p16(INK4a) as a complementary marker of high-grade intraepithelial lesions of the uterine cervix. I: Experience with squamous lesions in 189 consecutive cervical biopsies.

AIM: To test the usefulness of p16(INK4a) immunostaining for improving the diagnostic accuracy of cervical punch biopsies referred to a routine laboratory setting during the investigation of women with abnormal Papanicolaou smears. METHODS: A total of 188 consecutive and unselected colposcopically directed cervical biopsies and a single contemporaneous cervical polyp were accessioned prospectively over a 3-month period, step-serially sectioned and examined by H&E and immunostained for p16(INK4a). The clinical context, results of concurrent Papanicolaou smears/ThinPrep slides and Digene hybrid capture tests for high-risk human papillomavirus (HPV) subtypes, as well as follow-up cervical smears/ThinPrep, biopsies and loop excisions of transformation zones or cone biopsies were all correlated with the morphological and immunohistochemical findings. RESULTS: Seventy-seven biopsies (40.7%) displayed a high-grade squamous intraepithelial lesion (HGSIL; cervical intraepithelial neoplasia [CIN] 2-3), 27 (14.3%) showed a low grade squamous intraepithelial lesion (HPV +/- CIN1) and 85 (45%) showed a range of non-dysplastic (inflammatory or reactive) changes. Diffuse strong parabasal immunostaining for p16(INK4a), suggestive of integrated high-risk HPV DNA into the host genome, was observed in 81 biopsies (42.9%, including the cervical polyp) and correlated (>90%) with HGSIL in the H&E sections. Only one case revealed irreconcilable discordance between the histological features and this strong parabasal immunostaining pattern. Focal and weaker midzonal or superficial p16(INK4a) immunostaining, suggestive of episomal HPV infection, was noted in 19 biopsies (10%) and these biopsies exhibited a range of histological changes but predominantly low grade squamous intraepithelial lesion (LGSIL). No staining of the squamous epithelium was seen in 89 biopsies (47.1%). Again, only one case revealed irreconcilable discordance between the histological features and this negative immunostaining pattern. On review of all cases where discordant results were noted between the H&E appearances and expected p16(INK4a) immunostaining, we found 26 cases (13.7%) in which this discordance prompted justifiable modification of the original diagnosis. CONCLUSIONS: Thus, within a routine diagnostic laboratory, p16(INK4a) immunostaining appears to be a very useful adjunctive test in the examination of colposcopically directed cervical biopsies, in the diagnostic cascade of women investigated for abnormal Papanicolaou smears. It is possible, as further data accumulate concerning the importance of integration of high-risk HPV DNA into the host cell genome and the reliability with which this can be identified by p16(INK4a) immunostaining, that this will become the diagnostic 'lesion of interest', replacing the subjective histological grading of cervical dysplasia, in the management of such patients; i.e., the discriminatory watershed between continued surveillance and active intervention.

Human Papilloma Virus Identification in Breast Cancer Patients with Previous Cervical Neoplasia.

PURPOSE: Women with human papilloma virus (HPV)-associated cervical neoplasia have a higher risk of developing breast cancer than the general female population. The purpose of this study was to (i) identify high-risk HPVs in cervical neoplasia and subsequent HPV positive breast cancers which developed in the same patients and (ii) determine if these HPVs were biologically active. METHODS: A range of polymerase chain reaction and immunohistochemical techniques were used to conduct a retrospective cohort study of cervical precancers and subsequent breast cancers in the same patients. RESULTS: The same high-risk HPV types were identified in both the cervical and breast specimens in 13 (46%) of 28 patients. HPV type 18 was the most prevalent. HPVs appeared to be biologically active as demonstrated by the expression of HPV E7 proteins and the presence of HPV-associated koilocytes. The average age of these patients diagnosed with breast cancer following prior cervical precancer was 51 years, as compared to 60 years for all women with breast cancer (p for difference = 0.001). CONCLUSION: These findings indicate that high-risk HPVs can be associated with cervical neoplasia and subsequent young age breast cancer. However, these associations are unusual and are a very small proportion of breast cancers. These outcomes confirm and extend the observations of two similar previous studies and offer one explanation for the increased prevalence of serious invasive breast cancer among young women.

Asynchronous glands in the endometrium of women with recurrent reproductive failure.

Of 969 non-consecutive endometrial biopsies performed for investigation of recurrent reproductive failure, 20 cases (2.1%) showed the striking presence of retarded or asynchronous endometrial glands in otherwise unremarkable mid or late secretory endometrium. These glands were characterised by tall columnar cells with crowded nuclei showing increased reactivity for the proliferative marker MIB-1, occasional mitoses, greatly reduced or absent secretion, and persistent expression of oestrogen receptors and usually progesterone receptors and their isoforms typical of late proliferative phase endometrium. The nearby endometrial stromal cells exhibited no discernibly reduced reactivity for calretinin. These changes were seen in single glands (even portions of glands), or clusters of glands, adjacent to normal late secretory type endometrial glands and set in pseudodecidualised stroma characteristic of late luteal phase. Some examples also displayed adjacent glands with intermediate features and it is speculated that firstly, this is a relatively common phenomenon in women with recurrent miscarriage or implantation failure and with an unknown potential to affect implantation. Secondly, it is an intrinsic defect of the endometrium and can occur in sequential endometrial biopsies in the same patient. Thirdly, it differs from previously described patterns of so-called luteal phase defect or deficient secretory phase in that it occurs in the demonstrated presence of adequate progesterone effect on the endometrium and is associated with persistence rather than exaggerated down-regulation of receptors. Nevertheless, supplementary progesterone therapy (vaginal pessaries) for the first trimester appeared to have a beneficial therapeutic effect on reproductive outcome in these patients.

The distribution of immune cells and macrophages in the endometrium of women with recurrent reproductive failure I: Techniques.

Recurrent miscarriage affects approximately 1% of the population and in half of these cases no cause is found. Abnormally functioning immunocompetent cells, including natural killer (NK) cells, in the endometrium, are thought to be responsible for many such cases and treatment trials including oral prednisolone and intravenous immunoglobulins are now underway. Despite these encouraging developments, there is neither adequate standardization of counting uterine NK cells nor consensus as to what constitutes an abnormal level. To address this issue, immunohistochemistry was used to examine the presence and distribution of selected immune cells and macrophages in the endometrium from 222 women who had a routine endometrial biopsy for investigation of recurrent miscarriage or IVF failure, at various stages of the menstrual cycle, and accessioned prospectively over a 7-month period. Biopsies were examined by H+E and immunostained for CD8(+) T-cells, CD163(+) macrophages, CD56(+) NK cells, and CD57(+) cells. Cell numbers (expressed as immunopositive cells per mm(2)) were determined in the stroma of the functional layer of endometrium and the relative concentrations of some cell types (CD163(+) macrophages, CD56(+) NK cells) were expressed as a percentage of all stromal cells. Routine H+E sections revealed 12 patients with focal "endometritis" without plasma cells. CD8(+) T-cells showed focal perivascular aggregates in most instances, and non-random but scattered cells in all cases, with a twofold increase in the luteal phase. CD163(+) cells were distributed evenly throughout the superficial endometrial stroma and also present as single or clustered macrophages within the lumens of superficial glands, mostly in the luteal phase. CD56(+) NK cells showed "diffuse" but variable distribution throughout the functional layer and perivascular aggregates of various sizes in two thirds of cases. Raw cell counts were low and relatively stable in the proliferative phase, but increased somewhat during the first half of the secretory phase, while in the second half of secretory phase they increased six to tenfold. Percentage counts rose from approximately 5% of stromal cells in the early part of the secretory phase of the cycle to over 35% in premenstrual endometrium. CD57(+) cells were present in very low numbers in most cases. The study illustrates the complexity and variability of immune cell infiltration of endometrium. We stress the need for strict counting protocols and attention to histological criteria if any immunological perturbations potentially responsible for recurrent reproductive failure are to be identified. Reference ranges for individual cell types are only valid for individual "days" of a normalized menstrual cycle.