Periacinar Clefting and p63 Immunostaining in Prostatic Intraepithelial Neoplasia and Prostatic Carcinoma.

The aim of the present study was to correlate the presence and extent of retraction clefting and the expression of p63 in neoplastic glands and glands with prostatic intraepithelial neoplasia (PIN) in needle core biopsies. We analyzed needle core biopsies from 28 patients with PIN and 41 patients with adenocarcinoma. Neoplastic glands and those with PIN were analyzed on high power field (400x) and classified in three groups according to the extent of clefting. Immunohistochemical staining was performed following Microwave Streptavidin ImmunoPeroxidase (MSIP) protocol on DAKO TechMate Horizon automated immunostainer. Periacinar retraction clefting was significantly more prominent in prostatic carcinoma compared to PIN (p<0.0001) and nonneoplastic glands (p<0.0001). There was no difference between normal glands and PIN regarding clefting (p=0.8064). p63 was positive around the whole circumference in 12 out of 28 cases with PIN, and discontinuously positive in remaining 16 PIN cases suggesting initial disruption of the basal cell layer. p63 immunostaining was also positive in all nonneoplastic glands, and negative in all carcinomas. We conclude that retraction clefting was associated with cancer and lack of basal cells, but not with PIN. The relationship between clefting and p63 immunostaining in prostatic cancer should be further analyzed.

Superficial (early) endocervical adenocarcinoma in situ: a study of 12 cases and comparison to conventional AIS.

Although established histologic criteria for the diagnosis of endocervical adenocarcinoma in situ (AIS) have been published, some lesions are not readily classified or present with more subtle degrees of epithelial atypia. Lesions confined to the surface mucosa may be particularly challenging, possibly because they represent early disease. Twelve cases of superficial AIS (SAIS) confined to the surface mucosa or crypt openings culled from the in-house and consultation practices were examined histologically, immunostained for MIB-1 and p16, and analyzed (when possible) for HPV nucleic acids by DNA-DNA in situ hybridization (INFORM). The mean age was 26.7 years for SAIS versus 37.0 years for 42 consecutive cases of conventional AIS from the same practice (P < 0.001). Seven and five were biopsies and conization specimens, respectively. Five coexisted with CIN, four arose in endocervical papillae, and two arose in endocervical polyps. Nuclear hyperchromasia was conspicuous in 10 and mitoses were present in all; however, apoptosis was rare or absent in four, and six exhibited only mild nuclear atypia. Mib-1 staining exceeded 40% in 5 of 7 cases tested, and all (8 of 8) were strongly positive for p16(ink4). Five of five were positive for HPV by ISH with an "integrated" dot-like pattern. SAIS is an early variant of AIS that 1) occurs at a younger mean age, 2) exhibits variable atypia, and 3) arises adjacent to morphologically normal columnar epithelium. Diffuse p16 expression and integrated HPV pattern are identical to that seen in more extensive forms of the disease. Superficial AIS should be suspected in endocervical columnar epithelium with segmental nuclear hyperchromasia with mitotic activity, and confirmed by biomarker staining (p16 and Mib-1) if the pathologist is uncertain of the diagnosis.

Microglandular hyperplasia: a model for the de novo emergence and evolution of endocervical reserve cells.

BACKGROUND: Microglandular hyperplasia (MGH) of the cervix in human beings is associated early with gland proliferation and terminates in mature squamous metaplasia. Using antibodies to basal cell markers, we analyzed biopsies with MGH to profile the distribution and evolution of reserve cells and their relationship to these epithelial components. DESIGN: Serial sections of 24 MGHs were subdivided into (1) early MGH with microacinar proliferation, abundant subnuclear vacuoles, and a paucity of supporting stroma and (2) late MGH with more prominent supporting stroma and/or squamous metaplasia. Serial sections were stained with antibodies to p63, bcl-2, and keratin-5. RESULTS: Three patterns of p63 staining were observed corresponding to the age of the MGH: (1) scattered staining of columnar cells, (2) focal subcolumnar staining in a reserve cell distribution, and (3) linear subcolumnar arrays of p63-positive reserve cells that in some MGHs expanded into a squamous metaplasia. Early acinar proliferations showed weak and focal columnar cell staining followed by focal subcolumnar p63-positive cells. In late lesions, p63 staining was compartmentalized to the extraglandular (or subcolumnar) areas. Stainings of p63, bcl-2, and keratin-5 were concordant. Staining for keratin 14, which localizes to squamous cells, was variable. CONCLUSIONS: The immunohistochemical profile in MGH indicates that reserve cells are created in adulthood during specialized columnar proliferations. This columnar to reserve cell transition may produce a stable population of reserve cells or a transition to squamous metaplasia. Similar patterns are seen in cervical neoplasia, suggesting a link between benign and neoplastic cervical epithelial differentiation.

Vulvar acanthosis with altered differentiation: a precursor to verrucous carcinoma?

Verrucous carcinoma (VC) of the vulva is a rare variant of squamous cell carcinoma (SCC) of the vulva that afflicts older women and is characterized by a well-differentiated morphology with minimal nuclear atypia. The pathogenesis of VC is uncertain and a putative role for human papillomavirus (HPV) is doubtful. We analyzed 9 vulvar VCs from 7 patients diagnosed as VC of the vulva over the past 10 years at Brigham and Women's Hospital and Beth Israel Deaconess Medical Center. The patients ranged from 75 to 93 years in age (median, 83 years). One also involved the vagina and another coexisted with a keratinizing SCC. VC was associated with lichen sclerosus in 1 case; 7 others contained lichen simplex chronicus with verrucous architecture. In 7 cases, a distinctive noninvasive squamous epithelial proliferation, exhibiting a triad of marked acanthosis with variable verruciform architecture, loss of the granular cell layer with superficial epithelial cell pallor, and multilayered parakeratosis. We have designated these changes vulvar acanthosis with altered differentiation. In 5 of the 9 lesions, formalin-fixed, paraffin-embedded material was available for polymerase chain reaction analysis of HPV nucleic acids and all scored HPV negative. In conclusion, VC is a rare HPV-negative neoplasm that may be associated with other HPV-negative SCCs or its precursors, shares similar morphologic risk factors (lichen sclerosus and lichen simplex chronicus), and is frequently associated with an unusual intraepithelial lesion that can be distinguished from both classic and differentiated forms of vulvar intraepithelial neoplasia. The possibility that vulvar acanthosis with altered differentiation is a precursor to, or a risk factor for, vulvar carcinoma, merits further study.

Immunophenotypic and viral (human papillomavirus) correlates of vulvar seborrheic keratosis.

Human papillomavirus (HPV) infections of the genital mucosa classically present as warts (condylomata) and are traditionally defined by the presence of viral cytopathic effect (koilocytosis). In recent years, HPV has been detected in vulvar epithelial changes lacking koilocytosis, including squamous papillomas and lesions closely resembling seborrheic keratosis (SK). The purpose of this study was to determine the frequency and type of HPV associated with vulvar SK (VSK) and to compare expression of biomarkers (p16, Mib-1, and cyclin E) in these lesions. Sixty-seven biopsy specimens, including 25 VSKs, 10 nondiagnostic vulvar acanthoses, 12 fibroepithelial polyps (FEPs), and 20 nongenital cutaneous SKs (CSKs), were studied. Biopsy specimens were typed for HPV by polymerase chain reaction and immunostained with Mib-1, cyclin E, and p16(INK4) antibodies. Eighteen of 25 VSKs (72%), 0 of 10 nondiagnostic vulvar acanthuses (0%; P = 0.0001), 2 of 12 FEPs (16.7%; P = 0.004), and 3 of 20 CSKs (15%; P = 0.0002) scored HPV positive. Increased Mib-1 staining was significantly more common in VSKs than in other vulvar lesions, but not in CSKs; increased p16 and cyclin E staining was not more common. VSKs are morphologically and immunophenotypically similar to CSKs but distinct by their association with HPV. Unlike the cervix, p16 and cyclin E will not consistently distinguish VSKs from HPV-negative lesions due to underexpression in low-risk HPV infections (p16) and less-restricted expression in vulvar lesions (cyclin E). Whether CSKs are associated with other forms of HPV infection remains to be determined.

p16INK4A expression as biomarker for HPV 16-related vulvar neoplasias.

Up-regulation of p16INK4A is associated with high-risk human papillomavirus (HPV) in preinvasive and invasive cervical neoplasia. However, its expression in vulvar carcinomas, which have a diverse pathogenesis, has not been extensively studied. One hundred seventy-seven vulvar intraepithelial neoplasms (VIN), squamous cell carcinomas (SCC), and benign squamous epithelia were analyzed for p16 expression. RNA/RNA in situ hybridization was used to detect HPV 16 E6/E7 transcripts in 112. Ninety-five percent of VIN 3 and basaloid or warty SCCs (76/80) and 4% of keratinizing SCC (2/48) were moderately to strongly immunopositive for p16, which localized to nucleus and cytoplasm; 52/58 analyzed (90%) contained HPV 16 transcripts. The positive predictive value (PPV) of moderate to strong diffuse p16 immunostaining and HPV positivity for the diagnosis of VIN 3 and of basaloid or warty SCC was 97% and 95%, respectively. Conversely, 94% of keratinizing SCC contained heterogeneous staining, and when present, it was strictly cytoplasmic and frequently localized to the cells at the epithelial-stromal interface. Benign squamous epithelia were p16 negative, with the exception of lichen sclerosus, which contained focal and heterogeneously p16 positive in 42%. As in the cervix, intense diffuse p16 expression supports an HPV-related neoplastic process in vulvar neoplasia, irrespective of the level of differentiation. Up-regulation of p16 at the epithelial-stromal interface in HPV negative keratinizing SCCs is consistent with an HPV-independent response to alterations associated with invasion. These disparate patterns of p16 expression underscore 2 different mechanisms for p16 expression in HPV-related and HPV-unrelated vulvar carcinomas.

Human papillomaviruses, expression of p16, and early endocervical glandular neoplasia.

Adenocarcinoma in situ (ACIS) is the precursor of cervical adenocarcinoma (ACs), and its distinction from benign but morphologically atypical glandular epithelium may be difficult. The cyclin-dependent kinase inhibitor p16(ink4) is expressed in cervical squamous cell carcinomas, their precursors, and cervical ACs, and there is a strong relationship between p16 expression and the presence of human papillomavirus (HPV)-encoded E6/E7 transcription. This study analyzed 95 cases of benign and premalignant cervical glandular ACIS lesions for p16 antigen and the proliferative marker Ki-67; HPV E6/E7 transcripts were detected by RNA/RNA in situ hybridization. HPV 16 or 18 E6/E7 transcription and strong, diffuse p16 positivity were detected only in ACIS lesions. A high and moderate Ki-67 index was observed in 76% and 22% of ACIS, respectively. Thirty-three of 36 microglandular change, tubal, atypical tubal, and endometrial-type epithelia scored negative or weakly positive for p16. Distribution of staining in 3 strongly positive cases was heterogeneous. The diffuse distribution of p16 immunostaining in HPV16/18-positive glandular neoplasms supports a strong association with HPV infection and indicates that this biomarker may discriminate ACIS from its benign mimics. However, this distinction requires attention to staining distribution because p16 is focally expressed in tubal-endometrial epithelia and diffusely expressed in endometrium, indicating that in some cases the use of other biomarkers, such as Ki-67, may be necessary. Because endometrial glandular epithelia may also express p16, the diagnostic application of p16 immunohistochemistry to cytological samples is uncertain.

Type-specific HPV testing as a predictor of high-grade squamous intraepithelial lesion outcome after cytologic abnormalities.

OBJECTIVE: Human papillomavirus (HPV) risk assignment influences management after a cytologic diagnosis of atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL). This study addressed whether type-specific HPV testing predicts risk of biopsy outcome of high-grade cervical intraepithelial neoplasia (CIN 2,3). MATERIALS AND METHODS: A total of 162 ASCUS or LSIL diagnoses with colposcopic follow-up were evaluated and placed in 3 groups: Analysis 1: (high-risk HPVs including types 53 and 66; Analysis 2 (high-risk HPVs excluding types 53 and 66); and Analysis 3 (high-risk HPVs including type 53 and excluding type 66). RESULTS: CIN 2,3 biopsy results followed low-risk HPVs in 0%, 3%, and 0% of scenarios 1, 2, and 3, respectively; 21%, 40%, and 27% of smears were classified as low risk, respectively. Of HPV 53 infections, 13.6% had CIN 2,3 biopsy outcomes. CONCLUSIONS: Type-specific HPV testing accurately classifies a group of HPV-positive LSIL/ASCUS cases at low risk for CIN 2,3 at the first follow-up visit. Classifying HPV 53 as low-risk increases slightly the proportion of HSIL outcomes in the low-risk group, but may not increase cancer risk. HPV 53 merits designation as a high-risk HPV based only [corrected] on the proportion of CIN 2,3 in follow-up biopsy.

The role of CD10 staining in distinguishing invasive endometrial adenocarcinoma from adenocarcinoma involving adenomyosis.

Adenomyosis may be involved by endometrial adenocarcinoma, but in contrast to true myometrial invasion, the depth of an adenomyotic focus involved by carcinoma does not alter pathologic tumor staging. Therefore, distinction from carcinoma invading myometrium is clinically relevant. We hypothesized that CD10, a marker of non-neoplastic and neoplastic endometrial stroma, would highlight the stromal component of adenomyotic foci and be useful in this distinction. Thirty-nine cases of endometrial adenocarcinoma were analyzed and divided into three groups: I, invasive endometrial adenocarcinoma (n = 14); II, endometrial adenocarcinoma involving adenomyosis but without myometrial invasion (n = 18); and III, adenomyosis involved by endometrial adenocarcinoma with concomitant invasive component (n = 7). All cases of adenomyosis involved by endometrial adenocarcinoma demonstrated CD10 expression in the stromal cells of adenomyotic foci. Eleven of 21 cases (52%) of invasive adenocarcinoma also showed CD10 expression, at least focally, in cells immediately surrounding the infiltrating glands. Of these, two cases (from Group III) also had associated adenomyotic involvement by carcinoma. The remaining cases of invasive carcinoma were negative for CD10. Therefore, presence of CD10 staining immediately surrounding neoplastic glands does not equate with involvement of adenomyosis by endometrial adenocarcinoma. In contrast, absence of CD10 expression excludes involvement of adenomyosis by adenocarcinoma.

p63 Coordinates anogenital modeling and epithelial cell differentiation in the developing female urogenital tract.

p63 is a p53 homologue required for cutaneous development that is expressed in immature squamous epithelium and reserve cells of the cervix. Humans with p63 mutations exhibit defects in limb, accessory organ (skin appendage, breast, prostate), and genitourinary development. Because p63 expression patterns imply a strong role of the gene in the female genital tract development, newborn female p63-/-, +/-, and +/+ mice were examined in situ, dissected, and compared. Nuclear p63 protein was localized to the skin, vagina, bladder, urethra, and basal columnar cells of the caudal uterus in p63+/+ and +/- animals. p63-/- mice exhibited abnormal genital morphogenesis with hypoplastic genitalia, a single cloacal opening, and persistence of columnar epithelium at lower genital tract sites that normally undergo squamous and urothelial differentiation. The defects observed support p63-dependent pathways of genital tract development that permit externally, ectodermal basal cell replenishment integral to reciprocal epithelial stromal signaling, urorectal septation, and modeling of the external genitalia; and internally, the emergence of basal epithelial cell populations capable of divergent epithelial cell differentiation in the vagina, cervix, and urinary tract. Defects in the first pathway explain imperforate anus, vaginal septum, genital hypoplasia, and micropenis reported in humans with p63 mutations. The second is necessary for the generation of multipotential reserve cells in the cervix and may be operative in other epithelial stromal interactions integral to the emergence of uterine basal cells later in life.