Dyssynchronous secretory endometrial glands often show sporadically acquired progesterone nonresponsiveness.

Primary sporadic gene-inactivating events within the progesterone response cascade might explain the presence of individual dyssynchronous (outlier) glands commonly observed in a secretory background. We queried morphologically dyssynchronous glands in mid-secretory endometrium with a series of markers normally downregulated by progesterone. Seventy-nine mid-secretory endometrial biopsies were stained with hematoxylin and eosin, MIB-1, PAX2, estrogen and progesterone receptors, and PTEN. Aberrant staining of glands was independently scored for each marker. Outlier glands overlapping between stains were enumerated. A total of 63% of cases had hematoxylin and eosin stained outlier glands (average 9), which often demonstrated failed progesterone-mediated downregulation of PAX2 (43%), estrogen (40%), and/or progesterone receptors (28%). Aberrations of progesterone response was seen in 70% to 85% of cases overall, averaging 10 to 30 glands/affected case. The frequency and burden of affected glands was similar to that seen for primary inactivating events of the PAX2 and PTEN genes (35% and 41% of cases, respectively, averaging 32 and 38 glands per affected patient). Sporadic gene-inactivating events are common during endometrial regeneration, and may cause morphologic changes unmasked by the hormonal context. Some of these dyssynchronous "outlier" glands, whether evident on hematoxylin and eosin stain or not, have an interrupted progesterone response.

Emergence, involution, and progression to carcinoma of mutant clones in normal endometrial tissues.

Sporadic somatic inactivation of genes such as PTEN within histologically normal endometrium (latent precancers) is an early step in endometrial carcinogenesis. We have used clone-specific mutations of PTEN to determine the fate of latent precancers over time in women who do (high risk) and do not (low risk) develop endometrial neoplasia. PTEN immunohistochemistry was performed on 45 occurrences of endometrial neoplasia and their paired antecedent benign biopsies, along with age matched sample pairs from 167 patients who did not develop a neoplasm. When PTEN-deficient cells were present at both time points, DNA sequencing was performed to determine whether they were single or multiple independent events. Loss of PTEN protein in isolated glands was common in the initial normal biopsies of high- and low-risk groups (42% and 27%, respectively, P = 0.066). Protein-deficient glands have a tendency to disappear over time in low-risk women (P = 0.047) and, even when "persistent," are infrequently (19%, 3/16) confirmed to be the same clone. Similarly, only a small proportion (6.7%, 1/15) of latent precancers seen in high-risk women are the direct progenitors of subsequent neoplasia. There is a high rate of latent precancer turnover in both low- and high-risk patients, with rare long-term persistence of unique clones, which may or may not progress to a histologic lesion. The temporal dynamics of clonal emergence, persistence, and involution are sufficiently complex that in the individual patient, the presence of a latent precancer has an unknown contribution to long-term cancer risk.

Endometrial intraepithelial neoplasia with secretory differentiation: diagnostic features and underlying mechanisms.

Endometrial intraepithelial neoplasia (EIN) with secretory differentiation and ordinary EIN occurring in a secretory context are rare but recognized findings. We determined how often secretory differentiation in EIN was associated with evidence of circulating progestins in the background endometrium, and studied clinical characteristics and clinical outcomes of affected patients. We selected 41 patients with secretory differentiation in either the EIN itself (n=31) and/or background endometrium (n=38). Most (90%, 28/31) secretory EINs were associated with circulating progestins. Rare exceptions were observed, suggesting that secretory EIN may occur as a hormone-independent phenomenon. Circulating progestins are not sufficient, however, to induce EIN secretory differentiation, as 26% (10/38) of EIN within a secretory background were of the ordinary (non-secretory) type. EIN patients with secretory endometrium in the background are younger (averaging 45 years) than the aggregate group of all patients with EIN (53 years in previously published studies) and are often premenopausal with a cyclical source of endogenous progestins. Involution of EIN during follow-up was more frequent (81%, 17/21) for those with a secretory background at the time of initial EIN diagnosis compared with historical averages (25%, 36/142). These results suggest a potential role for endogenous progesterone, as well as therapeutic progestins, in modulating EIN outcomes.

Utility of PAX2 as a marker for diagnosis of endometrial intraepithelial neoplasia.

Diagnosis of endometrial intraepithelial neoplasia (EIN) requires learning new criteria. Two trainees rendered diagnoses based on biopsy findings, and then measured the effect of reviewing PAX2 on their interpretation. Fifty-two endometrial biopsy specimens diagnosed as having EIN were evaluated using EIN criteria. Background endometrial pattern, altered differentiation, and any features complicating diagnosis were noted. PAX2 stains were scored as confusing, helpful, or noncontributory. Fifty-two cases generated 104 passes; 82% were rediagnosed as EIN. The diagnosis was complicated because of altered differentiation (14%), EIN and background separation (13%), large lesions lacking background (11%), and secretory background (8%). PAX2 was most helpful in cases with secretory backgrounds and when EIN lacked adjacent normal tissue, and most confusing when scoring was ambiguous (14%). The diagnosis of EIN can be difficult when: (1) the lesion cannot be easily compared with background; (2) there is a confounding process; and (3) gland differentiation is altered. PAX2 can be of assistance in delimiting EIN lesions.

Digital quantification of precursor frequency in the fallopian tube and its significance.

A high frequency of precursor lesions is a risk factor for cancer in many organ systems but must be precisely quantified. Pelvic serous neoplasia is associated with an estimated increase in frequency of secretory cell outgrowths (SCOUTs) with loss of PAX2 protein (PAX2p) expression (PAX2p-null SCOUTs) in the fallopian tube. However, to confirm this, PAX2p-null SCOUTs must be precisely quantified relative to the epithelial surface. We developed a method by which fallopian tube sections were digitized using an iScan brightfield scanner (BioImagene) and uploaded in Adobe Photoshop CS3 Extended. Pixel length was translated into microns and epithelial length measured with the Magic Wand tool. SCOUTs were expressed as a function of total epithelial perimeter. Frequency, required perimeter length, topographic clustering tendency and effects of age were ascertained. SCOUT frequency per 10 cm was 0-4.60 for cases and 0-1.66 for controls, averaging 0.84 and 0.27, respectively, (P=0.007). Required perimeter length for SCOUT detection was less in serous cancer cases and topographic distribution followed a random pattern without aberrant clustering. Age was also associated with SCOUT frequency (P=0.025) and differences between cancers and controls were still significant after adjusting for age (P=0.001). We describe an efficient method for quantifying epithelial perimeter in the fallopian tube and verify its relevance to precursor frequency. This has important implications for assessing precursor frequency both in the fallopian tube and in other organs-such as prostate, pancreas and colon-where epithelial precursors are integral to carcinogenesis.

Joint loss of PAX2 and PTEN expression in endometrial precancers and cancer.

Latent endometrial carcinoma precancers are normal-appearing endometrial glands with sporadic loss of tumor suppressor gene function such as PTEN. Progression to carcinoma is inefficient and requires additional genetic damage that creates a histologic precursor lesion called endometrial intraepithelial neoplasia (EIN). In this study, we examined loss of PAX2 expression, a gene required for embryonic uterine development, during endometrial carcinogenesis. Normal proliferative, EIN, and malignant (endometrial adenocarcinoma) endometrial tissues were immunostained for PTEN and PAX2. Proliferative samples with loss of protein in at least one gland were scored as latent precancers. EIN and cancer lesions were scored by the majority pattern. Overall prevalence and topography of joint PAX2-PTEN expression loss was examined. The prevalence of PAX2 protein loss in the sequence of normal to precancer to cancer was 36%, 71%, and 77%, respectively, and for PTEN, it was 49%, 44%, and 68%, respectively. The normal endometrial prevalence of PAX2- or PTEN-deficient latent precancers was unaffected by biopsy indication, but increased significantly with age. Coincident loss of PAX2 and PTEN expression in an individual normal endometrium was seen in 21% of patients, but usually involved different glands. Coincident loss was more common in precancers (31%) and carcinoma (55%), in which case, both markers were protein null in an overlapping clonal distribution. PAX2 and PTEN protein loss occurs independently and accumulates with increasing age in latent precancers of normal premenopausal endometrium. Loss of function of both genes in an overlapping distribution characterizes the clinical emergence of a premalignant lesion which is carried forward to carcinoma.

High-grade fimbrial-ovarian carcinomas are unified by altered p53, PTEN and PAX2 expression.

High-grade endometrioid and serous carcinomas of the ovary and fallopian tube are responsible for the majority of cancer deaths and comprise a spectrum that includes early or localized (tubal intraepithelial carcinoma) and advanced (invasive or metastatic) disease. We subdivided a series of these tumors into three groups, (1) classic serous, (2) mixed serous and endometrioid and (3) endometrioid carcinomas and determined: (1) the frequencies of coexisting tubal intraepithelial carcinoma, (2) frequency of a dominant ovarian mass suggesting an ovarian origin and (3) immuno-localization of WT-1, p53, PTEN, PAX2 and p16(ink4). All tumors were analyzed for p53 mutations. Thirty six, 25 and 8% of groups 1-3 were associated with tubal intraepithelial carcinoma (P=0.09) and 34, 45 and 62% predominated in one ovary (P=0.028), respectively. Differences in frequencies of diffuse p53 immunostaining (85-93%), WT-1 (70-98%) and p16(ink4) positivity (69-75%) were not significant for all groups. Greater than 95% reduction in PAX2 and PTEN occurred in 67-75 and 5-12%, respectively; however, PAX2 and PTEN staining intensity, when present, was often heterogeneous, highlighting different tumor populations. PAX2 and PTEN expression were markedly reduced or absent in 12 of 12 and 4 of 12 tubal intraepithelial carcinomas. In summary, high-grade müllerian carcinomas share identical frequencies of altered or reduced expression of p53, PTEN and PAX2, all of which can be appreciated in tubal intraepithelial carcinomas. Because only a subset of these tumors appears to arise in the fallopian tube, attention to expression of these biomarkers in the ovary and other müllerian sites might facilitate the identification of other carcinogenic pathways. PAX2 and PTEN, in addition to p53 and p16(ink4), comprise a potentially important gene combination in high-grade pelvic carcinogenesis.

Differentiated vulvar intraepithelial neoplasia contains Tp53 mutations and is genetically linked to vulvar squamous cell carcinoma.

Differentiated vulvar intraepithelial neoplasia is a unique precursor to vulvar squamous cell carcinoma that is typically HPV-negative and frequently associated with nuclear p53 staining. These features imply a mode of pathogenesis involving somatic mutations. However, the genetic relationship of differentiated vulvar intraepithelial neoplasm and vulvar squamous cell carcinoma and the role of Tp53 mutations in this process have not been resolved. We analyzed 11 differentiated vulvar intraepithelial neoplasms and 6 associated vulvar squamous cell carcinomas. Sections were stained for p53 and p63 and DNA from multiple epithelial sites, representing normal control tissues (n=10), differentiated vulvar intraepithelial neoplasias (n=18), and vulvar squamous cell carcinomas (n=6), were obtained by laser capture microdissection, and sequenced for exons 2-11 of Tp53. Six of 10 cases contained at least one Tp53 mutation-positive differentiated vulvar intraepithelial neoplasia focus; 4 strongly p53 immuno-positive and 2 negative. Staining for p53 and p63 co-localized, targeting the immature epithelium, but surface epithelium was Tp53 mutation-positive. Four of five vulvar squamous cell carcinomas were Tp53 mutation-positive; two shared identical Tp53 mutation with adjacent differentiated vulvar intraepithelial neoplasia. Disparate foci of differentiated vulvar intraepithelial neoplasia often showed different mutations consistent with multiple neoplastic clones. Differentiated vulvar intraepithelial neoplasia is, with few exceptions, associated with Tp53 mutations and will be p53 immunopositive when missense mutations (versus some nonsense and all deletion mutations) are present. Multiple Tp53 mutations in different sites supports the presence of multiple independent genetic events, but shared Tp53 mutations in both differentiated vulvar intraepithelial neoplasia and vulvar squamous cell carcinoma support a genetic relationship between the two. The confinement of p53 staining to immature cell nuclei is consistent with maturation-dependent degradation of mutant p53 protein.