Diffuse P16 Research Articles

The association between expression of p53 and aggressiveness of serous adenocarcinoma of the uterine cervix

Background:Serous adenocarcinoma of the uterine cervix is an extremely rare variant of cervical adenocarcinoma. This study aimed to evaluate the clinicopathological and molecular features and outcomes of serous adenocarcinoma of the uterine cervix (SACC).Materials and Methods:This was a retrospective study conducted based on the clinical and pathological data of seven patients diagnosed with SACC after hysterectomy, who were evaluated at the gynecologic oncologic centers between 2010 and 2019.Results:Five cases were diagnosed at Stage IB and two at Stage IV. All patients underwent radical hysterectomy with bilateral salpingo-oophorectomy and subsequently received postoperative radiotherapy or chemotherapy. One patient showed persistent disease, and two patients suffered recurrence. Immunohistochemical study showed that three (43%) of the seven patients were positive for p53, and among these three patients, two with diffuse strong p53 expression experienced an aggressive course with recurrences at pelvic lymph nodes, lung, and brain.Conclusion:High p53 expression and advanced stage may be associated with poorer clinical outcomes in SACC, which suggest that immunohistochemistry may contribute to the prediction of prognosis.

A clinicopathological and molecular analysis of cervical carcinomas with basaloid features.

To investigate the relationship between adenoid basal carcinoma (ABC), adenoid cystic carcinoma (ACC) and squamous cell carcinoma (SCC) in the uterine cervix. We analysed the clinicopathological and molecular features in two pure ABCs, 15 SCCs with ABC-/ACC-like features and seven basaloid SCCs (BSCCs) by chart review, immunohistochemistry, human papillomavirus (HPV) RNA in-situ hybridisation and fluorescence in-situ hybridisation. All patients were alive with no evidence of disease, except for one patient with ACC-like features who died of disease at 18months post diagnosis. The mixed carcinomas comprised variable SCCs and ABC-/ACC-like components displaying vague transitional zones. All components consistently showed diffuse p16, p63 and SOX2, variable cytokeratin (CK)7 and CK17 and rare Ber-EP4 and MYB expression; there was a substantially lower Ki67 index in pure ABCs and the ABC-like components. The ACC-like components showed no myoepithelial differentiation (SMA, calponin and S100) and MYB gene fusions. CK7, CK17 and Ber-EP4 were characteristically stronger in BSCCs than in the mixed carcinomas (P<0.01). High-risk HPV (HR-HPV) E6/E7 mRNA was detected in 12 mixed carcinomas and seven BSCCs, but not in pure ABCs. The HR-HPV mRNA expression was higher in the SCC components and BSCCs than in the ABC-like components of mixed carcinomas (P<0.05). The ACC-like components in mixed carcinomas probably represent the morphological mimics of salivary ACCs. ABC-like components may be the potential precursor of the ACC-like and SCC components. HR-HPV oncogenes may play a role in the pathogenesis of SCCs with ABC-/ACC-like features.

Tissue Fixation Conditions for p16 Immunohistochemistry and Human Papillomavirus RNA In Situ Hybridization in Oropharyngeal Squamous Cell Carcinoma.

Human papillomavirus (HPV) has become a critical prognostic biomarker in oropharyngeal squamous cell carcinoma (OPSCC). While retrospective studies suggest that p16 immunohistochemistry and even HPV RNA in situ hybridization work well on tissues and tumors from a variety of labs and various fixation conditions, no formal study of fixation conditions has been performed to date. We took surgically resected specimens from three p16 and HPV RNA in situ hybridization positive OPSCC patients, divided their fresh tumors into small pieces, and varied the time to formalin fixation as 1, 3, 6, 24, and 48h. Tumors were either held moistened at room temperature or were refrigerated. After fixation and processing, routine hematoxylin and eosin slides were generated and p16 immunohistochemistry and RNA in situ hybridization performed. All three tumors were nonkeratinizing and had strong and diffuse p16 expression at immediate fixation, which, surprisingly, remained positive for all fixation times and conditions and despite significant degeneration at the later points for two of the patients while for one, the nuclear signal dropped out of most cells at early and mid time points, particularly at room temperature, causing false negatives. HPV RNA in situ hybridization stayed positive in all specimens up to 48h of cold ischemic time refrigerated and even at room temperature, except for overtly autolyzed tumor regions. These findings help to establish that, at least for standard nonkeratinizing, p16 and HPV RNA strongly positive OPSCC patients, and using the most common tests in clinical practice, relatively lenient time to fixation may be acceptable if it cannot be avoided. However, for some patients, p16 immunohistochemistry may be sensitive to signal loss with autolysis. HPV RNA in situ hybridization, in particular, seems remarkably resistant to pretest cold ischemic times.

Surrogate markers for high-risk human papillomavirus infection in oral epithelial dysplasia: A comparison of p16, Ki-67, and ProExC.

The aim of this study was to determine the utility of surrogate markers of human papillomavirus (HPV) infection in the diagnosis of HPV-associated oral epithelial dysplasia (OED). Twelve cases of oral dysplasia with histologic features of HPV infection were stained with surrogate markers for HPV (p16, Ki-67, and ProExC) on immunohistochemistry. A second group of 12 cases of oral dysplasia without histologic features of HPV infection was used for comparison. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to confirm the presence of high-risk HPV (HR HPV) in p16-positive cases. All of the surrogate markers showed a statistically significant association with HPV-positive OED (P < .001). The agreement between p16 and HPV positivity was the strongest (κ = 1.00), whereas Ki-67 showed very good association with HPV (κ = 0.83), and ProExC showed good association (κ = 0.75). In each case, the agreement was statistically significant (P < .001). Overall, each of the 3 markers showed good sensitivity; however, ProExC showed the lowest specificity. The clinicopathologic features of 12 cases of HPV OED are reported. Diffuse p16 positivity is an accurate and reliable method for predicting HR HPV infection in both high and low grade cases of epithelial dysplasia with histopathologic features of HPV OED. The use of Ki-67 and ProExC did not demonstrate any additional diagnostic benefit in the diagnosis HPV OED.

Endocervical Adenocarcinoma With Skip Lesion in a Fundal Endometrial Polyp Mimicking Endometrioid Adenocarcinoma Arising From Endometrial Polyp

Abstract Human papillomavirus–related adenocarcinomas account for approximately 20% of all cervical cancers. Most of them arise in the transformation zone but may be found exclusively in endocervical canal. Contiguous spread into the lower uterine segment is known to occur, but finding a “skip” lesion in a fundal endometrial polyp poses an interesting pathologic conundrum. We present a case of a 57-year-old woman with a history of postcoital bleeding and abnormal cervical smear. Cervical exam showed a 2-cm exophytic mass on anterior cervix. Biopsy revealed invasive endocervical adenocarcinoma. Patient underwent radical hysterectomy. Gross examination revealed large cervical mass without involvement of low uterine segment and a benign-appearing small uterine fundal polyp. Microscopic examination confirmed the deeply invasive endocervical adenocarcinoma, and the benign-appearing endometrial polyp had similar atypical glands on the polyp surface and admixed with benign endometrial glands. Both cervical tumor and the carcinoma involving endometrial polyp demonstrated identical immunohistochemistry showing diffuse positive p16 and CEA(m) and negative for ER and vimentin. The authors report a rare case of endocervical adenocarcinoma with skip lesion in the endometrial fundal polyp, and rare cases with skip lesion in the fallopian tubes from endocervical adenocarcinoma have been reported in the literature. High clinical suspicion and ancillary immunohistochemistry are necessary to differentiate the lesion from endometrioid or papillary carcinoma arising from endometrial polyp.

P14.117 Cost efficient test algorithm for molecular subgrouping of medulloblastomas for day-to-day practice in resource limited countries

Abstract INTRODUCTION Molecular classification of medulloblastomas(MB) is prognostically and therapeutically relevant and helps in better risk-stratification. Translation of this subgrouping to routine practice still remains a challenge. The most pathologist-accessible techniques for molecular subgrouping include immunohistochemistry(IHC), fluorescent in-situ hybridization(FISH) and Nanostring assay. Objectives:(1)Molecular subgrouping of MBs by IHC and FISH, and Nanostring Assay (2)To compare their efficacy against sequencing and DNA methylation, and cost for applicability in resource-constrained centers METHODS Ninety-five cases of MB with adequate tissue were included. Molecular subgrouping was performed by IHC for β-catenin, GAB1, YAP1, and p53; FISH for MYC amplification, and sequencing for CTNNB1, and by Nanostring Assay on the same set of MBs. Further, a subset of cases were subjected to 850k DNA methylation array. RESULTS IHC+FISH classified MBs into 15.8% WNT, 16.8% SHH, and 67.4% non-WNT/non-SHH subgroups; with MYC amplification identified in 20.3% cases of non-WNT/non-SHH. A single showed diffuse strong p53 positivity among the SHH subgroup. Nanostring successfully classified 91.5% MBs into 25.3% WNT, 17.2% SHH, 23% Group3 and 34.5% Group4. However, Nanostring assay failure was seen in eight cases, all of which were &gt;8-years-old formalin-fixed paraffin-embedded tissue blocks. Concordant subgroup assignment was noted in 88.5% cases, while subgroup switching was seen in 11.5% cases. Both methods showed prognostic correlation. Among the 5 discrepant cases, which switched to WNT subgroup by Nanostring, only 2 were found to have CTNNB1 mutation. Methylation profiling performed on discordant cases revealed 1 out of 4 extra WNT identified by Nanostring to be WNT, others aligned with IHC subgroups; extra SHH by Nanostring turned out to be SHH by methylation. CONCLUSIONS Both IHC supplemented by FISH and Nanostring are robust methods for molecular subgrouping, albeit with few disadvantages. IHC cannot differentiate between Groups 3 and 4, while Nanostring cannot classify older-archived tumors, and is not available at most centres. WNT subgroup with the best prognosis is best detected by IHC or IHC followed by sequencing for confirmation. Nanostring Assay is better suitable to separate Group 3, the worst prognostic group from Group 4. Thus, both the methods complement each other and can be used in concert for high confidence allotment of molecular subgroups in clinical practice. The cost of IHC plus Nanostring will almost be the same as IHC plus FISH. We recommend a cost-efficient algorithmic approach using histopathological subtype and IHC as the first step followed by Nanostring or FISH, wherever suitable.

Adult Granulosa Cell Tumor With High-grade Transformation: Report of a Series With FOXL2 Mutation Analysis.

Adult granulosa cell tumor (AGCT) is a low-grade malignant neoplasm with a significant propensity for late recurrence and metastasis. Almost all AGCTs are composed of cells with bland nuclear features and even when these tumors recur or metastasize, the nuclear features are almost always low-grade. We report 5 cases of AGCT in patients aged 37 to 88 years composed of areas of typical AGCT with low-grade morphology admixed with areas of high-grade morphology, with marked nuclear atypia, often with bizarre multinucleate cells and high mitotic activity; this is the first reported series of high-grade transformation in AGCTs. The high-grade areas often morphologically closely resembled juvenile granulosa cell tumor with abundant eosinophilic cytoplasm, significant mitotic activity, and intermediate sized follicles. Four cases were FIGO stage IA at diagnosis and 1 was stage IIIC with omental involvement. FOXL2 mutation analysis of both the morphologically low-grade and high-grade areas in 4 of 5 cases confirmed the presence of missense point mutation, c.402C>G, p.(Cys134Trp), providing conclusive evidence that the high-grade component represents transformation of typical AGCT rather than the coexistence of another sex cord-stromal tumor, such as juvenile granulosa cell tumor, which has been suggested for such neoplasms. In 3 of 4 cases where immunohistochemistry was undertaken, there was a striking difference between the p53 staining in the low-grade and high-grade components with wild-type staining in the former and diffuse mutation-type immunoreactivity in the latter, suggesting that TP53 mutation is likely to play a role in high-grade transformation. TP53 mutation analysis covering exons 4 to 10 was undertaken in 4 cases and TP53 mutations were identified in the high-grade component of 2 of the cases. In 1 case, there was diffuse block-type p16 staining in the high-grade component. Follow-up in the 4 stage IA neoplasms revealed no evidence of tumor recurrence in 3 (6 to 9 mo follow-up) while the other patient developed mediastinal, peritoneal, and pulmonary metastasis 17 months after diagnosis. High-grade transformation is uncommon in AGCTs and given that one of our cases was advanced stage at diagnosis, another exhibited widespread metastasis within a short period and there have been occasional case reports of aggressive behavior in AGCTs with high-grade transformation, this event may herald an aggressive clinical course.

Histologic Appearance and Immunohistochemistry of DNA Mismatch Repair Protein and p53 in Endometrial Carcinosarcoma: Impact on Prognosis and Insights Into Tumorigenesis.

Endometrial carcinosarcoma (ECS) is a rare and aggressive mixed-type epithelial and mesenchymal tumor. This study focused on the histologic appearance, loss of DNA mismatch repair (MMR) protein expression, and aberrant p53 expression in the epithelial component, and overall prognosis of 57 cases with ECS. Histologically, 21 and 36 cases exhibited low-grade (endometrioid grade 1 and 2) and high-grade (others) epithelial components, respectively. In a Kaplan-Meier analysis, patients with a high-grade epithelial component exhibited worse progression-free survival (PFS), compared with those with a low-grade component. Although the former group also exhibited worse overall survival, the difference was not significant. Thirty-six cases exhibited aberrant p53 expression. Of these, 5 cases exhibited focally aberrant p53 expression in carcinomatous components with diffuse aberrant p53 expression in mesenchymal components. Aberrant expression of p53 did not show significant association with prognosis. Six patients with MMR deficiency exhibited relatively better PFS. In conclusion, a low-grade epithelial component is a superior predictor of the PFS of ECS, compared with MMR protein and p53 expression status. In some cases of ECS, TP53 mutation may be a late event associated with histogenesis of the sarcomatous component.

Combination of MCM2 With Ki67 and p16 Immunohistochemistry Can Distinguish Uterine Leiomyosarcomas.

Our objective was to evaluate the diagnostic utility of 2 new proliferation markers, cyclin D1 and minichromosome maintenance complex component 2 (MCM2), in comparison with p16, p53, and Ki67 in differentiating the spectrum of smooth muscle tumors. An institutional database search from 2009 to 2017 identified 10 cases of uterine leiomyoma with bizarre nuclei (LBN), 12 smooth muscle tumors of uncertain malignant potential, and 13 leiomyosarcomas (LMS). Ten resected leiomyomas (LM) were included as controls. Immunohistochemistry was performed on the befitting representative block from each case. Ki67 was <10% in all LMs and LBNs, whereas >10% in all LMSs. Although wild-type in majority of cases, p53 was overexpressed in 38% of LMSs. Cyclin D1 nuclear positivity in LMs, LBNs, and smooth muscle tumors of uncertain malignant potentials ranged from 0% to 65% of neoplastic cells with mostly weak to moderate staining intensity. Instead, cyclin D1 expression was <5% in all LMSs. The ratio of MCM2 positivity exhibited a similar wide range (<1%-80%) in LMs, LBNs, and smooth muscle tumors of uncertain malignant potentials but interestingly, 92% (12/13) of LMSs were diffusely and strongly positive for MCM2 (>80% cell positivity). Overall, for diagnosis of LMS, the sensitivity for diffuse intense MCM2 staining was higher (92%) compared with diffuse staining for p16 (77%); however, specificity of MCM2 and p16 was comparable (94% and 97%, respectively). Herein, we describe the immunohistochemical profile of 2 new proliferation markers, cyclin D1 and MCM2 in uterine smooth muscle tumors. A combination of diffuse strong MCM2 and p16 reactivity with increased Ki67 index can reliably distinguish LMSs from benign histologic mimics.

Emerging relationships between papillary proliferation of the endometrium and endometrial carcinoma: evidence from an immunohistochemical and molecular analysis.

Papillary proliferation of the endometrium (PPE) is an uncommon lesion that frequently shows mucinous metaplasia. PPE occasionally has concurrent or preceding endometrial hyperplasia and carcinomas, but there is little molecular evidence to support the relationships between PPEs and endometrial neoplasia. In this study, we analyzed the clinicopathological and immunohistochemical features in 30 PPEs (22 simple PPEs and 8 complex papillary hyperplasia (CPH)). Hotspot mutations of KRAS, PI3KCA, AKT1, PTEN (exons 3, 5, and 7), and ARID1A (exons 1 and 14) were detected by pyrosequencing or bidirectional Sanger sequencing. We found that endometrial hyperplasia and carcinoma were more common in CPHs (4/6, 66.7%) than in simple PPEs (4/21, 19.0%) (p < 0.05). Compared with the adjacent normal endometrium, PPEs frequently showed loss of PAX2 (56.7%) and PTEN (10%) expression, diffuse p16 expression (36.7%), decreased PR expression (84.3%), and lower Ki67 labeling index (median 1%, range 1-15%). Simple PPEs and CPHs had similar immunohistochemical features (p > 0.05). KRAS mutations were identified in 14 PPEs and 1 concurrent endometrial carcinoma. The prevalence of KRAS mutations was not statistically different between simple PPEs (10/21, 45.5%) and CPHs (4/8, 50%) (p > 0.05), but was higher in PPEs displaying mucinous metaplasia (12/24, 50%) than in those without (2/6, 33.3%) (p< 0.05). One simple PPE with a KRAS mutation had an AKT1 mutation. No PPEs demonstrated mutations in PI3KCA, PTEN, and ARID1A. In conclusion, both simple PPE and CPH share some common molecular alterations with endometrial neoplasia, in which, KRAS mutations might be a driver.

Altered Rb, p16, and p53 expression is specific for porocarcinoma relative to poroma.

Porocarcinomas are rare aggressive carcinomas that harbor tumor suppressor mutations and must be distinguished from benign entities such as poromas. To determine whether altered expression of these genes was diagnostically informative, we examined p53, Rb, and p16 staining patterns in 15 poromas and 16 porocarcinomas. Poromas consistently displayed diffuse strong expression of Rb in all but one case that displayed focal loss (1/15, 7%), and no evidence of aberrancy in p53 or p16. Porocarcinomas displayed diffuse or focal loss of Rb expression in 9/16 (56%) cases, diffuse loss or overexpression of p53 in 8/15 (53%), and diffuse loss or overexpression of p16 in 6/14 (43%). Diffuse aberrancy in p53 and Rb expression correlated with tumor mutations in TP53 and RB1, respectively, whereas focal Rb loss was associated with wild type RB1. Diffuse p16 overexpression correlated with Rb loss rather than CDKN2A mutation. For porocarcinomas with all three stains evaluable, 10/13 (77%) displayed aberrancy in at least one marker. Our findings suggest that altered p53, p16, and/or Rb expression is relatively specific to porocarcinoma in comparison with poroma. Technical limitations of this panel, including possible focal Rb loss, must be kept in mind, especially in limited samples.

MON-309 Intratumoral Proliferation and Proinflammatory Macrophages in Obese and Non-Obese Patients with Different Molecular-Biological Types of Endometrial Cancer

Background and aims: Endometrial cancer (EC) is characterized in times of modern obesity epidemic by the rapid incidence increase and the recent update of the molecular-biological classification, which now distinguishes not two, as before, but four types of this tumor (Kandoth et al., 2013; Talhouk et al., 2017; Berstein et al., 2017) that need to be comprehensively studied. The objective of this work was to compare the proliferative potential of a tumor tissue (Ki67 index) and compare it with the expression of proinflammatory CD68-macrophages in obese and non-obese patients with different types of EC according to the PROMISE classification. Materials and methods: In all predominantly postmenopausal 216 treatment naive EC patients included in the study, the body mass index (BMI) was estimated due to which the females were divided into subgroups with BMI ≥30.0 and <30.0. According to genetic (high-resolution melting and sequencing) and immunohistochemical (DACO antibodies) analysis, endometrial carcinomas were classified in accordance with recommendations (Talhouk et al, 2017) into types with POLE gene mutations, defect of mismatch repair proteins (MMR-D), expression (positive or diffuse) of p53 oncoprotein and variant without characteristic molecular profile, WCMP. Immunohistochemistry was also used to evaluate the expression of Ki67 (DBS antibodies, clone SP6) and CD68 (DAKO antibodies, clone CD8/144B). Results: Obesity was more common in patients with a WCMP tumor type and, especially, in MMR-D group (BMI ratio ≥30.0/<30.0, respectively, 1.62 and 2.32). The highest Ki67 index values were detected with positive (59.3 ± 3.8%) and a combination of positive and diffuse (50.5 ± 3.7%) p53 expression, and the lowest (27.6 ± 1.7%) in the WCMP group. In all EC types (except for cases with p53+), Ki67 expression was lower at BMI ≥30.0, which was confirmed within this subgroup by negative Spearman correlation of Ki67 level with BMI value. CD68 expression showed a downward trend in the MMR-D and WCMP types (124.4 ± 7.8 and 115.0 ± 5.9 cond.un.) and a tendency to positive correlation with Ki67 in obese women belonging to the POLE and p53 groups. Conclusions: The findings suggest that the level of such important prognostic tumor markers as Ki67 and CD68 (Soeda et al., 2008) is characterized by a relationship with the presence/absence of obesity as well as with EC molecular-biological type, that expands target range for preventive and therapeutic interventions. Acknowledgement to grant 18-015-00026

Frequent and Specific Involvement of Changes of the p16-RB Pathway in Esophageal Neuroendocrine Carcinoma.

This study investigated the immunohistochemical expression of retinoblastoma (RB) protein and p16 protein in 10 neuroendocrine carcinomas (NECs), in comparison to two mixed-type NECs; 28 squamous cell carcinomas (SCCs), and 12 carcinosarcomas (CSs) from patients with esophageal cancer. Immunohistochemical staining was performed using the avidin-biotin complex detection method. The staining was evaluated as diffusely positive, heterogeneous (in 5-95% of tumor cells), or diffusely negative. The combination of a diffuse loss of RB and the diffuse overexpression of p16, which is found in highly aggressive malignant tumors and is considered to convincingly suggest changes in the p16-RB pathway, was found in all NECs (10/10). In contrast no mixed-type NECs, one SCC and one CS showed this finding. Coexisting intraepithelial carcinoma was detected in seven NECs and only one lesion showed the combination of diffuse RB loss and p16 overexpression. These data suggest that changes in the p16-RB pathway were universally and specifically involved in the development and invasion of esophageal NECs and that it may be a useful diagnostic marker and a potential therapeutic target.

Bartholin Gland Carcinoma: Clinicopathologic Features, Including p16 Expression and Clinical Outcome.

Bartholin gland carcinomas are rare forms of vulvar malignancy and it is unclear what proportion is associated with high-risk human papilloma virus (HPV) infection. Our hospital archives were searched for all cases of Bartholin gland carcinoma from 1984 to 2017 (n=16). We excluded 3 adenoid cystic carcinomas, which were the subject of a previous study, leaving 13 cases. We reviewed all slides and performed immunostains for p16 as a surrogate biomarker for high-risk HPV. There were 12 squamous cell carcinomas (SCCs), including 1 SCC with transitional-like morphology and 1 papillary SCC, and 1 adenocarcinoma. All SCCs showed diffuse and intense p16 expression consistent with the presence of HPV. The single case of poorly differentiated adenocarcinoma showed patchy staining. Patient age ranged from 38 to 72 yr (mean, 58.3 yr). Most tumors were low stage. All patients were treated with radical vulvectomy and inguinofemoral lymphadenectomy. Mean clinical follow-up was 53.7 mo (range, 3-181 mo), 9 patients were free of disease (75%), recurrence occurred in 3 cases, with death due to disease in 2 of the patients with recurrence, including the single patient with adenocarcinoma. All SCC of Bartholin gland expressed p16 diffusely and intensely regardless of histologic features and grade. Our results support the etiologic role of HPV in the pathogenesis of SCC of Bartholin gland. In this small study we observed SCC as the predominant histotype, and most tumors presented at early stage and were associated with relatively favorable outcomes.

High-Risk Human Papillomavirus E6/E7 mRNA Is Rarely Detected in Nonanogenital Cutaneous Squamous Cell Carcinoma: An RNA In Situ Hybridization-Based Tissue Microarray Study.

High-risk human papillomavirus (HR-HPV) is known to play an oncogenic role in squamous cell carcinoma (SCC) at certain anatomical sites, namely the uterine cervix, oropharynx, and anogenital skin. However, the association between HR-HPV and nonanogenital cutaneous SCC (CSCC) remains controversial. In this study, we addressed this controversy by performing HR-HPV E6/E7 mRNA in situ hybridization (ISH) on 243 CSCC samples. A cocktail of E6/E7 mRNA ISH probes, recognizing 18 HR-HPV genotypes, was applied to a tissue microarray of paraffin-embedded sections of 154 invasive and 89 in situ CSCC specimens. The anatomical sites of CSCC included the head and neck (n = 100), extremities (n = 100), trunk (n = 25), and anogenitalia (n = 18). We also investigated the correlation between the p16 expression and HR-HPV status by immunohistochemistry. The results of HR-HPV E6/E7 mRNA ISH showed that 5.8% (14/243) of all CSCC samples were positive for HR-HPV, including 66.7% (12/18) of the anogenital and only 0.9% (2/225) of the nonanogenital CSCC samples (P < 0.01). For the detection of diffuse p16 expression by immunohistochemistry, the sensitivity was 100% (14/14 HR-HPV-positive CSCC samples), and the specificity was 72.1% (165/229 HR-HPV-negative specimens). Thus, HR-HPV E6/E7 mRNA was rarely detected in nonanogenital CSCC, making it unlikely that the virus contributes to the pathogenesis of this malignancy. In addition, p16 immunoreactivity has a limited value as a surrogate marker for transcriptionally active HR-HPV in nonanogenital CSCC.

γH2AX can be a biomarker to distinguish between benign and malignant lesions in bile duct biopsy specimens

Introduction: The aim of this study was to determine whether γH2AX, a sensitive marker of DNA double-strand breaks, can be a biomarker to distinguish between benign and malignant lesions in bile duct biopsy specimens. Method: We examined biopsy specimens of 6 bile duct cancer tissues and 12 noncancerous bile duct tissues (inflammatory tissues, n = 7; normal tissues, n = 5). The diagnoses of these samples were confirmed by postoperative histological examination or clinical follow-up. Immunohistochemical examination of γH2AX, p53, and Ki67 was performed for each specimen. The nuclear staining pattern of γH2AX was classified into two categories: a focal staining pattern, characterized by focus formation in the nucleus, and a diffuse staining pattern, characterized by peripheral-nuclear or pan-nuclear staining. The labeling index (LI) of γH2AX-positive cells with the focal staining pattern, γH2AX-positive cells with the diffuse staining pattern, p53-positive cells, and Ki67-positive cells was calculated. Continuous variables were compared using the Mann-Whitney U test. Result: The LI of the γH2AX focal staining pattern was significantly higher in cancerous tissues than in noncancerous tissues (P < 0.01), while there was no significant difference in the LI of the γH2AX diffuse staining pattern, p53, and Ki67 between cancerous and noncancerous tissues (P = 0.62, 0.17, and 0.29, respectively). In cancerous tissues, the LI of the γH2AX focal staining pattern positively correlated with that of p53 (R = 0.65) but not with that of the γH2AX diffuse staining pattern and Ki67 (R = -0.41 and 0.48, respectively). In cancerous tissues, the LI of the γH2AX diffuse staining pattern had no correlation with that of p53 or Ki67 (R = 0.31 and 0.35, respectively). Conclusion: The LI of γH2AX-positive cells with a focal staining might be a useful biomarker to discriminate between benign and malignant lesions in bile duct biopsy specimens.

Human Papillomavirus-Driven Squamous Lesions: High-Risk Genotype Found in Conjunctival Papillomas, Dysplasia, and Carcinoma.

Human papillomavirus (HPV) is a causative agent for intraepithelial squamous neoplasms, particularly on mucosal surfaces. HPV has a well-established association with squamous cell carcinoma (SCC) of the oropharynx and genital tract, and recent studies suggest a potential role in ocular and periocular squamous neoplasms. Multiple high-risk HPV genotypes are associated with histologically similar squamous neoplasms, and some HPV genotypes have been differentially associated with high- or low-grade lesions. Squamous lesions were screened with immunohistochemical markers p16 and Ki-67 to compare expression in conjunctival papillomas (n = 21) to papillomas with high-grade dysplasia, SCC in situ, and invasive SCC (n = 40). Polymerase chain reaction was performed using the Roche COBAS HPV assay to identify the 14 most common high-risk HPV genotypes. Compared with squamous papillomas, the lesions showing high-grade dysplasia or worse expressed p16 with greater intensity and in a greater percentage of the lesion. A trend toward mild Ki-67 expression in papillomas versus marked Ki-67 expression in high-grade squamous lesions was also observed. HPV-16 was present in 7 of the SCC in situ and invasive SCC lesions but none of the papillomas. HPV may have an important role in squamous lesions of the conjunctiva. In addition to positive polymerase chain reaction results, strong and diffuse p16 expression with marked Ki-67 is strongly suggestive of an HPV-driven lesion.

Next-generation Sequencing Reveals Recurrent Somatic Mutations in Small Cell Neuroendocrine Carcinoma of the Uterine Cervix.

Small cell neuroendocrine carcinoma (SCNEC) of the uterine cervix is a rare but extremely aggressive tumor. While high-risk human papillomavirus (HPV) is involved at an early stage of oncogenesis in many tumors, additional driving events have been postulated to facilitate the progression of SCNECs. Identification of oncogenic drivers could guide targeted therapy of this neoplasm. Clinicopathologic features of 10 cervical SCNECs are reported. Analyses included immunohistochemical evaluation of p16, p53, synaptophysin, and chromogranin expression; in situ hybridizations and polymerase chain reaction for high-risk HPV and/or HPV 18; and next-generation sequencing based on a 637-gene panel. The patients ranged in age from 28 to 68 years (mean, 45.6 y; median, 40.5 y). All tumors had diffuse p16 and synaptophysin expression. All but 1 tumor was positive for chromogranin (extent of staining ranged from focal to diffuse). HPV 18 was detected in 6 tumors and HPV 35 in 1 tumor. At least 1 driver mutation was detected in 8 tumors. Four cases harbored TP53 somatic mutations, 3 of which correlated with an aberrant p53 staining pattern. Four PIK3CA mutations (p.G106A, p.N345T, p.E545K, and p.E545D) were detected in 3 tumors, 2 of which also harbored TP53 mutations. Oncogenic driver mutations involving KRAS, Erbb2, c-Myc, NOTCH1, BCL6, or NCOA3 were detected in 4 tumors. Mutations in caretaker tumor suppressors PTEN, RB1, BRCA1, BRCA2, and ARID1B were also identified in 4 tumors that commonly coharbored activating oncogenic mutations. Targeted next-generation gene sequencing identified genetic alterations involving the MAPK, PI3K/AKT/mTOR, and TP53/BRCA pathways in SCNECs. The presence of genetic alterations that are amenable to targeted therapy in SCNECs offers the potential for individualized management strategies for treatment of this aggressive tumor.

Acral Spitz Nevi: A Clinicopathologic Study of 50 Cases With Immunohistochemical Analysis of P16 and P21 Expression.

Spitz nevi on acral sites are rare and poorly documented. The combination of Spitzoid cytomorphology and atypical architectural features of the junctional component may lead to an erroneous diagnosis of melanoma. To study the clinicopthologic and immunohistochemical features, 50 Spitz nevi localized on the distal extremities were retrieved from departmental files. Clinical data and follow-up were obtained and the histologic features were analyzed. P16 and P21 immunohistochemical staining of the dermal component was compared with that of 10 acral lentiginous melanomas and 10 acral nevi. Acral Spitz nevi affected young adults (median: 24.5 y; range: 4 to 61 y) with a female predominence and a predilection for the feet. They were pigmented measuring 1 to 15 mm (median: 4 mm). Follow-up, available for 45 patients (median: 48 mo; range: 4 to 228 mo), revealed no local recurrence, metastasis or mortality. Histologically, acral Spitz nevi were composed of large epithelioid and/or spindled melanocytes. They were well circumscribed with occasional asymmetry. The junctional component was broad with a lentiginous and nested growth and shoulder formation. Marked pagetoid spread and transepidermal elimination of junctional nests were common features. Focal active regression of the dermal component was frequently noted but there was no nuclear pleomorphism or dermal mitotic activity. Acral Spitz nevi were characterized by strong and diffuse P16 and P21 expression, which differs from acral nevi and acral lentiginous melanoma. Acral Spitz nevi are a distinctive subgroup of Spitz nevi with benign behavior. Awareness of the subtle histologic differentiating features and the distinctive P16/P21 expression pattern allows separation from melanoma.

Normal ve tümöral orofaringeal dokuda in situ hibridizasyon ve p16 ekspresyonu ile human papilloma virüsü varlığının değerlendirilmesi ve klinikopatolojik önemi

Objective: The rise in the number of cancer cases with human papilloma virus (HPV)-positive squamous carcinoma of the oropharynx makes the detection of HPV clinically important. We aimed to investigate the HPV positivity in our patients who have oropharyngeal cancer and compare the two different HPV detection methods, which are HPV in situ hybridization (ISH) and p16 immunohistochemistry (IHC), and show the staining patterns. Methods: Twenty-three specimens of oropharyngeal cancer patients and ten tonsillectomy specimens that revealed no cancerous tissue (control group) were collected from retrospective file analysis. All specimens were evaluated by both p16 IHC and HPV ISH on paraffin blocks. Results: Seven of 23 cases showed p16 expression. Of all these 7 cases that showed p16 expression, six showed high p16 expression and one showed low p16 expression. All six cases that showed high p16 expression were HPV ISH (+). One case that showed low expression of p16 was HPV ISH (-). All cases that were p16 (+) showed diffuse p16 expression and none of the cases showed focal p16 expression. Conclusion: High p16 expression (>70%) is a reliable marker of HPV positivity. Combining p16 IHC with HPV ISH will further improve its specificity. All p16 positive cases showed diffuse p16 expression, thus did not show tumor heterogeneity, suggesting that even a biopsy specimen showing diffuse p16 expression shows p16 positivity of the whole tumoral tissue.