MM. Direct examination of the clonality of
carcinogen-induced colonic epithelial dysplasia in chimeric mice. J Natl
Cancer Inst 1986; 77: 967976.
22. Schmidt GH, Mead R. On the clonal origin of the tumourslessons from
studies of intestinal epithelium. BioEssays 1990; 12: 3743.
23. Steel CM. Peptide regulatory factors and malignancy. Lancet 1989; ii:
3034.
24. Hanby AM, Poulsom R, Singh S, et al. Hyperplastic polyps: a cell lineage
which both synthesizes and secretes trefoil-peptides and has phenotypic
similarity with the ulcer-associated cell lineage. Am J Pathol 1993; 142:
663668.
25. Ciccotosto GD, McLeish A, Hardy KJ, Shulkes A. Expression, processing,
and secretion of gastrin in patients with colorectal carcinoma. Gastroenterology 1995; 109: 11421153.
26. Powell SM, Petersen GM, Krush AJ, et al. Molecular diagnosis of familial
adenomatous polyposis. N Engl J Med 1993; 329: 19821987.
27. Westbrook CA, Chmura SJ, Arenas RB, Kim SY, Otto G. Human APC
gene expression in rodent epithelium in vivo using liposomal gene delivery.
Hum Mol Genet 1994; 3: 20052010.
28. Del Buono R, Wright NA. Approaches to gene therapy in the gut;
retrovirus mediated gene transfer in a model of gastrointestinal development. Gut 1993; 34: T107.

, . 185: 119122 (1998)