Changes in endometrial latent precancer rates, as detected by PTEN immunohistochemistry, were studied in untreated controls, and women either taking cyclic low dose oral progestins or having placement of a progestin impregnated IUD. All groups initially had comparable proportions of patients whose endometria contained PTEN null-glands, but these diverged significantly after therapy. The progestin impregnated IUD group, which delivered the highest local dose of progestins for the longest period, experienced a strong trend towards regression of pre-existing latent precancers, with all latent precancers seen at intake disappearing on post-therapy followup. Involution rates were modest and statistically insignificant for the other treatment and control groups.
Progestins are capable of inducing dose dependent apoptotic cell death of neoplastic endometrial cells grown in culture, but with a rapid extinction over a period of a few days. Subsequent withdrawal is accompanied by resumption of apoptotic cell death on a scale several orders of magnitude greater than achieved in the preceding steady state(18
). Dose and schedule of administration therefore interact in defining the net effect. Local delivery of progestins by placement of an impregnated intrauterine device provides a very high endometrial concentration of hormone while diminishing the complications of systemic distribution. Such devices have even been effective in treatment of established well differentiated endometrial adenocarcinoma(19
), and present non-surgical alternative therapies for management of premalignant endometrial lesions.
Declines in pre-existing latent precancers were not seen in endogenously driven normal menstrual cycles, and cyclic oral administration of low dose progestins. It is entirely possible that the cancer protective effects seen in low dose oral progestin and combination contraceptive administration require a longer duration to achieve than the relatively short term followup in this study. There was a measurable, but not statistically significant difference, however, in the emergence of new latent precancers during followup between these groups, with 21% of all cycling proliferative patients developing emergent latent precancers during followup, compared to only 4-9% in the more quiescent endometria of the three perimenopausal groups (Groups 1,2,3). This is no surprise as random mutagenesis, the presumed mechanism of origin for most latent precancers, is expected to occur in proportion to the mitotic activity of the source tissue(7
Despite a high rate of acquired PTEN mutation in histologically normal tissues, and a correspondingly low lifetime incidence of endometrial cancer, there are good reasons to link inactivation of PTEN to endometrial carcinogenesis. PTEN knockout mice develop endometrial carcinoma in 20% of cases.(10
) PTEN mutation is the most common genetic defect in endometrioid endometrial adenocarcinomas.(21
) Carry-forward of exact PTEN mutations seen in precancers to subsequent cancers in the same patient establishes lineage continuity between premalignant and malignant phases of disease(9
). Loss of PTEN tumor suppressor functions including Akt-dependent enabling of apoptosis and control of cell division rates, confer proliferative and survival advantages long associated with the neoplastic phenotype(1
We have previously used immunohistochemistry as a tool for detection of PTEN protein null endometrial glands, and know that these are due to irreversible changes in the structure of the PTEN gene itself, rather than transient alteration of protein expression from an intact gene. In our hands, loss of PTEN protein as assessed by immunohistochemistry is highly associated with presence of mutations of the PTEN gene or deletions of the 10q23 locus for PTEN(8
). In the case of small numbers of paraffin embedded endometrial glands isolated by laser capture microdissection, we have found that all PTEN expressing glands identified by immunohistochemistry have a wild-type (normal) genotype, whereas 84% of non-expressing samples have either a mutation or loss of at least one 10q23 heterozygous marker in the region of the PTEN
). A key aspect of achieving such a high concordance between genetic inactivating events and loss of protein by immunohistochemistry is use of appropriate antibodies (not all commercially available reagents meet this standard(24
Sampling error must be considered as a variable in monitoring of latent precancers for purposes of defining their fate in response to interventional therapies. Typically only a very small number of affected glands are present in a single biopsy, representing less than 2-3% of all present. While this is an advantage in providing abundant positively staining glands within the same specimen for interpretive comparison of loss of signal, they may be missed in a single specimen due to simple sampling error. This must account for some fraction of “emergent” precancers which were seen after therapy but missed at intake. For this reason, we have described these events as relative comparisons between therapeutic subgroups of patients which share the same sampling errors.
A potential problem in studying physically small premalignant lesions is that the biopsy itself may be destructive, removing the affected cells exclusive of a treatment effect. Data of persistence over time in normal proliferative endometrium suggests that sufficient PTEN-null glands usually remain after biopsy to be detected in future samples, as fully 83% (10 of 12) of those patients having a latent precancer at one timepoint will have a discernible latent precancer during followup(8
). As was the case with sampling error, comparison between groups of differently managed patients undergoing similar tissue sampling has controlled this variable.
Lastly, consistently defined hormonal exposures, such as those likely to change endometrial cancer risk, are difficult to achieve outside the controlled setting of a clinical trial. This was a problem in a previous retrospective study that showed a 90% rate of PTEN-null endometrial precancer involution, in response to vary doses and intervals of medically administered progestins(13
). The current study makes use of a systematic approach to therapy to generate more consistent groups for comparison.
This is a study exploring those biologic events which are effective during a preclinical phase and have the potential to alter the course of subsequent disease. Ultimately, long term patient outcomes, including a protracted followup interval after withdrawal of therapeutic hormones are an unmatchable gold standard for establishing therapeutic efficacy. We have shown that short term progestin administration, previously associated with long-term favorable clinical outcomes (cancer risk reduction), can increase the rate of involution of latent precancers while on therapy. This provides additional evidence that the preclinical phases of carcinogenesis have accessible short term laboratory endpoints that may be assessed to evaluate the impact of a variety of cancer prevention therapies.