An interest in possible relationships between megaloblastic cells
and cancerous ones dates back at least four decades. In 1954,
Massey and Rubin described the persistence of abnormal gastric
columnar cells in the stomachs of individuals with pernicious
anemia, even after the anemia had been successfully treated with
vitamin B12. Since the cytologic appearance of these cells
exhibited some characteristics of both megaloblastic and cancer
cells, and since several prior reports had observed an increased
incidence of gastric cancer in individuals with pernicious anemia,[2-4]
Massey and Rubin postulated that these abnormal cells might represent
a transitional cell type between cells that characterize the atrophic
gastric epithelium in pernicious anemia and gastric cancer cells.
The first studies to examine a relationship between folate-associated
megaloblastosis and cancer were conducted in the 1960s. Van Niekerk
noted several cytologic similarities between epithelial cells
of the uterine cervix from folate-deficient women and dysplastic
cervical cells. In both cases, the cells were larger than normal,
had nuclei that were disproportionately large compared to the
size of their cytoplasm, and had hyperchromatic chromatin. Nevertheless,
for many years, there was little evidence to suggest that the
cytologic similarity between megaloblastic and neoplastic cells
was anything more than a coincidence.
It was not until the 1970s and '80s that investigators began to
effectively question whether there might be a functional association
between megaloblastic and dysplastic cells. In the 1980s, investigators
at the University of Alabama conducted two prospective, controlled
clinical intervention trials in individuals with dysplastic changes
in the epithelium of the uterine cervix and metaplastic changes
in the bronchi. Both studies observed a significant degree
of attenuation in dysplasia or metaplasia after several months
of supplementation with pharmacologic doses of folic acid alone
(Figure 1) or in conjunction with pharmacologic doses of vitamin
B12. These findings lent further support to the concept that
folate and dysplasia might be functionally related, although it
remained unclear whether the alterations in cytology associated
with supplementation were truly an attenuation of dysplasia or
merely an attenuation of megaloblastosis masquerading as dysplasia.
Some studies of the uterine cervix highlight important features
that characterize the clinical literature on folate and carcinogenesis.
A large, recently published case-control study designed to examine
the various risk factors for cervical dysplasia is one such
example. Although this study did not find an overall association
between red blood cell (RBC) folate levels and dysplasia, subjects
with human papilloma virus-16 (HPV-16) infection of the cervix,
an important risk factor for cervical carcinogenesis, had a
fivefold greater risk of having dysplasia if they also had diminished
RBC folate levels. This finding suggests that the enhancement
of cervical carcinogenesis effected by diminished folate status
requires cofactors that predispose to carcinogenesis. Thus, diminished
folate status, by itself, is not a carcinogen; rather, it appears
to enhance an underlying predisposition to cancer.
Another interesting feature of this study is the fact that diminished
folate status was defined as an RBC folate level of < 660 nmol/L
(291 ng/mL)--a value that is well within the range of folate levels
conventionally accepted as normal and well above the threshold
below which an individual runs the risk of megaloblastic anemia.
This suggests that the effect of diminished folate status on carcinogenesis
does not require frank folate deficiency as it is usually defined;
rather, the data suggest that a modest diminution in folate levels
is all that is necessary to produce an effect. Such a relationship
also seems to exist in the colorectum.
As indicated above, much of the groundwork in this field was laid
in studies of the uterine cervix. Several case-control clinical
trials have examined the association between dietary folate intake
and cervical neoplasia (Table 1).[8,10-14] Although several of
these studies demonstrated that the relative risk of cervical
neoplasia is somewhat diminished in individuals who consume larger
quantities of folate, the effect uniformly fell short of statistical
significance when adjustment was made for confounding variables.
This negative consensus among case-control studies in cervical
neoplasia should not be construed as proof that no effect exists,
as these studies suffer from several limitations: In most instances,
folate status was assessed by estimating average dietary folate
intake by means of food frequency questionnaires that have not
been validated for folate intake. Also, none of the trials, except
the study by Butterworth et al, stratified patients for HPV infections.
These studies were therefore unable to explore possible synergism
between diminished folate intake and HPV, and such stratification
may be necessary to observe an effect of folate. Furthermore,
all of the studies, except that by Butterworth et al, examined
women with either carcinoma in situ or invasive cancer. These
represent the latter stages of neoplasia, by which time the progression
to cancer may be far enough advanced that there is little opportunity
for modulation by such factors as folate status.
Two intervention trials (see Table 2)[15,16] have attempted to
confirm the observations made in the University of Alabama study
described above. Both intervention trials examined larger populations
of women and followed the subjects over a longer period. In neither
trial were the investigators able to reproduce the regression
of dysplasia noted in the earlier study. However, on entry over
two-thirds of the subjects in the more recent trials had the lowest
grade of dysplasia--a disease stage known to have a rate of spontaneous
reversion to normal of more than 60%. This certainly may have
interfered with the ability of either study to observe a positive
effect of folate supplementation.
In summary, the results of case-control and intervention trials
in the cervix are not particularly compelling. However, the manner
in which these trials were performed precludes one from making
a definitive statement about whether alterations in folate status
truly modulate the process of carcinogenesis in the cervix.
Clinical observations from several different groups point to an
association between diminished folate status, assessed by either
dietary intake or measurement of blood levels, and an enhanced
risk of colorectal adenomas or cancer (Tables 3 and 4). Lashner
and colleagues first made this observation in a case-control study
of individuals afflicted with chronic ulcerative colitis--a
disease that poses not only a 10- to 40-fold increased risk of
colonic cancer but also a significant risk of folate deficiency.[19,20]
Individuals who had been not been taking folate supplements over
the long term had a rate of colonic neoplasia that was approximately
2-1/2-fold greater than the rate in those who had been taking
supplements. Furthermore, chronic administration of sulfasalazine,
a drug commonly used for the treatment of ulcerative colitis that
is well known to specifically inhibit both folate absorption[20,21]
and folate metabolism, was associated with a 50% increase
in the risk of dysplasia.
Both of these observations fell slightly short of statistical
significance. Nevertheless, the study established the importance
of examining this issue in the colon.
More recently, Lashner has confirmed these observations by prospectively
comparing RBC folate levels in ulcerative colitis patients who
harbor neoplastic changes in their colorectum with levels in age-,
gender-, and disease-matched controls without neoplastic lesions.
The mean RBC folate level was significantly lower in the individuals
with neoplasia than in the controls when adjustment was made for
confounding variables. It is also noteworthy that although the
group harboring neoplasia had diminished RBC folate levels compared
to the control group, the absolute folate level in the neoplasia
group (454 ng/mL) was well within the range of values that is
conventionally accepted as normal. This observation recapitulates
the earlier finding made in the cervix.
Epidemiologic studies conducted in the general population generally
support the hypothesis that diminished folate status is associated
with an increased rate of colorectal neoplasia, although the nature
of the relationship is different in each study. One study based
in western New York observed that increased habitual intake
of folate was associated with a decreased incidence of rectal,
but not colonic, cancer, whereas two studies examining the population
on the island of Majorca[25,26] observed reductions in relative
risk for colonic and rectal neoplasms combined.
A study conducted in a population from western Washington state
only assessed rates of colon cancer and did not observe a significant
reduction in risk with increased dietary folate intake. However,
a strong trend toward such an effect was present in women. Conversely,
in a large study from southern California, decreased RBC folate
levels were associated with a significantly higher risk of colorectal
adenomas in men, but no significant effect was observed in women.
To date, some of the most compelling epidemiologic evidence to
establish an association between folate status and colorectal
neoplasia in the general population has come from studies that
have carefully examined in-teractions between folate and other
dietary components, particularly alcohol.[29-31] Giovannucci et
al utilized large, prospectively analyzed populations to convincingly
support the hypothesis that increased folate intake is inversely
associated with the incidence of colorectal adenomatous polyps
and of colorectal cancers. The prospective design of the two
studies, the large size of the populations analyzed (25,474 and
47,931 subjects, respectively), and the effective control of several
confounding dietary variables add considerable strength to the
conclusions. In both studies, the superimposition of moderate
to high alcohol intake greatly increased the neoplastic risk of
a low-folate diet.
Similarly, using a nested case-control design among a prospectively
examined cohort of men, Glynn et al observed that neither blood
folate values nor dietary intake of folate alone was significantly
associated with colorectal cancer. However, the combination of
a high-alcohol, low-folate diet was associated with a highly significant,
fourfold increased risk of cancer.
These studies in the general population are particularly important
since they demonstrate an effect in people who clearly do not
have other conditions predisposing them to colorectal cancer.
Although the results from the epidemiologic studies pertaining
to folate and colorectal cancer are not uniformly positive, the
totality of the data provides a very persuasive argument for the
existence of such an effect; certainly the effect is much stronger
than that observed in the cervix.
Evidence from prospective, controlled, intervention trials are
apt to provide the most compelling proof for a causal relationship
between folate status and colorectal neoplasia, but results from
such trials are just beginning to emerge. Results from two small
intervention trials have been provocative, but nonetheless, inconclusive
Paspatis et al randomized 60 individuals to receive either 1 mg
of folic acid or a placebo daily following colonoscopic removal
of an adenomatous polyp. Colonoscopic follow-up at 12 and
24 months revealed a rate of polyp recurrence in the folate group
that was 40% to 60% of the rate observed in the placebo group.
Nevertheless, these results fell short of statistical significance,
perhaps because of the small size of the study population.
The second intervention trial, by Cravo et al, also utilized an
intermediary biomarker of colorectal cancer as the primary end
point, although a biochemical one. In this trial, 11 subjects
with colonic polyps and 11 subjects with colorectal adenocarcinoma
were randomized to receive either 10 mg or 0 mg of folic acid
daily for 6 months following removal of their neoplasm. The major
end point examined was genomic DNA methylation of normal-appearing
rectal mucosa, which has been proposed as an intermediary marker
of colorectal cancer.
Alterations in DNA methylation are commonly observed as an early
biochemical phenomenon in colonic carcinogenesis.[34-38] Genomic
hypomethylation of DNA has been noted uniformly in early, premalignant,
colonic neoplasms,[34,35] as has hypomethylation of certain proto-oncogenes,
such as K-ras. Genomic DNA hypomethylation has even been observed
to occur as a widespread phenomenon in normal-appearing colonic
mucosa of individuals harboring colorectal neoplasms. Therefore,
in addition to possibly serving as a mechanism by which folate
deficiency enhances carcinogenesis (a hypothesis that is discussed
later in this review), genomic DNA methylation may be a satisfactory
intermediary marker of colonic carcinogenesis.
Interestingly, DNA hypomethylation precedes most of the other
known molecular events in colorectal tumorigenesis, suggesting
that alterations in DNA methylation may play an active role in
the evolution of carcinogenesis. Therefore, it is of interest
to determine whether the DNA hypomethylation associated with early
dysplasia can be modified by intervention. The DNA hypomethylation
observed in early adenomatous polyps in humans is not likely a
consequence of folate deficiency. Thus, it is of considerable
interest that the trial by Cravo et al demonstrated that pharmacologic
doses of folic acid reversed the genomic DNA hypomethylation present
in the normal-appearing rectal epithelium of individuals harboring
either colorectal adenomas or cancers. This observation is
pertinent to the mechanism by which folate status modulates colorectal
carcinogenesis and, in addition, has important implications regarding
a potential role for folate in the prevention of colorectal cancer.
Lung, Esophagus, and Stomach
Chronic tobacco use is associated with diminished blood levels
of folate, as well as diminished concentrations of the vitamin
in the squamous mucosa of the oral cavity; this association is
seen even when dietary folate intake of smokers is the same as
that in nonsmoking controls.[40,41] Compared to nonsmokers, smokers
also display a greater prevalence of chromosomal aberrations in
oral mucosa cells.[41,42]
A single trial has examined the potential chemopreventive role
of folate in lung cancer. Heimburger et al performed a prospective,
placebo-controlled, intervention trial in 80 male smokers with
squamous metaplasia. Both folic acid and hydroxycobalamin (vitamin
B12) were concurrently administered to the experimental group.
After 4 months, a significant improvement in the severity of metaplasia
was observed in the experimental group but not in the controls.
Two studies performed in the tribal homelands of South Africa,
which contains regions with particularly high rates of esophageal
cancer, have begun to examine this issue in the esophagus.[43,44]
In both studies, there appeared to be an association between diminished
levels of serum folic acid and evidence of atypia and/or neoplasia
on esophageal cytopathology; lower levels of vitamins A, E and
B12 were also associated with dysplasia or cancer.
The sole intervention study in this organ to date was a randomized,
placebo-controlled trial among 3,318 Chinese individuals with
dysplastic esophageal cytopathology. The experimental group received
a daily multivitamin and multimineral preparation that included
800 mcg of folic acid. Follow-up at 6 years indicated that supplementation
caused a significant reversion of dysplastic cytology to normal;
however, supplementation did not confer protection against esophageal
cancer. It remains to be seen, therefore, whether the reversion
of dysplastic cytology will translate into a significant drop
in cancer rates with a longer follow-up.
A single case-control study in Italy has examined the relationship
between dietary folate intake and stomach cancer. Although
an inverse association was noted, this did not remain significant
after correction for other dietary variables was made.
In summary, data are insufficient at this point to make any firm
conclusions regarding folate and cancer in the lung, esophagus,
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