Edyta C. Pirog

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.

HPV Involvement in Head and Neck Cancers: Comprehensive Assessment of Biomarkers in 3680 Patients

BACKGROUND We conducted a large international study to estimate fractions of head and neck cancers (HNCs) attributable to human papillomavirus (HPV-AFs) using six HPV-related biomarkers of viral detection, transcription, and cellular transformation. METHODS Formalin-fixed, paraffin-embedded cancer tissues of the oral cavity (OC), pharynx, and larynx were collected from pathology archives in 29 countries. All samples were subject to histopathological evaluation, DNA quality control, and HPV-DNA detection. Samples containing HPV-DNA were further subject to HPV E6*I mRNA detection and to p16(INK4a), pRb, p53, and Cyclin D1 immunohistochemistry. Final estimates of HPV-AFs were based on HPV-DNA, HPV E6*I mRNA, and/or p16(INK4a) results. RESULTS A total of 3680 samples yielded valid results: 1374 pharyngeal, 1264 OC, and 1042 laryngeal cancers. HPV-AF estimates based on positivity for HPV-DNA, and for either HPV E6*I mRNA or p16(INK4a), were 22.4%, 4.4%, and 3.5% for cancers of the oropharynx, OC, and larynx, respectively, and 18.5%, 3.0%, and 1.5% when requiring simultaneous positivity for all three markers. HPV16 was largely the most common type. Estimates of HPV-AF in the oropharynx were highest in South America, Central and Eastern Europe, and Northern Europe, and lowest in Southern Europe. Women showed higher HPV-AFs than men for cancers of the oropharynx in Europe and for the larynx in Central-South America. CONCLUSIONS HPV contribution to HNCs is substantial but highly heterogeneous by cancer site, region, and sex. This study, the largest exploring HPV attribution in HNCs, confirms the important role of HPVs in oropharyngeal cancer and drastically downplays the previously reported involvement of HPVs in the other HNCs.

Human papillomavirus DNA prevalence and type distribution in anal carcinomas worldwide

Knowledge about human papillomaviruses (HPV) types involved in anal cancers in some world regions is scanty. Here, we describe the HPV DNA prevalence and type distribution in a series of invasive anal cancers and anal intraepithelial neoplasias (AIN) grades 2/3 from 24 countries. We analyzed 43 AIN 2/3 cases and 496 anal cancers diagnosed from 1986 to 2011. After histopathological evaluation of formalin‐fixed paraffin‐embedded samples, HPV DNA detection and genotyping was performed using SPF‐10/DEIA/LiPA25 system (version 1). A subset of 116 cancers was further tested for p16INK4a expression, a cellular surrogate marker for HPV‐associated transformation. Prevalence ratios were estimated using multivariate Poisson regression with robust variance in the anal cancer data set. HPV DNA was detected in 88.3% of anal cancers (95% confidence interval [CI]: 85.1–91.0%) and in 95.3% of AIN 2/3 (95% CI: 84.2–99.4%). Among cancers, the highest prevalence was observed in warty–basaloid subtype of squamous cell carcinomas, in younger patients and in North American geographical region. There were no statistically significant differences in prevalence by gender. HPV16 was the most frequent HPV type detected in both cancers (80.7%) and AIN 2/3 lesions (75.4%). HPV18 was the second most common type in invasive cancers (3.6%). p16INK4a overexpression was found in 95% of HPV DNA‐positive anal cancers. In view of the results of HPV DNA and high proportion of p16INK4a overexpression, infection by HPV is most likely to be a necessary cause for anal cancers in both men and women. The large contribution of HPV16 reinforces the potential impact of HPV vaccines in the prevention of these lesions.

Clinical, pathologic, and molecular features of early-onset colorectal carcinoma.

The incidence of colorectal carcinoma has increased among patients <40 years of age for unclear reasons. In this study, we describe the clinical, pathologic, and molecular features of colorectal carcinomas that developed in young patients. We compiled a study group of 24 patients <40 years of age with colorectal carcinoma, and 45 patients ≥40 years of age served as controls. Cases were evaluated for clinical risk factors of malignancy and pathologic features predictive of outcome. The tumors were immunohistochemically stained for O6-methylguanine methyltransferase, MLH-1, MSH-2, MSH-6, β-catenin, chemokine (C-X-C motif) receptor 4, epidermal growth factor receptor, TP53, p16, survivin, and α-methylacyl-CoA racemase; assessed for microsatellite instability and mutations in β-catenin, APC, EGFR, PIK3CA, KRAS, and BRAF; evaluated for micro-RNA expression (miR-21, miR-20a, miR-183, miR-192, miR-145, miR-106a, miR-181b, and miR-203); and examined for evidence of human papillomavirus infection. One study patient each had ulcerative colitis and hereditary nonpolyposis colorectal cancer. Ninety-two percent of tumors from young patients occurred in the distal colon (P=0.006), particularly the rectum (58%, P=0.02), and 75% were stage III or IV. Tumors from young patients showed more frequent lymphovascular (81%, P=0.03) and/or venous (48%, P=0.003) invasion, an infiltrative growth pattern (81%, P=0.03), and α-methylacyl-CoA racemase expression (83%, P=0.02) compared with controls. Carcinomas in this group showed significantly increased expression of miR-21, miR-20a, miR-145, miR-181b, and miR-203 (P≤0.005 for all comparisons with controls). These results indicate that early-onset carcinomas commonly show pathologic features associated with aggressive behavior. Posttranslational regulation of mRNA and subsequent protein expression may be particularly important to the development of colorectal carcinomas in young patients.

Unusual endocervical adenocarcinomas: an immunohistochemical analysis with molecular detection of human papillomavirus.

BackgroundEndocervical adenocarcinomas of the usual type are etiologically related to infection with oncogenic human papillomaviruses (HPVs). These tumors are typically diffusely positive for p16 and carcinoembryonic antigen (CEA) immunostains. The goal of our study was to determine the HPV status and immunohistochemical profiles of unusual histologic subtypes of endocervical adenocarcinoma. MethodsThe study consisted of a total of 26 cases of unusual subtypes including clear cell carcinoma (CCC, n=9), gastric-type adenocarcinoma (GAS, n=11), minimal deviation adenocarcinoma (MDA, n=3), mesonephric adenocarcinoma (MSN, n=1), serous adenocarcinoma (SER, n=1), and malignant mixed Müllerian tumor (n=1). In addition, 5 cases of usual-type endocervical adenocarcinoma (UEA) were included in the study as a control group. The cases were tested for HPV using SPF-10 PCR and LiPA assays, and immunostained for p16, HIK1083, hepatocyte nuclear factor 1-&bgr;, p53, CEA, estrogen receptor (ER), and progesterone receptor (PR). ResultsHPV DNA was not detected in any of the unusual adenocarcinoma subtypes, with the exception of a single case of SER in which HPV16 was detected. p16 positivity did not correlate with HPV status, as 42% of HPV-negative tumors showed patchy/diffuse p16 overexpression; however, p16 positivity was uncommon in GAS/MDA. HIK1083 positivity was limited to GAS and MDA, indicating relative specificity for tumors with gastric mucin expression. Hepatocyte nuclear factor 1-&bgr; was positive in the majority of CCCs and also in other tumor variants and in some UEA as well, indicating a lack of specificity for clear cell differentiation. CEA was consistently negative in CCCs and in a single MSN, but positive in GAS, MDA, SER, and UEA, suggesting that it may serve as a negative marker of clear cell differentiation. p53 was diffusely positive in almost half of the GAS cases, whereas UEA showed mostly negative staining and other variants showed focal staining. PR was negative in all variant cases and in all UEA. ER expression, although mostly negative, showed focal staining in a few variant cases and UEA. ConclusionsUnusual variants of endocervical adenocarcinoma are not related to HPV infection, with only rare exceptions, and p16 overexpression in non-UEA does not correlate with HPV status. Negative staining for PR and ER may serve as a general marker of endocervical neoplasia. GAS/MDA may be differentiated from all other adenocarcinomas with either positive HIK1083 stain or negative/focal p16 stain. Positive CEA stain differentiates GAS/MDA from CCC and negative PR and ER stains differentiate GAS/MDA from benign endocervical glands. CCC may be distinguished from all other adenocarcinomas, except MSN, with a negative CEA stain. Strong and diffuse p53 positivity in SER may be useful in differentiation from UEA. MSN may be identified with negative CEA, ER, and PR stains.

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 β-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 β-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.

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.

Diagnostic Accuracy of Cervical Low-Grade Squamous Intraepithelial Lesions Is Improved With MIB-1 Immunostaining.

There is considerable interobserver variation in the diagnosis of low-grade squamous intraepithelial lesion that involves mature squamous epithelium. Our aim was to evaluate the utility of MIB-1 immunostaining as an adjunct test to increase diagnostic accuracy. Consecutive cervical biopsies originally diagnosed as normal (n = 26) or low-grade squamous intraepithelial lesion (n = 23) were reviewed by three pathologists to obtain a consensus diagnosis. MIB-1 immunostaining was performed, and positive staining was defined as a cluster of at least two stained nuclei in the upper two thirds of the epithelial thickness. Human papillomavirus (HPV) DNA detection was performed using a polymerase chain reaction assay. All cases were subsequently reclassified as low-grade squamous intraepithelial lesion (LSIL) or normal (NL) when two or three of three gold standard criteria were satisfied (LSIL gold standard criteria = consensus diagnosis of LSIL, HPV+, MIB-1+; NL gold standard criteria = consensus diagnosis of NL, HPV−, MIB-1−). Using the gold standard diagnoses, we have identified that 14 normal cases (36%) were originally overdiagnosed as LSIL, and one LSIL case (10%) was originally underdiagnosed as normal. All MIB-1-positive cases were HPV+ and identified as LSIL in the consensus review. All MIB-1-negative cases were NL by gold standard criteria. The sensitivity (1.0) and the specificity (1.0) of MIB-1 staining for identifying LSIL were superior to the sensitivity (0.9) and the specificity (0.8) of HPV testing. In conclusion, MIB-1 is a highly sensitive and specific marker for identifying low-grade squamous intraepithelial lesion and is helpful in verifying the diagnosis of equivocal cases.

Low-Grade Vulvar and Vaginal Intraepithelial Neoplasia: Correlation of Histologic Features with Human Papillomavirus DNA Detection and MIB-1 Immunostaining

Histologic criteria of low-grade vulvar/vaginal intraepithelial neoplasia (VIN1/VAIN1) are well established; however, a significant interobserver variability in diagnosing VIN1/VAIN1 has been reported. The goal of this study was to evaluate the utility of MIB-1 immunostaining as an adjunct test to increase the diagnostic accuracy in equivocal cases of VIN1/VAIN1. The second goal was to examine the distribution of low- and high–oncogenic risk human papillomaviruses (HPVs) in VIN1/VAIN1 lesions. Consecutive vulvar/vaginal biopsies originally diagnosed as VIN1/VAIN1 (n = 43) or benign (n = 20) were reviewed by two pathologists to obtain a consensus diagnosis. The diagnosis was further confirmed with HPV testing using Short PCR Fragment 10 and Line Probe Assay. MIB-1 immunostaining was performed, and positive staining was defined as a cluster of two or more stained nuclei in the upper two thirds of the epithelial thickness. After verification of the diagnosis using the consensus histologic review and HPV detection as an objective confirmatory test, 31% of cases originally diagnosed as VIN1/VAIN1 were identified as being overdiagnosed. The sensitivity and the specificity of MIB-1 staining for identifying VIN1/VAIN1 were 0.96 and 0.90, respectively. Seventy percent of VIN1 cases were associated with low-risk viral types. In contrast, the majority (84%) of VAIN1 cases were associated with high-risk HPVs. In conclusion, MIB-1 staining is sensitive and specific for identifying VIN1/VAIN1, helpful in verifying the diagnosis in equivocal cases.