Ki-67, cyclin E, and p16INK4 are complimentary surrogate biomarkers for human papilloma virus-related cervical neoplasia.

Prior studies of Ki-67, cyclin E, and p16 expression have suggested that these biomarkers may be preferentially expressed in cervical neoplasia. This study examined and compared the distribution of staining for these three antigens in 1) normal and reactive epithelial changes, 2) diagnostically challenging cases (atypical metaplasia and atypical atrophy), 3) squamous intraepithelial lesions (SIL), and 4) high-and low-risk human papilloma virus (HPV) type-specific SIL. One hundred four epithelial foci from 99 biopsies were studied, including low-grade squamous intraepithelial lesions (LSIL; 24), high-grade squamous intraepithelial lesions (HSIL; 36), mature or immature (metaplastic) squamous epithelium (29), and atrophic or metaplastic epithelium with atypia (15). Cases were scored positive for Ki-67 expression if expression extended above the basal one third of the epithelium, for cyclin E if moderate to strong staining was present, and for p16 if moderate to strong diffuse or focal staining was present. HPV status was scored by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of extracted DNA. Immunohistochemical findings were correlated with histologic and viral data. Overall, a histologic diagnosis of SIL correlated strongly with all of the biomarkers used (p <0.001). Positive scores for Ki-67, cyclin E, and p16 were seen in 68.4%, 96.7%, and 100% of LSILs and 94.7%, 91.6%, and 100% of HSILs, respectively. Positive predictive values of these three biomarkers for HPV were 82.4%, 89.5%, and 91.4%, respectively. The positive predictive value for HPV of either cyclin E or p16 was 88.7%. Strong diffuse staining for p16 was significantly associated with high-risk HPV-associated lesions. Normal or reactive epithelial changes scored positive for the three biomarkers in 7.7%, 8.0%, and 12%, respectively. Limitations in specificity included minimal or no suprabasal staining for Ki-67 in immature condylomas and occasional suprabasal staining of reactive epithelial changes (10%), diffuse weak nuclear cyclin E staining in some normal or metaplastic epithelia, and diffuse weak basal p16 staining and occasional stronger focal positivity in normal epithelia. Ki-67, cyclin E, and p16 are complementary surrogate biomarkers for HPV-related preinvasive squamous cervical disease. (Because cyclin E and p16 are most sensitive for LSIL and HSIL [including high-risk HPV], respectively, use of these biomarkers in combination for resolving diagnostic problems, with an appreciation of potential background staining, is recommended.)     

The expression of key cell cycle markers and presence of human papillomavirus in squamous cell carcinoma of the tonsil

BACKGROUND: Chemical carcinogens induce squamous cell carcinoma (SCC) of the head and neck by targeting the p53 and the retinoblastoma (pRb) pathways. Human papillomavirus (HPV) might have an etiologic role in these cancers at particular sites. Few studies have compared cell cycle protein expression in HPV-positive and HPV-negative tumors in this region. METHODS: Fifty tonsil SCCs were analyzed for HPV by PCR and for expression of cell cycle proteins (p53, pRb, p16(INK4A), p21(CIP1/WAF1), p27(KIP1), and cyclinD1) by immunohistochemistry. RESULTS: HPV was present in 42%; almost all were type 16. There were statistical associations between HPV positivity and reduced expression of pRb and cyclinD1, overexpression of p16, and younger patient age. Tumor with down-regulated p27 tended to have down-regulated pRb and p21. CONCLUSIONS: HPV-positive tonsil SCCs have distinct molecular pathways. Their association with younger patient age suggests that they are biologically distinct from HPV-negative tumors.     

p16 overexpression identifies HPV-positive vulvar squamous cell carcinomas

Two types of vulvar squamous cell carcinomas (VSCCs) are recognized according to their relationship to human papillomavirus (HPV). Basaloid or warty carcinomas are considered HPV-associated tumors, whereas differentiated keratinizing neoplasms are considered non-HPV-associated. Recently, immunohistochemical detection of p16 and p53 has been proposed to differentiate these 2 types of VSCCs. We conducted a histologic study with immunohistochemical evaluation of p16 and p53 and HPV detection and typing by polymerase chain reaction using 2 different sets of primers in 92 cases of VSCCs to evaluate the usefulness of immunohistochemistry in the classification of VSCCs and to describe the clinico-pathologic characteristics of both types of VSCCs. HPV was detected in 16/92 (17.4%) specimens, HPV16 being identified in 75% of positive cases. A significant number of discrepancies between histology and HPV detection were observed, with 37.5% of HPV-positive tumors being keratinizing and 9.2% of HPV-negative carcinomas showing basaloid or warty features. Diffuse positivity for p16 and p53 was observed in 100% and 6.2% of HPV-positive tumors and in 2.3% and 64.5% of HPV-negative neoplasms, respectively. The sensitivity and specificity of p16 immunostaining to detect HPV-associated carcinomas (100% and 98.7%, respectively) were better than those of histologic criteria (93.8% and 35.5%) and of p53 negative stain (62.5% and 93.4%). Vulvar intraepithelial neoplasia grade 3 of basaloid/warty type was identified in 53.8% HPV-positive tumors, including 3 keratinizing tumors. All these cases were p16 positive and p53 negative. Vulvar intraepithelial neoplasia grade 3 of differentiated type was observed in 45.6% of HPV-negative cases; 90.8% of them were positive for p53 but all were negative for p16. No differences in age, stage, or development of recurrence were observed between HPV-positive and negative tumors. In summary, the current morphologic criteria to discriminate HPV-positive and negative VSCCs have a significant overlap. Immunostaining for p16 is a reliable marker for HPV-positive VSSCs, which improves the results of histologic classification.     

Association between human papillomavirus DNA and temporal arteritis

ABSTRACT: BACKGROUND: To examine the relationship between human papillomavirus (HPV) and giant cell arteritis (GCA) of the temporal artery. METHODS: The study group consisted of 22 cases of histologically positive/biopsy confirmed GCA. The control groups consisted of 21 histologically negative temporal artery biopsies and fifteen cases of vascular margins of nephrectomies. For detection of the presence of HPV, two methods were used: 1) polymerase chain reaction (PCR) with INNO-Lipa HPV Genotyping Extra 2) CervistaTM HPV HR. All cases were from the files of the Barnes-Jewish Hospital and Washington University in St. Louis. RESULTS: HPV DNA was detected by PCR and genotyping in 16 of 22 (73%) histologically positive cases of GCA and in only five of 21 (24%) histologically negative temporal artery biopsies. Among the vascular margin controls, only three of 15 (20%) were positive for HPV DNA. The second, independent method (CervistaTM) confirmed the aforesaid results with 100% concordance with the exception of three cases which had low genomic DNA for which it was not possible to perform the test. The differences in HPV positivity between the histologically positive and negative temporal artery biopsies and between the histologically positive temporal artery biopsies and controls were both statistically significant (p = 0.001 and 0.002, respectively) CONCLUSIONS: The results of our study revealed a statistically significant association between HPV positivity and biopsy confirmed temporal giant cell arteritis GCA (p = 0.001). Further studies are necessary to elucidate the pathophysiology underlying this association. KEY WORDS: Human Papillomavirus, Giant Cell Arteritis, Polymerase Chain Reaction.     

Utility of cell-cycle modulators to predict vascular invasion and recurrence after surgical treatment of hepatocellular carcinoma

BACKGROUND: The outcome of surgical treatment of hepatocellular carcinoma (HCC) could be improved by applying patient selection criteria based on tumoral aggressiveness. Here we analyzed the prognostic role of the expression of several genes involved in cell-cycle regulation in a group of patients with HCC. METHODS: We retrospectively studied 93 patients (67 transplanted and 26 resections) treated between 1996 and 2000. In micro-thick sections from paraffin-embedded tumoral tissues, the expression of p53, pRb, p16, and cyclin D1 was analyzed. A logistic regression model was used to detect factors related to vascular invasion. A Cox regression model was applied to identify pathologic and molecular factors with the capacity to predict the recurrence of HCC. RESULTS: Only tumor size>3 cm (odds ratio [OR]: 3.4; 95% CI: 1.2-9.9; P=0.019) and pRb expression (OR: 4.1; 95% CI: 1.02-17; P=0.053) were associated with an increased risk of vascular invasion. The regression model applied to the group of transplanted patients showed three factors that were independently related to recurrence: vascular invasion (OR 7.5; 95% CI: 1.1-51.8; P=0.039); pRb expression (OR: 11; 95% CI: 1.2-96.9; P=0.03); and p16 expression (OR: 69.7; 95% CI: 5.1-9448; P=0.001). In the group of resected patients, pRb expression was associated with higher risk of recurrence only in the univariate analysis (P=0.037). The multivariate analysis showed tumor size>3 cm (OR: 57.5; 95% CI: 1.1-51.8; P=0.039) and vascular invasion (OR: 6.1; 95% CI: 1.05-35.3; P=0.044) to be significantly associated with recurrence. CONCLUSIONS: Of the molecular factors studied, only pRb expression was useful as a predictive factor of vascular invasion in patients with HCC, and also of recurrence in transplanted patients with this carcinoma. pRb expression may be relevant to consider when selecting patients for resection and when identifying transplanted patients with a high risk of recurrence.     

Evidence That Alpha-9 Human Papillomavirus Infections are a Major Etiologic Factor for Oropharyngeal Carcinoma in Black South Africans

Human papillomavirus (HPV) infection, most commonly genotype 16 of the alpha-9 family, is implicated in the etiology of a subset of oropharyngeal squamous cell carcinomas (OPSC) worldwide. Data are scarce regarding OPSC in South Africans, and three prior studies suggest no significant etiologic role for HPV. We aimed to investigate for evidence of HPV etiology in OPSCs from black South Africans by polymerase chain reaction (PCR) methodologies with determination of HPV subtype by sequencing, in situ hybridization (ISH), and p16^INK4a immunohistochemistry (IHC), as a surrogate marker for an HPV-driven tumor. It was hypothesized that HPV-driven tumors would be positive by PCR plus IHC and/or ISH whereas OPSCs with HPV background infections (HPV-passenger) would be positive by PCR alone. Formalin-fixed, paraffin-embedded tissues from 51 OPSCs collected between 2005 and 2010 from 41 patients were analyzed for HPV by GP5+6+ PCR (targeting the HPV L1 region), pU-1M/pU-2R PCR (targeting the HPV E6/E7 region) and HPV-31 specific PCR (targeting the E5 region), chromogenic ISH, and p16^INK4a IHC. All cases positive by PCR were subject to sequencing to determine HPV genotype. The patient mean age was 58.0 years and 88 % were male. Of the 51 evaluable tumors, 48 (94.1 %) were positive for HPV DNA by PCR: 25 (49.1 %) met criteria for an HPV-driven tumor, 23 (45.1 %) for HPV-passenger, and 3 (5.9 %) were HPV-unrelated. Sequencing of the PCR-positive cases revealed the following genotypes: combined HPV-16 and 31 (41.7 %), HPV-31 (25.0 %), HPV-16 (22.9 %), combined HPV-16 and 18 (6.3 %), and a single case each of HPV 18 and HPV 33. Studies via ISH were negative in all cases. In accordance with worldwide trends but contrary to prior South African data, HPV likely plays an etiologic role in a significant subset (at least 49.1 %) of OPSC in black South Africans. We found that the alpha-9 HPV family, particularly HPV-16 and 31 either in combination or separately, to predominate in our sample tumors. The use of multiple PCR primers increased sensitivity of viral detection, and a HPV-31 specific primer confirmed the presence of this genotype in many samples. Further studies including HPV E6/E7 mRNA assays are needed to better elucidate the pathogenic role of HPV in black South African OPSCs.     

Molecular and immunohistochemical analysis of the prognostic value of cell-cycle regulators in urothelial neoplasms of the bladder

OBJECTIVE: To evaluate the prognostic and predictive value of molecular and immunohistochemical markers related to cell-cycle control in terms of recurrence, progression, and survival in urothelial neoplasms of the bladder (UNB). PATIENTS AND METHODS: Clinical and pathological findings of 84 patients with UNB were assessed. Homozygous deletion (HD) and promoter methylation of p14ARF, p15INK4B, p16INK4A, loss of heterozygosity of the locus 9p21, p53 mutations, and immunohistochemical expression of p53, p16, p14, p21, p27, pRb, Ki67, MDM2, and cyclin D1 proteins were evaluated in relation to overall survival (OS), recurrence-free survival (RFS), and progression-free survival (PFS). RESULTS: In the univariate analysis, RFS was shorter in cases with p14ARF (p=0.006), p15INK4B (p=0.003), p16INK4A (p=0.03) HD, low p14 immunoreactivity index (IRI) (p=0.01) and high Ki67 IRI (p=0.04); HD of the 9p21 locus genes and p14 IRI remained as independent prognostic factors for early UNB recurrence (p=0.006) whereas tumour stage (p=0.00001) and cyclin D1 IRI (p=0.049) were related to worse PFS in the multivariate analysis. In the univariate analysis, IRI for Ki67 (p=0.002), cyclin D1 (p=0.06), p53 (p=0.00008), p16 (p=0.02), p27 (p=0.0005) MDM2 (p=0.01) and p53 mutations (p=0.03) were related to poor OS, and only the Ki67 IRI retained their independent value in the multivariate analysis. CONCLUSION: 9p21 HD and p14 IRI constitute independent predictive factors for UNB recurrence and cyclin D1 IRI and tumour stage for progression. In addition, Ki67 IRI and tumour stage are independent prognostic factors for overall survival in UNB.     

Crypt dysplasia with surface maturation: a clinical, pathologic, and molecular study of a Barrett's esophagus cohort

Little is known regarding the significance of esophageal biopsies that show dysplasia-like atypia limited to the bases of the crypts, without involvement of the surface epithelium in Barrett's esophagus (BE). The aim of this study was to evaluate the clinical, pathologic, immunohistochemical, and molecular characteristics of basal crypt dysplasia-like atypia (BCDA) with surface maturation in surveillance endoscopic mucosal biopsies to gain insight into its biologic significance. The Seattle Barrett's Esophagus Project is a prospective cohort study in which patients and their biopsies have been evaluated prospectively for clinical, pathologic, and molecular markers. As part of continued surveillance of the cohort, 206 consecutive BE patients were evaluated prospectively for BCDA between July 1, 2001 and August 13, 2003; 15 patients had BCDA (prevalence rate = 7.3%). These 15 patients were evaluated for clinical, pathologic, and immunohistochemical (p53 and MIB-1) features during the study period (2001-2003) as well as associations with clinical, pathologic, and molecular markers [17p(TP53) loss of heterozygosity (LOH), 9p(p16) LOH, tetraploidy, and aneuploidy] that were detected previously in the same patients in the cohort study (1983-2001). All BE patients with BCDA (male-to female ratio, 12:3; mean age, 72 years; mean length of BE, 7.0 cm; mean duration of BE, 95.1 months), except 2 (87%), had dysplasia or adenocarcinoma detected in biopsies either prior to or concurrent to the one that contained BCDA. In contrast, only 112 of 191 (59%) controls had neoplasia during the same time period (59%, P = 0.05). The difference between BCDA and controls was particularly significant with regard to the association with high-grade dysplasia (P = 0.004). Compared with adjacent nonatypical and nondysplastic (metaplastic) BE, areas of BCDA showed a significantly elevated prevalence rate of p53 positivity (60% vs. 13%, P<0.02) and a significantly elevated total crypt and basal crypt MIB-1 proliferation rate (P<0.001). Indeed, the MIB-1 proliferation rate in the basal portion of the crypts in BCDA was similar to that detected in conventional low- or high-grade dysplasia. Patients with BCDA showed a significantly increased rate of 17p(TP53) LOH (P = 0.016), aneuploidy (P = 0.004), and a trend in increased 9p(p16) LOH (P = 0.08), compared with control patients without BCDA. The clinical, pathologic, immunohistochemical, and molecular abnormalities were similar in BCDA cases that were considered low-grade versus those considered high-grade by histologic evaluation, except that high-grade cases tended to be older (79 years vs. 68 years, P = 0.06). BCDA with surface maturation, in mucosal biopsies from patients with BE, is an uncommon but significant pathologic change that shows a variety of proliferative and molecular abnormalities and has a high association with conventional dysplasia and/or adenocarcinoma. Based on these findings, BCDA warrants further investigation as a possible subtype of true dysplasia despite the morphologic appearance of surface maturation.     

P16 and Ki67 immunostaining is a useful adjunct in the assessment of biopsies for HPV-associated anal intraepithelial neoplasia

P16 is a tumor suppressor gene product, shown to be overexpressed in most cervical carcinomas and dysplasias associated with high-risk human papilloma virus (HPV) infection. HPV is also associated with anal squamous dysplasias and carcinomas. Significant interobserver and intraobserver variation exists in the interpretation of biopsies for anal intraepithelial neoplasia (AIN). This study was undertaken to assess the potential role of p16 and Ki67 immunohistochemical expression in refining the diagnosis and grading of AIN.One-hundred and four anal biopsies from 74 patients were retrieved from the surgical pathology files of the department. After discrepancies were resolved and concurrence was achieved by at least 2 of 3 reviewing pathologists, the diagnoses were as follows: 37 negative, 12 condylomas without overt dysplasia, 14 AIN I, 25 AIN II, and 16 AIN III. p16 and Ki67 expression was evaluated by ABC immunoperoxidase staining whereas the presence of the high-risk subtypes of HPV virus was determined by in situ hybridization on a subset of the biopsies. Results were reviewed by 2 pathologists and positive and negative staining was correlated with H&E diagnoses. Nuclear and/or nuclear and cytoplasmic staining was considered as positive for p16 when present in >10% of squamous cells. Two patterns of positive p16 staining were observed: (1) "spotty" in which positive cells were scattered throughout the lesion and (2) "band" in which >90% of contiguous cells in the lesion stained positive. A band-like pattern of p16 immunoreactivity was seen in 21.4% AIN I, 80% AIN II, and 87.5% AIN III cases. None of the condylomas and only 1 of the negative cases showed a band of p16 positive staining. Spotty p16 immunoreactivity was observed in 8.1% negative, 8.3% condyloma, 14.3% AIN I, 12.0% AIN II, and 12.5% AIN III cases. More than 50% of nuclei stained positive for Ki67 in 28.6% AIN I, 48.0% AIN II, and 75.0% AIN III cases but in none of the negative or condyloma cases. On the basis of these results, a band-like pattern of p16 staining and Ki67 positivity in >50% of the squamous cell nuclei were strongly associated with high-grade AIN. Conversely, absence of a p16 band of positivity coupled with Ki67 positivity in <50% of nuclei was frequently associated with benign lesions. Band like p16 staining also correlated strongly with the presence of high-risk HPV. Most AIN I lesions stained similar to the nondysplastic cases. A small subset of biopsies studied did not conform to the pattern described above: 4 of 14 (28.6%) AIN I lesions showed a band-like pattern of p16 staining and/or >50% Ki67 positive nuclei. Two of these cases were positive for high-risk HPV DNA. 4 of 25 (16.0%) AIN II lesions comprising 9.8% of the 41 high-grade AINs (AIN II and III) showed spotty p16 positivity and <50% Ki67 positive nuclei. One was positive for high-risk HPV DNA. We conclude that when used together and evaluated in conjunction with H&E stained sections, p16 and Ki67 immunoexpression is a useful adjunct in the diagnosis and grading of AIN.     

p53 as a new prognostic factor for lymph node metastasis in penile carcinoma: analysis of 82 patients treated with amputation and bilateral lymphadenectomy

PURPOSE: Gold standard treatment for invasive penile carcinoma remains amputation and lymphadenectomy. This procedure has high morbidity and new prognostic factors on the incidence of metastasis would help select candidates to lymphadenectomy. Mutations in the p53 gene common in several neoplasms can be related to the prognosis. We studied 82 patients with penile carcinoma staged according to the 1978 TNM system who underwent amputation and bilateral lymphadenectomy to evaluate the prognostic value of immunohistochemical p53 staining in the primary tumor. MATERIALS AND METHODS: Immunoreactivity of p53 was studied with other clinicopathological variables, including patient age, stage, histological grade, tumor thickness, lymphatic and venous embolization, corpora cavernosa, corpus spongiosum and urethral infiltration, and human papillomavirus (HPV) status. We also determined its association with lymph node metastasis, the survival rate and the risk of death. In addition, we studied the association of p53 and HPV DNA with prognosis. All slides were reviewed by 1 pathologist. HPV DNA was detected by polymerase chain reaction using GP5/6+ generic primers. p53 expression was measured by immunohistochemical testing with monoclonal Clone DO-7 mouse anti-human p53 protein antibody (Dako A/S, Glostrup, Denmark). The Cox regression hazards method was used for multifactorial analysis. RESULTS: Nuclear accumulation of p53 was detected in 34 of 82 samples (41.5%). Clinical lymph node N stage (p = 0.045), lymphatic (p <0.001) and venous (p = 0.04) embolization by neoplastic cells, p53 positivity (p = 0.012) and p53 grade (p = 0.004) were significantly associated with lymph node metastasis. Followup was 0.1 to 453 months (mean 88.7). Multivariate analysis revealed that only lymphatic embolization (relative risk 9.4, 95% confidence interval [CI] 2.8 to 31.6) and p53 positivity (relative risk 4.8, 95% CI 1.6 to 14.9) were independent factors for lymph node metastasis. Patients with negative p53 had significantly better 5 and 10-year overall survival than those in whom tumors stained positive for p53 (64.5% and 54.6% versus 30.2% and 26.4%, respectively, p = 0.009). When tumors were p53 positive and HPV DNA positive, overall survival was worse. Multivariate analysis revealed that only age (relative risk 2.9, 95% CI 1.6 to 5.1) and lymph node metastasis (relative risk 3.2, 95% CI 1.8 to 5.8) were independent risk factors for death. CONCLUSIONS: Immunoreactivity of p53 is an independent factor for lymph node metastasis. The association of positive p53 with positive HPV DNA was related to a worse prognosis.     

Human papillomavirus hpv-16 DNA as an epitheliotropic virus that induces hyperproliferation in squamous penile tissue

The prevalence of human papillomavirus HPV-16DNA sequences in 57 penile carcinoma biopsies was examined using the polymerase chain reaction (PCR) with type specific internal probes, employing HPV consensus primers from the L1 region. The cases comprised 39 typical squamous cell carcinoma and 18 specimens with different subtype. PCR products were analyzed and HPV-16DNA was detected in a high percentage of specimens. Thirty-eight biopsies were HPV-16DNA positive. This determination was correlated with cellular differentiation and growth pattern. Our data corroborates that squamous cell carcinoma was invariably associated with HPV-16DNA.     

Detection of human papillomavirus in small cell carcinomas of the anus and rectum

Small cell carcinomas represent <1% of colorectal/anal carcinomas and have a poor prognosis. Anorectal squamous cell carcinomas are often associated with high-risk human papillomavirus (HPV) infection, similar to squamous and small cell carcinomas of the uterine cervix. In HPV infection, the oncoprotein E7 inactivates the tumor suppressor Rb, leading to p16 upregulation; however, in small cell carcinomas, the Rb pathway is often blocked by other mechanisms; thus, increased p16 may not indicate HPV infection. P16 immunohistochemistry (IHC) may have a limited role in evaluating small cell carcinomas for HPV infection. Formalin-fixed, paraffin-embedded tissue sections of previously diagnosed small cell carcinomas of the anus (n=5) and rectum (n=11) were subjected to IHC for p16, CDX2, and p63, followed by in situ hybridization for high-risk HPV. All (100%) cases of anal and rectal small cell carcinomas were positive for p16, and 100% of anal and 82% of rectal small cell carcinomas were positive for high-risk HPV. The majority of cases showed low or very low HPV copy numbers. In 6 cases, HPV was detected only by using the HPV-16 genotype-specific assay detecting very low copy numbers (1 to 2 viral copies). The majority of tumors expressed p63, which was more pronounced in the anal tumors. CDX2 expression was observed predominantly in rectal tumors. High-risk HPV can be detected using in situ hybridization in the majority of anorectal small cell carcinomas, which are uniformly p16 positive by IHC. HPV-targeted therapy could result in better control of these aggressive neoplasms.     

Expression patterns of cyclins D1, E and cyclin-dependent kinase inhibitors p21(Waf1/Cip1) and p27(Kip1) in urothelial carcinoma: correlation with other cell-cycle-related proteins (Rb, p53, Ki-67 and PCNA) and clinicopathological features

INTRODUCTION: The expression pattern of cyclins D1 and E, as well as cyclin-dependent kinase inhibitors p21(Wa1/Cip1) and p27(Kip1) and their relationship to tumour behaviour and patients' prognosis was examined in 142 urothelial cell carcinomas. The expression of these proteins was also analyzed along with other cell-cycle-related proteins such as: p53, pRb and the proliferation-associated indices Ki-67 and proliferating cell nuclear antigen (PCNA). PATIENTS AND METHODS: These molecule markers were localized immunochemically using the monoclonal antibodies anti-cyclin D1 (DCS-6), anti-cyclin E (13A3), anti-p21 (4D10), and anti-p27 (1B4) in 142 patients with urothelial cell carcinoma. RESULTS: Focal positivity (<10% of tumour cells) or the absence of cyclin D1 immunostaining was observed in 105/142 (73.9%) of the tumours. Cyclin D1 expression was correlated with tumour grade and stage as well as with the existence of in situ component. In addition, cyclin D1 expression was positively correlated with p21(Waf1/Cip1) and p27(Kip1) and inversely with the Ki-67 score. Focal positivity (<20% of tumour cells) or the absence of cyclin E immunoreactivity was observed in 105/142 (73.9%) in all cases. Cyclin E expression was correlated with tumour stage. A positive relationship between cyclin E expression and the two associated proliferating indices Ki-67 and PCNA, as well as with p53 and p27(Kip1) proteins expression was noted. Absence or focal positivity (<5% of tumour cells) of p21(Waf1/Cip1) was detected in 88/142 (62%) of the carcinomas. p21(Waf1/Cip1) expression was correlated with tumour grade and stage. A positive relationship of its expression cyclin D1, cyclin E, p27 and pRb expression was observed. Absence or focal immunostaining (<20% of tumour cells) of p27 protein was detected in 55/141 (39%) in all cases. p27(Kip1) expression was correlated with tumour grade as well as with cyclins D1 and E. The prognostic significance of cyclins D1, E and cyclin-dependent kinase inhibitors p21(Waf1/Cip1), p27(Kip1) in determining the risk of recurrence and progression with both univariate (log rank test) and multivariate (Cox regression) methods of analysis showed no statistically significance differences. CONCLUSION: These findings suggest that the level of the cell cycle regulators studied does not seem to have a clinical value in terms of predicting the risk of early recurrence and progression. In addition the interrelationship probably means their contribution to the regulation of cell growth through different pathways in bladder carcinogenesis.     

Small Biopsy Specimens Reliably Indicate p16 Expression Status of Oropharyngeal Squamous Cell Carcinoma

Human papillomavirus (HPV)—related oropharyngeal squamous cell carcinoma (SCC) is associated with favorable patient survival. Tumor HPV status at primary diagnosis is critical for proper management, and p16 immunohistochemistry (IHC) has emerged as a reliable, single, surrogate marker. It is not known, however, if small biopsy specimens are completely adequate for p16 evaluation. From a database of oropharyngeal SCC for which p16 IHC and histologic typing were already performed, all patients (32) who had available in-house primary tumor biopsy specimens and also subsequent surgical resections were analyzed. p16 IHC was performed along with histologic typing into: Type 1 keratinizing SCC, Type 2 nonkeratinizing SCC with maturation, and Type 3 nonkeratinizing SCC. Staining was graded on both biopsies and resections as follows: 0 = negative; 1+ = 1–25% of tumor cells positive; 2+ = 26–50%; 3+ = 51–75%; 4+ = 76–100%. Strictly considering p16 score, perfect biopsy-resection correlation was present in 28 of 32 cases (85%), including 6/9 (67%) Type 1, 6/7 (86%) Type 2, and 16/16 (100%) Type 3 cases. Considering p16 expression binarily as 51% tumor cell staining or more (3+ or 4+) being positive and lesser amounts (0, 1+, or 2+) as being negative, there was perfect biopsy-resection correlation for all 32 cases. With p16 expression in resection specimens considered the gold standard, p16 IHC in biopsies was both 100% sensitive and specific. Our results demonstrate that p16 staining in diagnostic biopsies reliably reflects whole tumor staining results, and suggest that biopsies do not suffer from false negatives or positives.     

Correlation between HPV sperm infection and male infertility

Human papillomavirus (HPV) is one of the most common sexually transmitted diseases which comprises a group of small DNA viruses that infect both cutaneous and mucous squamous epithelia. Liquid bead microarray technology (LBMA) were used to evaluate 24 HPV genotypes in confirmed fertile and infertile males of North China so that the effects of HPV infection on semen parameters and relationship with male infertility could be discussed. A total of 1138 subjects were recruited in this study; 142 were HPV-positive (12.48%). Among 523 confirmed fertile males, only 35 were HPV-positive (6.70%), and two of them had multiple infections. Among 615 infertile males, 107 were HPV-positive (17.4%), and 29 of them had multiple infections. Infertile males had a relatively high HPV infection rate compared with confirmed fertile males. Sperm progressive motility (PR) and the normal morphology rate were significantly decreased in HPV-positive subjects. HPV-45, HPV-52, HPV-18, HPV-59 and HPV-16 infections were more frequently in infertile males. Hence, HPV infection is closely related to male infertility which will decrease sperm PR and morphology. HPV-45, HPV-52, HPV-18, HPV-59 and HPV-16 infection seems to be major risk factors.     

Comparing Outcomes for Patients with Human Papillomavirus (HPV) Type 16 versus Other High-Risk HPV Types in Oropharyngeal Squamous Cell Carcinoma

Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is related to improved treatment outcomes. What remains unclear is whether all HPV DNA genotypes carry similar prognostic relevance. We aimed to evaluate disease control and survival outcomes by HPV DNA genotype. Patients with primary OPSCC without distant metastases treated with curative intent were retrospectively identified from an IRB-approved institutional database. Patients that underwent HPV DNA polymerase chain reaction (PCR) testing with available genotype were included and dichotomized by the presence of HPV type 16 (HPV-16) or other high-risk HPV genotype (HPV-non16). Overall survival (OS), disease-free survival (DFS), locoregional control (LRC) and distant control (DC) were determined using the Kaplan–Meier method and compared using the log-rank test. In our cohort of 193 patients treated from 2012 to 2018 with HPV DNA PCR, 10% were detected as HPV-non16 high-risk types. Patients with HPV-16 were significantly younger than those with HPV-non16, but no other baseline factors were associated with HPV-non16. With a median follow-up of 42.9 months, there were no significant differences in outcomes between the HPV-16 and HPV-non16 groups for 3-year OS (87.7% v. 73.6%), DFS (82.9% v. 68.7%), LRC (92.8% v. 88.5%) or DC (91% v. 89.2%). There is no statistically significant difference in outcomes between OPSCC with HPV-16 and HPV-non16 high-risk genotypes in our cohort, though trends of overall worse survival and disease-free survival in HPV-non 16 OPSCC were seen. Further studies with larger cohorts of patients with HPV-non 16-associated OPSCC are required to make definitive conclusions regarding the prognostic and clinical significance of HPV type.     

Overexpression of p16INK4 is a reliable marker of human papillomavirus-induced oral high-grade squamous dysplasia.

OBJECTIVE: Human papillomavirus (HPV) infection has been implicated in the development of high-grade squamous dysplasia and carcinoma of the oral cavity in the absence of other known risk factors such as smoking. HPV-induced oral dysplasia or carcinoma may be a unique tumor entity in terms of biologic behavior and treatment decisions. In detecting such cases, most reported studies have used techniques that are less sensitive than DNA amplification. Recent reports have suggested that overexpression of the p16INK4 protein is a surrogate marker of HPV-induced high-grade dysplasia or carcinoma. However, the correlation between expression of p16INK4 and the presence of HPV DNA as determined by polymerase chain reaction (PCR) amplification has not been previously reported. The purpose of this research was to determine if immunohistochemistry for p16 would serve as a marker of HPV-associated high-grade oral squamous dysplasia. STUDY DESIGN: Archival formalin-fixed, paraffin-embedded tissue sections from 41 cases of high-grade oral squamous dysplasia were randomly selected. Expression of p16INK4 protein was assessed by immunohistochemical analysis (16P04 Neomarkers, Fremont, CA). Strong and diffuse nuclear staining restricted to the dysplastic region in the epithelium was scored as positive for protein expression, whereas focal or weak nuclear or cytoplasmic staining was scored as negative. The presence of HPV was determined by microdissection, DNA extraction, and PCR DNA amplification using elongated primers that align with corresponding sequences of the L1 region of 23 mucosotropic HPV genotypes. The HPV type was determined by direct sequencing of the PCR product. Normal squamous epithelium was used as an internal negative control, and cases of severe cervical high-grade squamous dysplasia were used as a positive control for immunohistochemical staining and PCR. RESULTS: The results of immunohistochemical analysis for overexpression of p16INK4 were positive in 6 of the 41 tissue sections. The results of PCR DNA amplification were also positive for these 6 sections. HPV-16 was identified in 5 of the positive cases; in the other case, the viral strain could not be determined. CONCLUSIONS: Immunohistochemical detection of p16INK4 is a technically simple and potentially reliable assay for diagnosing cases of HPV-induced oral high-grade squamous dysplasia. Detecting such lesions may influence future therapeutic decisions.     

p53、p16、Cyclin D1表达与胃癌增殖细胞核抗原的关系

目的 探讨ｐ５３、ｐ１６、Ｃｙｃｌｉｎ Ｄ１表达与胃癌细胞增生状况的关系。方法 采用免疫组化ＡＢＣ法检测５８例胃癌中ｐ５３、ｐ１６、ＣｙｃｌｉｎＤ１和ＰＣＮＡ的表达状况,并对ＰＣＮＡ免疫阳性细胞作半定量分析。结果 ｐ５３、ｐ１６、Ｃｙｃｌｉｎ Ｄ１阳性表达率分别为５１．７％（３０／５８）、４８．３％（２８／５８）、５３．４％（３１／５８）;ｐ５３、Ｃｙｃｌｉｎ Ｄ１阳性组织中,单位面积ＰＣＮＡ阳性     

Differential Expression of p16INK4A and Cyclin D1 in Benign and Malignant Salivary Gland Tumors: A Study of 44 Cases

Salivary gland tumors (SGT) are a heterogeneous group of lesions. There is conflicting data concerning the molecular events involving the tumour suppressor retinoblastoma protein (pRb) pathway in these tumors. Few studies examined the alterations in components of the Rb pathway by immunohistochemical (IHC) methods in benign and malignant SGTs. Furthermore, recent evidence implicates human papillomavirus (HPV) in mucoepidermoid carcinoma (MEC) carcinogenesis. The purpose of our study is to examine p16^INK4A and cyclin D1 expression in a variety of benign and malignant salivary gland tumors, and to investigate p16^INK4A expression as a surrogate marker for HPV infection in MEC. Our series includes 30 malignant tumors [14 MEC, 6 acinic cell carcinomas (ACC), 5 polymorphous low grade adenocarcinomas (PLGA), 5 (AdCC)] and 14 benign tumors (4 benign cysts, 5 Warthin tumors and 5 pleomorphic adenomas (PA). All cases were tested by IHC for p16^INK4A and cyclin D1. Testing for HPV wide spectrum (HPV-WS) was performed by in situ hybridization in all MEC cases. Staining intensity was recorded semi quantitatively (on a scale from 0 to 4+). Fisher’s exact test and Pearson X² test with a p < 0.05 were used. Cyclin D1 and p16^INK4A are expressed similarly in malignant and benign tumors (p = 0.146 and p = 0.543, respectively). None of the MEC cases showed nuclear reactivity for HPV-WS. Statistical analysis showed positive correlation between cyclin D1 and p16^INK4A expression. Our findings suggest that p16^INK4A overexpression is likely secondary to cyclin D1 gene upregulation or amplification. Further molecular studies are warranted.