Grading immunohistochemical markers p16INK4a and HPV E4 identifies productive and transforming lesions caused by low- and high-risk HPV within high-grade anal squamous intraepithelial lesions.

OBJECTIVES: Because current guidelines recognise high-grade anal squamous intraepithelial lesions (HSILs) and low-grade SILs (LSILs), and recommend treatment of all HSILs although not all progress to cancer, this study aims to distinguish transforming and productive HSILs by grading immunohistochemical (IHC) biomarkers p16INK 4a (p16) and E4 in low-risk human papillomavirus (lrHPV) and high-risk (hr)HPV-associated SILs as a potential basis for more selective treatment. METHODS: Immunostaining for p16 and HPV E4 was performed and graded in 183 biopsies from 108 HIV-positive men who have sex with men. The causative HPV genotype of the worst lesion was identified using the HPV SPF10-PCR-DEIA-LiPA25 version 1 system, with laser capture microdissection for multiple infections. The worst lesions were scored for p16 (0-4) to identify activity of the hrHPV E7 gene, and panHPV E4 (0-2) to mark HPV production and life cycle completion. RESULTS: There were 37 normal biopsies, 60 LSILs and 86 HSILs, with 85% of LSILs caused by lrHPV and 93% of HSILs by hrHPV. No normal biopsy showed E4, but 43% of LSILs and 37% of HSILs were E4 positive. No differences in E4 positivity rates were found between lrHPV and hrHPV lesions. Most of the lesions caused by lrHPV (90%) showed very extensive patchy p16 staining; p16 grade in HSILs was variable, with frequency of productive HPV infection dropping with increasing p16 grade. CONCLUSIONS: Combined p16/E4 IHC identifies productive and nonproductive HSILs associated with hrHPV within the group of HSILs defined by the Lower Anogenital Squamous Terminology recommendations. This opens the possibility of investigating selective treatment of advanced transforming HSILs caused by hrHPV, and a 'wait and see' policy for productive HSILs. What's already known about this topic? For preventing anal cancer in high-risk populations, all patients with high-grade squamous intraepithelial lesions (HSILs) are treated, even though this group of lesions is heterogeneous, the histology is variable and regression is frequent. What does this study add? By adding human papillomavirus (HPV) E4 immunohistochemistry to p16 INK4a (p16), and grading expression of both markers, different biomarker expression patterns that reflect the heterogeneity of HSILs can be identified. Moreover, p16/E4 staining can separate high-risk HPV-associated HSILs into productive and more advanced transforming lesions, providing a potential basis for selective treatment.

Low- and high-risk human papillomavirus genotype infections in intra-anal warts in HIV-positive men who have sex with men.

BACKGROUND: Anogenital warts are often presumed to represent nondysplastic or low-grade anal intraepithelial neoplasia (LGAIN). We previously demonstrated that up to 20% of intra-anal warts in HIV-positive men who have sex with men (MSM) contain regions of high-grade AIN (HGAIN). OBJECTIVES: To determine the causative human papillomavirus (HPV) types of low- and high- grade dysplastic areas in warts from HIV-positive MSM. METHODS: A total of 42 intra-anal warts from 41 HIV-positive MSM were graded as nondysplastic, LGAIN or HGAIN. Whole-tissue sections (WTS) were analysed with the SPF10 polymerase chain reaction/LiPA25 HPV genotyping system. If the WTS contained multiple HPV types, dysplastic regions were isolated by laser capture microdissection (LCM) for HPV genotyping. RESULTS: Overall, 38 of 42 (91%) WTS tested positive for HPV DNA. Of these, 23 (61%) contained a single HPV type and 15 (39%) contained multiple HPV types. All LCM-selected regions contained no more than one HPV type. Ten of 42 (24%) WTS contained HGAIN disease, of which six (60%) were associated with a high-risk HPV (hrHPV) genotype. Twenty-three of 42 WTS contained LGAIN disease, of which two (9%) were associated with hrHPV. AIN lesions containing hrHPV types were identified using p16 staining. CONCLUSIONS: LGAIN lesions can be caused by high-risk HPV genotypes and vice versa. We therefore recommend routine follow-up and treatment of all dysplastic intra-anal warts for HIV-positive MSM.

Clinical outcomes of patients with adenocarcinoma in situ of the cervix treated by conization.

OBJECTIVE: To describe the clinical outcomes of histologically confirmed adenocarcinoma in situ (AIS) of the cervix treated with cervical conization. MATERIALS AND METHODS: A retrospective chart review of patients with histologically confirmed AIS from July 1998 to February 2011 included 52 patients. The rates of residual disease in subsequent excisions, the clinical recurrence rate, the average disease-free interval, and risk of progression to adenocarcinoma were described. The clinical outcomes of patients treated with cold knife cone (CKC) and loop electrosurgical excisional procedure (LEEP) were compared. RESULTS: Fifteen LEEPs and 37 CKC procedures were performed as initial treatment and 26 patients (50%) had positive margins. There was no significant difference in rate of positive margins between LEEP and CKC (40% vs. 54%, respectively. p = 0.55). LEEPs and CKCs resulted in similar volumes of cervical tissue resected (4.98 cm3 vs. 5.04 cm3, p = 0.40). Of patients with positive margins, ten underwent immediate hysterectomy, six underwent a second cone biopsy, seven were managed expectantly, and four were lost to follow up. Residual AIS was found in 47% (eight of 17) of repeat cone biopsy and hysterectomy specimens performed for positive cone margins. Of the 26 patients with negative cone margins, no residual or recurrent disease was found after an average follow-up of 32 months. CONCLUSIONS: A positive surgical margin was associated with residual disease in 47% of patients with AIS treated with conization. No patient with negative cone margins had recurrent or progressive disease. Cervical conization with negative margins appears to be a safe treatment option for patients with AIS but requires further investigation. CKC and LEEP were equally efficacious treatments in our study population.

Metabolism of dihydrotestosterone in human liver: importance of 3alpha- and 3beta-hydroxysteroid dehydrogenase.

This study compared the enzyme activity of 3alpha-hydroxysteroid dehydrogenase (3alphaHSD) and 3beta-hydroxysteroid dehydrogenase (3betaHSD) in the human liver. 3AlphaHSD was found in both microsomal and cytosolic liver fractions. Contrary to that in rat liver, microsomal 3alphaHSD activity was 12-fold higher than cytosolic 3alphaHSD activity, and 3alphaHSD was not inhibited by indomethacin (10 micromol/L). The rate of 5alpha-dihydrotestosterone (DHT) reduction to 5alpha-androstane-3alpha,17beta-diol (3alphaDIOL) by 3alphaHSD was 2 times higher than the rate of 3alphaDIOL oxidation to DHT. 3BetaHSD was present primarily in the microsomal fraction of the human liver, and the rate of DHT reduction to 5alpha-androstane-3beta,17beta-diol (3betaDIOL) by 3betaHSD was 3 times higher than the rate of 3betaHSD oxidation to DHT. When 3alphaHSD and 3betaHSD activities were compared, the rate of DHT reduction by 3betaHSD was 3-fold lower than the rate of DHT reduction by 3alphaHSD. No sex or age differences were found in either 3alphaHSD or 3betaHSD activity. As the activity of DHT-metabolizing enzymes is not sex dependent, the sex differences in plasma levels of 3alphaDIOL glucuronide probably reflect differences in DHT production rather than in DHT metabolism. Comparison of the activities of 3alphaHSD, 3betaHSD, and androgen UDP-glucuronyl transferase suggests that the major pathway of DHT metabolism in human liver involves 3alphaHSD reduction in the liver, followed by subsequent glucuronidation and clearance via the kidney.

Endometrial intraepithelial carcinoma with associated peritoneal carcinomatosis.

Endometrial intraepithelial carcinoma (EIC) is a recently described entity, defined as a noninvasive, cytologically malignant lesion that replaces the endometrial surface epithelium. EIC frequently coexists with uterine serous carcinoma (USC) and is hypothesized to be its precursor lesion. However, the clinical significance and biologic potential of finding EIC without USC is not known. We report three postmenopausal women with EIC alone who were found to have multiple, synchronous foci of extrauterine serous carcinoma at presentation. Because the clinical findings in these patients simulated primary peritoneal serous carcinoma (PSC), we compared the clinicopathologic features of these cases with a group of nine bona fide PSCs for which exhaustively sectioned endometria, fallopian tubes, and ovaries were available for review. The average age of the EIC patients was 73 years. Two patients presented with abdominal distention and one with vaginal bleeding. Hysterectomy in each case showed endometrial polyps with EIC, but without invasive USC, in a background of atrophic endometrium. Bilateral salpingo-oophorectomy and staging showed serous carcinoma involving the ovarian hilum, the surfaces of the fallopian tubes and ovaries, in addition to peritoneal carcinomatosis. p53 overexpression was observed in both EIC and the extrauterine deposits of serous carcinoma in each case. The average age of the PSC patients was 66 years. All nine patients presented with abdominal distention. EIC was not identified in any of the hysterectomy specimens. Bilateral salpingo-oophorectomies, omentectomies, and peritoneal biopsies showed peritoneal carcinomatosis, including bulky peritoneal tumor deposits, but only minimal ovarian surface involvement. p53 overexpression was observed in seven cases. These findings indicate that EIC without coincident USC can be associated with invasive, extrauterine serous carcinomatosis. We did not, however, find any significant differences between the clinicopathologic features of primary extrauterine serous carcinomas (PSCs) and those associated with EIC. We conclude that the finding of EIC in an endometrial curettage specimen should prompt a thorough search for an invasive uterine and/or extrauterine serous carcinoma. Conversely, an endometrial origin should be excluded in patients with peritoneal carcinomatosis.

MIB-1 immunostaining is a beneficial adjunct test for accurate diagnosis of vulvar condyloma acuminatum.

The histopathologic diagnosis of vulvar condyloma acuminatum is often based on architectural features that are not specific for human papillomavirus (HPV) infection. Because HPV-associated lesions show increased cellular proliferation, the authors evaluated the usefulness of MIB-1 immunostaining as an aid in the differential diagnosis of cases equivocal for condyloma. The MIB-1 immunostaining pattern was correlated with HPV DNA detection in condyloma acuminatum (CON-A; n = 15), "consistent with condyloma" (c/w CON-A; n = 26), fibroepithelial polyp (FEP; n = 14), and squamous papilloma (n = 10). HPV was detected in 100% of the CON-A cases, and all cases demonstrated MIB-1-positive nuclei in the upper two thirds of the epithelial thickness. With this definition of MIB-1 positivity, there was complete concordance between MIB-1 positivity and HPV detection for all cases (kappa = 0.88). Of the cases c/w CON-A, 17 of 26 (65%) were positive for both MIB-1 and HPV, and could be reclassified as CON-A, whereas 35% were identified as an overdiagnosis based on negative results. In addition, two cases of FEP were MIB-1 and HPV positive, and thus were identified as an underdiagnosis. These results suggest significant overdiagnosis of cases equivocal for condyloma, and indicate that MIB-1 immunostaining is a beneficial adjunctive test when the morphologic features are suggestive but not diagnostic for CON-A.

Androgen UDP-glucuronyl transferase activity is found primarily in the liver in the human.

In this study, androgen UDP-glucuronyl transferase (UDPGT) activity was determined in microsomes of human liver, skin, and prostate. Androgen UDPGT activity was highest in the liver microsomes using 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha DIOL), androsterone (AN) and dihydrotestosterone (DHT) as substrates. The Km and Vmax values and enzyme velocity at the physiological concentration of the substrates in the liver microsomes were similar for AN and 3 alpha DIOL. The values for AN were as follows: Km = 16.9 microM, Vmax = 3.77 nmol/min/mg protein, and UDPGT velocity = 0.62 pmol/min/mg protein. The values for 3 alpha DIOL were as follows: Km = 16.0 microM, Vmax = 5.61 nmol/min/mg protein, and UDPGT velocity = 0.42 pmol/min/mg protein. Androgen UDPGT activity was lower and showed less affinity for DHT (Km = 23.5 microM, Vmax = 0.84 nmol/min/mg protein, UDPGT velocity = 0.05 pmol/min/mg protein). No sex or age differences in 3 alpha DIOL-UDPGT activity were found in liver microsomes. The kinetic parameters and levels of androgen UDPGT activity in the skin and prostate samples were not determined because the levels of UDPGT activity were below the detection limit for our assay (0.003 pmol/min/mg protein). These results have important clinical implications. First, androgen UDPGT activity in the skin and prostate is much lower than the activity in the liver, suggesting that these tissues are not important sites for conjugation of androgens. Second, androgen UDPGT activity in the liver was not altered by sex or age in the 10 samples measured from men and women.

Androgen UDP-glucuronyl transferase activity is found primarily in liver in the rat.

UDP-glucuronyl transferase (UDPGT) activity was determined for androgens in tissue minces and microsomal fractions from the liver and extrahepatic tissues (kidney, skin, prostate, and preputial glands) of the male rat. Liver microsomes showed the highest UDPGT activity with each of the androgens tested (Vmax = 7, 3, and 10 nmol/minute/mg protein for testosterone, androsterone, and androstanediol, respectively). UDPGT activity (Vmax) for androstanediol in the liver was 10(2)-fold higher than in the kidney and 10(3)-fold higher than in the skin and prostate. UDPGT activity for androgens was not detected in microsomes from preputial glands. Furthermore, no body site distribution was found for androgen UDPGT activity in skin microsomes. The Michaelis-Menten constant (Km) for UDPGT in liver microsomes was 20.4, 12.2, and 2.2 microM, respectively, for testosterone, androstanediol, and androsterone. Kidney microsomes showed a Km of 19.4 and 26.9 microM, respectively, for androstanediol and androsterone. The Km for testosterone was very high in the kidney (138 microM), suggesting that it was a poor substrate. In microsomes from the skin and prostate, the Km was very high (range 43-162 microM) for all three androgen substrates, suggesting that these androgens were not the preferred substrates for UDPGT in these tissues. These results indicate that the liver was the main site of androgen UDPGT activity and the skin and prostate formed little, if any, androgen glucuronides. These results suggest that androstanediol glucuronide was formed primarily in the liver and may not be a reliable marker of peripheral androgen metabolism.

3 Alpha-hydroxysteroid dehydrogenase activity in rat liver and skin.

3 alpha-Hydroxysteroid dehydrogenase (3 alpha HSD) is one of the main enzymes involved in the metabolism of the active androgen, dihydrotestosterone (DHT). 3 alpha HSD catalyzes the reversible reduction of DHT to 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha DIOL). The equilibrium of 3 alpha HSD reductive and oxidative activity is an important factor in the regulation of intracellular levels of DHT. In this study, we determined the kinetic characteristics of 3 alpha HSD in the subcellular fractions of female rat liver and abdominal skin. The enzyme expressed its activity in the cytosol and microsomal fractions of both of these tissues. It showed higher activity with the phosphorylated cofactors, NADPH and NADP, and was inhibited by indomethacin. The Vmax values of 3 alpha HSD in the cytosol were 10-fold higher than the Vmax values in the microsomes in both the liver and skin. In both tissues, the Km values with DHT as the substrate (reductive) were lower than the Km with 3 alpha DIOL as the substrate (oxidative). Although the Vmax values of the oxidative reaction were higher than the Vmax values of the reductive reaction in both liver and skin, the low Km values and the higher Vmax/Km ratio for DHT indicated that the reduction of DHT to 3 alpha DIOL was the favored reaction. The enzyme kinetics of 3 alpha HSD suggest that neither tissue accumulates DHT, but promptly converts it to 3 alpha DIOL.(ABSTRACT TRUNCATED AT 250 WORDS)

Triiodothyronine and estradiol increase the serum level of androstanediol glucuronide but do not influence androgen UDP-glucuronyl transferase activity in the female rat.

In this study, adult female rats were treated with either estradiol, testosterone or triiodothyronine (T3). The mean +/- SD for serum levels of androstane-3 alpha,17 beta-diol glucuronide (ADIOL-G) in rats injected with estradiol (31.1 +/- 6.1 nmol/l) and T3 (28.7 +/- 6.1 nmol/l) increased three-fold in comparison to the controls (9.2 +/- 3.6 nmol/l; p < 0.01). There was no significant increase in ADIOL-G levels in rats injected with testosterone (12.0 +/- 3.4 nmol/l) even though serum testosterone levels increased more than 17-fold (1.2 +/- 0.3 nmol/l; p < 0.01). Testosterone levels remained below the detection level of the assay (0.07 nmol/l) in the control and the estradiol and T3 treatment groups. Androgen UDPGT activity in liver microsomes was not altered by treatment with testosterone, estradiol or T3. However, the UDPGT activity for p-nitrophenol increased two-fold in estradiol and T3-treated females (p < 0.01), but was not significantly altered by testosterone treatment. These results suggest that the activity of androgen UDPGT in liver is not the limiting factor in the increased serum levels of ADIOL-G observed on treatment with estradiol and T3.

Diagnostic and prognostic significance of the mitotic index in endometrial adenocarcinoma.

The aim of current study was to evaluate the diagnostic as well as the prognostic significance of the mitotic index (MI) in endometrial adenocarcinoma. We compared the MI in normal endometrium, endometrial hyperplasia, and endometrial adenocarcinoma. The mean MI in normal proliferative endometrium (4.35 +/- 3.4) was not significantly different from those in glandular hyperplasia (4.19 +/- 6.0) and well-differentiated adenocarcinoma (4.01 +/- 4.2). A significantly higher MI (10.7 +/- 8.2) was found only in poorly differentiated adenocarcinoma (P less than 0.05). Results of our work indicate that the MI cannot be used as a discriminating factor in the differential diagnosis of borderline cases of endometrial hyperplasia and endometrial adenocarcinoma. We examined the usefulness of the MI, grade of differentiation, and depth of invasion as the prognostic factors in endometrial adenocarcinoma. The significantly higher 5-year mortality rate was associated with an MI greater than 5, grade III of differentiation, and neoplastic invasion penetrating to the outer third of the myometrium. All of the patients with MI greater than 5 had tumors with the highest grade of differentiation and/or invasion involving the mid and outer third of myometrium. This suggests that the higher mortality of patients with an MI greater than 5 reflects the presence of anaplastic and/or highly invasive tumor.

Endometrial adenocarcinoma--lack of correlation between treatment delay and tumor stage.

OBJECTIVE: The objective was to examine why a proportion of patients with endometrioid adenocarcinoma (EMC) presents with advanced stage disease and whether the duration of bleeding prior to hysterectomy (treatment delay) is predictive of stage. STUDY DESIGN: A retrospective clinicopathologic study of 182 patients treated for EMC at Columbia-Presbyterian Medical Center in New York City between 1988 and 1996 was performed. RESULTS: Ninety percent of patients with EMC presented with abnormal uterine bleeding. The median time interval from onset of bleeding to biopsy diagnosis was 2.5 months (range, 2 weeks to 60 months) and 3 weeks from biopsy to hysterectomy. No correlation between the duration of symptoms and tumor stage was found (P = 0.87). There was, however, significant correlation between poor tumor differentiation and the advanced stage of the disease (P = 0.0001). CONCLUSIONS: Stage at presentation in patients with EMC correlates with tumor differentiation but not with time interval from bleeding to definitive therapy.

Mutational analysis of the CTNNB1 and APC genes in uterine endometrioid carcinoma.

Despite recent studies, the molecular genetic events responsible for the development of uterine endometrioid carcinoma (UEC) remain incompletely characterized. Mutations in the beta-catenin (CTNNB1) gene have been recently reported in a small percentage of UECs and in the endometrioid variant of ovarian carcinoma suggesting that the Wnt signal transduction pathway is involved in the development of female genital tract tumors with endometrioid morphology. The Wnt pathway is a critical pathway in the development of colorectal cancer (CRC) with mutations occurring in the beta-catenin (CTNNB1) or adenomatous polyposis coli (APC) genes in 10 to 15% and 85% of cases, respectively. Because UEC and CRC share other molecular genetic alterations and histologic features and previous studies of UEC have not reported an analysis of the APC gene, we chose to further elucidate the role of the Wnt pathway in UEC. To this end, we analyzed 32 cases of UEC for mutations of the CTNNB1 and APC genes. Mutations of CTNNB1 were present in six of 32 (18%) cases: four grade 1 carcinomas, one grade 2, and one grade 3 carcinoma. Five missense mutations were identified, three involving Ser/Thr phosphorylation sites and two adjacent to a Ser phosphorylation site. One case contained a deletion encompassing codons 34 to 37, which includes a Ser phosphorylation site. No mutations resulting in truncation of the APC protein were found. Our results support a role for the Wnt signaling pathway via mutation of CTNNB1, but not APC, in the development of a subset of UECs.

Detection and typing of human papillomavirus DNA in penile carcinoma: evidence for multiple independent pathways of penile carcinogenesis.

To clarify the role of human papillomavirus (HPV) in penile cancer we evaluated the prevalence of HPV DNA in different histological subtypes of penile carcinoma, dysplasia, and condyloma using a novel, sensitive SPF10 HPV polymerase chain reaction assay and a novel genotyping line probe assay, allowing simultaneous identification of 25 different HPV types. Formalin-fixed, paraffin-embedded tissue samples were collected from the United States and Paraguay. HPV DNA was detected in 42% cases of penile carcinoma, 90% cases of dysplasia, and 100% cases of condyloma. There were significant differences in HPV prevalence in different histological cancer subtypes. Although keratinizing squamous cell carcinoma and verrucous carcinoma were positive for HPV DNA in only 34.9 and 33.3% of cases, respectively, HPV DNA was detected in 80% of basaloid and 100% of warty tumor subtypes. There was no significant difference in HPV prevalence between cases from Paraguay and the United States. In conclusion, the overall prevalence of HPV DNA in penile carcinoma (42%) is lower than that in cervical carcinoma (approximately 100%) and similar to vulvar carcinoma (approximately 50%). In addition, specific histological subtypes of penile cancer--basaloid and warty--are consistently associated with HPV, however, only a subset of keratinizing and verrucous penile carcinomas is positive for HPV DNA, and thus these two tumor groups seem to develop along different pathogenetic pathways.

Prevalence of human papillomavirus DNA in different histological subtypes of cervical adenocarcinoma.

The prevalence of human papilloma virus (HPV) DNA in different histological subtypes of cervical adenocarcinoma and related tumors was examined using formalin-fixed, paraffin-embedded tissue samples from 105 primary cervical adenocarcinomas and adenosquamous carcinomas. Broad-spectrum HPV DNA amplification and genotyping was performed with the SPF10 primer set and line probe assay (LiPA), respectively. HPV DNA was detected in 82 of 90 (91%) mucinous adenocarcinomas, encompassing endocervical, intestinal, and endometrioid histological subtypes, and in nine of nine adenosquamous tumors (100%). HPV DNA was not detected in any nonmucinous adenocarcinomas including clear cell, serous, and mesonephric carcinomas (0/6). The most common viral types detected in adenocarcinoma were HPV 16 (50%) and HPV 18 (40%), followed by HPV 45 (10%), HPV52 (2%), and HPV 35 (1%). Multiple HPV types were detected in 9.7% of the cases. In conclusion, mucinous adenocarcinomas and adenosquamous carcinomas of the cervix demonstrate a very high prevalence of HPV DNA, similar to that reported for cervical squamous cell carcinoma. Only rare histological variants of cervical adenocarcinoma seem unrelated to HPV infection.